CN114437226A

CN114437226A - Methods of making bispecific antibodies

Info

Publication number: CN114437226A
Application number: CN202011223835.5A
Authority: CN
Inventors: 姜有为; 徐飞; 李发慧; 郑奔; 刘金贵
Original assignee: Hangzhou Genekine Biotech Co ltd
Current assignee: Hangzhou Genekine Biotech Co ltd
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2022-05-06
Also published as: WO2022095977A1

Abstract

The present invention provides a method for increasing the rate of correct heavy and light chain pairing in the production of bispecific antibodies, comprising the step of eliminating the native interchain disulfide bond by amino acid substitution in the CH1-CL domain of one Fab arm, while forming an engineered interchain disulfide bond by amino acid substitution. The method of the invention further comprises the step of reversing the charge of a pair of amino acids of the CH1-CL domain of the Fab arm by amino acid substitution. The method of the invention can obviously improve the correct pairing rate of the heavy chain and the light chain in the production of the bispecific antibody, and is suitable for various antibody types.

Description

Methods of making bispecific antibodies

Technical Field

The invention relates to the field of biotechnology; in particular, the present invention relates to novel methods for making bispecific antibodies and uses thereof.

Background

The recombinant monoclonal antibody (mAb) can be combined with antigen with high efficiency and high specificity, and provides exciting opportunities for the fields of biomedicine and biotechnology. Many therapeutic monoclonal antibodies have been successfully used to treat a variety of diseases, including cancer, immune diseases and infections. Furthermore, bispecific antibodies (bsAbs) targeting more than one antigen have shown great potential, which will maximize the benefits of antibody therapy.

Antibodies are proteins (commonly called immunoglobulins, abbreviated as Ig) that recognize and specifically bind to antigens. In most mammals, including humans and mice, an immunoglobulin consists of two identical heavy chains and two identical light chains. Each timeThe heavy and light chains can be divided into two parts: constant region (C) and variable region (V). Based on the differences in the structure of the constant region of the heavy chain of an antibody, immunoglobulins can be assigned to five classes: IgA, IgD, IgE, IgG and IgM. Each class may have kappa (. kappa.) or lambda (. lamda.) light chains. Based on the heavy chain structure, human IgG can be divided into four subclasses: IgG1, IgG2, IgG3, and IgG 4. Full-length IgG comprises two identical heavy chains and two identical light chains. The amino-terminal (N-terminal) region of the heavy chain is the variable domain (VH), and the remainder is the heavy chain constant domain (CH). The heavy chain constant region of human IgG consists of three domains, CH1, CH2, and CH 3. The region between the CH1 and CH2 domains is called the hinge region. The light chain is the variable region (VL) domain in the amino-terminal half, and the light chain is the constant region (CL) domain in the carboxy-terminal (C-terminal) half. Both the variable and constant regions of the heavy and light chains are structurally folded into functional units called domains. Each heavy chain binds to the light chain via disulfide bonds and non-covalent interactions to form heterodimers. The two light-heavy chain heterodimers are linked by disulfide bonds in the hinge regions of the two heavy chains, forming a complex Y-shaped antibody (fig. 1). Papain digestion of IgG (molecular weight 150kDa) produced two identical fragments retaining antigen binding activity, called Fab fragment (molecular weight 45kDa), and one crystallizable fragment (Fc fragment, molecular weight 50 kDa). Pepsin digestion of IgG produces a protein called F (ab')₂The fragment of (molecular weight 100kDa), consisting of two Fab fragments (FIG. 2). The two arms of the Y-shaped antibody are also referred to as Fab arms. The Fab arm consists of a heavy chain variable region and a CH1 domain with its paired light chain.

IgG1, IgG2, IgG3, and IgG4 showed the greatest sequence diversity in the hinge region (fig. 3), their interchain disulfide bond structures, showing many similarities and differences. The two heavy chains are connected at the hinge region by a different number of interchain disulfide bonds: IgG1 and IgG4 were 2, IgG2 was 4, and IgG3 was 11 interchain disulfide bonds. The light chain of IgG1 was linked to the heavy chain by an interchain disulfide bond between kappa and the cysteine at the C-terminus of the CL domain of the lambda chain (cysteine at position 214 in the kappa light chain [ EU numbering ], cysteine at position 214 in the lambda light chain [ Kabat numbering ]) and the cysteine at the C-terminus of the CH1 domain of the heavy chain (cysteine at position 220 in the heavy chain [ EU numbering ]). Unlike IgG1, the light chain of IgG2, IgG3, or IgG4 was linked to the heavy chain by an interchain disulfide bond between the C-terminal cysteine of the CL domain in the kappa and lambda chains and the N-terminal cysteine of the CH1 domain in the heavy chain (cysteine at position 131 in the heavy chain [ EU numbering ]) (fig. 1, a and B). Although the positions of cysteines in the amino acid sequence differ between IgG1 and other subtypes (IgG2, IgG3, IgG4), their spatial positions are similar to form interchain disulfide bonds. The disulfide bond between the light and heavy chains is important for Fab stability. For a structural and functional review of immunoglobulins, reference may be made to Immunology (Janis Kuby, w.h.freeman and Company, New York, 1997).

Although antibodies are symmetric immunoglobulins with two identical heavy and light chains, bispecific antibodies may be asymmetric structures with two different heavy chains, each of which is associated with a different light chain. Thus, antibodies are bivalent and monospecific and capable of specific binding to two identical antigens simultaneously, but bispecific antibodies are monovalent and bispecific and are capable of binding to two different antigens or epitopes.

Two major difficulties remain to be solved for the efficient production of bispecific antibodies in the intact IgG format. One is how to ensure that heavy chain a against epitope a only pairs with heavy chain b against epitope b (heterodimerization between heavy chains ab) and that homodimerization between two identical heavy chains of the antibody (a and a, b and b) is prevented. Another difficulty is how to ensure that a heavy chain a is only paired with its own light chain a, but not with the light chain b of other epitopes; meanwhile, the heavy chain B is only paired with the light chain B of the user and is not paired with the light chain A. Some methods for generating bispecific antibodies can be found in review articles, Brinkmann U. & Kontermann R.E., The labeling of bispecific antibodies, mAbs,9(2017), 182-.

Antibody heavy chain heterodimerization is usually generated using the so-called "knob-into-hole" (KIH) method. In this method, heavy chain A has a T366W "knob" (knob) mutation in the CH3 domain, while heavy chain B has a T366S/L368A/Y407V "hole" (holes) mutation in the CH3 domain [ EU numbering ]. These mutations create asymmetric, spatially complementary structures in the CH3 domains of both the a and b heavy chains, promoting heavy chain heterodimerization through hydrophobic interactions.

Another approach utilizes electrostatic interactions to promote heavy chain heterodimerization through electrostatic attraction and to avoid homodimerization between identical heavy chains through electrostatic repulsion. There is a charge interaction between the CH3-CH3 domains of the two heavy chains of the antibody, K409 and D399. In this approach, heavy chain a has K409D, K392D mutations in the CH3 domain and heavy chain b has D399K, E356K mutations in the CH3 domain [ EU numbering ]. These amino acid mutations promote heterodimerization of the antibody heavy chains A and B by electrostatic interaction, and inhibit homodimerization between the same heavy chains.

Although antibody heavy chain heterodimerization can be promoted and homodimerization between identical heavy chains can be inhibited by amino acid mutations in the CH3 domain, these approaches fail to avoid the light chain mismatch problem. When two antibody heavy chains form heterodimers, two different light chains can be combined with the heavy chains in four different combinations, only one of which is a correctly paired bispecific antibody, and the other three are mismatched heavy and light chains.

A straightforward approach to avoid the light chain mismatch problem is to use a common light chain. This requires extensive screening work to find a common light chain that can pair with two different heavy chains and retain its high affinity for two different antigens. However, this approach may not be feasible for all antibodies.

Based on rational design, it is possible to develop other methods to achieve the correct pairing of heavy and light chains. The main principle is to change the heavy and light chain domains in one Fab arm while leaving the other Fab arm wild-type, which may facilitate pairing of the light chain with its own heavy chain.

Proper pairing of heavy and light chains can be facilitated by interchanging corresponding domains between the heavy and light chains of the antibody (CrossMab technology), by which wild-type light chains can be paired with wild-type heavy chains and interchanged light chains paired with the corresponding interchange heavy chains. However, domain swapping of the heavy and light chains may be detrimental to the structural stability of the bispecific antibody.

Another approach to the problem of light chain mismatches is to alter the domains of the light and heavy chains based on structural design. The association of the light chain with its own heavy chain is facilitated by altering the interaction between the variable (VH-VL) and constant (CH1-CL) domains of one Fab arm. However, altering the variable domain of the Fab may have a greater effect on its binding to the antigen.

Pairing of the light chain with its own heavy chain can be facilitated by replacing the native interchain disulfide bond in the CH1-CL domain with an engineered disulfide bond. Structural modeling predicts three groups of positions in the CH1-CL domain, where new interchain disulfide bonds may be formed by the introduction of a pair of cysteines. Comparing the three groups of cysteine mutations shows that one group of cysteine mutations can efficiently promote the pairing of the light chain and the heavy chain thereof, and the correct pairing rate reaches 98%. In this set of mutations, phenylalanine at position 126 of the heavy chain (H) and serine at position 121 of the light chain (L) [ EU numbering ] were substituted with cysteine (H: F126C/L: S121C; DuetMab technique). In addition to this group of cysteine mutations, it is currently unknown whether any other cysteine mutation can form a new interchain disulfide bond, facilitating pairing of the light chain with its own heavy chain.

Disclosure of Invention

The invention aims to provide a brand-new preparation method of a bispecific antibody, and the correct pairing rate of a heavy chain and a light chain of the bispecific antibody obtained by the method is obviously improved.

In a first aspect, the present invention provides a method for increasing the correct pairing rate of heavy and light chains during the manufacture of a bispecific antibody, said method comprising the steps of:

in the CH1-CL domain of one Fab arm, the natural interchain disulfide bond is eliminated by amino acid substitution, while the engineered interchain disulfide bond is formed by amino acid substitution.

In a preferred embodiment, the bispecific antibody is derived from any one of IgG, IgA, IgD, IgE and IgM; preferably, derived from an IgG molecule; more preferably, the IgG molecules are derived from human or non-human, e.g. primate or rodent IgG molecules.

In a preferred embodiment, the bispecific antibody is derived from human IgG1, IgG2, IgG3, IgG 4.

In a preferred embodiment, the cysteine is substituted with an amino acid other than cysteine (e.g., a cysteine pair mutated to valine or serine, IgG1 heavy chain C220V, IgG2, IgG3, or IgG4 heavy chain C131S, kappa light chain C214V [ EU numbering ], lambda light chain C214V [ Kabat numbering ]) to eliminate the native interchain disulfide bond, and a cysteine pair is substituted for the amino acid of a pair of non-cysteines to form an engineered interchain disulfide bond.

In a preferred embodiment, the amino acids that form the engineered interchain disulfide bond are located predominantly in the following three regions of the heavy chain domain of IgG 1: F126-T135, G166-T187, K218-S219; the following four regions of the kappa CL domain: S114-S121, N158-T164, T172-T180 and F209-E213.

In particular embodiments, the amino acids in the CH1-CL domain of IgG1 that form the engineered interchain disulfide bond are selected from the following table:

in specific embodiments, the amino acids in the CH1-CL domain of IgG1 that form the engineered interchain disulfide bond are selected from the group consisting of: the amino acid [ EU numbering ] selected in the heavy chain was mutated to cysteine/the amino acid [ EU numbering ] selected in the kappa light chain was mutated to cysteine: F126/F118, L128/P120, A129/P120, P130/F118, S132/F118, K133/S121, S134/F118, G166/T172, G166/S174, G166/T180, H168/T180, F170/Q160, F170/T164, F170/T172, F170/S174, F170/S176, F170/T180, P171/T172, P171/S174, P171/T180, V173/S174, Q175/S176, Q175/T180, S176/S162, S181/T172, S181/S176, S181/T180, S183/S114, S183/F118, S183/Q160, S183/S174, S183/T178, S183/T180, V185/T172, V185/S174, V187/S185, V187/T185, T185/T185, T178/T178, S183/S178, S185/S178, T172, S185/S178, T174, S187/S178, S185/T172, S, S219C/F116C, S219C/F118C, S219C/P120C.

In a preferred embodiment, the amino acids that form the engineered interchain disulfide bond are located predominantly in the following three regions of the heavy chain domain of IgG 1: F126-S136, G166-T187, V215-S219; the following four regions of the lambda CL domain: S114-S121, G158-Q167, K172-S180 and V209-S215.

In particular embodiments, the amino acid residues forming the engineered interchain disulfide bond in the CH1 domain and the corresponding CL domain of IgG1 are selected from the following table:

in specific embodiments, the amino acid residues that form the engineered interchain disulfide bond in the CH1 domain and the corresponding CL domain of IgG1 are selected from the group consisting of: a mutation of a selected amino acid [ EU numbering ] in the heavy chain to cysteine/a selected amino acid [ Kabat numbering ] in the lambda light chain to cysteine;

S132C/S121C、K133C/T116C、K133C/P211C、S136C/S121C、F170C/G158C、P171C/T162C、P171C/P164C、S176C/T162C、L179C/G158C、S181C/P164C、V215C/T116C、E216C/F118C。

in a preferred embodiment, the amino acids that form the engineered interchain disulfide bond are located predominantly in the following two regions of the heavy chain domain of IgG 4: F126-E137, G166-P189; the following four regions of the kappa CL domain: S114-S121, N158-E165, T172-S182 and F209-E213.

In particular embodiments, the amino acids in the CH1-CL domain of IgG4 that form the engineered interchain disulfide bond are selected from the following table:

in specific embodiments, the amino acids in the CH1-CL domain of IgG4 that form the engineered interchain disulfide bond are selected from the group consisting of: the mutation of the selected amino acid [ EU numbering ] in the heavy chain to cysteine/the selected amino acid [ EU numbering ] in the kappa light chain to cysteine; A129/F209, A129/N210, P130/F116, P130/F118, P130/P119, P130/N210, P130/R211, S132/S114, S132/F116, S132/F118, S132/P120, S132/R211, S132/E213, R133/P119, R133/R211, R133/E213, T135/F116, T135/P120, G166/T178, H168/N158, F170/F182, V173/Q160, V173/S162, Q175/T180, S181/T172, S181/S176, S183/N158, S183/S176, V185/E165, V185/T178.

In a preferred embodiment, the amino acids that form the engineered interchain disulfide bond are located predominantly in the following two regions of the heavy chain domain of IgG 4: F126-E137, H168-T187; the following four regions of the lambda CL domain: S114-S121, G158-Q167, K172-S180 and V209-S215.

in a specific embodiment, the method further comprises the steps of: the charge of a pair of amino acids of the CH1-CL domain of the Fab arm is reversed by amino acid substitution.

In particular embodiments, the wild-type positively charged lysine at position 213 of the heavy chain is substituted with a negatively charged amino acid (e.g., K213E, K213D); the wild-type negatively charged glutamate at position 123 of the light chain is substituted with a positively charged amino acid (e.g., E123K, E123R).

In a preferred embodiment, in the production of IgG1 kappa bispecific antibodies, correct pairing of heavy and light chains is achieved by combining charge reversal (e.g., K213D/E123K) with different cysteine pairs to form engineered disulfide bonds in the CH1-CL domain of the Fab arm:

S132C/F116C、V173C/N158C、S131C/P120C、S132C/S114C、S132C/P120C、K133C/F116C、K133C/P120C、S134C/P120C、T135C/S114C、G166C/S176C、G166C/T178C、H168C/T172C、H168C/S174C、H168C/T178C、V173C/T180C、Q175C/N158C、Q175C/T172C、S176C/N158C、S176C/Q160C、G178C/N158C、G178C/S162C、G178C/T164C、S181C/S174C、S183C/P120C、S183C/S162C、V185C/S114C、V185C/P120C、V185C/S176C、T187C/P120C、T187C/S176C、K218C/S114C、K218C/P120C，

(Each pair of cysteines is listed in such a way that the wild type amino acid [ EU numbering ] mutation in the heavy chain is cysteine/wild type amino acid [ EU numbering ] mutation in the kappa chain is cysteine).

In a preferred embodiment, in the production of IgG1 lambda bispecific antibodies, correct pairing of heavy and light chains is achieved by combining charge reversal (e.g. K213D/E123K) with different cysteine pairs to form engineered disulfide bonds in the CH1-CL domain of the Fab arm, including:

L128C/T116C、A129C/P211C、A129C/T212C、P130C/A210C、P171C/T163C、E216C/T116C、P217C/S215C、K218C/F118C、K218C/P119C

(Each pair of cysteine mutations are listed in such a way that the wild type amino acid [ EU numbering ] mutation in the heavy chain is cysteine/wild type amino acid [ Kabat numbering ] mutation in the lambda chain is cysteine.

In particular embodiments, proper pairing of the heavy and light chains can also be achieved by combining charge reversal (e.g., K213D/E123K) with the other cysteine pairs listed above, such that an engineered disulfide bond is formed in the CH1-CL domain of the Fab arm.

In particular embodiments, the formation of the engineered interchain disulfide bond by amino acid substitution and the charge reversal of a pair of amino acid residues of the CH1-CL domain of a Fab arm by amino acid substitution may occur on the same Fab arm or on different Fab arms.

In a preferred embodiment, the method further comprises making one heavy chain having a T366W "knob" mutation in the CH3 domain and a T366S/L368A/Y407V "hole" mutation in the CH3 domain of the other heavy chain; and/or, the heavy chain A CH3 domain has K409D, K392D mutations (EU numbering), and the heavy chain B CH3 domain has D399K, E356K mutations.

In a second aspect, the present invention provides a method for increasing the correct pairing rate of heavy and light chains during the manufacture of a bispecific antibody, said method comprising the steps of: charge-reversing a pair of amino acids of the CH1-CL domain of the Fab arm by amino acid substitution, wherein the wild-type positively charged lysine at position 213 of the heavy chain is substituted with a negatively charged amino acid (e.g., K213E, K213D); the wild-type negatively charged glutamic acid at position 123 of the light chain is substituted with a positively charged amino acid (e.g., E123K, E123R).

In a third aspect, the present invention provides a method of making a bispecific antibody comprising the step of employing the method of the first or second aspect in the manufacture of a bispecific antibody in order to increase the correct pairing rate of the heavy and light chains in the bispecific antibody.

In a fourth aspect, the present invention provides a bispecific antibody prepared by the method of the third aspect, or having an improved correct heavy and light chain pairing rate as defined in the first or second aspect.

In a preferred embodiment, the antibody is a bispecific antibody specifically obtained in the examples.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

These drawings illustrate various aspects of the technology and are not limiting. The drawings are not to scale for clarity and ease of illustration, and in some instances various aspects may be exaggerated or enlarged to facilitate understanding of particular aspects.

FIG. 1 schematic domain diagrams of IgG1 and IgG4 antibodies. IgG antibodies are Y-type tetramers with two heavy chains (longer) and two light chains (shorter). The light chain is linked to the heavy chain by an interchain disulfide bond (-S-S-) in the domains of CL and CH 1. The two heavy chains are joined together at the hinge region by interchain disulfide bonds (-S-S-). VL: light chain variable domain, CL: light chain constant domain, VH: heavy chain variable domain, CH 1: heavy chain constant domain 1, CH 2: heavy chain constant domain 2, CH 3: heavy chain constant domain 3.

FIG. 2 is a schematic representation of the structure of an IgG fragment. Papain digestion of IgG yields two identical Fab fragments (retaining antigen binding activity) and one Fc fragment. Pepsin digestion of IgG to F (ab')₂A fragment consisting of two Fab-like fragments.

FIG. 3 amino acid sequence of hinge region of human IgG1, IgG2, IgG3, and IgG 4. "_" indicates the cysteines that form disulfide bonds between the two heavy chains. Sequences are obtained from the IMGT database (www.imgt.org).

FIG. 4 amino acid sequence comparison and EU numbering of CH1 domains of human IgG1, IgG2, IgG3 and IgG 4. "" indicates sequence identity. ": means that one of the following groups is completely conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, or FYW. "." indicates that one of the following groups is completely conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, or HFY. CH1 sequences of human IgG1(P01857), IgG2(P01859), IgG3(P01860) and IgG4(P01861) were obtained from the Uniprot database (www.uniprot.org). The Clustal Omega program in Uniprot was used for amino acid sequence comparison.

FIG. 5 comparison of the amino acid sequences of CL domains of human antibody light chains kappa (. kappa.) [ EU numbering ] and lambda (. lamda.) [ Kabat numbering ]. "" indicates sequence identity. ": means that one of the following groups is completely conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, or FYW. "." indicates that one of the following groups is completely conserved: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, or HFY. CL sequences for human antibody kappa light chain (P01834) and lambda light chain (P0DOY3) were obtained from the Uniprot database (www.uniprot.org). The Clustal Omega program in Uniprot was used for amino acid sequence comparison.

FIG. 6 schematic representation of bispecific IgG structure. (A) The mutant interchain disulfide bonds in the antibody 1Fab arm and the wild type interchain disulfide bonds in the antibody 2Fab arm are shown. The "knob" mutation in the heavy chain of antibody 1 and the "hole" mutation in the heavy chain of antibody 2 promote heavy chain heterodimerization. RF mutations in the antibody 2 heavy chain were used to remove antibody homodimers in protein a purification. The 6xHis tag at the C-terminus of antibody 2 heavy chain was used for western blotting. (B) The mutant interchain disulfide bonds and charge reversals in the Fab arm of antibody 1 are shown, as well as the wild type interchain disulfide bonds and residue charges in the Fab arm of antibody 2.

FIG. 7 ELISA identifies a library of bispecific IgG1(kappa) cysteine mutations in which the native interchain disulfide bond in the CH1-CL domain of one Fab arm was replaced by an engineered disulfide bond (each pair of cysteine mutations is listed in such a way that the heavy chain wild amino acid [ EU numbering ] is mutated to cysteine/kappa light chain wild amino acid [ EU numbering ] is mutated to cysteine) and WT represents the two native interchain disulfide bonds in the two Fab arms.

FIG. 8 ELISA identifies bispecific IgG1(Kappa) cysteine and charge mutation libraries in which the CH1-CL domains K213/E123 in one Fab arm are reversed to opposite charges and the native interchain disulfide bonds are replaced by engineered disulfide bonds (charge reversals are listed in the following manner: K213[ EU numbering ] mutations in the heavy chain to D or E123[ EU numbering ] mutations in the E/light chain to K or R (K213D/E123K, K213E/E123K, K213D/E123R, K213E/E123R). Each pair of cysteine mutations is listed in the following manner: wild amino acid [ EU numbering ] mutation in the heavy chain to cysteine/wild amino acid [ EU numbering ] in the Kappa light chain to cysteine, WT represents the two native interchain disulfide bonds in both Fab arms.

FIG. 9 ELISA identifies a library of bispecific IgG1(lambda) cysteine mutations in which the native interchain disulfide bond in the CH1-CL domain of one Fab arm was replaced by an engineered disulfide bond (each pair of cysteine mutations is listed in such a way that the heavy chain wild amino acid [ EU numbering ] was mutated to cysteine/lambda light chain amino acid [ Kabat numbering ] was mutated to cysteine, and WT represents the two native interchain disulfide bonds in both Fab arms.

FIG. 10 ELISA identifies bispecific IgG1(lambda) cysteine and charge mutation libraries in which the CH1-CL domains K213/E123 in one Fab arm are reversed to opposite charges and the native interchain disulfide bonds are replaced by engineered disulfide bonds (charge reversals are listed in the following manner: K213[ EU numbering ] mutation in the heavy chain to E123[ Kabat numbering ] mutation in the D/light chain to K (K213D/E123K). Each pair of cysteine mutations is listed in the following manner: heavy chain wild amino acid [ EU numbering ] mutation to cysteine/lambda light chain wild amino acid [ Kabat numbering ] mutation to cysteine, WT represents the two native interchain disulfide bonds in both Fab arms.

FIG. 11 ELISA identifies bispecific IgG1(kappa/lambda) in which the native interchain disulfide bond in the CH1-CL domain of one Fab arm was replaced by an engineered interchain disulfide bond (each pair of cysteine mutations is listed in the following manner: heavy chain wild amino acid [ EU numbering ] mutated to cysteine/kappa light chain wild amino acid [ EU numbering ] mutated to cysteine, WT represents the two native interchain disulfide bonds on both Fab arms.

Figure 12 ELISA identifies bispecific IgG1 in which the CH1-CL domain K213/E123 in one Fab arm was reversed to opposite charges and the natural interchain disulfide bond in the CH1-CL domain of the other Fab arm was replaced by an engineered disulfide bond (charge reversal is listed in the following manner: K213 in the heavy chain was mutated to E123 in the D/light chain to K (K213D/E123K). each pair of cysteine mutations is listed in the following manner: heavy chain wild amino acid [ EU numbering ] was mutated to cysteine/wild amino acid [ EU numbering ] to cysteine, WT indicates that there is a natural interchain disulfide bond in both Fab arms, while no charge reversal mutation is present.

FIG. 13 ELISA identifies a library of bispecific IgG4(kappa) cysteine mutations in which the native interchain disulfide bond in the CH1-CL domain of one Fab arm was replaced by an engineered disulfide bond (each pair of cysteine mutations is listed in the manner that heavy chain wild amino acid [ EU numbering ] mutated to cysteine/kappa light chain wild amino acid [ EU numbering ] mutated to cysteine. K147D/T129R represents a lysine mutation at position 147 to aspartic acid (K147D) in CH1 of one Fab arm and a threonine mutation at position 129 to arginine (T129R) in CL.

Detailed Description

The inventors have conducted extensive and intensive studies to provide a method for preparing a bispecific antibody capable of binding two different antigens and related compositions. In particular, the invention provides a method for producing a bispecific antibody from two existing antibodies, comprising mutating an amino acid at the interface of the heavy and light chain. Such mutations include the substitution of cysteine with other amino acids to eliminate the native interchain disulfide bond, and the substitution of cysteine with other amino acids to form an engineered interchain disulfide bond. On the basis of these mutations, it is also possible to include changes in the charge interactions by amino acid mutations. In the presence of these mutations, bispecific antibodies can be produced in which the heavy chain is preferentially paired with its own light chain and mismatches between heavy and light chains can be prevented. Thus, the present invention has been completed.

Definition of terms

Unless defined otherwise, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art.

As used herein, a term in the singular may use the corresponding term in the plural as appropriate, and vice versa.

As used herein, the term "amino acid" or "amino acid residue" includes natural amino acids and unnatural amino acids.

Amino acids are generally referred to herein by the well-known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, are generally referred to by the accepted single-letter code.

The term "amino acid mutation" herein includes amino acid substitutions, insertions and/or deletions in the polypeptide sequence. As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a polypeptide sequence with another amino acid.

As used herein, the term "natural" or "wild-type" amino acid refers to an amino acid residue that is naturally present at a particular position in a polypeptide and that has not been modified by mutation.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a chain of amino acids of any length. The protein may be produced by any method known in the art for protein synthesis, in particular by chemical synthesis or by recombinant expression techniques.

As used herein, the terms "vector" and "plasmid" in protein expression are used interchangeably.

As used herein, "cell", "cell line" and "cell culture" in the expression of a protein are used interchangeably.

"cell transformation" refers to the introduction of foreign DNA into a cell. Usually as a result of integration of the foreign DNA into the genome or introduction of a self-replicating plasmid.

Transformation and transfection of host cells may be performed according to methods well known to those skilled in the art. Suitable transformation methods include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun techniques, calcium phosphate precipitation, direct microinjection, and the like. The choice of method will generally depend on the type of cell being transformed and the environment in which the transformation is to take place. A general discussion of these methods can be found in the literature (Ausubel, et al, Short Protocols in Molecular Biology, Wiley & Sons, 1995).

Yeast transformation can be performed by various methods including the spheroplast method, electroporation, polyethylene glycol method, alkali metal cation method, etc. (Gregg JM, Pichia Protocols, Totowa, New Jersey: Humanna Press, 2010).

As used herein, the term "antigen" refers to any substance that specifically binds to an antibody. For example, the antigen may be a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing.

As used herein, the term "epitope" refers to a molecular site on an antigen that is recognized and bound by a particular antibody.

As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense. Antibodies are proteins (immunoglobulins) that recognize and specifically bind to antigens. Antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) comprising at least two different epitope binding domains, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, antibody fragments, fusion proteins comprising an antigen-binding portion of an antibody, and any other modified antibody molecule comprising an antigen-binding site. The antibody may be derived from any mammal, including but not limited to humans, monkeys, goats, horses, rabbits, dogs, cats, mice, chickens, camels, sharks, or other animals.

As used herein, the term "recombinant antibody" is intended to include all antibodies produced by a host cell, such as a yeast or CHO cell, transfected with a recombinant expression vector.

Immunoglobulins consist of two identical heavy chains and two identical light chains. Immunoglobulins can be classified into five classes, IgA, IgD, IgE, IgG and IgM, based on their heavy chain constant region structure. Each class may have kappa (. kappa.) or lambda (. lamda.) light chains. Based on their heavy chain structure, human IgG can be divided into four subclasses, IgG1, IgG2, IgG3, IgG 4. The "antibody" of the present invention may be of any class or subclass. Preferably, the antibody of the invention is a human IgG.

Herein, the amino acid sequence numbering of the CH1, CH2, CH3 heavy chain constant regions of human IgG (IgG1, IgG2, IgG3 and IgG4) is by the "EU numbering system" (Edelman GM et al, Proc Natl Acad Sci USA,63(1):78-85 (1969)). IMGT database (

International ImmunoGeneTiCs

) The amino acid sequences of the CH1, hinge, CH2 and CH3 constant regions of human IgG1 are listed in their entirety with the corresponding numbering.

For the human IgG kappa light chain constant region, the amino acid sequence numbering is also used in the "EU numbering system". The IMGT database fully lists the amino acid sequences and corresponding numbering for the human kappa light chain constant region.

For the Human IgG lambda light chain constant region, the amino acid sequence numbering used is the "Kabat numbering system" (Kabat EA et al, sequences of proteins of immunological interest.5th Edition-US Department of Health and Human Services, NIH publication,91-3242 (1991)). The IMGT database lists the human lambda light chain constant region amino acid sequences in their entirety and the corresponding numbering.

Human IgG1 heavy chain constant region and hinge region boundaries are defined in the IMGT database as follows: the CH1 constant region is defined as amino acids 118 to 215, the hinge region is defined as amino acids 216 to 230, the CH2 constant region is defined as amino acids 231 to 340, and the CH3 constant region is defined as amino acids 341 to 447. The amino acid sequence is highly conserved in the CH1 constant regions of IgG1, IgG2, IgG3, and IgG4 (fig. 4). However, unlike the hinge region definition of IMGT, the hinge region on the spatial structure of IgG1 is defined as amino acids 221-. Thus, in the present invention, the CH1 constant region of IgG1 is defined as amino acids 118 to 220, the hinge region is defined as amino acids 221-237, and the CH2 constant region is defined as amino acids 238 to 340[ EU numbering ]. The human kappa light chain constant region is defined as amino acids 108 to 214[ EU numbering ], and the human lambda light chain constant region is defined as amino acids 107A-215[ Kabat numbering ], as shown in FIG. 5.

Kabat lists a number of amino acid sequences of each subtype of antibody and lists the most common amino acid at each position in each subtype, thereby listing conserved sequences. Kabat numbers each amino acid in the listed sequences, and this numbering has become standard in the art. It will be appreciated that due to the presence of allotypic and allelic variations in the population, the wild type amino acid residues at these positions may differ from the amino acids listed, and thus there may be individual amino acid differences between the presented sequence and the prior art sequence.

The position of a particular amino acid can vary among the four IgG subtypes and between IgA, IgD, IgE, and IgM. Thus, a particular amino acid position is not limited to that particular position of that amino acid in an immunoglobulin, but rather, should include all corresponding amino acid positions in an immunoglobulin.

The definition of the corresponding amino acids for each domain of an IgG molecule may vary by one skilled in the art. Thus, the N-or C-terminus of the above domains may be extended or shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9 or even 10 amino acids.

Herein, the mutation of the amino acid is represented by the following method: wild amino acids, amino acid positions, mutated amino acids. Amino acids are indicated by single letter codes, and the positions of the amino acids are determined using the EU numbering system (IgG heavy chain constant region and kappa light chain constant region), and the Kabat numbering system (Lambda light chain constant region). For example, H (C220V) indicates that the wild cysteine at position 220 in the antibody heavy chain (H) was mutated to valine.

The term "full-length IgG" or "intact IgG" of the present invention refers to structurally intact IgG, but it may not have all the functions of IgG. Full-length IgG comprises two heavy chains and two light chains. Each heavy chain binds to the light chain through interchain disulfide bonds and non-covalent interactions, forming heterodimers. The two heavy chains are linked by interchain disulfide bonds at the hinge region.

As used herein, the term "disulfide bond" includes a covalent bond formed between two cysteine residues. A cysteine has a thiol group which can form a disulfide bond with a thiol group of another cysteine. "intrachain disulfide" refers to a disulfide bond formed between two cysteines within the same protein chain. "interchain disulfide" refers to a disulfide bond formed between two cysteines on different chains of the same protein or between two cysteines of different proteins. In the present invention, the terms "cysteine pair" or "cysteine pair" have the same meaning and are thus used interchangeably, both referring to two cysteine residues capable of forming a disulfide bond.

As used herein, the term "protein domain" refers to a portion of a protein that can be folded sterically, has a biological function, and can exist independently of the rest of the protein. Similarly, the term "CH 1-CL domain" as used herein refers to the protein structure of an antibody that is composed of the heavy chain CH1 domain interacting with the light chain CL domain.

As used herein, the term "interface" refers to the region where the separate protein domains are in contact with each other.

The term "antibody mutant" includes antibodies that do not occur in nature, as well as other non-wild type antibodies in which at least one amino acid or amino acid side chain structure differs from the amino acid of a wild-type antibody.

As used herein, the term "antibody mutant" also includes other forms of antibodies that do not naturally occur, such as bispecific antibodies and antibody fragments (e.g., Fab, F (ab') 2, etc.).

The terms "Fab portion", "Fab arm", "Fab" or "arm" are used interchangeably herein.

As used herein, "charge reversal" refers to the replacement of an amino acid residue with a charge that is opposite to the charge of the amino acid residue. For example, the wild-type positively charged lysine at position 213 in the CH1 domain was substituted with a negatively charged amino acid (K213E or K213D); the wild-type negatively charged glutamate at position 123 in the CL domain was substituted with a positively charged amino acid (E123K or E123R).

In some aspects, the antibodies provided herein are bispecific. As used herein, "bispecific antibodies" (bsAbs) are antibodies that have binding specificity for at least two different antigens or at least two different epitopes within the same antigen. Antibodies are symmetric immunoglobulins with two identical heavy and light chains, but bispecific antibodies may be asymmetric with two different heavy chains, each paired with its own light chain. Thus, an antibody is bivalent and monospecific, meaning that it specifically binds to two identical antigens or epitopes simultaneously, but a bispecific antibody is monovalent and bispecific, each Fab arm specifically binding to a different antigen or epitope, respectively.

Bispecific antibodies of the invention and methods of making the same

Based on the KIH technique of heavy chain heterodimerization, we constructed a library of IgG mutants with the following substitutions in the CH1 and CL domains of the first Fab arm: (a) wild cysteine was mutated to other amino acids to eliminate the disulfide bond between heavy and light chains; (b) the predicted wild amino acid pair is mutated to a cysteine pair for forming an engineered interchain disulfide bond; (c) the naturally positively charged lysine at position 213 of the CH1 domain was mutated to a negatively charged amino acid (K213E, K213D) substitution; the naturally negatively charged glutamate mutation at position 123 in the CL domain is substituted with a positively charged amino acid (E123K, E123R). The corresponding amino acids in the CH1 and CL domains of the second Fab arm of the IgG were not mutated (wild type, WT). Like mammalian cells, glycoengineered pichia pastoris can correctly perform cellular biological mechanisms such as protein folding, disulfide bond formation, and glycosylation modification (Ellgaard l.and Helenius a., Quality control in the endoplastic reticulum, nat. rev. mol. cell biol.4(2003) 181-. Thus, libraries of IgG mutants can be expressed in Glycoengineered Pichia pastoris (Glycoengineered Pichia pastoris) and the expressed IgG mutants can be screened by ELISA to determine which cysteine pairs introduced by the mutation can form engineered interchain disulfide bonds, facilitating proper pairing of heavy and light chains in bispecific antibodies.

The bispecific antibody and the preparation method thereof can be applied to various human IgG subtypes (IgG1, IgG2, IgG3 and IgG4) and other classes of Ig. In another aspect, bispecific antibodies of the invention and methods of making the same can be applied to different subclasses of non-human (e.g., primate or rodent) IgG (e.g., murine IgG1, IgG2a, IgG2b, or IgG3 antibodies).

In some aspects, the invention provides methods of producing bispecific antibodies. Bispecific antibodies can be made as full length antibodies or antibody fragments, such as F (ab') 2. In addition, the bispecific antibodies provided herein are readily expressed, stable and low in immunogenicity. The bispecific antibody structures described herein provide a good platform for the generation of bispecific antibodies that can achieve the advantages associated with bispecific antibodies while reducing potential therapeutic risks. In certain aspects, a bispecific antibody can specifically bind two different antigens or two different epitopes on the same antigen. The two Fab arms of a bispecific antibody typically comprise two different variable regions. In some aspects, the binding affinities of the two Fab arms to two independent antigens can be about the same. In some aspects, the binding affinity of the two Fab arms to two independent antigens can be different. In some aspects, the binding affinities of the two Fab arms for two independent epitopes on the same antigen can be about the same. In some aspects, the binding affinities of the two Fab arms to two separate epitopes on the same antigen can be different. In other aspects, the two Fab arms can have the same specificity (e.g., bind the same or overlapping epitopes), but can differ in binding affinity. In some aspects, two antibodies with different in vivo potency can be combined into a bispecific antibody, where one Fab arm has high affinity and the other Fab arm has low affinity, which may prevent overdosing or underdosing of one of the arms.

In certain aspects, provided herein are "cysteine mutant" polypeptides (e.g., heavy and light chains) for use in generating bispecific antibodies and preventing heavy and light chain mismatches. In some aspects, wild cysteines in the heavy and light chains of antibody a to antigen (or epitope) a are mutated to other amino acids to eliminate the native interchain disulfide bonds, and wild amino acids are mutated to cysteines to form engineered interchain disulfide bonds. Such mutations provided herein are located in the CH1 and CL domains. Antibody b heavy and light chains against antigen b have no cysteine mutations. The four polypeptides (heavy chain and light chain) can be polymerized together by cysteine mutation of the heavy chain and light chain of antibody A, so that the heavy chain of antibody A is correctly paired with the light chain, the heavy chain of antibody B is correctly paired with the light chain, and the heavy chain of antibody A is prevented from being mismatched with the light chain of antibody B, and the heavy chain of antibody B is mismatched with the light chain of antibody A. As used herein, the term "cysteine unmutated" polypeptide (e.g., heavy and light chains) means that the heavy and light chains contain wild-type cysteines forming the native interchain disulfide bonds. Such "cysteine mutations" and "unmutated" heavy and light chains may comprise other mutations, e.g., mutations in the Fc region described herein and/or known in the art, to promote heavy chain heterodimerization.

In some aspects, the wild cysteine forming the disulfide bond between CH1 and CL chains in the heavy chain of IgG1, IgG2, IgG3, or IgG4 is mutated to another amino acid. Wild cysteine at position 220 [ EU numbering ] of IgG1 heavy chain was mutated to another amino acid. Wild cysteines at position 131 [ EU numbering ] of the heavy chains of IgG2, IgG3 and IgG4 were mutated to other amino acids. In some aspects, the wild cysteine forming the interchain disulfide bond between CH1 and CL in the IgG kappa and lambda light chains is mutated to another amino acid. IgG kappa light chain 214[ EU numbering ] wild cysteine was mutated to other amino acids. The wild cysteine at position 214[ Kabat numbering ] of the IgG lambda light chain was mutated to another amino acid. In some aspects, such other amino acids include naturally occurring and/or non-canonical amino acids. Other amino acids that occur in nature include glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, methionine, histidine, lysine, arginine, glutamic acid, aspartic acid, glutamine, asparagine, phenylalanine, tyrosine, and tryptophan. Non-classical amino acids include, but are not limited to, ornithine, diaminobutyric acid, norleucine, pyran alanine, thiophene alanine, naphthalene alanine, and phenyl glycine. Preferred other amino acids are valine, serine or alanine.

In some aspects, wild amino acids in the CH1 and CL domains of IgG1(kappa) are mutated to different cysteine pairs. Table 1 summarizes the mutations of wild amino acids to different cysteine pairs in IgG1(kappa) that can form engineered interchain disulfide bonds that promote proper pairing of heavy and kappa light chains in bispecific antibody production.

List 1: IgG1(kappa) cysteine pairs were mutated to form engineered interchain disulfide bonds, facilitating proper pairing of the heavy and kappa light chains.

F126C/F118C、L128C/P120C、A129C/P120C、P130C/F118C、S132C/F118C、K133C/S121C、S134C/F118C、G166C/T172C、G166C/S174C、G166C/T180C、H168C/T180C、F170C/Q160C、F170C/T164C、F170C/T172C、F170C/S174C、F170C/S176C、F170C/T180C、P171C/T172C、P171C/S174C、P171C/T180C、V173C/S174C、Q175C/S174C、Q175C/S176C、Q175C/T180C、S176C/S162C、S181C/T172C、S181C/S176C、S181C/T180C、S183C/S114C、S183C/F118C、S183C/Q160C、S183C/S174C、S183C/T178C、S183C/T180C、V185C/T172C、V185C/S174C、V185C/T178C、V185C/T180C、T187C/S114C、T187C/T172C、T187C/S174C、T187C/T178C、K218C/F118C、S219C/F116C、S219C/F118C、S219C/P120C

(Each pair of cysteine mutations are listed in the following manner: the heavy chain wild amino acid [ EU numbering ] mutation is cysteine/kappa chain wild amino acid [ EU numbering ] mutation is cysteine,. for example, F126C/F118C indicates that F (phenylalanine) at position 126 of the heavy chain is mutated to C (cysteine)/F (phenylalanine) at position 118 of the kappa light chain is mutated to (cysteine).

In some aspects, wild amino acids in the CH1 and CL domains of IgG1(lambda) are mutated to different cysteine pairs. Table 2 summarizes IgG1(lambda) wild amino acids mutated to different cysteine pairs, thereby enabling the formation of engineered interchain disulfide bonds that promote proper pairing of heavy and lambda light chains in bispecific antibody production.

List 2: the IgG1(lambda) cysteine pair was mutated to form engineered interchain disulfide bonds, promoting proper pairing of heavy and lambda light chains.

S132C/S121C、K133C/T116C、K133C/P211C、S136C/S121C、F170C/G158C、P171C/T162C、P171C/P164C、S176C/T162C、L179C/G158C、S181C/P164C、V215C/T116C、E216C/F118C

(Each pair of cysteine mutations are listed in the following manner: heavy chain wild amino acid [ EU numbering ] mutation to cysteine/lambda chain wild amino acid [ Kabat numbering ] mutation to cysteine. for example: S132C/S121C indicates that the S (serine) mutation at position 132 of the heavy chain is to C (cysteine)/S (serine) mutation at position 121 of the lambda light chain is to C (cysteine).

Provided herein are bispecific antibodies having cysteine mutations in the CH1 and CL domains, which mutated antibodies may further comprise one or more mutations in the Fc region described below. An Fc region comprising one or more mutations is referred to herein as a "mutated Fc region". The interface between a pair of antibody Fc can be mutated to promote heavy chain heterodimerization, including but not limited to "KIH" and electrostatic interaction mutations.

In some aspects, provided herein are mutant antibodies having an engineered interchain disulfide bond in the CH1 and CL domains of one Fab arm, and a native interchain disulfide bond in the CH1 and CL domains of the other Fab arm. Typically, the CH1 and CL domains of one Fab arm are mutated as follows: wild cysteine was mutated to other amino acids and wild amino acid was mutated to cysteine, thereby forming a new disulfide bond in the CH1 and CL domains to replace the native disulfide bond. For example, tables 1 and 2 summarize the mutation of IgG1 wild amino acids to a pair of cysteines to form engineered interchain disulfide bonds in the CH1 and CL domains.

In certain aspects, provided herein are "charge mutant" polypeptides (e.g., heavy and light chains) for use in generating bispecific antibodies and preventing mispairing of heavy and light chains. In some aspects, a wild lysine positively charged at position 213[ EU numbering ] of the heavy chain of antibody a against antigen a is mutated to a negatively charged amino acid, e.g., aspartic acid and glutamic acid (K213E, K213D) substitutions. The negatively charged wild-type glutamic acid at position 123 of the antibody A light chain [ EU numbering in kappa, Kabat numbering in lambda ] was mutated to positively charged amino acids, such as lysine and arginine (E123K, E123R). Such mutations provided herein that alter charge polarity are located in the CH1 and CL domains. The antibody, the B heavy and light chains, directed against antigen B, do not have these "charge mutations". The four polypeptides (heavy chain and light chain) can be polymerized together by "charge mutation" of the heavy chain and light chain of antibody A, so that the heavy chain of antibody A is correctly paired with the light chain, the heavy chain of antibody B is correctly paired with the light chain, and the heavy chain of antibody A is prevented from being mismatched with the light chain of antibody B, and the heavy chain of antibody B is mismatched with the light chain of antibody A. As used herein, the term "unmutated" means that the heavy and light chains do not comprise mutations that alter the polarity of the charge of the wild-type amino acids in CH1 and CL as described herein. Such "charge mutated" and "unmutated" heavy and light chains may comprise other mutations, e.g., mutations in the Fc region described herein and/or known in the art, to facilitate heavy chain heterodimerization.

Provided herein are bispecific antibodies having charge mutations in the CH1 and CL domains, which mutated antibodies may further comprise one or more mutations in the Fc region described below (mutated Fc region) to promote heavy chain heterodimerization, including but not limited to "KIH" and electrostatic interaction mutations.

In certain aspects, provided herein are "cysteine and charge mutated" polypeptides (e.g., heavy and light chains) for use in generating bispecific antibodies and preventing heavy and light chain mismatches. In some aspects, the antibody a heavy chain CH1 domain directed to antigen a comprises the following mutations: (a) wild-type cysteine was mutated to other amino acids (e.g., IgG1C 220V; IgG2, IgG3, and IgG 4C 131S) to eliminate the native interchain disulfide bond, and wild-type amino acids were mutated to cysteine (as listed in tables 1, 2, 3, 4, 5) to form an engineered interchain disulfide bond; (b) the wild lysine with a positive charge at position 213 is mutated to a negatively charged amino acid (e.g. K213D, K213E). The CL domain of the antibody nail light chain directed against antigen nail comprises the following mutations: (a) wild-type cysteine was mutated to other amino acids (e.g., kappa and lambda light chain C214V) to eliminate the native interchain disulfide bond, and wild-type amino acids were mutated to cysteine (as listed in tables 1, 2, 3, 4, 5) to form a new interchain disulfide bond; (b) the negatively charged wild-type glutamate at position 123 is mutated to a positively charged amino acid (e.g., E123K, E123R). Antibody b heavy and light chains directed against antigen b do not have these "cysteine and charge mutations". These four polypeptides (heavy and light chains) can be aggregated together by "cysteine and charge mutation" of the heavy and light chains of antibody a, so that the heavy chain of antibody a is correctly paired with its light chain, and the heavy chain of antibody b is correctly paired with its light chain, while preventing the heavy chain of antibody a from being mismatched with the light chain of antibody b, and the heavy chain of antibody b from being mismatched with the light chain of antibody a. As used herein, the term "unmutated" refers to heavy and light chains that do not comprise cysteine and charge mutations in the CH1 and CL domains described herein. Such "cysteine and charge mutations" and "unmutated" heavy and light chains may comprise other mutations, such as mutations in the Fc region described herein and/or known in the art, to promote heavy chain heterodimerization. Table 3 summarizes the IgG1(kappa) wild amino acid mutations to different cysteine pairs that, under the synergistic effect of "charge mutations", form engineered interchain disulfide bonds that promote proper pairing of the heavy and kappa light chains.

List 3: IgG1(kappa) wild amino acid was mutated to different cysteine pairs, which under the synergistic effect of "charge mutations" could form engineered interchain disulfide bonds, promoting the correct pairing of heavy and light chains.

S132C/F116C、V173C/N158C、S131C/P120C、S132C/S114C、S132C/P120C、K133C/F116C、K133C/P120C、S134C/P120C、T135C/S114C、G166C/S176C、G166C/T178C、H168C/T172C、H168C/S174C、H168C/T178C、V173C/T180C、Q175C/N158C、Q175C/T172C、S176C/N158C、S176C/Q160C、G178C/N158C、G178C/S162C、G178C/T164C、S181C/S174C、S183C/P120C、S183C/S162C、V185C/S114C、V185C/P120C、V185C/S176C、T187C/P120C、T187C/S176C、K218C/S114C、K218C/P120C

(Each pair of cysteine mutations are listed in the following manner: heavy chain wild amino acid [ EU numbering ] mutation to cysteine/kappa chain wild amino acid [ EU numbering ] mutation to cysteine).

In some aspects, "cysteine and charge mutations" may be applied to the CH1 and CL domains of IgG1 (lambda). Table 4 summarizes the IgG1(lambda) wild amino acid mutations into different cysteine pairs that, under the synergistic effect of "charge mutations", can form engineered interchain disulfide bonds that promote proper pairing of the heavy and lambda light chains.

List 4: IgG1(lambda) wild amino acid mutations to different cysteine pairs, in the "charge mutations" (e.g., K213D/E123K) under the synergistic effect, can form engineered interchain disulfide bonds, promote the heavy and light chain correct pairing.

(each pair of cysteine mutations are listed in the following manner: heavy chain wild amino acid [ EU numbering ] mutation to cysteine/lambda chain wild amino acid [ Kabat numbering ] mutation to cysteine).

In some aspects, wild amino acids in the CH1 and CL domains of IgG4(kappa) are mutated to a pair of cysteines. Table 5 summarizes the mutation of the wild amino acids into different cysteine pairs in IgG4(kappa) that can form engineered interchain disulfide bonds that promote the correct pairing of IgG4 heavy and kappa light chains.

List 5: IgG4(kappa) cysteine pair mutations formed engineered interchain disulfide bonds in the CH1 and CL domains, facilitating proper pairing of heavy and kappa light chains in bispecific antibody production.

A129C/F209C、A129C/N210C、P130C/F116C、P130C/F118C、P130C/P119C、P130C/N210C、P130C/R211C、S132C/S114C、S132C/F116C、S132C/F118C、S132C/P120C、S132C/R211C、S132C/E213C、R133C/P119C、R133C/R211C、R133C/E213C、T135C/F116C、T135C/P120C、G166C/T178C、H168C/N158C、F170C/F182C、V173C/Q160C、V173C/S162C、Q175C/S162C、Q175C/T180C、S181C/T172C、S181C/S176C、S183C/N158C、S183C/S176C、V185C/E165C、V185C/T178C

(Each pair of cysteine mutations are listed in the following manner: the heavy chain wild amino acid [ EU numbering ] mutation to cysteine/kappa light chain wild amino acid [ EU numbering ] mutation to cysteine).

In some aspects, cysteine pair mutations as listed in tables 1, 2 and 5 can also form engineered interchain disulfide bonds under the synergistic effect of "charge mutations" (e.g., K213D/E123K) that promote proper pairing of heavy and light chains in bispecific antibody production.

In certain aspects, bispecific antibodies provided herein in which the CH1 and CL domains of antibody a have a "cysteine and charge mutation" comprise: (a) wild-type cysteine was mutated to other amino acids to eliminate the native interchain disulfide bond, and wild-type amino acids were mutated to different cysteine pairs (as listed in tables 1, 2, 3, 4 and 5) to form an engineered interchain disulfide bond; (b) wild lysine with positive charge at position 213 in CH1 was mutated to a negatively charged amino acid (e.g. K213D, K213E); the negatively charged wild-type glutamate at position 123 in CL is mutated to a positively charged amino acid (e.g., E123K, E123R). Antibody b does not have these "cysteine and charge mutations" in the CH1 and CL domains. Antibodies a and b may further comprise one or more mutations in the Fc region described below (mutated Fc region) to promote heavy chain heterodimerization, including but not limited to "KIH" and electrostatic interaction mutations.

In some aspects, bispecific antibodies provided herein have cysteine mutations in the CH1 and CL domains of antibody a, and charge mutations in the CH1 and CL domains of antibody b. The CH1 and CL domains of antibody a contain the following mutations: wild-type cysteine was mutated to other amino acids to eliminate the native interchain disulfide bond, and wild-type amino acids were mutated to cysteine pairs (as shown in tables 1, 2, 3, 4 and 5) to form new interchain disulfide bonds. The CH1 and CL domains of antibody b contain the following mutations: wild lysine with positive charge at position 213 in CH1 was mutated to a negatively charged amino acid (e.g. K213D, K213E); the negatively charged wild-type glutamate at position 123 in CL was mutated to a positively charged amino acid (e.g., E123K, E123R). Antibodies a and b may further comprise one or more mutations in the Fc region described below (mutated Fc region) to promote heavy chain heterodimerization, including but not limited to "KIH" and electrostatic interaction mutations.

In an advantageous aspect, the bispecific antibody of the invention is derived from a human IgG molecule. Bispecific antibodies of the invention can generally be produced from any suitable type of immunoglobulin. In another aspect, bispecific antibodies of the invention are derived from non-human (e.g., primate or rodent) IgG molecules (e.g., murine IgG1, IgG2a, IgG2b, or IgG3 antibodies).

The host cell line for antibody expression is preferably a mammalian cell; they have the correct cellular biological mechanisms for steric folding, disulfide bond formation and glycosylation modification. Such mammalian host cells include, but are not limited to: CHO (Chinese hamster ovary), 293 (human kidney), CVI (monkey kidney cell line), COS (CVI with SV 40T antigen), R1610 (Chinese hamster fibroblast), BALBC/3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2/0 (mouse myeloma), and RAJI (human lymphocyte), etc. CHO cells are a particularly preferred expression system. Methods for producing recombinant antibodies in these cells are described in review articles, such as: makrides, S.C, Components of vectors for gene transfer and expression in mammalian cells protein Expr. purify.17 (1999) 183-.

The host cell line used for antibody expression may also preferably be yeast. Pichia pastoris (Pichia pastoris) and glycosyl engineered Pichia pastoris are more preferred host cells. Yeasts include, but are not limited to: pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium graminearum. Like mammalian cells, yeast cells have cellular biological mechanisms for proper steric folding, disulfide bond formation, and glycosylation modification. However, the N-glycosylation machinery of yeast and mammalian cells is not exactly the same. The same N-glycosylation initiation step and modification process are carried out on the nascent peptide chain while the nascent peptide chain is synthesized by the endoplasmic reticulum cavity by the mammalian cells and the yeast cells, firstly, the precursor oligosaccharide G1c₃Man₉GlcNAc₂Is linked to Asn residue in Asn-X-Thr/Ser (X is any amino acid except Pro) conserved sequence of nascent peptide chain, and then under the action of glycoside hydrolase such as glucoside hydrolase I, glucoside hydrolase II and mannoside hydrolase I, the sugar chain of protein is processed to form Man₈GlcNAc₂The sugar chain structure, and then the protein with the sugar chain is transported to the golgi. However, in mammalian cells and yeast Golgi, the further modification of protein sugar chains is completely different. Man on protein in Golgi body of mammalian cell₈GlcNAc₂The sugar chain is firstly removed three mannose under the action of mannoside hydrolase I (MnsI) to form Man₅GlcNAc₂A sugar chain structure; then adding an N-acetylglucosamine under the action of N-acetylglucosamine transferase I (GnTI) to form GlcNAcMan₅GlcNAc₂A sugar chain structure; then under the action of mannoside hydrolase II (MnsII), two more mannoside hydrolases are removedMannose, forming GlcNAcMan₃GlcNAc₂A sugar chain structure; adding an additional N-acetylglucosamine under the action of N-acetylglucosamine transferase II (GnTII) to form GlcNAc₂Man₃GlcNAc₂A sugar chain structure; finally processing to form Gal under the action of galactosyltransferase (GalT) and Sialyltransferase (ST)₂GlcNAc₂Man₃GlcNAc₂And Sia₂Gal₂GlcNAc₂Man₃GlcNAc₂Complex sugar chain structure. But in the Golgi body of yeast cells, under the action of alpha-l, 6-mannosyltransferase (Ochlp) coded by OCH1 gene, Man on the protein₈GlcNAc₂The sugar chain first accepts an alpha-l, 6-mannose to form Man₉GlcNAc₂The sugar chain structure is then subjected to the action of various other mannosyltransferases to continue the addition of mannose to form a high mannose type sugar chain structure (Kornfeld, R).&Kornfeld, S.Assembly of antisense-linked oligonucleotides, Annu.Rev.biochem.54, 631-664,1985). In order to express an antibody having a human N-glycosylation structure using yeast, the yeast needs to be subjected to glycosylation engineering. In addition to deletion of endogenous OCH1 and BMT genes, stable expression of a number of exogenous genes is also desired, including mannosidase I (MnsI), N-acetylglucosamine transferase (GnTI), mannosidase II (MnsII), N-acetylglucosamine transferase (GnTII), galactosyltransferase (GalT), Sialyltransferase (ST), and six biosynthetic genes to supply galactose and sialic acid. Glycosylation engineered Pichia pastoris has been successfully used for antibody production (Li H, Sethuraman N, Stadheim TA, Zha D, Priz B et al (2006) Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24: 210-.

The invention has the advantages that:

1. the method can obviously improve the correct pairing rate of the heavy chain and the light chain in the production of the bispecific antibody;

2. the methods of the invention are applicable to a variety of antibody types, including but not limited to IgG, IgA, IgD, IgE, and IgM; more preferably IgG1, IgG2, IgG3, IgG 4;

3. the method of the invention can directly combine two antibodies into a bispecific antibody.

4. The bispecific antibodies of the invention are purified in a manner similar to that of monoclonal antibodies.

5. The bispecific antibody of the invention has complete immunoglobulin structure, good stability and small immunogenicity.

The technical solution of the present invention is further described below with reference to specific embodiments, but the following examples are not intended to limit the present invention, and all of the various application methods adopted according to the principles and technical means of the present invention belong to the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Material

Chemicals, enzymes, media and solutions used to create, validate and apply libraries are commonly used and well known to those skilled in the art of molecular and cellular biology; they are available from a number of companies, including Thermo Fisher Scientific, Invitrogen, Sigma, New England BioLabs, Takara Biotechnology, Toyobo, TransGen Biotechnology, Vazyme Biotechnology, and general Biotechnology, among others. Many of which are provided in kit form. DNA can be chemically synthesized by some of these companies. Antibody sequence data was mainly from the Uniprot database (www.uniprot.org) and IMGT database (www.imgt.org).

Method

Unless otherwise indicated, the methods used in the present invention, including Polymerase Chain Reaction (PCR), restriction enzyme cloning, DNA purification, bacterial, yeast and eukaryotic cell culture, transformation, transfection and western blotting are performed by standard methods well known to those skilled in the art of molecular and cellular biology. The amino acid mutations (e.g., substitutions, deletions, and insertions) described herein can be made using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis, or cassette mutagenesis (see generally Sambrook J et al (Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Autosubel F M et al. Current Protocols in Molecular Biology, Wiley Interscience, 2010; and Greg JM (Pichia Protocols, Totowa, New Jersey: Humanna Press, 2010).

DH5 alpha competent cells were used for plasmid construction and amplification according to the manufacturer's (Vazyme Biotech) protocol.

The strains were grown in Luria-Bertani (LB) medium (10g/L trypsin, 5g/L yeast extract and 5g/L sodium chloride) or LB plates (10g/L trypsin, 5g/L yeast extract and 5g/L sodium chloride, 20g/L agar) containing the appropriate antibiotics. The following concentrations of antibiotics were added: 100mg/L ampicillin, 50mg/L kanamycin, 25mg/L Zeocin and 100mg/L blasticidin.

Transformation of Pichia strains was performed by electroporation using a MicroPulser (TM) electroporation apparatus according to the manufacturer's protocol (BioRad).

Pichia strains were grown and selected on YPD medium (10g/L yeast extract, 20g/L peptone, 20g/L glucose) and YPD plates (10g/L yeast extract, 20g/L peptone, 20g/L glucose, 20g/L agar). The following concentrations of antibiotics were added: 250mg/L sulfuric acid G-418, 100mg/L Zeocin and 300mg/L blasticidin.

Pichia pastoris vegetative strains were cultured and selected on amino acid free YNB medium (6.7g/L yeast nitrogen base, 20g/L glucose) and amino acid free YNB plates (6.7g/L yeast nitrogen base, 20g/L glucose, 20g/L agar) with appropriate supplementation of amino acids as required.

Pichia strains were grown in BMGY medium (10g/L yeast extract, 20g/L peptone, 100mM potassium phosphate, pH 6.0, 13.4g/L yeast nitrogen base, 0.4mg/L biotin, and 10ml/L glycerol) and antibody expression was then induced in BMMY medium (10g/L yeast extract, 20g/L peptone, 100mM potassium phosphate, pH 6.0, 13.4g/L yeast nitrogen base, 0.4mg/L biotin, and 10ml/L methanol).

Example 1 design of IgG1(Kappa) CH1 and CL Domain "cysteine mutations" library

The CH1 domain sequences of IgG1, IgG2, IgG3, and IgG4 are highly conserved. The light chain is linked to the heavy chain by interchain disulfide bonds in the CH1-CL domain. In the present invention, we wished to replace the natural interchain disulfide bond by an engineered interchain disulfide bond by mutating the CH1-CL domain of IgG. The cysteine mutation sites in the CH1 and CL domains that might interact to form interchain disulfide bonds were analyzed and designed using the Fab crystal structure (PDB code: 1VGE) of human IgG1(kappa) from the protein database as a representative structure. In which the cysteine pairs forming the native interchain disulfide bond were mutated to valine and new cysteine pairs were introduced at different positions of the CH1-CL domain to form engineered interchain disulfide bonds, thereby designing IgG1(kappa) mutant libraries. Table 6 lists the introduction of new cysteine pairs at different amino acid positions in the CH1-CL domain of IgG1(Kappa), which may form engineered interchain disulfide bonds.

TABLE 6 cysteine mutation library of IgG1(kappa) CH1-CL domain. The regions where cysteine mutations aggregate are referred to as a group. The CH1 domain and the CL domain are possibly formed with interchain disulfide bond, and the groups are respectively listed correspondingly.

Example 2 construction and expression of IgG1(Kappa) "cysteine mutation" library

Trastuzumab (Trastuzumab, ERBB2 Ab) heavy chain (HoleRF-His) comprising a T366S/L368A/Y407V "pore" mutation, H435R, Y436F (RF) mutation [ EU numbering ] and a C-terminal 6XHis tag was used as a representation of the IgG1(Kappa) heavy chain (SEQ ID NO: 1). Trastuzumab heavy chain (HolerF-His) codon optimized DNA was synthesized and used as template for PCR amplification (SEQ ID NO: 2).

PCR 1, TraF (SEQ ID NO:3, with Xho I restriction enzyme site) and TraR (SEQ ID NO:4, with Not I restriction enzyme site) primer pairs were used for PCR amplification of trastuzumab heavy chain (HoleRF-His), using the synthesized DNA as template. The PCR product was digested with Xho I and Not I and inserted into the same digestion site in pPIC9(Invitrogen) to construct an expression vector for trastuzumab heavy chain (HoleRF-His), which was designated pPIC9-TraH (HoleRF-His).

The wild-type trastuzumab kappa light chain was used as a representative of the IgG1(kappa) light chain (SEQ ID NO: 5). Trastuzumab kappa light chain codon-optimized DNA was synthesized and used as a template for PCR amplification (SEQ ID NO: 6).

PCR 2, Tra κ F (SEQ ID NO:7, with Xho I restriction enzyme site) and Tra κ R (SEQ ID NO:8, with Not I restriction enzyme site) primer pairs were used for PCR amplification of the trastuzumab Kappa light chain using the synthesized trastuzumab Kappa light chain as a template. The PCR product was digested with Xho I and Not I and inserted into the same digestion site of pPICZ α a (invitrogen) to construct an expression vector for trastuzumab kappa light chain, designated pPICZ α -Tra κ.

An ARG2-ADH1TT fragment (SEQ ID NO:9) of Pichia pastoris ARG 23 'and 5' homologous sequences (with Afe I restriction enzyme site and ADH1 terminator sequence) was synthesized and used as template for PCR amplification.

PCR 3, ARG 2F (SEQ ID NO:10, with BamH I restriction site) and ADH1TT R (SEQ ID NO:11, with BamH I restriction site) primer pairs were used for PCR amplification, with the synthesized ARG2-ADH1TT fragment as template. The PCR product was digested with BamH I restriction enzyme and inserted into the same site in pPICZ α -Tra κ to give pPICZ α -Tra κ -ARG2 expression vector for integration at the Pichia ARG2 locus.

PCR 4, PICZ F (SEQ ID NO:12) and PICZ R (SEQ ID NO:13) primer pairs were used to PCR amplify a pPICZ α linear fragment without Zeocin, with the pPICZ α vector used as the template.

PCR 5, Kan F (SEQ ID NO:14) and Kan R (SEQ ID NO:15) primer pairs were used for PCR amplification of the kanamycin fragment, and pPIC9K vector (Invitrogen) was used as a template.

The kanamycin fragment was inserted into the pPICZ α linear fragment using the ClonExpress II one-step cloning kit (Vazyme) to generate the pEG vector, in which Zeocin in the pPICZ α vector was replaced with kanamycin.

PCR 6, EG F (SEQ ID NO:16) and EG R (SEQ ID NO:17) primer pairs were used for PCR amplification of pEG linear fragments, with the pEG vector used as a template.

The Clazakizumab (IL6 Ab) heavy chain (Knob) containing the T366W "Knob" mutation and the C220V mutation [ EU numbering ] was used as a representation of the IgG1(Kappa) heavy chain (SEQ ID NO: 18). Codon-optimized DNA for the heavy chain of clazakizumab (Knob) was synthesized and used as a template for PCR amplification (SEQ ID NO: 19).

PCR 7, heavy chain N end primer ClaH-Nt (SEQ ID NO:20) and C end primer ClaH-Ct (SEQ ID NO:21) and corresponding reverse primer (R) in Table 7, and corresponding forward primer (F) constitute different primer pairs for PCR amplification of N end and C end heavy chains, using the synthesized clazakizumab heavy chain (Knob) as template.

PCR 8, using a ClaH-Nt and ClaH-Ct primer pair, the N-and C-terminal heavy chain PCR products were ligated by overlap extension PCR. In this way, a clazakizumab heavy chain mutant was generated that contained a wild amino acid mutation to cysteine, a T366W "knob" mutation, and a C220V mutation.

TABLE 7 PCR primers for mutation of wild amino acid to cysteine in Clazakizumab heavy chain [ EU numbering ]

The clazakizumab heavy chain mutant was inserted into the pEG linear fragment using the ClonExpress II one-step cloning kit (Vazyme) to generate a library of expression vectors named pEG-ClaH (X # C) (ClaH: clazakizumab heavy chain, (X # C): number # wild amino acid mutated to cysteine). For example, pEG-ClaH (L128C) indicates that leucine at position 128 of the heavy chain is mutated to cysteine. Each vector can express the clazakizumab heavy chain, which contains a T366W "knob" mutation, a C220V mutation, and the wild amino acid mutation listed in table 6 to cysteine. One vector, pEG-ClaH, expresses clazakizumab heavy chain, which contains a T366W "knob" mutation and a wild cysteine at position 220, which is capable of forming a native interchain disulfide bond.

An ARG4-ADH1TT fragment (SEQ ID NO:53) was synthesized with Pichia pastoris ARG 43 'and 5' homologous sequences, as well as the Sma I restriction enzyme site and ADH1 terminator sequence, and used as a template for PCR amplification.

PCR 9, ARG 4F (SEQ ID NO:54, with a BamH I restriction site) and ADH1TT R (SEQ ID NO:55, with a BamH I restriction site) primer pairs were used to PCR amplify the ARG4-ADH1TT fragment using the synthetic ARG4-ADH1TT fragment as a template. The PCR product was digested with BamH I restriction enzyme and inserted into the same site of pPIC6 α (Invitrogen) to produce a pPIC6 α -ARG4-ADH1TT expression vector, which can be integrated at the pichia pastoris ARG4 locus.

A Clazakizuzumab kappa light chain containing a C214V mutation was used as a representation of an IgG1 kappa (kappa) light chain (SEQ ID NO: 56). Codon-optimized DNA for the light chain of clazakizumab kappa with the C214V mutation was synthesized and used as a template for PCR amplification (SEQ ID NO: 57).

PCR 10, light chain N-terminal primer Cla κ -Nt (SEQ ID NO:58 with Xho I restriction site) and light chain C-terminal primer Cla κ -Ct (SEQ ID NO:59 with Not I restriction site) constituting different primer pairs with the corresponding reverse primer (R) and forward primer (F) in Table 8, respectively, for PCR amplification of N-and C-terminal kappa light chains, with the synthesized clazakizumab kappa chain as a template.

PCR 11, using the primer pair Cla κ -Nt and Cla κ -Ct, ligated the PCR products of the N-and C-terminal kappa light chains by overlap extension PCR. It produced a clazakizumab kappa light chain mutant that contained the wild amino acid mutation to cysteine and the C214V mutation. The C-terminal wild amino acid can be mutated to cysteine by direct PCR amplification of the clazakizumab kappa light chain using Cla κ -Nt and the corresponding reverse primer (R) in table 8. The clazakizumab kappa chain with the wild cysteine at position 214 can be directly PCR amplified using the Cla kappa-Nt and Cla kappa-V214C reverse primers (R).

TABLE 8 PCR primers for mutation of the wild amino acid to cysteine in the Clazakizumab kappa (kappa) light chain [ EU numbering ]

The Clazakizumab kappa light chain mutant was digested with Xho I and Not I, respectively, and inserted into the same digestion site of pPIC6 α -ARG4-ADH1TT to generate a library of kappa light chain mutant expression vectors designated pPIC6-ARG4-Cla kappa (X # C) (Cla kappa: Clazakizumab kappa light chain, (X # C): number # wild amino acid mutated to cysteine). For example, pPIC6-ARG4-Cla κ (S114C) represents a mutation of the wild serine at position 114 of the kappa light chain to cysteine. As shown in table 8, each vector can express a clazakizumab kappa light chain containing a C214V mutation and a wild amino acid mutation to cysteine, and one vector can express a clazakizumab kappa chain with a wild cysteine at position 214.

The method described in Chinese patent application Nos. 201510220631.9, 201710331370.7 and 201810108390.2 is adopted to construct a glycosyl-engineered Pichia pastoris strain GS2-1(his4, PpBMT2-SfMNS1:: och1, ScMNN10-AtMNS1:: pno1) (PpBMT2-SfMNS1 is a fusion protein comprising N-terminal 1-74 amino acids of Pichia pastoris BMT2 and MnsI catalytic domain 164-amino acids of Spodoptera frugiperda, ScMNN10-AtMNS1 is a fusion protein comprising N-terminal 1-116 amino acids of Saccharomyces cerevisiae MNN10 and MnsI catalytic domain 78-560 amino acids of Arabidopsis thaliana). The structure of the protein N-glycans expressed with the GS2-1 strain comprises mainly Man5GlcNAc 2.

The expression vector for pPICZ α -Tra κ -ARG2 was linearized with the restriction enzyme Afe I within the ARG 23 'and 5' homology sequences and electroporated into GS 2-1. The linear expression vector was integrated at the ARG2 locus by recombination of ARG 25 'and 3' homologous sequences. Transformed cells were grown on YPD plates supplemented with 100mg/L Zeocin. This resulted in a novel expression strain GS2-Tra κ, which can express trastuzumab wild-type kappa light chain.

The pPIC9-TraH (HoleRF-His) expression vector was linearized with the restriction enzyme Sal I, electroporated into the GS2-Tra kappa strain, and integrated at the His4 locus. Transformed cells were selected on YNB plates. This resulted in a novel expression strain GS2-TraH κ to express the trastuzumab wild-type light chain and the Fc mutated heavy chain, comprising a T366S/L368A/Y407V "well" mutation, a H435R, Y436F (RF) mutation and a C-terminal 6xHis tag.

Each expression vector of the clazakizumab heavy chain mutant library pEG-ClaH (X # C) was linearized with restriction enzyme Pme I, electroporated into the GS2-TraH kappa strain, and integrated at the AOX1 locus. Transformed cells were selected on YPD plates supplemented with 250mg/L G-418 sulfate. This resulted in a library of expression strains GS2-TraH κ -ClaH (X # C). These strains can express trastuzumab with a wild-type light chain and Fc mutated heavy chain, as well as clazakizumab heavy chain with a cysteine mutation in CH1 and a "knob" mutation in Fc.

Each expression vector of the clazakizumab kappa light chain mutant library pPIC6-ARG4-Cla kappa (X # C) was linearized in ARG 43 'and 5' homology sequences with the restriction enzyme Sma I, and then electroporated into the expression strain library GS2-TraH kappa-ClaH (X # C). The linear expression vector was integrated at the ARG4 locus by recombination of ARG 43 'and 5' homologous sequences. Transformed cells were grown on YPD plates supplemented with 300mg/L blasticidin. This produced an expression strain library GS 2-TraH-. kappa.ClaH (X # C). kappa.X # C. These strains can express asymmetric bispecific antibodies. In asymmetric bispecific antibodies, one half of the bispecific antibody is the clazakizumab heavy and light chains with engineered interchain disulfide bonds in the CH1 and CL domains of the Fab arm, and "knob" mutations in the Fc. The other half is trastuzumab heavy and light chains with native interchain disulfide bonds in the CH1 and CL domains of the Fab arms, a "hole" and RF mutation in the Fc, a 6xHis tag at the C-terminus of the heavy chain,

the GS2-TraH kappa-ClaH (X # C) kappa (X # C) -expressing strain was cultured in BMGY medium at 24 ℃ and 240rpm with shaking for 72 hours. Cells were then pelleted by centrifugation at 3000g for 5 minutes, resuspended in BMMY medium, and cultured with shaking at 24 ℃ and 240rpm, with methanol (1% concentration) added to the medium each day, and expression was induced continuously for 72 hours. Subsequently, the culture supernatant was harvested by centrifugation (3000g, 10 min) and the supernatant was frozen at-20 ℃ until next use.

Example 3 screening identification of IgG1(Kappa) "cysteine mutation" library

The concentration of bispecific antibody in the culture supernatant was measured by ELISA. Briefly, 100. mu.L/well of 5. mu.g/mL AGL protein (Leeanntech), 50mM sodium carbonate buffer (pH 9.6) was added to a 96-well plate (Maxisorp Nunc-Immuno, Thermo Scientific) and coated overnight at 4 ℃. After washing the plates 3 times with PBS-T (PBS containing 0.05% Tween-20), the plates were blocked with 2% skim milk powder in PBS-T for 1 hour at 37 ℃. After washing the plates 3 times with PBS-T, 100. mu.L/well of human IgG standard (starting concentration 2.5ug/ml) was added and the reaction was performed as 1: the culture supernatant was 2-diluted in a gradient and incubated at 37 ℃ for 1 hour. Plates were washed 3 times with PBS-T and added to 100. mu.L/well in PBS-T and 0.5% skim milk as 1: AGL-HRP (Leeanntech) at 5000 dilution. The plates were incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T, and 100. mu.L/well of TMB (Leeanntech) was added. Then 100. mu.L/well of 2M H was added₂SO₄The colorimetric reaction was stopped and the absorbance of each well was measured at 450nm (A450 nm).

Bispecific antibodies were purified by protein a affinity chromatography using MabSelect SuRe resin (GE Healthcare) according to the manufacturer's instructions. Since the RF mutation in trastuzumab heavy chain eliminates its binding to protein a, the well-well heavy chain homodimers and half-antibodies (one well heavy and light chain) and like by-products of trastuzumab can be easily removed from bispecific antibodies in protein a purification. Briefly, the collected supernatant was mixed with MabSelect SuRe resin and shaken at room temperature for 1 hour, then the MabSelect SuRe resin was washed with 25mM sodium phosphate buffer ph7.0, 1M sodium chloride. Bispecific antibody was eluted from MabSelect SuRe resin with 50mM sodium citrate (pH 3.0) and neutralized to pH6.5 with 1M disodium hydrogen phosphate (pH 8.9).

In the present invention, we used an ELISA method to compare whether different interchain disulfide bonds in bispecific antibodies could promote the correct pairing of heavy and light chains. Bispecific antibodies with correctly paired heavy and light chains can bind to the antigen and produce a signal in an ELISA, but bispecific antibodies with mismatched heavy and light chains cannot bind to the antigen and do not produce a signal in an ELISA. Under the same concentration conditions of bispecific antibody, it binds to antigen and produces high signal in ELISA, indicating high correct pairing of heavy and light chains, and conversely low correct pairing of heavy and light chains.

Protein a purified bispecific antibody was purified with PBS as 1: after 2-step dilution, the mixture was divided into two parts, one part for measuring the concentration of the antibody by ELISA and the other part for measuring the binding of the antibody to the antigen by ELISA. ELISA measures the concentration of purified antibody. Briefly, 100. mu.L/well of 5. mu.g/mL AGL protein, 50mM sodium carbonate buffer (pH 9.6) was added to the plate and coated overnight at 4 ℃. After washing the plates 3 times with PBS-T, the plates were blocked with 2% nonfat dry milk in PBS-T for 1 hour at 37 ℃. After washing the plate 3 times with PBS-T, a gradient of 100. mu.L/well PBS diluted purified bispecific antibody was added and incubated at 37 ℃ for 1 hour. The plate was washed 3 times with PBS-T and 100. mu.L/well AGL-HRP diluted 1:5000 in PBS-T was added. The plate was incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T, and 100. mu.L/well of TMB was added. Then 100. mu.L/well 2M H was added₂SO₄The colorimetric reaction was stopped and the absorbance of each well was measured at 450nm (A450 nm). The concentration of bispecific antibody can be expressed by the absorbance value measured by ELISA reaction corresponding to the concentration.

ELISA measures the binding of antibodies to antigens. Briefly, 100. mu.L/well of 1. mu.g/mL human HER2/ErbB2 protein (His-tag) (Nano Biological), 50mM sodium carbonate buffer (pH 9.6) was added to the plate and coated overnight at 4 ℃. After washing the plates 3 times with PBS-T, the plates were blocked with 2% skim milk in PBS-T for 1 hour at 37 ℃. After washing the plate 3 times with PBS-T, a gradient of 100. mu.L/well PBS diluted purified bispecific antibody was added and incubated at 37 ℃ for 1 hour. Plates were washed 3 times with PBS-T, and 100. mu.L/well was added to make 1: AGL-HRP at 5000 dilution. The plate was incubated at 37 ℃ for 1 hour, washed 3 times with PBS-T, and 100. mu.L/well of TMB was added. Then 100. mu.L/well 2M H was added₂SO₄The colorimetric reaction was stopped and the absorbance of each well was measured at 450nm (A450 nm). The amount of bispecific antibody bound to the antigen can beExpressed as the absorbance value (A450nm) measured by the ELISA reaction corresponding thereto.

In asymmetric bispecific antibodies, half of the bispecific antibodies have trastuzumab heavy and light chains with natural interchain disulfide bonds in the Fab arm CH1 and CL domains, a "pore" and RF mutation in the heavy chain Fc, a 6xHis tag at the C-terminus of the heavy chain, and the other half contains clazakizumab heavy and light chains with engineered interchain disulfide bonds in the Fab arm CH1 and CL domains, and a "knob" mutation in the heavy chain Fc. According to reports, when phenylalanine at position 126 in clazakizumab heavy chain CH1 is mutated to cysteine (F126C) and serine at position 121 in light chain CL is mutated to cysteine (S121C), an engineered interchain disulfide bond can be formed, and the asymmetric bispecific antibody can have 98% of the heavy chain and the light chain correctly paired. This asymmetric bispecific antibody was designated herein as TraH κ -ClaH (F126C) κ (S121C), abbreviated F126C/S121C, and used as a positive control. However, when both Fab arms have a native interchain disulfide bond, only 25% of the heavy and light chains of the bispecific antibody formed are correctly paired. The bispecific antibody is designated herein as TraH-. kappa. -ClaH-. kappa.abbreviated as WT and used as a negative control (Yariv Mazor, Vaheh Oganysian, Chunning Yang, Anna Hansen, Jihong Wang, Hongji Liu, Kris Sachsenseier, Marcia Carlson, Dhanesh V Gadre, Martin Jack Borrak, Xiang-Qing Yu, William Dall' Acqua, Herren Wu, and Partha Sarathi Chowdour, bs mA 7,377-389; 2015).

Bispecific antibodies with correctly paired heavy and light chains can bind to the antigen and produce a signal in an ELISA, but bispecific antibodies with mismatched heavy and light chains cannot bind to the antigen and do not produce a signal in an ELISA. The concentration of bispecific antibody was expressed by horizontal axis ELISA absorbance value (a450nm) and the amount of bispecific antibody bound to antigen was expressed by vertical axis ELISA absorbance value (a450nm), and the data was processed with Excel to obtain a scatter plot. Within the range of effective ELISA color values, the higher the curve position, i.e., the higher the color value of the ELISA reaction of the bispecific antibody with the same amount of antigen under the same concentration condition, the higher the correct pairing rate of the heavy chain and the light chain of the bispecific antibody, and the lower the mismatch rate. The lower the curve position, the lower the correct pairing rate of the heavy and light chains of the bispecific antibody, and the higher the mismatch rate. As shown in fig. 7, the positive control F126C/S121C for the reported bispecific antibody had 98% of the correct heavy and light chain pairing and thus showed a very high antibody/antigen binding ELISA absorbance value (vertical axis) under the same antibody concentration conditions (horizontal axis ELISA absorbance value), whereas the negative control WT for the bispecific antibody was expected to have only 25% of the correct heavy and light chain pairing and thus showed a very low antibody/antigen binding ELISA absorbance value. Under the same antibody concentration conditions (cross-axis ELISA absorbance values), many bispecific antibodies we constructed had the same or higher antibody/antigen binding absorbance values as the positive control F126C/S121C. This indicates that our bispecific antibodies have 98% or more of the heavy and light chains correctly paired. Thus, in IgG1 kappa bispecific antibody production, correct heavy and light chain pairing can be achieved by replacing the native interchain disulfide bonds with different cysteine pairings, forming engineered disulfide bonds in the CH1-CL domain of one Fab arm, including:

F126C/F118C、L128C/P120C、A129C/P120C、P130C/F118C、S132C/F118C、K133C/S121C、S134C/F118C、G166C/T172C、G166C/S174C、G166C/T180C、H168C/T180C、F170C/Q160C、F170C/T164C、F170C/T172C、F170C/S174C、F170C/S176C、F170C/T180C、P171C/T172C、P171C/S174C、P171C/T180C、V173C/S174C、Q175C/S174C、Q175C/S176C、Q175C/T180C、S176C/S162C、S181C/T172C、S181C/S176C、S181C/T180C、S183C/S114C、S183C/F118C、S183C/Q160C、S183C/S174C、S183C/T178C、S183C/T180C、V185C/T172C、V185C/S174C、V185C/T178C、V185C/T180C、T187C/S114C、T187C/T172C、T187C/S174C、T187C/T178C、K218C/F118C、S219C/F116C、S219C/F118C、S219C/P120C、

(Each pair of cysteine mutations are listed in the following manner: the heavy chain wild amino acid [ EU numbering ] mutation to cysteine/kappa light chain wild amino acid [ EU numbering ] mutation to cysteine)

Example 4 construction and expression of IgG1(Kappa) "cysteine and Charge mutation" library

In the IgG1(Kappa) CH1 and CL domains, a pair of charged amino acids (K213 in the heavy chain and E123 in the Kappa light chain, EU numbering) participate in charge-charge interactions. This pair of amino acids is conserved among IgG1, IgG2, IgG3 and IgG4, and works the same. At neutral pH (pH 7.0), aspartic acid (D) and glutamic acid (E) are negatively charged, and lysine (K), arginine (R), and histidine (H) are positively charged. Amino acids of opposite charge may interact attractively, while amino acids of similar charge may interact repulsively. As shown in Table 9, mutations in the CH1 and CL domains of antibody A heavy and light chains that invert the charge polarity of the amino acids (e.g., K213D and E123K) may result in unfavorable mutual repulsion between the mismatched heavy and light chains (e.g., the negative charge of the antibody A heavy chain K213D mutation is repulsive to the negative charge of antibody B light chain E123; the positive charge of the antibody A light chain E123K mutation is repulsive to the positive charge of antibody B heavy chain K213). Introduction of mutated interchain disulfide bonds or/and charge reversals in the CH and CL domains may facilitate proper pairing of heavy and light chains in bispecific antibodies.

TABLE 9 mutation of charge polarity reversal in heavy and light chains.

PCR 1, EG F and EG R primer pairs were used to PCR amplify the pEG linear fragment (as described in example 2) using the pEG vector as a template.

PCR 2 was performed using ClaH-Nt and ClaH-K213D R primer pairs (SEQ ID NO:84), ClaH-K213D F (SEQ ID NO:85) and ClaH-Ct primers for PCR amplification of the N-and C-terminal heavy chains, respectively, using the expression vectors of the clazakizumab mutant library pEG-ClaH (X # C) as templates. The N-and C-terminal heavy chains were connected by overlap extension PCR using a ClaH-Nt and ClaH-Ct primer pair. This resulted in a mutated clazakizumab heavy chain comprising a T366W "knob" mutation, a C220V mutation, a cysteine mutation, and a K213D charge mutation.

PCR 3, PCR amplification of the N-terminal and C-terminal heavy chains was performed using ClaH-Nt and ClaH-K213D R primer pairs, ClaH-K213E F (SEQ ID NO:86) and ClaH-Ct primer pairs, respectively, using each expression vector of the clazakizumab heavy chain mutant library pEG-ClaH (X # C) as a template. The N-terminal and C-terminal heavy chains were connected by overlap extension PCR using a ClaH-Nt and ClaH-Ct primer pair. This resulted in a mutated clazakizumab heavy chain comprising a T366W "knob" mutation, a C220V mutation, a cysteine mutation, and a K213E charge mutation.

The mutated clazakizumab heavy chain was inserted into the pEG linear fragment using ClonExpress II one-step cloning kit (Vazyme) to construct a library of clazakizumab mutation expression vectors, named pEG-ClaH (X # CD) and pEG-ClaH (X # CE). Each vector can express a mutated clazakizumab heavy chain comprising a T366W "knob" mutation, a C220V mutation, a wild amino acid mutation to cysteine, and a K213D or K213E mutation.

PCR 4, N-terminal and C-terminal Kappa light chains were PCR amplified using the corresponding primer pairs in Table 10-1, respectively, using the expression vectors pPIC 6. alpha. -Cla. Kappa. (X # C) of the Kappa light chain mutant library as templates. The N-and C-terminal kappa chains were ligated by overlap extension PCR using a Cla kappa-Nt and Cla kappa-Ct primer pair. This resulted in a mutated clazakizumab kappa chain comprising the C214V mutation, the cysteine mutation and the E123K mutation.

TABLE 10-1 Clazakizumab light chain N-and C-terminal PCR amplification primer pairs

PCR 5, N-terminal and C-terminal Kappa light chains were PCR amplified using the corresponding primer pairs in Table 10-2, respectively, using the expression vector pPIC6 alpha-Cla Kappa (X # C) of each Kappa light chain mutant library as a template. The N-and C-terminal kappa chains were ligated by overlap extension PCR using a Cla kappa-Nt and Cla kappa-Ct primer pair. This resulted in a mutated clazakizumab kappa chain comprising the C214V mutation, the cysteine mutation and the E123R mutation.

TABLE 10-2 Clazakizumab light chain N-and C-terminal PCR amplification primer pairs

The mutated clazakizumab kappa chain was digested with Xho I and Not I and inserted into the same digestion site of pPIC6 α -ARG4 to generate a library of kappa light chain mutation expression vectors designated pPIC6 α -ARG4-Cla κ (X # CK) and pPIC6 α -ARG4-Cla κ (X # CR). Each vector may express a mutated clazakizumab kappa light chain comprising a C214V mutation, a cysteine mutation, and an E123K or E123R mutation.

Each of the expression vectors pEG-ClaH (X # CD) and pEG-ClaH (X # CE) of the clazakizumab heavy chain mutant library was linearized with restriction enzyme Pme I, electroporated into the GS2-TraH κ strain, and integrated at the AOX1 locus. Transformed cells were selected on YPD plates supplemented with 250mg/L G-418 sulfate. This resulted in the production of expression strain libraries GS2-TraH κ -ClaH (X # CD), GS2-TraH κ -ClaH (X # CE).

Each expression vector of the clazakizumab kappa light chain mutation library pPIC6 alpha-ARG 4-Cla kappa (X # CK) and pPIC6 alpha-ARG 4-Cla kappa (X # CR) was linearized with Sma I and electroporated into expression strain libraries GS2-TraH kappa-ClaH (X # CD) and GS2-TraH kappa-ClaH (X # CE). The linear expression vector is integrated at the ARG4 locus. Transformed cells were grown on YPD plates supplemented with 300mg/L blasticidin. Thus, expression strain libraries GS2-TraH κ -ClaH (X # CD) κ (X # CK), GS2-TraH κ -ClaH (X # CD) κ (X # CR), GS2-TraH κ -ClaH (X # CE) κ (X # CK), and GS2-TraH κ -ClaH (X # CE) κ (X # CR) were generated. These strains can express asymmetric bispecific antibodies. In asymmetric bispecific antibodies, one half of the bispecific antibody is the clazakizumab heavy and light chains, with the CH1 and CL domains of the Fab arms having engineered interchain disulfide bonds and/or a pair of charge mutations, with a "knob" mutation in the Fc; the other half is trastuzumab heavy and light chains with native interchain disulfide bonds in the CH1 and CL domains of the Fab arms, a "hole" and RF mutation in the Fc, and a 6xHis tag at the C-terminus of the heavy chain.

The expression strains were cultured in BMGY medium and antibody expression was induced in BMMY medium as described in example 2. The supernatant harvested by centrifugation was frozen at-20 ℃ until next use.

Example 5 screening identification of IgG1(Kappa) "cysteine and Charge mutation" libraries

The bispecific antibody was purified by protein a affinity chromatography and the concentration of the purified antibody was determined by ELISA as described in example 3.

As described in example 3, ELISA was used to compare whether different interchain disulfide bonds and charge mutations in bispecific antibodies comprising a novel pair of cysteines and charge reversal mutations (e.g., K213D and E123K) in the CH1 and CL domains could promote correct pairing of heavy and light chains. As shown in fig. 8A, under the same antibody concentration conditions (horizontal axis ELISA absorbance values), the bispecific antibody positive control F126C/S121C had 98% of the heavy and light chains correctly paired, with very high antibody/antigen binding ELISA absorbance values (vertical axis), whereas the bispecific antibody negative control WT had only 25% of the heavy and light chains correctly paired, with very low antibody/antigen binding absorbance values (vertical axis). Under the same antibody concentration conditions (horizontal axis ELISA absorbance values), two bispecific antibodies comprising charge reversals (K213D/E123K, K213E/E123K) in the CH1-CL domain of the Fab arm had higher antibody/antigen binding absorbance values than the positive control (F126C/S121C). However, the antibody/antigen binding absorbance values of the other two bispecific antibodies comprising charge reversals (K213D/E123R, K213E/E123R) in the CH1-CL domain of the Fab arm were significantly lower than the positive control F126C/S121C. Thus, in IgG bispecific antibody production, correct pairing of heavy and light chains can be facilitated by the use of charge reversal mutations (e.g., K213D/E123K, K213E/E123K) (each pair of charge reversal mutations is listed in such a way that lysine K at position 213 of the heavy chain is mutated to a negatively charged amino acid D or glutamic acid E at position 123 of the E/light chain is mutated to a positively charged amino acid K or R).

To evaluate the effect of a novel pair of cysteines in the CH1 and CL domains of a Fab arm in combination with charge reversal on the correct pairing of heavy and light chains, a bispecific antibody TraH κ -ClaH (S132C) κ (F116C), abbreviated as S132/F116C and the following four bispecific antibodies with any mutations were used:

TraH kappa-ClaH (S132C, K213D) kappa (F116C, E123K), abbreviated as S132C, K213D/F116C, E123K;

TraH kappa-ClaH (S132C, K213E) kappa (F116C, E123K), abbreviated as S132C, K213E/F116C, E123K;

TraH kappa-ClaH (S132C, K213D) kappa (F116C, E123R), abbreviated as S132C, K213D/F116C, E123R,

TraH kappa-ClaH (S132C, K213E) kappa (F116C, E123R), abbreviated as S132C, K213E/F116C, E123R.

As shown in fig. 8B, the bispecific antibody S132C/F116C containing a new pair of cysteines in the CH1-CL domain of one Fab arm had similar antibody/antigen binding absorbance values as the positive control F126C/S121C under the same antibody concentration conditions (horizontal axis ELISA absorbance values). 4 bispecific antibodies S132C, K213D/F116C, E123K, S132C, K213E/F116C, E123K, S132C, K213D/F116C, E123R, S132C, K213E/F116C, E123R containing novel cysteine pairings and charge reversals can further increase the antibody/antigen binding absorbance values.

To evaluate the effect of a new pair of cysteines and charge reversals in the CH1 and CL domains of one Fab arm on the correct pairing of the heavy and light chains, a bispecific antibody (TraH κ -ClaH (V173C) κ (N158C), abbreviated as V173C/N158C and the following four bispecific antibodies were used as further examples:

TraH kappa-ClaH (V173C, K213D) kappa (N158C, E123K), abbreviated as V173C, K213D/N158C, E123K;

TraH kappa-ClaH (V173C, K213E) kappa (N158C, E123K), abbreviated as V173C, K213E/N158C, E123K;

TraH kappa-ClaH (V173C, K213D) kappa (N158C, E123R), abbreviated as V173C, K213D/N158C, E123R;

TraH kappa-ClaH (V173C, K213E) kappa (N158C, E123R), abbreviated as V173C, K213E/N158C, E123R.

As shown in fig. 8C, bispecific antibody V173C/N158C containing a novel pair of cysteines in the CH1-CL domain of one Fab arm had lower antibody/antigen binding absorbance values than the positive control F126C/S121C under the same antibody concentration conditions (horizontal axis ELISA absorbance values). Two bispecific antibodies V173C, K213D/N158C, E123K containing a novel cysteine pair and charge reversal; V173C, K213E/N158C, E123K had higher antibody/antigen binding absorbance values than the positive control F126C/S121C. The other two bispecific antibodies containing novel cysteine pairings and charge reversals V173C, K213D/N158C, E123R; V173C, K213E/N158C, E123R) had higher antibody/antigen binding absorbance values than bispecific antibody V173C/N158C, but lower antibody/antigen binding absorbance values than the positive control F126C/S121C. These examples show that charge reversal mutations in the heavy chain K213 and light chain E123 in one Fab arm CH1-CL domain can further improve the correct pairing of heavy and light chains of bispecific antibodies with cysteine pair mutations. It appears that the charge inversion of K213D/E123K and K213E/E123K has a significantly greater improvement effect than the charge inversion of K213D/E123R, K213E/E123R.

In the present invention, the charge reversal of K213D/E123K in the CH1-CL domain of one Fab arm was applied to improve the correct pairing of the heavy and light chains of other bispecific antibodies with different cysteine mutations. As shown in fig. 8D, the bispecific antibody containing a cysteine mutation in the CH1-CL domain of one Fab arm had similar or lower antibody/antigen binding absorbance values as the positive control F126C/S121C under the same antibody concentration conditions (horizontal axis ELISA absorbance values). The corresponding bispecific antibody containing cysteine and the charge mutation of K213D/E123K had higher antibody/antigen binding absorbance values than the positive control F126C/S121C. Thus, in the production of IgG kappa bispecific antibodies, to further facilitate the correct pairing of heavy and light chains to form engineered disulfide bonds, the charge mutations of K213D/E123K and the different cysteine pair mutations can be combined in the CH1-CL domain of one Fab arm, including but not limited to:

(Each pair of cysteine mutations are listed in the following manner: the heavy chain wild amino acid [ EU numbering ] mutation to cysteine/kappa light chain wild amino acid [ EU numbering ] mutation to cysteine, EU numbering).

If necessary, the different cysteine pair mutations identified in example 3 and the K213D/E123K charge mutations can be combined together in the CH1-CL domain of one Fab arm in order to further facilitate the correct pairing of the heavy and light chains to form engineered disulfide bonds.

Example 6 design of IgG1(lambda) CH1 and CL Domain "cysteine mutation" library

The IgG lambda light chain is linked to the heavy chain by an interchain disulfide bond between the CH1 and CL domains. In the present invention, we mutated the CH1-CL domain of IgG (lambda), mutated the cysteine pair that forms the native interchain disulfide bond to valine, and introduced a new cysteine pair to form the engineered interchain disulfide bond, replacing the native interchain disulfide bond. We used the Fab crystal structure (PDB code: 2FB4) of human IgG1(lambda) from the protein database as a representative structure to analyze and design cysteine mutation sites in the CH1 and CL domains that might interact to form interchain disulfide bonds. These cysteine mutation sites in the CH1 and CL domains are listed in table 11 for the construction of an IgG1(lambda) "cysteine mutation" library.

TABLE 11 cysteine mutagenesis library of IgG1(lambda) CH1-CL domain. The regions where cysteine mutations aggregate are referred to as a group. The CH1 domain and the CL domain are possibly formed with interchain disulfide bond, and the groups are respectively listed correspondingly.

Example 7 construction and expression of IgG1(lambda) cysteine mutation library

The heavy chain of Fezakinumab (IL22 antibody) comprising a T366S/L368A/Y407V "pore" mutation, H435R, Y436F (RF) mutation and a 6XHis tag at the C-terminus (HoleRF-His) was used as a representation of the IgG1 heavy chain (SEQ ID NO: 102). Fezakinumab heavy chain codon optimized DNA (HoleRF-His) was synthesized and used as a template for PCR amplification (SEQ ID NO: 103).

PCR 1, FezH F (SEQ ID NO:104, primers with Xho I restriction enzyme site) and FezH R (SEQ ID NO:105, primers with Not I restriction enzyme site) primer pairs were used for PCR amplification of the Fezakinumab heavy chain (HoleRF-His), using the synthesized DNA as template. The PCR product was digested with Xho I and Not I, and inserted into the same digestion site of pPIC9(Invitrogen) to construct an expression vector for the Fezakinumab heavy chain (HoleRF-His), which was designated pPIC9-FezH (HoleRF-His).

The Fezakinumab light chain was used as a representative IgG1 lambda (. lamda.) light chain (SEQ ID NO: 106). Codon-optimized DNA for the Fezakinumab lambda light chain (SEQ ID NO:107) was synthesized and used as a template for PCR amplification.

PCR 2Fez λ F (SEQ ID NO:108, with Xho I restriction enzyme site) and Fez λ R (SEQ ID NO:109, with Not I restriction enzyme site) primer pairs were used for PCR amplification of the fezakinumab lambda light chain, using the synthesized fezakinumab light chain as template. The PCR product was digested with Xho I and Not I and inserted into the same digestion site of pPICZ α -Tra κ -ARG2 (as described in example 2) to construct an expression vector for the fezakinumab light chain, designated pPICZ α -Fez λ -ARG 2.

PCR 3, EG F and EG R primer pairs were used to PCR amplify the pEG linear fragment with the pEG vector as template.

The otelixizumab (CD3 mab) heavy chain (knob) containing the T366W "knob" mutation and the C220V mutation was used as another representation of the IgG1 heavy chain (SEQ ID NO: 110). Codon-optimized DNA for the otelixizumab heavy chain (knob) was synthesized and used as template for PCR amplification (SEQ ID NO: 111).

In PCR 4, the heavy chain N-terminal primer OteH-Nt (SEQ ID NO:112) and the C-terminal primer OteH-Ct (SEQ ID NO:113) and the corresponding reverse primer (R) and forward primer (F) in Table 12 respectively form different primer pairs for PCR amplification of N-terminal and C-terminal heavy chains, and synthesized otelixizumab heavy chains (knobs) are used as templates.

PCR 5, PCR products of the N-and C-terminal heavy chains were ligated by overlap extension PCR using the OteH-Nt and OteH-Ct primer pairs. In this way, an otelixizumab heavy chain mutant was generated comprising a wild amino acid mutation to cysteine, a T366W "knob" mutation and a C220V mutation.

The otelixizumab heavy chain mutant was inserted into the pEG linear fragment using the ClonExpress II one-step cloning kit (Vazyme) to generate an expression vector library named pEG-OteH (X # C) (OteH: oteliximab heavy chain, (X # C): wild amino acid at position # was mutated to cysteine).

Table 12 PCR primers for the mutation of the wild amino acid of the otecixizumab heavy chain to cysteine (EU numbering).

Mutations	Reverse primer (R)	Forward primer (F)
			OteH-F126C	OteH-F126C R(SEQ ID NO:114)	OteH-F126C F(SEQ ID NO:115)
OteH-L128C	OteH-F126C R(SEQ ID NO:114)	OteH-L128C F(SEQ ID NO:116)
			OteH-A129C	OteH-F126C R(SEQ ID NO:114)	OteH-A129C F(SEQ ID NO:117)
OteH-P130C	OteH-F126C R(SEQ ID NO:114)	OteH-P130C F(SEQ ID NO:118)
			OteH-S131C	OteH-F126C R(SEQ ID NO:114)	OteH-S131C F(SEQ ID NO:119)
OteH-S132C	OteH-F126C R(SEQ ID NO:114)	OteH-S132C F(SEQ ID NO:120)
			OteH-K133C	OteH-F126C R(SEQ ID NO:114)	OteH-K133C F(SEQ ID NO:121)
OteH-S134C	OteH-F126C R(SEQ ID NO:114)	OteH-S134C F(SEQ ID NO:122)
			OteH-T135C	OteH-F126C R(SEQ ID NO:114)	OteH-T135C F(SEQ ID NO:123)
OteH-S136C	OteH-F126C R(SEQ ID NO:114)	OteH-S136C F(SEQ ID NO:124)
			OteH-G166C	OteH-G166C R(SEQ ID NO:125)	OteH-G166C F(SEQ ID NO:126)
OteH-H168C	OteH-G166C R(SEQ ID NO:125)	OteH-H168C F(SEQ ID NO:127)
			OteH-F170C	OteH-G166C R(SEQ ID NO:125)	OteH-F170C F(SEQ ID NO:128)
OteH-P171C	OteH-G166C R(SEQ ID NO:125)	OteH-P171C F(SEQ ID NO:129)
			OteH-V173C	OteH-G166C R(SEQ ID NO:125)	OteH-V173C F(SEQ ID NO:130)
OteH-Q175C	OteH-G166C R(SEQ ID NO:125)	OteH-Q175C F(SEQ ID NO:131)
			OteH-S176C	OteH-S176C R(SEQ ID NO:132)	OteH-S176C F(SEQ ID NO:133)
OteH-S177C	OteH-S176C R(SEQ ID NO:132)	OteH-S177C F(SEQ ID NO:134)
			OteH-G178C	OteH-S176C R(SEQ ID NO:132)	OteH-G178C F(SEQ ID NO:135)
OteH-L179C	OteH-S176C R(SEQ ID NO:132)	OteH-L179C F(SEQ ID NO:136)
			OteH-S181C	OteH-S181C R(SEQ ID NO:137)	OteH-S181C F(SEQ ID NO:138)
OteH-S183C	OteH-S181C R(SEQ ID NO:137)	OteH-S183C F(SEQ ID NO:139)
			OteH-V185C	OteH-S181C R(SEQ ID NO:137)	OteH-V185C F(SEQ ID NO:140)
OteH-T187C	OteH-S181C R(SEQ ID NO:137)	OteH-T187C F(SEQ ID NO:141)
			OteH-V215C	OteH-V215C R(SEQ ID NO:142)	OteH-V215C F(SEQ ID NO:143)
OteH-E216C	OteH-V215C R(SEQ ID NO:142)	OteH-E216C F(SEQ ID NO:144)
			OteH-P217C	OteH-V215C R(SEQ ID NO:142)	OteH-P217C F(SEQ ID NO:145)
OteH-K218C	OteH-V215C R(SEQ ID NO:142)	OteH-K218C F(SEQ ID NO:146)
			OteH-S219C	OteH-V215C R(SEQ ID NO:142)	OteH-S219C F(SEQ ID NO:147)
OteH-V220C	OteH-V215C R(SEQ ID NO:142)	OteH-V220C F(SEQ ID NO:148)

An Otelixizumab lambda (lambda) light chain containing a C214V mutation was used as another representation of an IgG1 lambda light chain (SEQ ID NO: 149). An otelixizumab lambda light chain codon-optimized DNA (SEQ ID NO:150) was synthesized and used as a template for PCR amplification.

PCR 6, light chain N-terminal primer Ote lambda-Nt (SEQ ID NO:151, with Xho I restriction enzyme site) and light chain C-terminal primer Ote lambda-Ct (SEQ ID NO:152, with Not I restriction enzyme site) composed different primer pairs with the corresponding reverse primer (R) and forward primer (F) in Table 13, respectively, for PCR amplification of N-and C-terminal lambda light chains using the synthesized otelixizumab lambda chains as templates.

PCR 7, PCR products of N-and C-terminal lambda light chains were ligated by overlap extension PCR using primer pairs Ote lambda-Nt and Ote lambda-Ct. In this way, an otelixizumab lambda light chain mutant comprising a mutation of the wild amino acid to cysteine and a C214V mutation was generated. The atelixizumab lambda chain can be directly PCR amplified using Ote λ -Nt and the corresponding reverse primer (R) in table 13, mutating the C-terminal wild amino acid to cysteine. Direct PCR amplification using Ote λ -Nt and Ote λ -V214C reverse primers (R) generated an Otelixizumab lambda chain with a wild type cysteine at position 214 (Kabat numbering).

TABLE 13 PCR primers for wild amino acid mutation to cysteine in the Otelixizumab lambda (λ) light chain [ Kabat numbering ].

Mutations	Reverse primer (R)	Forward primer (F)
			Oteλ-S114C	Oteλ-S114C R(SEQ ID NO:153)	Oteλ-S114C F(SEQ ID NO:154)
Oteλ-T116C	Oteλ-S114C R(SEQ ID NO:153)	Oteλ-T116C F(SEQ ID NO:155)
			Oteλ-F118C	Oteλ-S114C R(SEQ ID NO:153)	Oteλ-F118C F(SEQ ID NO:156)
Oteλ-P119C	Oteλ-S114C R(SEQ ID NO:153)	Oteλ-P119C F(SEQ ID NO:157)
			Oteλ-S121C	Oteλ-S114C R(SEQ ID NO:153)	Oteλ-S121C F(SEQ ID NO:158)
Oteλ-G158C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-G158C F(SEQ ID NO:160)
			Oteλ-E160C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-E160C F(SEQ ID NO:161)
Oteλ-T162C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-T162C F(SEQ ID NO:162)
			Oteλ-T163C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-T163C F(SEQ ID NO:163)
Oteλ-P164C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-P164C F(SEQ ID NO:164)
			Oteλ-S165C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-S165C F(SEQ ID NO:165)
Oteλ-Q167C	Oteλ-G158C R(SEQ ID NO:159)	Oteλ-Q167C F(SEQ ID NO:166)
			Oteλ-K172C	Oteλ-K172C R(SEQ ID NO:167)	Oteλ-K172C F(SEQ ID NO:168)
Oteλ-A174C	Oteλ-K172C R(SEQ ID NO:167)	Oteλ-A174C F(SEQ ID NO:169)
			Oteλ-S176C	Oteλ-K172C R(SEQ ID NO:167)	Oteλ-S176C F(SEQ ID NO:170)
Oteλ-Y178C	Oteλ-K172C R(SEQ ID NO:167)	Oteλ-Y178C F(SEQ ID NO:171)
			Oteλ-S180C	Oteλ-K172C R(SEQ ID NO:167)	Oteλ-S180C F(SEQ ID NO:172)
Oteλ-V209C	Oteλ-V209C R(SEQ ID NO:173)
			Oteλ-A210C	Oteλ-A210C R(SEQ ID NO:174)
Oteλ-P211C	Oteλ-P211C R(SEQ ID NO:175)
			Oteλ-T212C	Oteλ-T212C R(SEQ ID NO:176)
Oteλ-E213C	Oteλ-E213C R(SEQ ID NO:177)
			Oteλ-V214C	Oteλ-V214C R(SEQ ID NO:178)
Oteλ-S215C	Oteλ-S215C R(SEQ ID NO:179)

The Otelixizumab lambda light chain mutant was digested with Xho I and Not I and inserted into the same digestion site of pPIC6 α -ARG4 to generate a library of lambda light chain mutant expression vectors, designated pPIC6-ARG4-Ote λ (X # C) (Ote λ: otecixizumab lambda light chain, (X # C): wild amino acid mutation at # to cysteine, Kabat numbering). For example, pPIC6-ARG4-Otel λ -S114C indicates that the wild serine at position 114 of the light chain of otelixizumab lambda was mutated to cysteine. As shown in table 13, each vector can express an otelixizumab lambda light chain comprising a C214V mutation and a wild amino acid mutation to a cysteine, and one vector can express an otelixizumab lambda chain having a wild cysteine at position 214.

The ARG2 homologous sequence was digested with the restriction enzyme Afe I, the pPICZ α -Fez λ -ARG2 expression vector was linearized, electroporated into GS2-1, and integrated at the ARG2 locus by recombination of the ARG 25 'and 3' homologous sequences, as described in example 3. Transformed cells were grown on YPD plates supplemented with 100mg/L Zeocin. This resulted in the novel expression strain GS 2-Fez. lambda.

The pPIC9-FezH (HoleRF-His) expression vector was linearized with the restriction enzyme Sal I, electroporated into the GS2-Fez lambda strain, and integrated into the His4 locus of the Pichia pastoris genome. Transformed cells were selected on YNB plates. This gave rise to the novel expression strain GS2-FezH lambda.

Each expression vector of the library of the otelixizumab heavy chain mutant pEG-OteH (X # C) was linearized with the restriction enzyme Pme I, electroporated into the GS2-FezH lambda strain, and integrated into the AOX1 locus of the Pichia genome. Transformed cells were selected on YPD plates supplemented with 250mg/L G-418 sulfate. This produced the expression strain library GS2-FezH κ -OteH (X # C).

Each expression vector of the otalixizumab lambda chain mutant library pPIC6-ARG4-Ote lambda (X # C) was linearized within the ARG 43 'and 5' homology sequences using restriction enzyme Sma I and electroporated into the expression strain library GS2-FezH lambda-OteH (X # C). The linear expression vector was integrated at the ARG4 locus by recombination of ARG 43 'and 5' homologous sequences. The transformed cells were grown on YPD plates supplemented with 300mg/L blasticidin, thus giving rise to the expression strain library GS 2-FezH. lamda. -OteH (X # C) lamda (X # C). They can express asymmetric bispecific antibodies, half of which are the otecixizumab heavy and light chains with engineered interchain disulfide bonds in the CH1 and CL domains of the Fab arm and "knob" mutations in the Fc. The other half is the Fezakinumab heavy and light chains, with native interchain disulfide bonds in the CH1 and CL domains of the Fab arms, a "hole" and RF mutation in the Fc, and a 6xHis tag at the C-terminus of the heavy chain.

Example 8 screening to identify IgG1(lambda) "cysteine mutation" library

As described in example 3, the bispecific antibody was purified by protein a affinity chromatography and the purified antibody concentration was determined by ELISA.

Protein A purified bispecific antibody was diluted with PBS at a 1:2 gradient and split into two portions, one portion being assayed for antibody concentration by ELISA on plates coated with AGL protein and the other portion being assayed for antibody binding to antigen by ELISA on plates coated with human interleukin 22(IL22) (Sino Biological), as described in example 3.

According to the report, when phenylalanine 126 position in Otelixizumab heavy chain CH1 is mutated to cysteine (F126C, EU numbering) and serine 121 position in lambda light chain CL is mutated to cysteine (S121C, Kabat numbering), an engineered interchain disulfide bond can be formed to make the heavy chain and light chain pair correctly. This asymmetric bispecific antibody was designated herein as FezH λ -OteH (F126C) λ (S121C), abbreviated as F126C/S121C, and used as a positive control. However, when both Fab arms have a native interchain disulfide bond, only 25% of the heavy and light chains of the bispecific antibody formed are correctly paired. The bispecific antibody is designated herein as FezH λ -OteH λ, abbreviated WT, and used as a negative control (Yariv Mazor, Valeh Oganesian, Chunning Yang, Anna Hansen, Jihong Wang, Hongji Liu, Kris Sachsenseier, Marcia Carlson, Dhanesh V Gadre, Martin Jack Borrak, Xiang-Qing Yu, William Dall' Acqua, Herren Wu, and Partha Sarathi Chowdhury, mA 7,377-389; 2015).

As described in example 3, ELISA methods were used to identify engineered interchain disulfide bonds in bispecific antibodies that can promote proper pairing of heavy and light chains.

As shown in fig. 9, the bispecific antibody positive control F126C/S121C showed a very high antibody/antigen binding ELISA absorbance value (vertical axis) but the bispecific antibody negative control WT showed a very low antibody/antigen binding ELISA absorbance value under the same antibody concentration conditions (horizontal axis ELISA absorbance value). Under the same antibody concentration conditions (horizontal axis ELISA absorbance values), many bispecific antibodies we constructed had the same or higher antibody/antigen binding absorbance values as the positive control F126C/S121C. This indicates that the heavy and light chains of our bispecific antibody are able to pair correctly. Thus, in the production of IgG1(lambda) bispecific antibodies, correct heavy and light chain pairing can be achieved by replacing the natural interchain disulfide bond with a different cysteine pairing to form an engineered disulfide bond in the CH1-CL domain of one Fab arm, including:

(each pair of cysteine mutations are listed in the following manner: the heavy chain wild amino acid [ EU numbering ] mutation to cysteine/lambda light chain wild amino acid [ Kabat numbering ] mutation to cysteine).

Example 9 construction and expression of IgG1(lambda) "cysteine and Charge mutation" library

PCR 1, EG F and EG R primer pairs were used for PCR amplification of pEG linear fragments using the pEG vector as template (as described in example 2). "

PCR 2 was performed using the primer pairs shown in Table 14-1 for PCR amplification of the N-and C-terminal heavy chains, respectively, using each expression vector of the otteliximab mutant library pEG-OteH (X # C) as a template. The N-and C-terminal heavy chains were joined by overlap extension PCR using the OteH-Nt and OteH-Ct primer pairs, thus generating a mutated otecixizumab heavy chain comprising a T366W "knob" mutation, a C220V mutation, a wild amino acid mutation to cysteine, and a K213D mutation.

TABLE 14-1 Oceliximab heavy chain N-and C-terminal PCR amplification primer pairs

PCR 3 was performed using the primer pairs shown in Table 14-2 for PCR amplification of the N-and C-terminal heavy chains, and each expression vector of the otecizumab mutant library pEG-OteH (X # C) was used as a template. The N-and C-terminal heavy chains were joined by overlap extension PCR using the OteH-Nt and OteH-Ct primer pairs, thus generating a mutated otecixizumab heavy chain comprising a T366W "knob" mutation, a C220V mutation, a wild amino acid mutation to cysteine, and a K213E mutation.

TABLE 14-2 Oceliximab heavy chain N-and C-terminal PCR amplification primer pairs

The mutated otecixizumab heavy chain was inserted into the pEG linear fragment using the ClonExpress II one-step cloning kit (Vazyme) to construct a library of otecixizumab mutant expression vectors, named pEG-OteH (X # CD) and pEG-OteH (X # CE).

PCR 4, PCR amplification of the N-terminal and C-terminal lambda light chains using the corresponding primer pairs in Table 15, respectively, using each expression vector of the otalixizumab mutant library pPIC6 α -ARG4-OteH λ (X # C) as a template. The N-and C-terminal lambda light chains were joined by overlap extension PCR using the Ote λ -Nt and Ote λ -Ct primer pairs, thus generating a mutated otecixizumab lambda light chain comprising a T366W "knob" mutation, a C220V mutation, a wild amino acid mutation to cysteine, and an E123K mutation.

TABLE 15 Oceliximab light chain N-and C-terminal PCR amplification primer pairs

The mutated otecixizumab lambda light chain was digested with Xho I and Not I and inserted into the same digestion site of pPIC6 α -ARG4 to generate a library of lambda light chain mutant expression vectors designated pPIC6 α -ARG4-Ote λ (X # CK). Each vector can express a mutant otecizumab lambda light chain comprising a C214V mutation, a wild amino acid mutation to cysteine, and E123K.

Each expression vector of the library of the otelixizumab heavy chain mutant pEG-OteH (X # CD) was linearized with the restriction enzyme Pme I, electroporated into the GS2-FezH lambda strain, and integrated at the AOX1 locus in the Pichia genome. Transformed cells were selected on YPD plates supplemented with 250mg/L G-418 sulfate, thus generating a library of expression strains (GS 2-FezH. lamda. -OteH (X # CD).

Each expression vector of the library of otelixizumab lambda light chain mutants, pPIC6 α -ARG4-Ote λ (X # CK), was linearized in ARG 43 'and 5' homologous sequences with the restriction enzyme Sma I, electroporated into the expression strain library GS2-FezH λ -OteH (X # CD), and integrated into the ARG4 locus. The transformed cells were grown on YPD plates supplemented with 300mg/L blasticidin, thus giving rise to the expression strain library GS 2-FezH. lamda. -OteH (X # CD). lamda. (X # CK) for expression of asymmetric bispecific antibodies. In asymmetric bispecific antibodies, half are the otecixizumab heavy and light chains, with the CH1-CL domain of the Fab arm having an engineered interchain disulfide bond and a pair of charge reversal mutations, and a "knob" mutation in the Fc; the other half was the Fezakinumab heavy and light chains, with the CH1-CL domain of the Fab arm having the natural interchain disulfide bond, the "well" and RF mutation in the Fc, and the 6xHis tag at the C-terminus of the heavy chain, and the expression strain was cultured in BMGY medium and induced to express antibody in BMMY medium as described in example 2. The supernatant harvested by centrifugation was frozen at-20 ℃ until next use.

Example 10 screening identification of IgG1(lambda) "cysteine and Charge mutation" library

As shown in fig. 10, the positive control F126C/S121C had a very high absorbance value (vertical axis) of antibody/antigen binding ELISA, but the negative control WT had a very low absorbance value of antibody/antigen binding ELISA, under the same concentration conditions of antibody (horizontal axis ELISA). Bispecific antibodies containing a cysteine mutation in the CH1-CL domain of one Fab arm have similar or lower antibody/antigen binding absorbance values than the positive control F126C/S121C. The corresponding bispecific antibody containing cysteine and a charge mutation of K213D/E123K had higher antibody/antigen binding absorbance values than the positive control F126C/S121C. Thus, in the production of IgG lambda bispecific antibodies, to further achieve proper pairing of the heavy and light chains, forming engineered disulfide bonds, K213D/E123K charge mutations can be combined with different cysteine pairs in the CH1-CL domain of one Fab arm, including:

(each pair of cysteine mutations are listed in the following manner: the heavy chain wild amino acid [ EU numbering ] mutation is cysteine/lambda light chain wild amino acid [ Kabat numbering ] mutation is cysteine).

Example 11 expression of bispecific antibody IgG1(kappa/lambda)

The expression vector pEG-ClaH (X # C) for the clazakizumab heavy chain mutant was linearized with the restriction enzyme Pme I, electroporated into the GS2-FezH λ strain (as described in example 7), and integrated into the Pichia genome at the AOX1 locus. Transformed cells GS2-FezH lambda-ClaH (X # C) were selected on YPD plates supplemented with 500mg/L G-418 sulfate. Subsequently, the expression vector pPIC6-ARG4-Cla κ (X # C) for the clazakizumab kappa light chain mutant was linearized with the restriction enzyme Sma I, electroporated into the GS2-FezH λ -ClaH (X # C) strain, and integrated into the ARG4 locus. Transformed cells were grown on YPD plates supplemented with 300mg/L blasticidin. Thus, the expression strain GS2-FezH lambda-ClaH (X # C) kappa (X # C) capable of expressing the asymmetric bispecific antibody was produced. In asymmetric bispecific antibodies, half were clazakizumab heavy and kappa light chains, with the CH1-CL domain of the Fab arm having an engineered interchain disulfide bond, with a "knob" mutation in the Fc, and the other half were fezakinumab heavy and lambda light chains, with the native interchain disulfide bond in the CH1-CL domain of the Fab arm, a "hole" and RF mutation in the Fc, and a 6xHis tag at the C-terminus of the heavy chain.

Protein a purified bispecific antibody was used as described in example 3 with PBS as 1: after 2-step dilution, the samples were divided into two portions, one portion of the antibody concentration was measured by ELISA on plates coated with AGL protein, and the other portion of the antibody binding to antigen was measured by ELISA on plates coated with human interleukin 22(IL22) (Sino Biological).

Representative examples are two bispecific anti-FezH λ -ClaH (K218C) κ (F118C) and FezH λ -ClaH (S132C) κ (F116C), correct pairing of light and heavy chains in bispecific IgG kappa/lambda containing a new pair of cysteine mutations (K218C/F118C or S132C/F116C) in the Fab arm heavy chain CH1 and kappa light chain CL domains of clazakizumab. In both of these asymmetric bispecific antibodies, half were the clazakizumab heavy and kappa light chains, which contained a new pair of cysteines in the Fab arms (K218C/F118C or S132C/F116C), and there was a "knob" mutation in the Fc. The other half was a fezakinumab heavy chain and a lambda light chain with native interchain disulfide bonds in their Fab arms, a "hole" and RF mutation in the Fc, and a 6xHis tag at the heavy chain C-terminus, as shown in fig. 11, these two IgG1(kappa/lambda) bispecific antibodies had similar antibody/antigen binding absorbance values to the positive control F126C/S121C under the same antibody concentration conditions (horizontal axis ELISA absorbance values).

These examples demonstrate that in IgG1 kappa/lambda bispecific antibody production, correct pairing of heavy and light chains can be achieved by mutating the CH1-CL domain of one Fab arm with cysteine pairs to form engineered disulfide bonds, replacing the native interchain disulfide bonds. If necessary, correct pairing of heavy and light chains can also be achieved by binding the charge mutations of K213D/E123K and the different cysteine pairings in the CH1-CL domain of the Fab arm to form engineered disulfide bonds.

Example 12 production of bispecific IgG1 at different Fab arms at the cysteine mutation and Charge mutation

PCR 1 was performed using synthetic trastuzumab heavy chain (HolerRF-His) DNA as a template, and using primer pairs of TraH F and ClaH-K213D R and ClaH-K213D F and TraH R for PCR amplification of the N-and C-terminal heavy chains. The N-and C-terminal heavy chains were connected by overlap extension PCR using a TraH F and TraH R primer pair.

PCR 2 was performed using the synthetic trastuzumab heavy chain (HolerRF-His) DNA as a template, and using primer pairs of TraH F and ClaH-K213E R and ClaH-K213E F and TraH R for PCR amplification of the N-and C-terminal heavy chains. The N-and C-terminal heavy chains were ligated by overlap extension PCR using TraH F and TraH R primers.

PCR

1 and 2 produced mutated trastuzumab heavy chains whose CH1 domain contained either the K213D or K213E mutations, the T366S/L368A/Y407V "pore" mutations in the Fc domain, the H435R, Y436F (RF) mutations, the 6xHis tag at the C-terminus.

The mutated trastuzumab heavy chain was digested with Xho I and Not I and inserted into the same digestion site of pPIC9(Invitrogen) to construct expression vectors for trastuzumab heavy chain, designated pPIC9-trah (d) and pPIC9-trah (e). Both vectors can express mutated trastuzumab heavy chains comprising either K213D or K213E mutations in the CH1 domain, T366S/L368A/Y407V "pore" mutations in the Fc domain, H435R, Y436F (RF) mutations and a 6xHis tag at the C-terminus.

PCR 3, using the synthetic trastuzumab kappa chain as template, PCR amplified N-and C-terminal kappa chains using the primer set Tra kappa F and Cla kappa-E123K R and the primer set Cla kappa-E123K F and Tra kappa R. The N-terminal and C-terminal kappa chains were ligated by overlap extension PCR using the Tra κ F and Tra κ R primer pairs.

PCR 4, using the synthetic trastuzumab kappa chain as a template, PCR amplified N-and C-terminal kappa chains with the primer set Tra kappa F and Cla kappa-E123R R and the primer set Cla kappa-E123R F and Tra kappa R. The N-terminal and C-terminal kappa chains were ligated by overlap extension PCR using the Tra κ F and Tra κ R primer pairs.

PCR 3 and 4 products were digested with Xho I and Not I and inserted into the same digestion site of pPICZ α -Tra κ -ARG2 to construct expression vectors for trastuzumab kappa chain, designated pPICZ α -Tra κ (K) -ARG2 and pPICZ α -Tra κ (R) -ARG 2. Both vectors can express mutated trastuzumab kappa chains that contain either the E123K or E123R mutation in the CL domain.

Expression vectors for pPICZ α -Tra κ (K) -ARG2 and pPICZ α -Tra κ (R) -ARG2 were linearized with Afe I, electroporated into GS2-1 and integrated at the ARG2 locus as described in example 2. Transformed cells were grown on YPD plates supplemented with 100mg/L Zeocin. Thus, novel expression strains GS2-Tra kappa (K) and GS2-Tra kappa (R) were produced, which can express the trastuzumab kappa light chain.

Expression vectors for pPIC9-TraH (D) and pPIC9-TraH (E) were linearized with Sal I, electroporated into the GS 2-TraK (K) and GS 2-TraK (R) strains, and integrated into the his4 locus. Transformed cells were selected on YNB plates. Thus, strains of GS2-TraH (D) kappa (K), GS2-TraH (D) kappa (R), GS2-TraH (E) kappa (K) and GS2-TraH (E) kappa (R) were produced to express mutated trastuzumab.

Certain expression vectors of the library of clazakizumab heavy chain mutants pEG-ClaH (X # C) were linearized with Pme I, electroporated into strains GS2-TraH (D) kappa (K), GS2-TraH (D) kappa (R), GS2-TraH (E) kappa (K) and GS2-TraH (E) kappa (R), and integrated at the AOX1 locus. Transformed cells were selected on YPD plates supplemented with 250mg/L G-418 sulfate. This produced a library of expression strains:

GS2-TraH(D)κ(K)-ClaH(X#C)、

GS2-TraH(D)κ(R)-ClaH(X#C)、

GS2-TraH(E)κ(K)-ClaH(X#C)、

GS2-TraH(E)κ(R)-ClaH(X#C)。

expression vectors for the clazakizumab kappa chain mutant library pPIC6-ARG4-Cla kappa (X # C) were linearized with Sma I, electroporated into the above expression strain library, and integrated into the ARG4 locus. The transformed strains were grown on YPD plates supplemented with 300mg/L blasticidin. This thus produced a library of expression strains:

GS2-TraH(D)κ(K)-ClaH(X#C)κ(X#C)、

GS2-TraH(D)κ(R)-ClaH(X#C)κ(X#C)、

GS2-TraH(E)κ(K)-ClaH(X#C)κ(X#C)、

GS2-TraH(E)κ(R)-ClaH(X#C)κ(X#C)。

Protein a purified bispecific antibody was used as described in example 3 with PBS as 1: after 2-step dilution, the solution was divided into two portions, one portion was measured for antibody concentration by ELISA on plates coated with AGL protein, and the other portion was measured for binding of antibody to antigen by ELISA on plates coated with human HER2/ErbB2 protein.

The following three groups of bispecific antibodies were utilized as representative examples:

TraH (D) kappa (K) -ClaH (S219C) kappa (P120C) abbreviated as S219C/P120C, K213D/E123K;

TraH (E) kappa (K) -ClaH (S219C) kappa (P120C) abbreviated as S219C/P120C, K213E/E123K;

TraH (D) kappa (R) -ClaH (S219C) kappa (P120C) abbreviated as S219C/P120C, K213D/E123R;

TraH (E) kappa (R) -ClaH (S219C) kappa (P120C) abbreviated as S219C/P120C, K213E/E123R;

TraH (D) K (K) -ClaH (K218C) K (F118C) abbreviated as K218C/F118C, K213D/E123K;

TraH (E) kappa (K) -ClaH (K218C) kappa (F118C) abbreviated as K218C/F118C, K213E/E123K;

TraH (D) kappa (R) -ClaH (K218C) kappa (F118C) abbreviated as K218C/F118C, K213D/E123R;

TraH (E) kappa (R) -ClaH (K218C) kappa (F118C) abbreviated as K218C/F118C, K213E/E123R,

TraH (D) kappa (K) -ClaH (V173C) kappa (N158C) abbreviated as V173C/N158C, K213D/E123K;

TraH (E) kappa (K) -ClaH (V173C) kappa (N158C) abbreviated as V173C/N158C, K213E/E123K;

TraH (D) kappa (R) -ClaH (V173C) kappa (N158C) abbreviated as V173C/N158C, K213D/E123R;

TraH (E) kappa (R) -ClaH (V173C) kappa (N158C), abbreviated as V173C/N158C, K213E/E123R.

In these asymmetric bispecific antibodies, half are the clazakizumab heavy and kappa light chains, which contain a new pair of cysteines in the Fab arm CH1-CL domain (S219C/P120C, K218C/F118C, or V173C/N158C), containing a "knob" mutation in the "Fc; the other half is trastuzumab heavy and light chains, with the natural interchain disulfide bonds retained in their Fab arms, but Fab arm CH1 contains charge mutations of K213D or K213E, charge mutations of E123K or E123R in Fab arm CL, a "hole" and RF mutation in Fc, a 6xHis tag at the C-terminus of the heavy chain,

as shown in fig. 12, all of these bispecific antibodies had higher antibody/antigen binding absorbance values (vertical axis ELISA absorbance values) than the positive control F126C/S121C under the same antibody concentration conditions (horizontal axis ELISA absorbance values).

These examples demonstrate that in IgG bispecific antibody production, correct pairing of heavy and light chains can be achieved by charge mutation of the CH1-CL domain of one Fab arm, and mutation of cysteine pairs into engineered disulfide bonds in the CH1-CL domain of the other Fab arm, instead of the natural interchain disulfide bonds.

Example 13 design of IgG4(Kappa) CH1 and CL Domain "cysteine mutations" library

Although the CH1 domain amino acid sequences and spatial structures of IgG1, IgG2, IgG3, and IgG4 are very similar, the cysteine pairs that form the native disulfide bonds differ in position. The light chain of IgG1 formed an interchain disulfide bond between the kappa and C-terminal cysteines of the CL domain of the lambda chain (cysteine at position 214 of the kappa light chain [ EU numbering ]; cysteine at position 214 of the lambda light chain [ Kabat numbering ]) and the C-terminal cysteine of the CH1 domain of the heavy chain (cysteine at position 220 of the heavy chain [ EU numbering ]). In contrast, the light chain of IgG2, IgG3, or IgG4 formed an interchain disulfide bond with the cysteine C-terminal to the CL domain in the kappa and lambda chains and the cysteine N-terminal to the CH1 domain in the heavy chain (cysteine at position 131 of heavy chain [ EU numbering ]) (fig. 1, a and B). Although the positions of cysteines in the amino acid sequence differ between IgG1 and other subtypes (IgG2, IgG3, IgG4), their spatial positions are similar to form interchain disulfide bonds. To understand where IgG2, IgG3, or IgG4 might form other disulfide bonds, we used Fab crystal structure (PDB code: 5DK3) of human IgG4(kappa) from protein databases as a representative structure, analyzed and designed cysteine mutation sites in CH1 and CL domains that might interact to form interchain disulfide bonds, designed libraries of IgG4(kappa) mutants in which heavy and light chain cysteine pairs that form native interchain disulfide bonds are mutated to serine and valine, respectively, and introduced new cysteine pairs at different positions in CH1-CL domain to form engineered interchain disulfide bonds. Table 16 lists the introduction of new cysteine pairs at different amino acid positions in the CH1-CL domain of IgG4(Kappa), which may form engineered interchain disulfide bonds.

TABLE 16 cysteine mutation library of IgG4(kappa) CH1-CL domain. The regions where cysteine mutations aggregate are referred to as a group. The CH1 domain and the CL domain are possibly formed with interchain disulfide bond, and the groups are respectively listed correspondingly.

Example 14 construction and expression of IgG4(Kappa) cysteine mutation library

Ixekizumab (IL-17A antibody) heavy chain (HoleRF-His) comprising a T366S/L368A/Y407V "well" mutation, H435R, Y436F (RF) mutation (EU numbering) and a 6XHis tag at the C-terminus was used as a representation of the IgG4 heavy chain (SEQ ID NO: 202). Ixekizumab heavy chain codon-optimized DNA (HoleRF-His) was synthesized and used as template for PCR amplification (SEQ ID NO: 203).

PCR 1, IxeH F (SEQ ID NO:204, primers with Xho I restriction enzyme site) and IxeH R (SEQ ID NO:205, primers with Not I restriction enzyme site) primer pairs were used for PCR amplification of Ixekizumab heavy chain (HoleRF-His), using the synthesized DNA as template. The PCR product was digested with Xho I and Not I and inserted into the same digestion site of pPIC9(Invitrogen) to construct an expression vector for Ixekizumab heavy chain (HoleRF-His), designated pPIC9-IxeH (HoleRF-His).

Ixekizumab light chain was used as a representative of the IgG4 kappa (kappa) light chain (SEQ ID NO: 206). Codon-optimized DNA for Ixekizumab kappa light chain (SEQ ID NO:207) was synthesized and used as a template for PCR amplification.

PCR 2 Ixe kF (SEQ ID NO:208, with Xho I restriction enzyme sites) and Ixe kR (SEQ ID NO:209, with Not I restriction enzyme sites) primer pairs were used for PCR amplification of Ixekizumab kappa light chain using the synthesized Ixekizumab light chain as template. The PCR product was digested with Xho I and Not I and inserted into the same digestion site of pPICZ α -Tra κ -ARG2 (as described in example 2) to construct an expression vector for the Ixekizumab light chain, designated pPICZ α -Ixe κ -ARG 2.

An olykizumab (IL6 mab) heavy chain (knob) containing the T366W "knob" mutation and the C131S mutation [ EU numbering ] was used as another representation of the IgG4 heavy chain (SEQ ID NO: 210). Codon-optimized DNA (SEQ ID NO:211) of the olykizumab heavy chain (knob) was synthesized and used as template for PCR amplification.

In PCR 4, heavy chain N-terminal primer OloH-Nt (SEQ ID NO:212) and C-terminal primer OloH-Ct (SEQ ID NO:213) and corresponding reverse primer (R) and forward primer (F) in Table 17 respectively form different primer pairs for PCR amplification of N-terminal and C-terminal heavy chains, and synthesized olykizumab heavy chain (knob) is used as a template.

TABLE 17 PCR primers for mutation of wild amino acid to cysteine in the Clazakizumab heavy chain (EU numbering)

PCR 5, PCR products of the N-and C-terminal heavy chains were ligated by overlap extension PCR using the OloH-Nt and OloH-Ct primer pairs. In this way, an olokizumab heavy chain mutant was generated comprising a wild amino acid mutation to cysteine, a T366W "knob" mutation and a C131S mutation. One of them contains the T366W "knob" mutation and the wild cysteine at position 134, and is capable of forming a natural interchain disulfide bond.

PCR 6, heavy chain N-terminal primer OloH-Nt and OloH-K147D R (SEQ ID NO:246) primer pair and OloH-K147D F (SEQ ID NO:247) and C-terminal primer OloH-Ct primer pair were used for PCR amplification of N-terminal and C-terminal heavy chains, respectively, using a heavy chain containing a T366W "knob" mutation and a 134-position wild cysteine as a template.

PCR 7, PCR products of the N-and C-terminal heavy chains were ligated by overlap extension PCR using the OloH-Nt and OloH-Ct primer pairs. In this way, an olykizumab heavy chain mutant was generated which comprises the K147D mutation, the T366W "knob" mutation and the wild cysteine at position 134, capable of forming the natural interchain disulfide bond

The olykizumab heavy chain mutant was inserted into the pEG linear fragment using the ClonExpress II one-step cloning kit (Vazyme) to generate a library of expression vectors designated pEG-OloH (X # C) (OloH: olykizumab heavy chain, (X # C): wild amino acid at position # was mutated to cysteine). One of the expression vectors is pEG-OloH (K147D) (OloH: olokizumab heavy chain, K147D: 147 lysine mutated to aspartic acid).

Olokizuzumab kappa (kappa) light chain containing the C214V mutation was used as another representation of IgG4 kappa light chain (SEQ ID NO: 248). Olokizumab kappa light chain codon-optimized DNA (SEQ ID NO:249) was synthesized and used as a template for PCR amplification.

PCR 8, light chain N-terminal primer Olo κ -Nt (SEQ ID NO:250 with Xho I restriction site) and light chain C-terminal primer Olo κ -Ct (SEQ ID NO:251 with Not I restriction site) constituting different primer pairs with the corresponding reverse primer (R) and forward primer (F) in Table 18, respectively, for PCR amplification of N-and C-terminal kappa light chains using the synthesized Olokizumab kappa chain as a template.

TABLE 18 PCR primers for the mutation of the wild amino acid to cysteine in the Olokizumab kappa (kappa) light chain [ EU numbering ]

Mutations	Reverse primer (R)	Forward primer (F)
			Oloκ-S114C	Oloκ-S114C R(SEQ ID NO:252)	Oloκ-S114C F(SEQ ID NO:253)
Oloκ-F116C	Oloκ-S114C R(SEQ ID NO:252)	Oloκ-F116C F(SEQ ID NO:254)
			Oloκ-F118C	Oloκ-S114C R(SEQ ID NO:252)	Oloκ-F118C F(SEQ ID NO:255)
Oloκ-P119C	Oloκ-S114C R(SEQ ID NO:252)	Oloκ-P119C F(SEQ ID NO:256)
			Oloκ-P120C	Oloκ-S114C R(SEQ ID NO:252)	Oloκ-P120C F(SEQ ID NO:257)
Oloκ-S121C	Oloκ-S114C R(SEQ ID NO:252)	Oloκ-S121C F(SEQ ID NO:258)
			Oloκ-N158C	Oloκ-N158C R(SEQ ID NO:259)	Oloκ-N158C F(SEQ ID NO:260)
Oloκ-Q160C	Oloκ-N158C R(SEQ ID NO:259)	Oloκ-Q160C F(SEQ ID NO:261)
			Oloκ-S162C	Oloκ-N158C R(SEQ ID NO:259)	Oloκ-S162C F(SEQ ID NO:262)
Oloκ-V163C	Oloκ-N158C R(SEQ ID NO:259)	Oloκ-V163C F(SEQ ID NO:263)
			Oloκ-T164C	Oloκ-N158C R(SEQ ID NO:259)	Oloκ-T164C F(SEQ ID NO:264)
Oloκ-E165C	Oloκ-N158C R(SEQ ID NO:259)	Oloκ-E165C F(SEQ ID NO:265)
			Oloκ-T172C	Oloκ-T172C R(SEQ ID NO:266)	Oloκ-T172C F(SEQ ID NO:267)
Oloκ-S174C	Oloκ-T172C R(SEQ ID NO:266)	Oloκ-S174C F(SEQ ID NO:268)
			Oloκ-S176C	Oloκ-T172C R(SEQ ID NO:266)	Oloκ-S176C F(SEQ ID NO:269)
Oloκ-T178C	Oloκ-T172C R(SEQ ID NO:266)	Oloκ-T178C F(SEQ ID NO:270)
			Oloκ-T180C	Oloκ-T172C R(SEQ ID NO:266)	Oloκ-T180C F(SEQ ID NO:271)
Oloκ-S182C	Oloκ-T172C R(SEQ ID NO:266)	Oloκ-S182C F(SEQ ID NO:272)
			Oloκ-F209C	Oloκ-F209C R(SEQ ID NO:273)
Oloκ-N210C	Oloκ-N210C R(SEQ ID NO:274)
			Oloκ-R211C	Oloκ-R211C R(SEQ ID NO:275)
Oloκ-G212C	Oloκ-G212C R(SEQ ID NO:276)
			Oloκ-E213C	Oloκ-E213C R(SEQ ID NO:277)
Oloκ-V214C	Oloκ-V214C R(SEQ ID NO:278)

PCR 9 PCR products of the N-and C-terminal light chains were ligated by overlap extension PCR using primer pairs Olo kappa-Nt and Olo kappa-Ct. In this way, an Olokizumab kappa light chain mutant comprising a mutation of the wild amino acid to cysteine and a C214V mutation was generated. The Olokizumab kappa light chain can be directly PCR amplified using Olo kappa-Nt and the corresponding reverse primer (R) in Table 18, mutating the C-terminal wild amino acid to cysteine. Direct PCR amplification using the reverse primer (R) Olo K-Nt and Olo K-V214C produced an Olokizumab kappa light chain with a wild-type cysteine at position 214, capable of forming native interchain disulfide bonds.

PCR 10, a light chain N-terminal primer Olo kappa-Nt and Olo kappa-T129R R (SEQ ID NO:279) primer pair and Olo kappa-T129R F (SEQ ID NO:280) and a C-terminal primer Olo kappa-Ct primer pair were used for PCR amplification of the N-and C-terminal light chains, respectively, using Olokizuzumab kappa light chain with a wild-type cysteine at position 214 as a template.

PCR 11, PCR products of the N-and C-terminal heavy chains were ligated by overlap extension PCR using primer pairs Olo κ -Nt and Olo κ -Ct. In this way an olykizumab light chain mutant was generated which contained the T129R mutation and the wild cysteine at position 214 capable of forming a native interchain disulfide bond

The Olokizumab kappa light chain mutant was digested with Xho I and Not I and inserted into the same digestion site of pPIC6 α -ARG4 to generate a library of light chain mutant expression vectors, designated pPIC6-ARG4-Olo κ (X # C) (Olo κ: Olokizumab kappa light chain, (X # C): wild amino acid at # was mutated to cysteine).

The ARG2 homologous sequence was digested with restriction enzyme Afe I, the pPICZ α -Ixe κ -ARG2 expression vector was linearized, electroporated into GS2-1, and integrated at the ARG2 locus by recombination of the ARG 25 'and 3' homologous sequences as described in example 2. Transformed cells were grown on YPD plates supplemented with 100mg/L Zeocin. This gave rise to the novel expression strain GS2-Ixe K.

The pPIC9-IxeH (HoleRF-His) expression vector was linearized with the restriction enzyme Sal I, electroporated into the GS2-Ixe K strain, and integrated into the His4 locus of the Pichia pastoris genome. Transformed cells were selected on YNB plates. This gave rise to the novel expression strain GS2-IxeH κ.

Each expression vector of the Olokizumab heavy chain mutant library pEG-OloH (X # C) was linearized with the restriction enzyme Pme I, electroporated into the GS2-IxeH kappa strain, and integrated into the AOX1 locus of the Pichia genome. Transformed cells were selected on YPD plates supplemented with 250mg/L G-418 sulfate. This produced the expression strain library GS2-IxeH κ -OloH (X # C).

Each expression vector of the Olokizumab kappa light chain mutant library pPIC6-ARG4-Olo kappa (X # C) was linearized within the ARG 43 'and 5' homology sequences using restriction enzyme Sma I and electroporated into the expression strain library GS2-IxeH kappa-OloH (X # C). The linear expression vector was integrated at the ARG4 locus by recombination of ARG 43 'and 5' homologous sequences. The transformed cells were grown on YPD plates supplemented with 300mg/L blasticidin, thus giving rise to the expression strain library GS2-IxeH κ -OloH (X # C) κ (X # C). They can express asymmetric bispecific antibodies, half of which are the Olokizumab heavy and light chains with engineered interchain disulfide bonds in the CH1 and CL domains of the Fab arms and "knob" mutations in the Fc. The other half is the Ixekizumab heavy and light chains with native interchain disulfide bonds in the CH1 and CL domains of the Fab arms, a "hole" and RF mutation in the Fc, and a 6xHis tag at the C-terminus of the heavy chain.

Example 15 screening and identification of IgG4(Kappa) cysteine mutation library

Protein a purified bispecific antibody was used as described in example 3 with PBS as 1: after 2-step dilution, the samples were divided into two portions, one portion of the bispecific antibody was directly coated on the plate, and the antibody concentration was measured by ELISA using anti-Fc-HRP (Invitrogen), and the other portion of the bispecific antibody was coated on the plate with human interleukin 17A (IL17A) (Sino Biological) and the binding of the antibody to the antigen was measured by ELISA.

According to the report, when the 147 th lysine in Olokizumab heavy chain CH1 is mutated to aspartic acid (K147D), the 129 th threonine in kappa light chain CL is mutated to arginine (T129R) [ EU numbering]And can promote the correct pairing of the heavy chain and the light chain to reach 80-99%. This asymmetric bispecific antibody was designated IxeH κ -OloH (K147D) κ (T129R), abbreviated as K147D/T129R, and used as a positive control. (Maximiian)

Carolin Sellmann,Daniel Maresch,Claudia Halbig,Stefan Becker,Lars Toleikis,

Hock,and Florian Rüker,Protein Engineering,Design&Selection 30,685-696,2017). We found that the F126C/S121C cysteine pair mutation reported in the literature failed to efficiently form interchain disulfide bonds in IgG4(kappa) and failed to serve as a positive control.

The engineered interchain disulfide bonds in bispecific antibodies were identified using ELISA methods for promoting proper pairing of heavy and light chains as described in example 3.

As shown in fig. 13, the bispecific antibody positive control (K147D/T129R) showed very high antibody/antigen binding ELISA absorbance values (vertical axis) under the same antibody concentration conditions (horizontal axis ELISA absorbance values). Under the same antibody concentration conditions (horizontal axis ELISA absorbance values), many bispecific antibodies we constructed had the same or higher antibody/antigen binding absorbance values as the positive control K147D/T129R. This indicates that the heavy and light chains of our bispecific antibody are able to pair correctly. Thus, in IgG4(kappa) bispecific antibody production, correct heavy and light chain pairing can be achieved by replacing the natural interchain disulfide bond with a different cysteine pairing to form an engineered disulfide bond in the CH1-CL domain of one Fab arm, including:

(Each pair of cysteine mutations are listed in the following manner: IgG4 heavy chain wild amino acid [ EU numbering ] mutation to cysteine/kappa light chain wild amino acid [ EU numbering ] mutation to cysteine).

Example 16 design of IgG4(lambda) CH1 and CL Domain chain "cysteine mutation" libraries

We used the Fab crystal structure (PDB code: 5GKS) of human IgG2(lambda) from the protein database as a representative structure, analyzed and designed cysteine mutation sites in the CH1 and CL domains that might interact to form interchain disulfide bonds, designed a library of IgG4(kappa) mutants in which the heavy and light chain cysteine pairs forming the native interchain disulfide bonds were mutated to serine and valine, respectively, and introduced new cysteine pairs at different positions in the CH1-CL domain to form engineered interchain disulfide bonds. Table 19 lists the introduction of new cysteine pairs at different amino acid positions in the CH1-CL domain of IgG4(lambda), which may form engineered interchain disulfide bonds.

TABLE 19 cysteine mutagenesis library of IgG4(lambda) CH1-CL domain. The regions where cysteine mutations aggregate are referred to as a group. The CH1 domain and the CL domain are possibly formed with interchain disulfide bond, and the groups are respectively listed correspondingly.

The sequences used in the present invention are shown below:

SEQ ID NO:1

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGHHHHHH

SEQ ID NO:2

GAGGTCCAGTTGGTCGAGTCTGGTGGTGGTTTGGTTCAGCCAGGTGGATCTTTGAGATTGTCTTGTGCAGCATCTGGATTCAACATTAAAGATACTTACATTCATTGGGTTAGACAGGCACCAGGTAAAGGTTTGGAGTGGGTTGCTAGAATTTACCCAACTAACGGTTACACTAGATACGCTGATTCTGTCAAGGGTAGATTCACTATTTCTGCTGATACATCTAAAAACACTGCTTACTTGCAAATGAACTCTTTGAGAGCTGAAGATACAGCCGTTTACTATTGCTCCAGATGGGGAGGAGACGGATTTTACGCTATGGATTACTGGGGACAAGGTACTTTGGTTACTGTTTCTTCTGCTTCTACTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTACTTCTGGAGGAACTGCCGCCTTGGGATGTTTGGTCAAGGATTATTTCCCTGAGCCTGTCACAGTTTCTTGGAACTCTGGTGCATTGACATCTGGTGTTCACACATTCCCAGCAGTCTTGCAATCTTCTGGTTTGTACTCTTTGTCTTCTGTCGTTACAGTCCCTTCTTCTTCTTTGGGTACTCAAACCTACATCTGTAATGTTAATCATAAGCCTTCTAACACAAAAGTTGATAAGAAAGTCGAGCCAAAATCTTGCGACAAAACACATACCTGCCCACCATGTCCAGCTCCTGAGTTGTTGGGAGGTCCTTCTGTTTTTTTGTTCCCACCTAAGCCAAAGGATACATTGATGATTTCCAGAACTCCTGAAGTTACATGTGTCGTTGTCGATGTTTCTCATGAAGATCCTGAGGTTAAATTCAATTGGTACGTTGATGGTGTCGAAGTTCATAACGCTAAGACTAAACCAAGAGAAGAACAGTACAATTCTACTTATAGAGTCGTCTCTGTTTTGACCGTTTTGCATCAAGATTGGTTGAACGGAAAGGAGTATAAGTGCAAAGTTTCTAATAAGGCTTTGCCTGCCCCTATTGAGAAGACCATTTCTAAGGCTAAAGGTCAGCCTAGAGAACCTCAAGTTTACACTTTGCCACCTTCCAGAGAGGAAATGACTAAGAACCAGGTCTCTTTGtctTGCgctGTTAAGGGTTTTTACCCTTCTGACATCGCTGTTGAGTGGGAATCTAACGGACAGCCTGAAAATAACTATAAAACAACACCTCCAGTTTTGGATTCTGACGGTTCTTTCTTTTTGgttTCTAAGTTGACAGTCGATAAGTCCAGATGGCAACAAGGAAACGTCTTTTCTTGTTCTGTTATGCATGAAGCCTTGCATAATagatttACCCAGAAATCTTTGTCTTTGTCTCCAGGTcatcatcatcatcatcat

SEQ ID NO:3

ccgCTCGAGAAAAGAGAGGCTGAAGCTGAGGTCCAGTTGGTCGAGTCT

SEQ ID NO:4

ataagaatGCGGCCGCtcaatgatgatgatgatgatgACCTGGAGACAAAGACAAAGA

SEQ ID NO:5

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:6

GATATTCAAATGACTCAATCTCCTTCTTCTTTGTCTGCTTCTGTCGGAGATAGAGTCACTATCACTTGTAGAGCTTCTCAAGACGTCAACACCGCTGTTGCTTGGTATCAGCAAAAGCCAGGTAAGGCTCCAAAATTGTTGATTTACTCTGCATCTTTTTTGTACTCTGGTGTCCCTTCCAGATTCTCTGGTTCTAGGTCTGGAACCGATTTTACTTTGACTATCTCTTCTTTGCAGCCTGAAGATTTCGCAACTTACTACTGTCAACAGCATTACACTACTCCACCAACTTTTGGTCAAGGAACTAAAGTTGAGATTAAGAGAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTCCATCTGACGAGCAGTTGAAGTCTGGTACAGCATCTGTTGTCTGTTTGTTGAACAACTTCTACCCAAGAGAAGCAAAGGTTCAATGGAAGGTTGACAACGCCTTGCAATCTGGTAACTCTCAGGAATCTGTTACTGAACAAGATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGACATTGTCTAAGGCTGATTACGAAAAGCATAAGGTTTACGCTTGTGAAGTTACTCATCAAGGATTGTCTTCTCCTGTCACAAAATCTTTTAACAGAGGTGAGTGT

SEQ ID NO:7

CCGCTCGAGAAAAGAGAGGCTGAAGCTGATATTCAAATGACTCAATCTCCTTC

SEQ ID NO:8

ATAAGAATGCGGCCGCTCAACACTCACCTCTGTTAAAAGA

SEQ ID NO:9

AGACGAGATCAATTAGAACCTGTTTTGGCAATAACCGAGGATTAGGAACAAAGTCCGGGTAATTATGCGACTCTCTCTTTTTCATATGCAGGTCGAGCAAGAATCTTGTTTTTCGTGGTGGGACTGGGCAGCAATTAACAACCAATCGGCACTTGCAATAAGTCGATTAGCGCATGGTGGGGGACAGTAGATAGCTGCTGAAATTTTTGGGTGCGGACAATTTCAAGAGTCTGAGGCCCTGCTCTCACTAGCAGTCAGAATCATCTGCCTCACATACAGTCTCCTGATGCTGCTACTTACTTGGCAAGCGATGATGTTTACACAATTGCAGCTTTTTAGTTGCTGTCATGGCTCTAGAATTCCTTGGCCTTTTATCAGCATCTCAGGCAACAATGTGAAATGCGCACCTTCAGATTTTTATATTGGTGGACGGAAATGGTAGGAAGTAGTGAAGAAAAGGTAGCGCGAGGGCATTGTCCCCCCATGCGAAGAAAAATTACTAGCATAAAAAAATGGAAACAAGTGACCTCCGCAGCATGTCTGCCCACTTTATACTGAAACTACATTCTTGCTAATGGATCGTAAAACACTATCACCTGGTATGTCGTAATGGACTTGATGGCGTGTACTTTGCTTTACTGAGTTCTAGTTGCCTGCTCAGTGCCGAAGAACATGGGGCTCCATGTGTATTGTATTCTCTTATCTAGAACAATCGAAGCGCTACTGACTTGAAATCCTTGAATGATAGACATAGATTGGACCATGACAAGGAGAACTTATTCACAATTGAAAAGTACATTTTCATTGACCCATTGGGAGGTATCCCATCTTTGGAGAGGTACAAAAGTGCACATGTATATATCAATCTATTACAAGAGTATGAAGACATTGTGTCGGAGCTTTACATAGGGTTCTTAAAGACTGGAGAAAGGGACCAGCATTTGAAGAACTTAAACTTGCTTCAGAAATTGTTGCAGGTAACCACAGATGCATCAGGAATAGTTACTACTCCTCAAATCGCCATGTTGAATCAGACCGACCGATTCACCAATCCAATAATTTACAATGTCTTAACCGATAGGCCGACAATATCATCGTCATTACCGGTTGATTTGAAAAAGACCCCTTTGCTAAACACTTCAATCATTAGGAGAGGCGTACCGGTTGAAGTTTATGTGGACGAATCATCTGACAAAAGTGGGCTGTGCCTAGACTCTCTTCTGAAACGAGGAGCTTTAGACTTAGAAAAGCTTAAGAATGTGATCGATTTGTCGTTTCGAAAGGACTTGAATATGAAAAAGTACCTAGCCAGAGTAAAGAACAATGTTGCAGCTATCTTAATCGCTGGAGATTACGAAGGCGTGATCATAGTTACTTGGGAGGTAACGGATGAAGAAAAGCCGCAGAAAATAGCTTATTTAGATAAGTTTGCAGTGTCTCCTAAGGCCCAAGGATCGACAGGGGTTGCCGATGTTCTTTTCAAGTCATTATTGTCCAATTTTGAGAACGAATTGTTCTGGAGATCTCGATCTAATAATCCAGTGAACAAATGGTACTTTGAACGGAGCAAAGGTTCTCTTACTGTTACTGGCACAAATTGGAAATGCTTCTACACCGGCAAGAACTATCCTTCATTGGATAGAATGAAGGGCTATTTCAACATCTGTGAGAGAATCCAACCTTCCTGGAATGGATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGAGCAAATGCCTGCAAATCGCTCCCCATTTCACCCAATTGTAGATATGCTAACTCCAGCAATGAGTTGATGAATCTCGGTGTGTATTTTATGTCCTCAGAGGACAA

SEQ ID NO:10

GACCTTCGTTTGTGCGGATCCAGACGAGATCAATTAGAACCTG

SEQ ID NO:11

GCTATGGTGTGTGGGGGATCCTTGTCCTCTGAGGACATAAAATAC

SEQ ID NO:12

GGAGACCAACATGTGAGCAAAAG

SEQ ID NO:13

GGATCCGCACAAACGAAGGTC

SEQ ID NO:14

cgtttgtgcggatccGACCTGCAGGGGGGGGGGGG

SEQ ID NO:15

CACATGTTGGTCTCCGCGCCAGCAACCGCACCTGTG

SEQ ID NO:16

CATCATCATCATCATCATTGAGTTTG

SEQ ID NO:17

AGCTTCAGCCTCTCTTTTCTCGAG

SEQ ID NO:18

EVQLVESGGGLVQPGGSLRLSCAASGFSLSNYYVTWVRQAPGKGLEWVGIIYGSDETAYATSAIGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDSSDWDAKFNLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:19

GAGGTCCAGTTGGTCGAGTCTGGTGGTGGTTTGGTTCAGCCAGGTGGATCTTTGAGATTGTCTTGTGCAGCATCTGGATTCTCTTTGTCTAACTACTACGTTACTTGGGTTAGACAGGCACCAGGTAAAGGTTTGGAGTGGGTTGGTATTATTTACGGTTCTGATGAAACTGCTTACGCTACTTCTGCTATTGGTAGATTCACTATTTCTAGAGATAACTCTAAAAACACTTTGTACTTGCAAATGAACTCTTTGAGAGCTGAAGATACAGCCGTTTACTATTGCGCTAGAGATGATTCTTCTGATTGGGATGCTAAGTTTAACTTGTGGGGACAAGGTACTTTGGTTACTGTTTCTTCTGCTTCTACTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTACTTCTGGAGGAACTGCCGCCTTGGGATGTTTGGTCAAGGATTATTTCCCTGAGCCTGTCACAGTTTCTTGGAACTCTGGTGCATTGACATCTGGTGTTCACACATTCCCAGCAGTCTTGCAATCTTCTGGTTTGTACTCTTTGTCTTCTGTCGTTACAGTCCCTTCTTCTTCTTTGGGTACTCAAACCTACATCTGTAATGTTAATCATAAGCCTTCTAACACAAAAGTTGATAAGAAAGTCGAGCCAAAATCTgttGACAAAACACATACCTGCCCACCATGTCCAGCTCCTGAGTTGTTGGGAGGTCCTTCTGTTTTTTTGTTCCCACCTAAGCCAAAGGATACATTGATGATTTCCAGAACTCCTGAAGTTACATGTGTCGTTGTCGATGTTTCTCATGAAGATCCTGAGGTTAAATTCAATTGGTACGTTGATGGTGTCGAAGTTCATAACGCTAAGACTAAACCAAGAGAAGAACAGTACAATTCTACTTATAGAGTCGTCTCTGTTTTGACCGTTTTGCATCAAGATTGGTTGAACGGAAAGGAGTATAAGTGCAAAGTTTCTAATAAGGCTTTGCCTGCCCCTATTGAGAAGACCATTTCTAAGGCTAAAGGTCAGCCTAGAGAACCTCAAGTTTACACTTTGCCACCTTCCAGAGAGGAAATGACTAAGAACCAGGTCTCTTTGTGGTGCTTGGTTAAGGGTTTTTACCCTTCTGACATCGCTGTTGAGTGGGAATCTAACGGACAGCCTGAAAATAACTATAAAACAACACCTCCAGTTTTGGATTCTGACGGTTCTTTCTTTTTGTATTCTAAGTTGACAGTCGATAAGTCCAGATGGCAACAAGGAAACGTCTTTTCTTGTTCTGTTATGCATGAAGCCTTGCATAATCATTACACCCAGAAATCTTTGTCTTTGTCTCCAGGT

SEQ ID NO:20

AGAGAGGCTGAAGCTGAGGTCCAGTTGGTCGAGTCTGGTG

SEQ ID NO:21

TCAATGATGATGATGATGATGtcaACCTGGAGACAAAGACAAAGATTTC

SEQ ID NO:22

GACAGATGGTCCCTTAGTAGAAGC

SEQ ID NO:23

CTAAGGGACCATCTGTCtgtCCATTG

SEQ ID NO:24

CTAAGGGACCATCTGTCTTTCCAtgtGCACCATCTTC

SEQ ID NO:25

CTAAGGGACCATCTGTCTTTCCATTGtgtCCATCTTCTAAA

SEQ ID NO:26

CTAAGGGACCATCTGTCTTTCCATTGGCAtgtTCTTCTAAATCTACTTC

SEQ ID NO:27

CTAAGGGACCATCTGTCTTTCCATTGGCACCAtgtTCT

SEQ ID NO:28

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTtgtAAATC

SEQ ID NO:29

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTtgtTCTACT

SEQ ID NO:30

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAAtgtACTTCTGGAG

SEQ ID NO:31

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTtgtTCTGGAGGAACT

SEQ ID NO:32

AGATGTCAATGCACCAGAGTTCC

SEQ ID NO:33

GGTGCATTGACATCTtgtGTTCAC

SEQ ID NO:34

GGTGCATTGACATCTGGTGTTtgtACATTC

SEQ ID NO:35

GGTGCATTGACATCTGGTGTTCACACAtgtCCAGCA

SEQ ID NO:36

GGTGCATTGACATCTGGTGTTCACACATTCtgtGCAGTC

SEQ ID NO:37

GGTGCATTGACATCTGGTGTTCACACATTCCCAGCAtgtTTGCAA

SEQ ID NO:38

GGTGCATTGACATCTGGTGTTCACACATTCCCAGCAGTCTTGtgtTCTTCT

SEQ ID NO:39

TTGCAAGACTGCTGGGAATGTGTG

SEQ ID NO:40

CCAGCAGTCTTGCAAtgtTCTGGTTTG

SEQ ID NO:41

CCAGCAGTCTTGCAATCTtgtGGTTTGTAC

SEQ ID NO:42

CCAGCAGTCTTGCAATCTTCTtgtTTGTACTCTTTGTC

SEQ ID NO:43

CCAGCAGTCTTGCAATCTTCTGGTtgtTACTCTTTGTCTTC

SEQ ID NO:44

GTACAAACCAGAAGATTGCAAGAC

SEQ ID NO:45

CAATCTTCTGGTTTGTACtgtTTGTC

SEQ ID NO:46

CAATCTTCTGGTTTGTACTCTTTGtgtTCTGTC

SEQ ID NO:47

CAATCTTCTGGTTTGTACTCTTTGTCTTCTtgtGTTACAGTCCCTTC

SEQ ID NO:48

CAATCTTCTGGTTTGTACTCTTTGTCTTCTGTCGTTtgtGCTCCT

SEQ ID NO:49

TGGCTCGACTCTCTTATCAAC

SEQ ID NO:50

AAGAAAGTCGAGCCAtgtTCTGTTGAC

SEQ ID NO:51

AAGAAAGTCGAGCCAAAAtgtGTTGAC

SEQ ID NO:52

AAGAAAGTCGAGCCAAAATCTTGCGAC

SEQ ID NO:53

AGGTTTTATACTGAGTTTGTTAATGATACAATAAACTGTTATAGTACATACAATTGAAACTCTCTTATCTATACTGGGGGACCTTCTCGCAGAATGGTATAAATATCTACTAACTGACTGTCGTACGGCCTAGGGGTCTCTTCTTCGATTATTTGCAGGTCGGAACATCCTTCGTCTGATGCGGATCTCCTGAGACAAAGTTCACGGGTATCTAGTATTCTATCAGCATAAATGGAGGACCTTTCTAAACTAAACTTTGAATCGTCTCCAGCAGCATCCTCGCATAATCCTTTTGTCATTTCCTCTATGTCTATTGTCACTGTGGTTGGCGCATCAAGAGTCGTCCTTCTGTAAACCGGTACAGAATTCCTACCACTAGAAGCTTGAAATGGGGAGGGTTTCAGCTTTGTATCCCGATACTGTGCTTTAAAAAGGGAGTCCAAACTGAAATCTTTTTCGGAATCATTGGATGATACCTCTGTATTAGATCTCCTATGTATCGGTTTCCTCGGGTAGATAGAACTGTCGACAGAGTCTCTAGCAAATTGAGAATTGGCTTTAGACTTCGGAGAAGTAGGTGACGATGTGGTAGATATATCAGCAGATGAAAAGACTGAATTTTTTCTCTCTGGTGATGAGTTGATTCCCTCTTGGTGTTGATATCTTGAACCGGGCTCATGTTGAACCTTTGAATTCAAAAAGGTAGCTTGAAGTTGCTCATTGACAGCATCTGCCACGTCGTATCTTGCAACATCTTTGGGAAACTGATACGAAGTGTTCAACATCTTTGTTGCGGTATGACAAGAGCACTTCGTTGTACTTTTATCAGAGAAAAGAAACACCTCAATTATGGTATTTAGGTTTATATATTACGCAAATTCTATTAGAAAACCCGGGAGCTGGAGCTTTGGCTGGTCATCCTTATGGAATTGATCGTGAATACATTGCTGAGAGATTAGGGTTTGATTCTGTTATTGGTAATTCTTTGGCCGCTGTTTCAGACAGAGATTTTGTAGTCGAAACCATGTTCTGGTCTTCGTTGTTTATGAATCATATTTCTCGATTCTCAGAAGATTTGATCATTTACTCCACTGGAGAGTTTGGATTTATCAAGTTGGCAGATGCTTATTCTACTGGATCTTCTCTGATGCCTACAAAAAAAAACCCAGACTCTTTGGAGTTATTGAGGGGTAAATCTGGTAGATGTTTTGGGGCCTTGGCTGGTTTCCTCATGTCTATTAAGTCCATTCCGTCAACCTATAACAAAGATATGCAAGAGGATAAGGAGCCTTTATTTGATACTCTAATCACTGTAGAGCACTCGATTTTGATAGCATCCGGTGTAGTTTCTACCTTGAACATTGATGCCGAACGAATGAAGAATGCTCTAACTATGGATATGCTGGCTACAGATCTTGCCGACTATTTAGTTAGAAGGGGAGTTCCATTCAGAGAAACTCACCACATTTCTGGTGAATGTGTCAGACAAGCCGAGGAGTTGAACCTTTCTGGTATTGATCAGTTGTCCCTCGAACAATTGAAATCCATTGACTCCCGTTTTGAGGCTGATGTGGCTTCAACGTTTGACTTTGAAGCCAGTGTTGAAAAAAGAACTGCCACCGGAGGAACTTCTAAGACTGCTGTTTTAAAGCAATTGGATGCACTGAATGAAAAGCTAGAGTCTTGAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGAGCAAATGCCTGCAAATCGCTCCCCATTTCACCCAATTGTAGATATGCTAACTCCAGCAATGAGTTGATGAATCTCGGTGTGTATTTTATGTCCTCAGAGGACAA

SEQ ID NO:54

GACCTTCGTTTGTGCGGATCCAGGTTTTATACTGAGTTTGTTA

SEQ ID NO:55

GCTATGGTGTGTGGGGGATCCTTGTCCTCTGAGGACATAAAATAC

SEQ ID NO:56

AIQMTQSPSSLSASVGDRVTITCQASQSINNELSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQGYSLRNIDNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEV

SEQ ID NO:57

GCTATTCAAATGACTCAATCTCCTTCTTCTTTGTCTGCTTCTGTCGGAGATAGAGTCACTATCACTTGTCAAGCTTCTCAATCTATTAACAACGAATTGTCTTGGTATCAGCAAAAGCCAGGTAAGGCTCCAAAATTGTTGATTTACAGAGCATCTACTTTGGCTTCTGGTGTCCCTTCCAGATTCTCTGGTTCTGGTTCTGGAACCGATTTTACTTTGACTATCTCTTCTTTGCAGCCTGATGATTTCGCAACTTACTACTGTCAACAGGGTTACTCTTTGAGAAACATTGATAACGCTTTTGGTGGTGGAACTAAAGTTGAGATTAAGAGAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTCCATCTGACGAGCAGTTGAAGTCTGGTACAGCATCTGTTGTCTGTTTGTTGAACAACTTCTACCCAAGAGAAGCAAAGGTTCAATGGAAGGTTGACAACGCCTTGCAATCTGGTAACTCTCAGGAATCTGTTACTGAACAAGATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGACATTGTCTAAGGCTGATTACGAAAAGCATAAGGTTTACGCTTGTGAAGTTACTCATCAAGGATTGTCTTCTCCTGTCACAAAATCTTTTAACAGAGGTGAGgtt

SEQ ID NO:58

AGAGAGGCTGAAGCTGCTATTCAAATGACTCAATCTCC

SEQ ID NO:59

TCAATGATGATGATGATGATGTCAaacCTCACCTCTGTTAAAAGATTTTG

SEQ ID NO:60

AGGTGCAGCGACAGTTCTAGTAATTTG

SEQ ID NO:61

GAACTGTCGCTGCACCTtgtGTTTTC

SEQ ID NO:62

GAACTGTCGCTGCACCTTCTGTTtgtATTTTTCCTCCATC

SEQ ID NO:63

GAACTGTCGCTGCACCTTCTGTTTTCATTtgtCCTCCATCTGAC

SEQ ID NO:64

GAACTGTCGCTGCACCTTCTGTTTTCATTTTTtgtCCATCTGACGAGCAG

SEQ ID NO:65

GAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTtgtTCTGACGAGCAGTTG

SEQ ID NO:66

GAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTCCAtgtGACGAGCAGTTG

SEQ ID NO:67

ACCAGATTGCAAGGCGTTGTCAAC

SEQ ID NO:68

GCCTTGCAATCTGGTtgtTCTCAGGAATCTG

SEQ ID NO:69

GCCTTGCAATCTGGTAACTCTtgtGAATCTGTTACTG

SEQ ID NO:70

GCCTTGCAATCTGGTAACTCTCAGGAAtgtGTTACTGAAC

SEQ ID NO:71

GCCTTGCAATCTGGTAACTCTCAGGAATCTGTTtgtGAACAAGATTCTAAG

SEQ ID NO:72

AGAATCCTTAGAATCTTGTTCAG

SEQ ID NO:73

GATTCTAAGGATTCTtgtTACTCTTTGTC

SEQ ID NO:74

GATTCTAAGGATTCTACTTACtgtTTGTCTTC

SEQ ID NO:75

GATTCTAAGGATTCTACTTACTCTTTGtgtTCTACA

SEQ ID NO:76

GATTCTAAGGATTCTACTTACTCTTTGTCTTCTtgtTTGACA

SEQ ID NO:77

GATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGtgtTTGTCTAAGGCTG

SEQ ID NO:78

TCAATGATGATGATGATGATGTCAaacCTCACCTCTGTTACAAGA

SEQ ID NO:79

TCAATGATGATGATGATGATGTCAaacCTCACCTCTACAAAAAG

SEQ ID NO:80

TCAATGATGATGATGATGATGTCAaacCTCACCACAGTTAAAAG

SEQ ID NO:81

TCAATGATGATGATGATGATGTCAaacCTCACATCTGTTAAAAG

SEQ ID NO:82

TCAATGATGATGATGATGATGTCAaacACAACCTCTGTTAAAAG

SEQ ID NO:83

TCAATGATGATGATGATGATGTCAACACTCACCTCTGTTAAAAG

SEQ ID NO:84

ATCAACTTTTGTGTTAGA

SEQ ID NO:85

TCTAACACAAAAGTTGATGATAAAGTCGAGCCAAAA

SEQ ID NO:86

TCTAACACAAAAGTTGATGAAAAAGTCGAGCCAAAA

SEQ ID NO:87

AGATGGAGGAAAAAT

SEQ ID NO:88

ATTTTTCCTCCATCTGACAAGCAGTTGAAGTCTGGT

SEQ ID NO:89

ACATGGAGGAAAAATGAAAAC

SEQ ID NO:90

ATTTTTCCTCCATGTGACAAGCAGTTGAAGTCTGGT

SEQ ID NO:91

AGAACAAGGAAAAATGAAAAC

SEQ ID NO:92

ATTTTTCCTTGTTCTGACAAGCAGTTGAAGTCTGGT

SEQ ID NO:93

AGATGGACAAAAAATGAAAAC

SEQ ID NO:94

ATTTTTTGTCCATCTGACAAGCAGTTGAAGTCTGGT

SEQ ID NO:95

AGATGGAGGACAAATGAAAAC

SEQ ID NO:96

ATTTGTCCTCCATCTGACAAGCAGTTGAAGTCTGGT

SEQ ID NO:97

ATTTTTCCTCCATCTGACAGACAGTTGAAGTCTGGT

SEQ ID NO:98

ATTTTTCCTCCATGTGACAGACAGTTGAAGTCTGGT

SEQ ID NO:99

ATTTTTCCTTGTTCTGACAGACAGTTGAAGTCTGGT

SEQ ID NO:100

ATTTTTTGTCCATCTGACAGACAGTTGAAGTCTGGT

SEQ ID NO:101

ATTTGTCCTCCATCTGACAGACAGTTGAAGTCTGGT

SEQ ID NO:102

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWVGWINPYTGSAFYAQKFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREPEKFDSDDSDVWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGHHHHHH

SEQ ID NO:103

CAAGTTCAATTGGTTCAATCAGGAGCTGAAGTTAAGAAGCCAGGTGCTTCCGTTAAGGTTTCTTGTAAGGCTTCAGGATACACTTTTACTAACTACTACATGCATTGGGTTAGACAAGCTCCAGGACAAGGTTTGGAATGGGTTGGATGGATTAACCCATACACTGGGAGTGCTTTTTACGCTCAAAAGTTTAGAGGTAGAGTTACTATGACTAGAGATACTTCTATTTCCACTGCTTACATGGAATTGTCGCGTTTGAGATCAGATGATACTGCTGTTTACTACTGTGCTAGAGAACCTGAAAAGTTTGATTCCGATGATTCTGATGTTTGGGGTAGAGGTACTTTGGTTACTGTTTCTTCTGCTTCTACTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTACTTCTGGAGGAACTGCCGCCTTGGGATGTTTGGTCAAGGATTATTTCCCTGAGCCTGTCACAGTTTCTTGGAACTCTGGTGCATTGACATCTGGTGTTCACACATTCCCAGCAGTCTTGCAATCTTCTGGTTTGTACTCTTTGTCTTCTGTCGTTACAGTCCCTTCTTCTTCTTTGGGTACTCAAACCTACATCTGTAATGTTAATCATAAGCCTTCTAACACAAAAGTTGATAAGAAAGTCGAGCCAAAATCTTGCGACAAAACACATACCTGCCCACCATGTCCAGCTCCTGAGTTGTTGGGAGGTCCTTCTGTTTTTTTGTTCCCACCTAAGCCAAAGGATACATTGATGATTTCCAGAACTCCTGAAGTTACATGTGTCGTTGTCGATGTTTCTCATGAAGATCCTGAGGTTAAATTCAATTGGTACGTTGATGGTGTCGAAGTTCATAACGCTAAGACTAAACCAAGAGAAGAACAGTACAATTCTACTTATAGAGTCGTCTCTGTTTTGACCGTTTTGCATCAAGATTGGTTGAACGGAAAGGAGTATAAGTGCAAAGTTTCTAATAAGGCTTTGCCTGCCCCTATTGAGAAGACCATTTCTAAGGCTAAAGGTCAGCCTAGAGAACCTCAAGTTTACACTTTGCCACCTTCCAGAGAGGAAATGACTAAGAACCAGGTCTCTTTGTCTTGCGCTGTTAAGGGTTTTTACCCTTCTGACATCGCTGTTGAGTGGGAATCTAACGGACAGCCTGAAAATAACTATAAAACAACACCTCCAGTTTTGGATTCTGACGGTTCTTTCTTTTTGGTTTCTAAGTTGACAGTCGATAAGTCCAGATGGCAACAAGGAAACGTCTTTTCTTGTTCTGTTATGCATGAAGCCTTGCATAATAGATTTACCCAGAAATCTTTGTCTTTGTCTCCAGGTcatcatcatcatcatcat

SEQ ID NO:104

CCGCTCGAGAAAAGAGAGGCTGAAGCTCAAGTTCAATTGGTTCAATCAG

SEQ ID NO:105

ATAGTTTAGCGGCCGCTCAATGATGATGATGATGATGACCTGGAGACAAAGA

SEQ ID NO:106

QAVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYGVHWYQQLPGTAPKLLIYGDSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDNSLSGYVFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:107

CAAGCTGTTTTGACTCAACCACCATCCGTTTCAGGGGCGCCTGGTCAAAGAGTTACTATTTCATGTACTGGGTCCTCATCCAACATTGGTGCTGGATACGGTGTTCATTGGTATCAACAATTGCCAGGAACTGCTCCTAAGTTGTTGATTTACGGTGATTCTAACAGACCATCTGGTGTTCCAGATAGATTTTCCGGATCTAAGTCCGGAACTTCTGCTTCCTTGGCTATTACTGGTTTGCAAGCTGAAGATGAAGCTGATTACTACTGTCAATCATACGATAACTCCTTGTCCGGATACGTTTTTGGTGGAGGAACTCAATTGACTGTTTTGGGACAACCAAAGGCTGCTCCATCCGTTACTTTGTTTCCACCATCTTCAGAAGAATTGCAAGCTAACAAGGCTACTTTGGTTTGTTTGATTTCCGATTTTTACCCAGGAGCTGTTACTGTTGCTTGGAAGGCTGATTCCTCACCAGTTAAGGCTGGTGTTGAAACTACTACTCCATCTAAGCAATCAAACAACAAGTACGCTGCTTCATCATACTTGTCCTTGACTCCAGAACAATGGAAGTCACATAGATCATACTCATGTCAAGTTACTCATGAAGGAAGTACTGTTGAAAAGACTGTTGCTCCAACTGAATGTTCT

SEQ ID NO:108

CCGCTCGAGAAAAGAGAGGCTGAAGCTCAAGCTGTTTTGACTCAACC

SEQ ID NO:109

AAGGAAAAAAGCGGCCGCTTAAGAACATTCAGTTGGAGCAAC

SEQ ID NO:110

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:111

GAGGTCCAGTTGTTGGAGTCTGGTGGTGGTTTGGTTCAGCCAGGTGGATCTTTGAGATTGTCTTGTGCAGCATCTGGATTCACTTTTTCTTCTTTTCCAATGGCTTGGGTTAGACAGGCACCAGGTAAAGGTTTGGAGTGGGTTTCTACTATTTCTACTTCTGGTGGTAGAACTTACTACAGAGATTCTGTCAAGGGTAGATTCACTATTTCTAGAGATAACTCTAAAAACACTTTGTACTTGCAAATGAACTCTTTGAGAGCTGAAGATACAGCCGTTTACTATTGCGCTAAGTTTAGACAATACTCTGGTGGTTTTGATTACTGGGGACAAGGTACTTTGGTTACTGTTTCTTCTGCTTCTACTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTACTTCTGGAGGAACTGCCGCCTTGGGATGTTTGGTCAAGGATTATTTCCCTGAGCCTGTCACAGTTTCTTGGAACTCTGGTGCATTGACATCTGGTGTTCACACATTCCCAGCAGTCTTGCAATCTTCTGGTTTGTACTCTTTGTCTTCTGTCGTTACAGTCCCTTCTTCTTCTTTGGGTACTCAAACCTACATCTGTAATGTTAATCATAAGCCTTCTAACACAAAAGTTGATAAGAAAGTCGAGCCAAAATCTGTTGACAAAACACATACCTGCCCACCATGTCCAGCTCCTGAGTTGTTGGGAGGTCCTTCTGTTTTTTTGTTCCCACCTAAGCCAAAGGATACATTGATGATTTCCAGAACTCCTGAAGTTACATGTGTCGTTGTCGATGTTTCTCATGAAGATCCTGAGGTTAAATTCAATTGGTACGTTGATGGTGTCGAAGTTCATAACGCTAAGACTAAACCAAGAGAAGAACAGTACAATTCTACTTATAGAGTCGTCTCTGTTTTGACCGTTTTGCATCAAGATTGGTTGAACGGAAAGGAGTATAAGTGCAAAGTTTCTAATAAGGCTTTGCCTGCCCCTATTGAGAAGACCATTTCTAAGGCTAAAGGTCAGCCTAGAGAACCTCAAGTTTACACTTTGCCACCTTCCAGAGAGGAAATGACTAAGAACCAGGTCTCTTTGTGGTGCTTGGTTAAGGGTTTTTACCCTTCTGACATCGCTGTTGAGTGGGAATCTAACGGACAGCCTGAAAATAACTATAAAACAACACCTCCAGTTTTGGATTCTGACGGTTCTTTCTTTTTGTATTCTAAGTTGACAGTCGATAAGTCCAGATGGCAACAAGGAAACGTCTTTTCTTGTTCTGTTATGCATGAAGCCTTGCATAATCATTACACCCAGAAATCTTTGTCTTTGTCTCCAGGT

SEQ ID NO:112

AGAGAGGCTGAAGCTGAGGTCCAGTTGTTGGAGTCTG

SEQ ID NO:113

TCAATGATGATGATGATGATGtcaACCTGGAGACAAAGACAAAGATTTC

SEQ ID NO:114

GACAGATGGTCCCTTAGTAGAAGC

SEQ ID NO:115

CTAAGGGACCATCTGTCtgtCCATTG

SEQ ID NO:116

CTAAGGGACCATCTGTCTTTCCAtgtGCACCATCTTC

SEQ ID NO:117

CTAAGGGACCATCTGTCTTTCCATTGtgtCCATCTTCTAAATC

SEQ ID NO:118

CTAAGGGACCATCTGTCTTTCCATTGGCAtgtTCTTCTAAATCTACTTC

SEQ ID NO:119

CTAAGGGACCATCTGTCTTTCCATTGGCACCAtgtTCTAAATCTACT

SEQ ID NO:120

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTtgtAAATCTACT

SEQ ID NO:121

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTtgtTCTACTTCTGGAG

SEQ ID NO:122_

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAAtgtACTTCTGGAG

SEQ ID NO:123

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTtgtTCTGGAGGAACT

SEQ ID NO:124

CTAAGGGACCATCTGTCTTTCCATTGGCACCATCTTCTAAATCTACTtgtGGAGGAACT

SEQ ID NO:125

AGATGTCAATGCACCAGAGTTCC

SEQ ID NO:126

GGTGCATTGACATCTtgtGTTCACACATTCCCA

SEQ ID NO:127

GGTGCATTGACATCTGGTGTTtgtACATTCCCAGC

SEQ ID NO:128

GGTGCATTGACATCTGGTGTTCACACAtgtCCAGCAGTCTTGC

SEQ ID NO:129

GGTGCATTGACATCTGGTGTTCACACATTCtgtGCAGTCTTGCAA

SEQ ID NO:130

GGTGCATTGACATCTGGTGTTCACACATTCCCAGCAtgtTTGCAATCTTCT

SEQ ID NO:131

GGTGCATTGACATCTGGTGTTCACACATTCCCAGCAGTCTTGtgtTCTTCTGGTTTG

SEQ ID NO:132

TTGCAAGACTGCTGGGAATGTGTG

SEQ ID NO:133

CCAGCAGTCTTGCAAtgtTCTGGTTTG

SEQ ID NO:134

CCAGCAGTCTTGCAATCTtgtGGTTTGTACTCTTTG

SEQ ID NO:135

CCAGCAGTCTTGCAATCTTCTtgtTTGTACTCTTTGTC

SEQ ID NO:136

CCAGCAGTCTTGCAATCTTCTGGTtgtTACTCTTTGTCTTC

SEQ ID NO:137

GTACAAACCAGAAGATTGCAAGAC

SEQ ID NO:138

CAATCTTCTGGTTTGTACtgtTTGTCTTCTGTC

SEQ ID NO:139

CAATCTTCTGGTTTGTACTCTTTGtgtTCTGTCGTTACAG

SEQ ID NO:140

CAATCTTCTGGTTTGTACTCTTTGTCTTCTtgtGTTACAGTCCCTTC

SEQ ID NO:141_

CAATCTTCTGGTTTGTACTCTTTGTCTTCTGTCGTTtgtGTCCCTTCTTCTTC

SEQ ID NO:142

TTTCTTATCAACTTTTGTGTTAG

SEQ ID NO:143

CAAAAGTTGATAAGAAAtgtGAGCCAAAATCTG

SEQ ID NO:144

CAAAAGTTGATAAGAAAGTCtgtCCAAAATCTGTTG

SEQ ID NO:145

CAAAAGTTGATAAGAAAGTCGAGtgtAAATCTGTTGAC

SEQ ID NO:146

AAGAAAGTCGAGCCAtgtTCTGTTGACAAAACA

SEQ ID NO:147

AAGAAAGTCGAGCCAAAAtgtGTTGACAAAACA

SEQ ID NO:148

AAGAAAGTCGAGCCAAAATCTtgcGACAAAACACATA

SEQ ID NO:149.

DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEVS

SEQ ID NO:150

GATATTCAATTGACTCAACCTAACTCAGTTTCCACTTCCTTGGGTTCGACTGTTAAGTTGTCATGTACTTTGTCCTCTGGTAACATTGAAAACAACTACGTTCATTGGTATCAATTGTACGAAGGTAGATCCCCTACTACTATGATTTACGATGATGATAAGAGACCTGATGGAGTTCCTGATAGATTTTCCGGTTCTATTGATAGAAGCTCTAACTCCGCTTTTTTGACTATTCATAACGTTGCTATTGAAGATGAAGCTATTTACTTTTGTCATTCTTACGTTTCTTCCTTTAACGTTTTTGGAGGTGGAACTAAGTTGACTGTTTTGAGACAACCAAAGGCTGCTCCATCCGTTACTTTGTTTCCACCATCTTCAGAAGAATTGCAAGCTAACAAGGCTACTTTGGTTTGTTTGATTTCCGATTTTTACCCAGGAGCTGTTACTGTTGCTTGGAAGGCTGATTCCTCACCAGTTAAGGCTGGTGTTGAAACTACTACTCCATCTAAGCAATCAAACAACAAGTACGCTGCTTCATCATACTTGTCCTTGACTCCAGAACAATGGAAGTCACATAGATCATACTCATGTCAAGTTACTCATGAAGGAAGTACTGTTGAAAAGACTGTTGCTCCAACTGAAgttTCT

SEQ ID NO:151

AGAGAGGCTGAAGCTGATATTCAATTGACTCAAC

SEQ ID NO:152

TCAATGATGATGATGATGATGTCAAGAAACTTCAGTTGGAGCAAC

SEQ ID NO:153

TGGAGCAGCCTTTGGTTGTCTC

SEQ ID NO:154

CCAAAGGCTGCTCCAtgtGTTACTTTGTTTCCACC

SEQ ID NO:155

CCAAAGGCTGCTCCATCCGTTtgtTTGTTTCCACCATCTTC

SEQ ID NO:156

CCAAAGGCTGCTCCATCCGTTACTTTGtgtCCACCATCTTCAG

SEQ ID NO:157

CCAAAGGCTGCTCCATCCGTTACTTTGTTTtgtCCATCTTCAGAAGAATTG

SEQ ID NO:158

CCAAAGGCTGCTCCATCCGTTACTTTGTTTCCACCAtgtTCAGAAGAATTG

SEQ ID NO:159

AGCCTTAACTGGTGAGGAATCAGC

SEQ ID NO:160

TCACCAGTTAAGGCTtgtGTTGAAACTACTACTC

SEQ ID NO:161

TCACCAGTTAAGGCTGGTGTTtgtACTACTACTCCATCTAAG

SEQ ID NO:162

TCACCAGTTAAGGCTGGTGTTGAAACTtgtACTCCATCTAAGCAATC

SEQ ID NO:163

TCACCAGTTAAGGCTGGTGTTGAAACTACTtgtCCATCTAAGCAATCA

SEQ ID NO:164

TCACCAGTTAAGGCTGGTGTTGAAACTACTACTtgtTCTAAGCAATCAAAC

SEQ ID NO:165

TCACCAGTTAAGGCTGGTGTTGAAACTACTACTCCAtgtAAGCAATC

SEQ ID NO:166

TCACCAGTTAAGGCTGGTGTTGAAACTACTACTCCATCTAAGtgtTCAAACAACAAGTA

SEQ ID NO:167

GTTGTTTGATTGCTTAGATGGAG

SEQ ID NO:168

TAAGCAATCAAACAACtgtTACGCTGCTTCATCATAC

SEQ ID NO:169

TAAGCAATCAAACAACAAGTACtgtGCTTCATCATACTTGTC

SEQ ID NO:170

TAAGCAATCAAACAACAAGTACGCTGCTtgtTCATACTTGTCCTTGAC

SEQ ID NO:171

TAAGCAATCAAACAACAAGTACGCTGCTTCATCAtgtTTGTCCTTGACTCCAG

SEQ ID NO:172

TAAGCAATCAAACAACAAGTACGCTGCTTCATCATACTTGtgtTTGACTCCAGAACAAT

SEQ ID NO:173

CAATGATGATGATGATGATGTCAAGAAACTTCAGTTGGAGCacaAGTCTTTTCAACAG

SEQ ID NO:174

CAATGATGATGATGATGATGTCAAGAAACTTCAGTTGGacaAACAGTCTTTTCAACA

G

SEQ ID NO:175

TCAATGATGATGATGATGATGTCAAGAAACTTCAGTacaAGCAACAGTCTTTTCAAC

SEQ ID NO:176

TCAATGATGATGATGATGATGTCAAGAAACTTCacaTGGAGCAACAGTCTTTTC

SEQ ID NO:177

TCAATGATGATGATGATGATGTCAAGAAACacaAGTTGGAGCAACAGTC

SEQ ID NO:178

TCAATGATGATGATGATGATGTCAAGAacaTTCAGTTGGAGCAAC

SEQ ID NO:179

TCAATGATGATGATGATGATGTCAacaAACTTCAGTTGGAGCAAC

SEQ ID NO:180

ATCAACTTTTGTGTTAGAAGGC

SEQ ID NO:181

TAACACAAAAGTTGATgatAAAGTCGAGCCAAAATCTG

SEQ ID NO:182

TAACACAAAAGTTGATgatAAAtgtGAGCCAAAATCTG

SEQ ID NO:183

TAACACAAAAGTTGATgatAAAGTCtgtCCAAAATCTG

SEQ ID NO:184

TAACACAAAAGTTGATgatAAAGTCGAGtgtAAATCTG

SEQ ID NO:185

TAACACAAAAGTTGATgatAAAGTCGAGCCAtgtTCTG

SEQ ID NO:186

TAACACAAAAGTTGATgatAAAGTCGAGCCAAAAtgtG

SEQ ID NO:187

ATCAACTTTTGTGTTAGAAGGC

SEQ ID NO:188

TAACACAAAAGTTGATgaaAAAGTCGAGCCAAAATCTG

SEQ ID NO:189

TAACACAAAAGTTGATgaaAAAtgtGAGCCAAAATCTG

SEQ ID NO:190

TAACACAAAAGTTGATgaaAAAGTCtgtCCAAAATCTG

SEQ ID NO:191

TAACACAAAAGTTGATgaaAAAGTCGAGtgtAAATCTG

SEQ ID NO:192

TAACACAAAAGTTGATgaaAAAGTCGAGCCAtgtTCTG

SEQ ID NO:193

TAACACAAAAGTTGATgaaAAAGTCGAGCCAAAAtgtG

SEQ ID NO:194

TGAAGATGGTGGAAACAAAGTAAC

SEQ ID NO:195

GTTTCCACCATCTTCAaagGAATTGCAAGCTAACAAGGCTAC

SEQ ID NO:196

TGAAGATGGTGGAAACAAAGTAACACA

SEQ ID NO:197

TGAAGATGGTGGAAACAAACAAAC

SEQ ID NO:198

GTTAGCTTGCAATTCcttTGAACATGGTGGAAACAAAG

SEQ ID NO:199

GAATTGCAAGCTAACAAGGCTAC

SEQ ID NO:200

GTTAGCTTGCAATTCcttTGAAGATGGACAAAACAAAG

SEQ ID NO:201

GTTAGCTTGCAATTCcttTGAAGATGGTGGACACAAAG

SEQ ID NO:202

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSLGHHHHHH

SEQ ID NO:203

CAAGTTCAATTGGTTCAATCCGGAGCTGAAGTTAAGAAGCCTGGGTCATCCGTTAAGGTTTCATGTAAGGCTTCAGGATACTCTTTTACTGATTACCATATTCATTGGGTTAGACAAGCTCCAGGTCAAGGTTTGGAATGGATGGGAGTTATTAACCCTATGTACGGAACTACTGATTACAACCAAAGATTTAAGGGTAGAGTTACTATTACTGCTGATGAATCCACTTCCACTGCTTACATGGAATTGTCCTCTTTGAGATCAGAAGATACTGCTGTTTACTACTGTGCTAGATACGATTACTTTACTGGAACTGGTGTTTACTGGGGACAAGGAACTTTGGTTACTGTTTCTTCCGCTTCCACTAAGGGACCATCCGTTTTTCCATTGGCTCCATGTTCTAGATCCACTTCCGAATCAACTGCTGCTTTGGGATGTTTGGTTAAGGATTACTTTCCAGAACCAGTTACTGTTTCATGGAACTCAGGAGCTTTGACTTCCGGAGTTCATACTTTTCCAGCTGTTTTGCAATCCTCAGGATTGTACTCTTTGTCTTCAGTTGTTACTGTTCCATCTTCTTCCTTGGGTACTAAGACTTACACTTGTAACGTTGATCATAAGCCATCTAACACTAAGGTTGATAAGAGAGTTGAATCTAAGTACGGACCTCCATGTCCACCATGTCCAGCTCCTGAATTTTTGGGTGGACCATCCGTTTTTTTGTTTCCACCAAAGCCAAAGGATACTTTGATGATTTCTAGAACTCCTGAAGTTACTTGTGTTGTTGTTGATGTTTCACAAGAAGATCCTGAAGTTCAATTTAACTGGTACGTTGATGGAGTTGAAGTTCATAACGCTAAGACTAAGCCTAGAGAAGAACAATTTAACTCCACTTACAGAGTTGTTTCCGTTTTGACTGTTTTGCATCAAGATTGGTTGAACGGTAAGGAATACAAGTGTAAGGTTTCTAACAAGGGATTGCCATCCTCTATTGAAAAGACTATTTCAAAGGCTAAGGGTCAACCTAGAGAACCTCAAGTTTACACTTTGCCACCTTCACAAGAAGAAATGACTAAGAACCAAGTTTCCTTGtctTGTgctGTTAAGGGATTTTACCCATCCGATATTGCTGTTGAATGGGAATCAAACGGTCAACCTGAAAACAACTACAAGACTACTCCACCAGTTTTGGATTCTGATGGATCATTTTTTTTGgttTCTAGATTGACTGTTGATAAGTCTAGATGGCAAGAAGGTAACGTTTTTTCATGTTCCGTTATGCATGAAGCTTTGCATAACagatttACTCAAAAGTCCTTGTCCTTGTCATTGGGAcatcatcatcatcatcat

SEQ ID NO:204

CCGCTCGAGAAAAGAGAGGCTGAAGCTCAAGTTCAATTGGTTCAATCCG

SEQ ID NO:205

AAGGAAAAAAGCGGCCGCTTAATGATGATGATGATGATGTCCCAATGACAAGGACAAG

SEQ ID NO:206.

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:207

GATATTGTTATGACTCAAACTCCATTGTCCTTGTCCGTTACTCCAGGTCAACCTGCTTCTATTTCATGTAGATCATCTAGGTCCTTGGTTCATTCCAGAGGTAACACTTACTTGCATTGGTACTTGCAAAAGCCAGGACAATCACCACAATTGTTGATTTACAAGGTTTCTAACAGATTTATTGGTGTTCCTGATAGATTTTCCGGATCTGGATCAGGAACTGATTTTACTTTGAAGATTTCGCGGGTTGAAGCTGAAGATGTTGGAGTTTACTACTGTTCCCAATCCACTCATTTGCCTTTTACTTTTGGTCAAGGAACTAAGTTGGAAATTAAGAGAACTGTTGCTGCACCTTCTGTTTTCATTTTTCCTCCATCTGACGAGCAGTTGAAGTCTGGTACAGCATCTGTTGTCTGTTTGTTGAACAACTTCTACCCAAGAGAAGCAAAGGTTCAATGGAAGGTTGACAACGCCTTGCAATCTGGTAACTCTCAGGAATCTGTTACTGAACAAGATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGACATTGTCTAAGGCTGATTACGAAAAGCATAAGGTTTACGCTTGTGAAGTTACTCATCAAGGATTGTCTTCTCCTGTCACAAAATCTTTTAACAGAGGTGAGTGT

SEQ ID NO:208

CCGCTCGAGAAAAGAGAGGCTGAAGCTGATATTGTTATGACTCAAACTC

SEQ ID NO:209

AAGGAAAAAAGCGGCCGCTTAACACTCACCTCTGTTAAAAG

SEQ ID NO:210

EVQLVESGGGLVQPGGSLRLSCAASGFNFNDYFMNWVRQAPGKGLEWVAQMRNKNYQYGTYYAESLEGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARESYYGFTSYWGQGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLCCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

SEQ ID NO:211.

GAAGTTCAATTGGTTGAATCTGGAGGTGGATTGGTTCAACCTGGTGGATCATTGAGATTGTCATGTGCTGCTTCTGGATTTAACTTTAACGATTACTTTATGAACTGGGTTAGACAAGCTCCAGGAAAGGGATTGGAATGGGTTGCTCAAATGAGAAACAAGAACTACCAATACGGTACTTACTACGCTGAATCCTTGGAAGGTAGATTTACTATTTCTAGAGATGATTCAAAGAACTCCTTGTACTTGCAAATGAACTCATTGAAGACTGAAGATACTGCTGTTTACTACTGTGCTAGAGAATCATACTACGGATTTACTTCTTACTGGGGACAAGGAACTTTGGTTACTGTTTCTTCCGCTTCCACTAAGGGACCATCCGTTTTTCCATTGGCTCCATCTTCTAGATCCACTTCCGAATCAACTGCTGCTTTGGGATGTTTGGTTAAGGATTACTTTCCAGAACCAGTTACTGTTTCATGGAACTCAGGAGCTTTGACTTCCGGAGTTCATACTTTTCCAGCTGTTTTGCAATCCTCAGGATTGTACTCTTTGTCTTCAGTTGTTACTGTTCCATCTTCTTCCTTGGGTACTAAGACTTACACTTGTAACGTTGATCATAAGCCATCTAACACTAAGGTTGATAAGAGAGTTGAATCTAAGTACGGACCTCCATGTCCACCATGTCCAGCTCCTGAATTTTTGGGTGGACCATCCGTTTTTTTGTTTCCACCAAAGCCAAAGGATACTTTGATGATTTCTAGAACTCCTGAAGTTACTTGTGTTGTTGTTGATGTTTCACAAGAAGATCCTGAAGTTCAATTTAACTGGTACGTTGATGGAGTTGAAGTTCATAACGCTAAGACTAAGCCTAGAGAAGAACAATTTAACTCCACTTACAGAGTTGTTTCCGTTTTGACTGTTTTGCATCAAGATTGGTTGAACGGTAAGGAATACAAGTGTAAGGTTTCTAACAAGGGATTGCCATCCTCTATTGAAAAGACTATTTCAAAGGCTAAGGGTCAACCTAGAGAACCTCAAGTTTACACTTTGCCACCTTCACAAGAAGAAATGACTAAGAACCAAGTTTCCTTGtgtTGTTTGGTTAAGGGATTTTACCCATCCGATATTGCTGTTGAATGGGAA

SEQ ID NO:212

AGAGAGGCTGAAGCTGAAGTTCAATTGGTTGAATCTG

SEQ ID NO:213

TCAATGATGATGATGATGATGTTATCCCAATGACAAGGAC

SEQ ID NO:214

AACGGATGGTCCCTTAGTGGAAGC

SEQ ID NO:215

TAAGGGACCATCCGTTtgtCCATTGGCTCCA

SEQ ID NO:216

TAAGGGACCATCCGTTTTTCCAtgtGCTCCATCTTCTAG

SEQ ID NO:217

TAAGGGACCATCCGTTTTTCCATTGtgtCCATCTTCTAGATC

SEQ ID NO:218

TAAGGGACCATCCGTTTTTCCATTGGCTtgtTCTTCTAGATCCAC

SEQ ID NO:219

TAAGGGACCATCCGTTTTTCCATTGGCTCCAtgtTCTAGATCCAC

SEQ ID NO:220

TAAGGGACCATCCGTTTTTCCATTGGCTCCATCTtgtAGATCCACTTC

SEQ ID NO:221

AGAAGATGGAGCCAATGGAAAAAC

SEQ ID NO:222

TTGGCTCCATCTTCTtgtTCCACTTCCGAATC

SEQ ID NO:223

TTGGCTCCATCTTCTAGAtgtACTTCCGAATC

SEQ ID NO:224

TTGGCTCCATCTTCTAGATCCtgtTCCGAATCAACTGC

SEQ ID NO:225

TTGGCTCCATCTTCTAGATCCACTtgtGAATCAACTGCTGC

SEQ ID NO:226

TTGGCTCCATCTTCTAGATCCACTTCCtgtTCAACTGCTGCTTTG

SEQ ID NO:227

GGAAGTCAAAGCTCCTGAGTTCCA

SEQ ID NO:228

GGAGCTTTGACTTCCtgtGTTCATACTTTTC

SEQ ID NO:229

GGAGCTTTGACTTCCGGAGTTtgtACTTTTCCAGCTG

SEQ ID NO:230

GGAGCTTTGACTTCCGGAGTTCATACTtgtCCAGCTGTTTTG

SEQ ID NO:231

GGAGCTTTGACTTCCGGAGTTCATACTTTTtgtGCTGTTTTGCAATCCTC

SEQ ID NO:232

GGAGCTTTGACTTCCGGAGTTCATACTTTTCCAGCTtgtTTGCAATCCTCAGG

SEQ ID NO:233

AACAGCTGGAAAAGTATGAACTC

SEQ ID NO:234

ACTTTTCCAGCTGTTtgtCAATCCTCAGGATTG

SEQ ID NO:235

ACTTTTCCAGCTGTTTTGtgtTCCTCAGGATTGT

SEQ ID NO:236

ACTTTTCCAGCTGTTTTGCAAtgtTCAGGATTGTACTC

SEQ ID NO:237

ACTTTTCCAGCTGTTTTGCAATCCtgtGGATTGTACTCTTTG

SEQ ID NO:238

ACTTTTCCAGCTGTTTTGCAATCCTCAtgtTTGTACTCTTTGTC

SEQ ID NO:239

ACTTTTCCAGCTGTTTTGCAATCCTCAGGAtgtTACTCTTTGTCTTC

SEQ ID NO:240

GTACAATCCTGAGGATTGCAAAAC

SEQ ID NO:241

TCCTCAGGATTGTACtgtTTGTCTTCAG

SEQ ID NO:242

TCCTCAGGATTGTACTCTTTGtgtTCAGTTGTTAC

SEQ ID NO:243

TCCTCAGGATTGTACTCTTTGTCTTCAtgtGTTACTGTTCCATCTTC

SEQ ID NO:244

TCCTCAGGATTGTACTCTTTGTCTTCAGTTGTTtgtGTTCCATCTTCTTC

SEQ ID NO:245

TCCTCAGGATTGTACTCTTTGTCTTCAGTTGTTACTGTTtgtTCTTCTTCCTTGGGTAC

SEQ ID NO:246

AACCAAACATCCCAAAGCAGCAG

SEQ ID NO:247

CTTTGGGATGTTTGGTTgatGATTACTTTCCAGAA

SEQ ID NO:248

DIQMTQSPSSLSASVGDRVTITCQASQDIGISLSWYQQKPGKAPKLLIYNANNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNSAPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEV

SEQ ID NO:249

GATATTCAAATGACTCAATCACCATCCTCCTTGTCTGCTTCCGTTGGAGATAGAGTTACTATTACTTGTCAAGCTTCCCAAGATATTGGAATTTCATTGTCATGGTATCAACAAAAGCCAGGTAAGGCTCCTAAGTTGTTGATTTACAACGCTAACAACTTGGCTGATGGTGTTCCATCTAGATTTTCTGGGAGTGGTTCTGGAACTGATTTTACTTTGACTATTTCTTCATTGCAACCTGAAGATTTTGCTACTTACTACTGTTTGCAACATAACTCAGCTCCATACACTTTTGGTCAAGGAACTAAGTTGGAGATTAAGAGAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTCCATCTGACGAGCAGTTGAAGTCTGGTACAGCATCTGTTGTCTGTTTGTTGAACAACTTCTACCCAAGAGAAGCAAAGGTTCAATGGAAGGTTGACAACGCCTTGCAATCTGGTAACTCTCAGGAATCTGTTACTGAACAAGATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGACATTGTCTAAGGCTGATTACGAAAAGCATAAGGTTTACGCTTGTGAAGTTACTCATCAAGGATTGTCTTCTCCTGTCACAAAATCTTTTAACAGAGGTGAGGTT

SEQ ID NO:250

AGAGAGGCTGAAGCTGATATTCAAATGACTCAATC

SEQ ID NO:251

TCAATGATGATGATGATGATGTTAAACCTCACCTCTGTTAAAAGATTTTG

SEQ ID NO:252

AGGTGCAGCGACAGTTCTCTTAATC

SEQ ID NO:253

GAACTGTCGCTGCACCTtgtGTTTTC

SEQ ID NO:254

GAACTGTCGCTGCACCTTCTGTTtgtATTTTTCCTCCATC

SEQ ID NO:255

GAACTGTCGCTGCACCTTCTGTTTTCATTtgtCCTCCATCTGAC

SEQ ID NO:256

GAACTGTCGCTGCACCTTCTGTTTTCATTTTTtgtCCATCTGACGAGCAG

SEQ ID NO:257

GAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTtgtTCTGACGAGCAGTTG

SEQ ID NO:258

GAACTGTCGCTGCACCTTCTGTTTTCATTTTTCCTCCAtgtGACGAGCAGTTG

SEQ ID NO:259

ACCAGATTGCAAGGCGTTGTCAAC

SEQ ID NO:260

GCCTTGCAATCTGGTtgtTCTCAGGAATCTG

SEQ ID NO:261

GCCTTGCAATCTGGTAACTCTtgtGAATCTGTTACTG

SEQ ID NO:262

GCCTTGCAATCTGGTAACTCTCAGGAAtgtGTTACTGAAC

SEQ ID NO:263

GCCTTGCAATCTGGTAACTCTCAGGAATCTtgtACTGAACAAGATTC

SEQ ID NO:264

GCCTTGCAATCTGGTAACTCTCAGGAATCTGTTtgtGAACAAGATTCTAAG

SEQ ID NO:265

GCCTTGCAATCTGGTAACTCTCAGGAATCTGTTACTtgtCAAGATTCTAAG

SEQ ID NO:266

AGAATCCTTAGAATCTTGTTCAG

SEQ ID NO:267

GATTCTAAGGATTCTtgtTACTCTTTGTC

SEQ ID NO:268

GATTCTAAGGATTCTACTTACtgtTTGTCTTC

SEQ ID NO:269

GATTCTAAGGATTCTACTTACTCTTTGtgtTCTACA

SEQ ID NO:270

GATTCTAAGGATTCTACTTACTCTTTGTCTTCTtgtTTGACA

SEQ ID NO:271

GATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGtgtTTGTCTAAGGCTG

SEQ ID NO:272

GATTCTAAGGATTCTACTTACTCTTTGTCTTCTACATTGACATTGtgtAAGGCTG

SEQ ID NO:273

TCAATGATGATGATGATGATGTCAAACCTCACCTCTGTTacaAGATTTTGT

SEQ ID NO:274

TCAATGATGATGATGATGATGTCAAACCTCACCTCTacaAAAAGATTTTGT

SEQ ID NO:275

TCAATGATGATGATGATGATGTCAAACCTCACCacaGTTAAAAGATTTTGT

SEQ ID NO:276

TCAATGATGATGATGATGATGTCAAACCTCacaTCTGTTAAAAG

SEQ ID NO:277

TCAATGATGATGATGATGATGTCAAACacaACCTCTGTTAAAAG

SEQ ID NO:278

TCAATGATGATGATGATGATGTCAacaCTCACCTCTGTTAAAAG

SEQ ID NO:279

ACCAGACTTCAACTGCTCGTCAGA

SEQ ID NO:280

GCAGTTGAAGTCTGGTagaGCATCTGTTGTCTG

all documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Hangzhou Jingyin Biotechnology Ltd

<120> Process for producing bispecific antibody

<130> P2020-1206

<160> 280

<170> SIPOSequenceListing 1.0

<210> 1

<211> 455

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly His His His His His His

450 455

<210> 2

<211> 1365

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gaggtccagt tggtcgagtc tggtggtggt ttggttcagc caggtggatc tttgagattg 60

tcttgtgcag catctggatt caacattaaa gatacttaca ttcattgggt tagacaggca 120

ccaggtaaag gtttggagtg ggttgctaga atttacccaa ctaacggtta cactagatac 180

gctgattctg tcaagggtag attcactatt tctgctgata catctaaaaa cactgcttac 240

ttgcaaatga actctttgag agctgaagat acagccgttt actattgctc cagatgggga 300

ggagacggat tttacgctat ggattactgg ggacaaggta ctttggttac tgtttcttct 360

gcttctacta agggaccatc tgtctttcca ttggcaccat cttctaaatc tacttctgga 420

ggaactgccg ccttgggatg tttggtcaag gattatttcc ctgagcctgt cacagtttct 480

tggaactctg gtgcattgac atctggtgtt cacacattcc cagcagtctt gcaatcttct 540

ggtttgtact ctttgtcttc tgtcgttaca gtcccttctt cttctttggg tactcaaacc 600

tacatctgta atgttaatca taagccttct aacacaaaag ttgataagaa agtcgagcca 660

aaatcttgcg acaaaacaca tacctgccca ccatgtccag ctcctgagtt gttgggaggt 720

ccttctgttt ttttgttccc acctaagcca aaggatacat tgatgatttc cagaactcct 780

gaagttacat gtgtcgttgt cgatgtttct catgaagatc ctgaggttaa attcaattgg 840

tacgttgatg gtgtcgaagt tcataacgct aagactaaac caagagaaga acagtacaat 900

tctacttata gagtcgtctc tgttttgacc gttttgcatc aagattggtt gaacggaaag 960

gagtataagt gcaaagtttc taataaggct ttgcctgccc ctattgagaa gaccatttct 1020

aaggctaaag gtcagcctag agaacctcaa gtttacactt tgccaccttc cagagaggaa 1080

atgactaaga accaggtctc tttgtcttgc gctgttaagg gtttttaccc ttctgacatc 1140

gctgttgagt gggaatctaa cggacagcct gaaaataact ataaaacaac acctccagtt 1200

ttggattctg acggttcttt ctttttggtt tctaagttga cagtcgataa gtccagatgg 1260

caacaaggaa acgtcttttc ttgttctgtt atgcatgaag ccttgcataa tagatttacc 1320

cagaaatctt tgtctttgtc tccaggtcat catcatcatc atcat 1365

<210> 3

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccgctcgaga aaagagaggc tgaagctgag gtccagttgg tcgagtct 48

<210> 4

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ataagaatgc ggccgctcaa tgatgatgat gatgatgacc tggagacaaa gacaaaga 58

<210> 5

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 6

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gatattcaaa tgactcaatc tccttcttct ttgtctgctt ctgtcggaga tagagtcact 60

atcacttgta gagcttctca agacgtcaac accgctgttg cttggtatca gcaaaagcca 120

ggtaaggctc caaaattgtt gatttactct gcatcttttt tgtactctgg tgtcccttcc 180

agattctctg gttctaggtc tggaaccgat tttactttga ctatctcttc tttgcagcct 240

gaagatttcg caacttacta ctgtcaacag cattacacta ctccaccaac ttttggtcaa 300

ggaactaaag ttgagattaa gagaactgtc gctgcacctt ctgttttcat ttttcctcca 360

tctgacgagc agttgaagtc tggtacagca tctgttgtct gtttgttgaa caacttctac 420

ccaagagaag caaaggttca atggaaggtt gacaacgcct tgcaatctgg taactctcag 480

gaatctgtta ctgaacaaga ttctaaggat tctacttact ctttgtcttc tacattgaca 540

ttgtctaagg ctgattacga aaagcataag gtttacgctt gtgaagttac tcatcaagga 600

ttgtcttctc ctgtcacaaa atcttttaac agaggtgagt gt 642

<210> 7

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccgctcgaga aaagagaggc tgaagctgat attcaaatga ctcaatctcc ttc 53

<210> 8

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ataagaatgc ggccgctcaa cactcacctc tgttaaaaga 40

<210> 9

<211> 1997

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agacgagatc aattagaacc tgttttggca ataaccgagg attaggaaca aagtccgggt 60

aattatgcga ctctctcttt ttcatatgca ggtcgagcaa gaatcttgtt tttcgtggtg 120

ggactgggca gcaattaaca accaatcggc acttgcaata agtcgattag cgcatggtgg 180

gggacagtag atagctgctg aaatttttgg gtgcggacaa tttcaagagt ctgaggccct 240

gctctcacta gcagtcagaa tcatctgcct cacatacagt ctcctgatgc tgctacttac 300

ttggcaagcg atgatgttta cacaattgca gctttttagt tgctgtcatg gctctagaat 360

tccttggcct tttatcagca tctcaggcaa caatgtgaaa tgcgcacctt cagattttta 420

tattggtgga cggaaatggt aggaagtagt gaagaaaagg tagcgcgagg gcattgtccc 480

cccatgcgaa gaaaaattac tagcataaaa aaatggaaac aagtgacctc cgcagcatgt 540

ctgcccactt tatactgaaa ctacattctt gctaatggat cgtaaaacac tatcacctgg 600

tatgtcgtaa tggacttgat ggcgtgtact ttgctttact gagttctagt tgcctgctca 660

gtgccgaaga acatggggct ccatgtgtat tgtattctct tatctagaac aatcgaagcg 720

ctactgactt gaaatccttg aatgatagac atagattgga ccatgacaag gagaacttat 780

tcacaattga aaagtacatt ttcattgacc cattgggagg tatcccatct ttggagaggt 840

acaaaagtgc acatgtatat atcaatctat tacaagagta tgaagacatt gtgtcggagc 900

tttacatagg gttcttaaag actggagaaa gggaccagca tttgaagaac ttaaacttgc 960

ttcagaaatt gttgcaggta accacagatg catcaggaat agttactact cctcaaatcg 1020

ccatgttgaa tcagaccgac cgattcacca atccaataat ttacaatgtc ttaaccgata 1080

ggccgacaat atcatcgtca ttaccggttg atttgaaaaa gacccctttg ctaaacactt 1140

caatcattag gagaggcgta ccggttgaag tttatgtgga cgaatcatct gacaaaagtg 1200

ggctgtgcct agactctctt ctgaaacgag gagctttaga cttagaaaag cttaagaatg 1260

tgatcgattt gtcgtttcga aaggacttga atatgaaaaa gtacctagcc agagtaaaga 1320

acaatgttgc agctatctta atcgctggag attacgaagg cgtgatcata gttacttggg 1380

aggtaacgga tgaagaaaag ccgcagaaaa tagcttattt agataagttt gcagtgtctc 1440

ctaaggccca aggatcgaca ggggttgccg atgttctttt caagtcatta ttgtccaatt 1500

ttgagaacga attgttctgg agatctcgat ctaataatcc agtgaacaaa tggtactttg 1560

aacggagcaa aggttctctt actgttactg gcacaaattg gaaatgcttc tacaccggca 1620

agaactatcc ttcattggat agaatgaagg gctatttcaa catctgtgag agaatccaac 1680

cttcctggaa tggataagcg aatttcttat gatttatgat ttttattatt aaataagtta 1740

taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct 1800

tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg 1860

ctcttattga ccacacctct accggcatgc cgagcaaatg cctgcaaatc gctccccatt 1920

tcacccaatt gtagatatgc taactccagc aatgagttga tgaatctcgg tgtgtatttt 1980

atgtcctcag aggacaa 1997

<210> 10

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gaccttcgtt tgtgcggatc cagacgagat caattagaac ctg 43

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gctatggtgt gtgggggatc cttgtcctct gaggacataa aatac 45

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggagaccaac atgtgagcaa aag 23

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggatccgcac aaacgaaggt c 21

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgtttgtgcg gatccgacct gcaggggggg ggggg 35

<210> 15

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cacatgttgg tctccgcgcc agcaaccgca cctgtg 36

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

catcatcatc atcatcattg agtttg 26

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agcttcagcc tctcttttct cgag 24

<210> 18

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Val Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 19

<211> 1347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gaggtccagt tggtcgagtc tggtggtggt ttggttcagc caggtggatc tttgagattg 60

tcttgtgcag catctggatt ctctttgtct aactactacg ttacttgggt tagacaggca 120

ccaggtaaag gtttggagtg ggttggtatt atttacggtt ctgatgaaac tgcttacgct 180

acttctgcta ttggtagatt cactatttct agagataact ctaaaaacac tttgtacttg 240

caaatgaact ctttgagagc tgaagataca gccgtttact attgcgctag agatgattct 300

tctgattggg atgctaagtt taacttgtgg ggacaaggta ctttggttac tgtttcttct 360

gcttctacta agggaccatc tgtctttcca ttggcaccat cttctaaatc tacttctgga 420

ggaactgccg ccttgggatg tttggtcaag gattatttcc ctgagcctgt cacagtttct 480

tggaactctg gtgcattgac atctggtgtt cacacattcc cagcagtctt gcaatcttct 540

ggtttgtact ctttgtcttc tgtcgttaca gtcccttctt cttctttggg tactcaaacc 600

tacatctgta atgttaatca taagccttct aacacaaaag ttgataagaa agtcgagcca 660

aaatctgttg acaaaacaca tacctgccca ccatgtccag ctcctgagtt gttgggaggt 720

ccttctgttt ttttgttccc acctaagcca aaggatacat tgatgatttc cagaactcct 780

gaagttacat gtgtcgttgt cgatgtttct catgaagatc ctgaggttaa attcaattgg 840

tacgttgatg gtgtcgaagt tcataacgct aagactaaac caagagaaga acagtacaat 900

tctacttata gagtcgtctc tgttttgacc gttttgcatc aagattggtt gaacggaaag 960

gagtataagt gcaaagtttc taataaggct ttgcctgccc ctattgagaa gaccatttct 1020

aaggctaaag gtcagcctag agaacctcaa gtttacactt tgccaccttc cagagaggaa 1080

atgactaaga accaggtctc tttgtggtgc ttggttaagg gtttttaccc ttctgacatc 1140

gctgttgagt gggaatctaa cggacagcct gaaaataact ataaaacaac acctccagtt 1200

ttggattctg acggttcttt ctttttgtat tctaagttga cagtcgataa gtccagatgg 1260

caacaaggaa acgtcttttc ttgttctgtt atgcatgaag ccttgcataa tcattacacc 1320

cagaaatctt tgtctttgtc tccaggt 1347

<210> 20

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

agagaggctg aagctgaggt ccagttggtc gagtctggtg 40

<210> 21

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tcaatgatga tgatgatgat gtcaacctgg agacaaagac aaagatttc 49

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gacagatggt cccttagtag aagc 24

<210> 23

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctaagggacc atctgtctgt ccattg 26

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ctaagggacc atctgtcttt ccatgtgcac catcttc 37

<210> 25

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ctaagggacc atctgtcttt ccattgtgtc catcttctaa a 41

<210> 26

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ctaagggacc atctgtcttt ccattggcat gttcttctaa atctacttc 49

<210> 27

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ctaagggacc atctgtcttt ccattggcac catgttct 38

<210> 28

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ctaagggacc atctgtcttt ccattggcac catcttgtaa atc 43

<210> 29

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ctaagggacc atctgtcttt ccattggcac catcttcttg ttctact 47

<210> 30

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ctaagggacc atctgtcttt ccattggcac catcttctaa atgtacttct ggag 54

<210> 31

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ctaagggacc atctgtcttt ccattggcac catcttctaa atcttgttct ggaggaact 59

<210> 32

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

agatgtcaat gcaccagagt tcc 23

<210> 33

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ggtgcattga catcttgtgt tcac 24

<210> 34

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ggtgcattga catctggtgt ttgtacattc 30

<210> 35

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

ggtgcattga catctggtgt tcacacatgt ccagca 36

<210> 36

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ggtgcattga catctggtgt tcacacattc tgtgcagtc 39

<210> 37

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ggtgcattga catctggtgt tcacacattc ccagcatgtt tgcaa 45

<210> 38

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ggtgcattga catctggtgt tcacacattc ccagcagtct tgtgttcttc t 51

<210> 39

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ttgcaagact gctgggaatg tgtg 24

<210> 40

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ccagcagtct tgcaatgttc tggtttg 27

<210> 41

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ccagcagtct tgcaatcttg tggtttgtac 30

<210> 42

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ccagcagtct tgcaatcttc ttgtttgtac tctttgtc 38

<210> 43

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ccagcagtct tgcaatcttc tggttgttac tctttgtctt c 41

<210> 44

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

gtacaaacca gaagattgca agac 24

<210> 45

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

caatcttctg gtttgtactg tttgtc 26

<210> 46

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

caatcttctg gtttgtactc tttgtgttct gtc 33

<210> 47

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

caatcttctg gtttgtactc tttgtcttct tgtgttacag tcccttc 47

<210> 48

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

caatcttctg gtttgtactc tttgtcttct gtcgtttgtg ctcct 45

<210> 49

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

tggctcgact ctcttatcaa c 21

<210> 50

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

aagaaagtcg agccatgttc tgttgac 27

<210> 51

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

aagaaagtcg agccaaaatg tgttgac 27

<210> 52

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

aagaaagtcg agccaaaatc ttgcgac 27

<210> 53

<211> 1987

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

aggttttata ctgagtttgt taatgataca ataaactgtt atagtacata caattgaaac 60

tctcttatct atactggggg accttctcgc agaatggtat aaatatctac taactgactg 120

tcgtacggcc taggggtctc ttcttcgatt atttgcaggt cggaacatcc ttcgtctgat 180

gcggatctcc tgagacaaag ttcacgggta tctagtattc tatcagcata aatggaggac 240

ctttctaaac taaactttga atcgtctcca gcagcatcct cgcataatcc ttttgtcatt 300

tcctctatgt ctattgtcac tgtggttggc gcatcaagag tcgtccttct gtaaaccggt 360

acagaattcc taccactaga agcttgaaat ggggagggtt tcagctttgt atcccgatac 420

tgtgctttaa aaagggagtc caaactgaaa tctttttcgg aatcattgga tgatacctct 480

gtattagatc tcctatgtat cggtttcctc gggtagatag aactgtcgac agagtctcta 540

gcaaattgag aattggcttt agacttcgga gaagtaggtg acgatgtggt agatatatca 600

gcagatgaaa agactgaatt ttttctctct ggtgatgagt tgattccctc ttggtgttga 660

tatcttgaac cgggctcatg ttgaaccttt gaattcaaaa aggtagcttg aagttgctca 720

ttgacagcat ctgccacgtc gtatcttgca acatctttgg gaaactgata cgaagtgttc 780

aacatctttg ttgcggtatg acaagagcac ttcgttgtac ttttatcaga gaaaagaaac 840

acctcaatta tggtatttag gtttatatat tacgcaaatt ctattagaaa acccgggagc 900

tggagctttg gctggtcatc cttatggaat tgatcgtgaa tacattgctg agagattagg 960

gtttgattct gttattggta attctttggc cgctgtttca gacagagatt ttgtagtcga 1020

aaccatgttc tggtcttcgt tgtttatgaa tcatatttct cgattctcag aagatttgat 1080

catttactcc actggagagt ttggatttat caagttggca gatgcttatt ctactggatc 1140

ttctctgatg cctacaaaaa aaaacccaga ctctttggag ttattgaggg gtaaatctgg 1200

tagatgtttt ggggccttgg ctggtttcct catgtctatt aagtccattc cgtcaaccta 1260

taacaaagat atgcaagagg ataaggagcc tttatttgat actctaatca ctgtagagca 1320

ctcgattttg atagcatccg gtgtagtttc taccttgaac attgatgccg aacgaatgaa 1380

gaatgctcta actatggata tgctggctac agatcttgcc gactatttag ttagaagggg 1440

agttccattc agagaaactc accacatttc tggtgaatgt gtcagacaag ccgaggagtt 1500

gaacctttct ggtattgatc agttgtccct cgaacaattg aaatccattg actcccgttt 1560

tgaggctgat gtggcttcaa cgtttgactt tgaagccagt gttgaaaaaa gaactgccac 1620

cggaggaact tctaagactg ctgttttaaa gcaattggat gcactgaatg aaaagctaga 1680

gtcttgagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat 1740

aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag 1800

taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga 1860

ccacacctct accggcatgc cgagcaaatg cctgcaaatc gctccccatt tcacccaatt 1920

gtagatatgc taactccagc aatgagttga tgaatctcgg tgtgtatttt atgtcctcag 1980

aggacaa 1987

<210> 54

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

gaccttcgtt tgtgcggatc caggttttat actgagtttg tta 43

<210> 55

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

gctatggtgt gtgggggatc cttgtcctct gaggacataa aatac 45

<210> 56

<211> 217

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Arg Asn

85 90 95

Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Val

210 215

<210> 57

<211> 651

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gctattcaaa tgactcaatc tccttcttct ttgtctgctt ctgtcggaga tagagtcact 60

atcacttgtc aagcttctca atctattaac aacgaattgt cttggtatca gcaaaagcca 120

ggtaaggctc caaaattgtt gatttacaga gcatctactt tggcttctgg tgtcccttcc 180

agattctctg gttctggttc tggaaccgat tttactttga ctatctcttc tttgcagcct 240

gatgatttcg caacttacta ctgtcaacag ggttactctt tgagaaacat tgataacgct 300

tttggtggtg gaactaaagt tgagattaag agaactgtcg ctgcaccttc tgttttcatt 360

tttcctccat ctgacgagca gttgaagtct ggtacagcat ctgttgtctg tttgttgaac 420

aacttctacc caagagaagc aaaggttcaa tggaaggttg acaacgcctt gcaatctggt 480

aactctcagg aatctgttac tgaacaagat tctaaggatt ctacttactc tttgtcttct 540

acattgacat tgtctaaggc tgattacgaa aagcataagg tttacgcttg tgaagttact 600

catcaaggat tgtcttctcc tgtcacaaaa tcttttaaca gaggtgaggt t 651

<210> 58

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

agagaggctg aagctgctat tcaaatgact caatctcc 38

<210> 59

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

tcaatgatga tgatgatgat gtcaaacctc acctctgtta aaagattttg 50

<210> 60

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

aggtgcagcg acagttctag taatttg 27

<210> 61

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

gaactgtcgc tgcaccttgt gttttc 26

<210> 62

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

gaactgtcgc tgcaccttct gtttgtattt ttcctccatc 40

<210> 63

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

gaactgtcgc tgcaccttct gttttcattt gtcctccatc tgac 44

<210> 64

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

gaactgtcgc tgcaccttct gttttcattt tttgtccatc tgacgagcag 50

<210> 65

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

gaactgtcgc tgcaccttct gttttcattt ttccttgttc tgacgagcag ttg 53

<210> 66

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

gaactgtcgc tgcaccttct gttttcattt ttcctccatg tgacgagcag ttg 53

<210> 67

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

accagattgc aaggcgttgt caac 24

<210> 68

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

gccttgcaat ctggttgttc tcaggaatct g 31

<210> 69

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

gccttgcaat ctggtaactc ttgtgaatct gttactg 37

<210> 70

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

gccttgcaat ctggtaactc tcaggaatgt gttactgaac 40

<210> 71

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

gccttgcaat ctggtaactc tcaggaatct gtttgtgaac aagattctaa g 51

<210> 72

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

agaatcctta gaatcttgtt cag 23

<210> 73

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

gattctaagg attcttgtta ctctttgtc 29

<210> 74

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

gattctaagg attctactta ctgtttgtct tc 32

<210> 75

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

gattctaagg attctactta ctctttgtgt tctaca 36

<210> 76

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

gattctaagg attctactta ctctttgtct tcttgtttga ca 42

<210> 77

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

gattctaagg attctactta ctctttgtct tctacattgt gtttgtctaa ggctg 55

<210> 78

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

tcaatgatga tgatgatgat gtcaaacctc acctctgtta caaga 45

<210> 79

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

tcaatgatga tgatgatgat gtcaaacctc acctctacaa aaag 44

<210> 80

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

tcaatgatga tgatgatgat gtcaaacctc accacagtta aaag 44

<210> 81

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

tcaatgatga tgatgatgat gtcaaacctc acatctgtta aaag 44

<210> 82

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

tcaatgatga tgatgatgat gtcaaacaca acctctgtta aaag 44

<210> 83

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

tcaatgatga tgatgatgat gtcaacactc acctctgtta aaag 44

<210> 84

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

atcaactttt gtgttaga 18

<210> 85

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

tctaacacaa aagttgatga taaagtcgag ccaaaa 36

<210> 86

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

tctaacacaa aagttgatga aaaagtcgag ccaaaa 36

<210> 87

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

agatggagga aaaat 15

<210> 88

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

atttttcctc catctgacaa gcagttgaag tctggt 36

<210> 89

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

acatggagga aaaatgaaaa c 21

<210> 90

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

atttttcctc catgtgacaa gcagttgaag tctggt 36

<210> 91

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

agaacaagga aaaatgaaaa c 21

<210> 92

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

atttttcctt gttctgacaa gcagttgaag tctggt 36

<210> 93

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

agatggacaa aaaatgaaaa c 21

<210> 94

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

attttttgtc catctgacaa gcagttgaag tctggt 36

<210> 95

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

agatggagga caaatgaaaa c 21

<210> 96

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

atttgtcctc catctgacaa gcagttgaag tctggt 36

<210> 97

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

atttttcctc catctgacag acagttgaag tctggt 36

<210> 98

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

atttttcctc catgtgacag acagttgaag tctggt 36

<210> 99

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

atttttcctt gttctgacag acagttgaag tctggt 36

<210> 100

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

attttttgtc catctgacag acagttgaag tctggt 36

<210> 101

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

atttgtcctc catctgacag acagttgaag tctggt 36

<210> 102

<211> 456

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 102

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Pro Tyr Thr Gly Ser Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Pro Glu Lys Phe Asp Ser Asp Asp Ser Asp Val Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly His His His His His His

450 455

<210> 103

<211> 1368

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

caagttcaat tggttcaatc aggagctgaa gttaagaagc caggtgcttc cgttaaggtt 60

tcttgtaagg cttcaggata cacttttact aactactaca tgcattgggt tagacaagct 120

ccaggacaag gtttggaatg ggttggatgg attaacccat acactgggag tgctttttac 180

gctcaaaagt ttagaggtag agttactatg actagagata cttctatttc cactgcttac 240

atggaattgt cgcgtttgag atcagatgat actgctgttt actactgtgc tagagaacct 300

gaaaagtttg attccgatga ttctgatgtt tggggtagag gtactttggt tactgtttct 360

tctgcttcta ctaagggacc atctgtcttt ccattggcac catcttctaa atctacttct 420

ggaggaactg ccgccttggg atgtttggtc aaggattatt tccctgagcc tgtcacagtt 480

tcttggaact ctggtgcatt gacatctggt gttcacacat tcccagcagt cttgcaatct 540

tctggtttgt actctttgtc ttctgtcgtt acagtccctt cttcttcttt gggtactcaa 600

acctacatct gtaatgttaa tcataagcct tctaacacaa aagttgataa gaaagtcgag 660

ccaaaatctt gcgacaaaac acatacctgc ccaccatgtc cagctcctga gttgttggga 720

ggtccttctg tttttttgtt cccacctaag ccaaaggata cattgatgat ttccagaact 780

cctgaagtta catgtgtcgt tgtcgatgtt tctcatgaag atcctgaggt taaattcaat 840

tggtacgttg atggtgtcga agttcataac gctaagacta aaccaagaga agaacagtac 900

aattctactt atagagtcgt ctctgttttg accgttttgc atcaagattg gttgaacgga 960

aaggagtata agtgcaaagt ttctaataag gctttgcctg cccctattga gaagaccatt 1020

tctaaggcta aaggtcagcc tagagaacct caagtttaca ctttgccacc ttccagagag 1080

gaaatgacta agaaccaggt ctctttgtct tgcgctgtta agggttttta cccttctgac 1140

atcgctgttg agtgggaatc taacggacag cctgaaaata actataaaac aacacctcca 1200

gttttggatt ctgacggttc tttctttttg gtttctaagt tgacagtcga taagtccaga 1260

tggcaacaag gaaacgtctt ttcttgttct gttatgcatg aagccttgca taatagattt 1320

acccagaaat ctttgtcttt gtctccaggt catcatcatc atcatcat 1368

<210> 104

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

ccgctcgaga aaagagaggc tgaagctcaa gttcaattgg ttcaatcag 49

<210> 105

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

atagtttagc ggccgctcaa tgatgatgat gatgatgacc tggagacaaa ga 52

<210> 106

<211> 217

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 106

Gln Ala Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asn Ser

85 90 95

Leu Ser Gly Tyr Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 107

<211> 651

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

caagctgttt tgactcaacc accatccgtt tcaggggcgc ctggtcaaag agttactatt 60

tcatgtactg ggtcctcatc caacattggt gctggatacg gtgttcattg gtatcaacaa 120

ttgccaggaa ctgctcctaa gttgttgatt tacggtgatt ctaacagacc atctggtgtt 180

ccagatagat tttccggatc taagtccgga acttctgctt ccttggctat tactggtttg 240

caagctgaag atgaagctga ttactactgt caatcatacg ataactcctt gtccggatac 300

gtttttggtg gaggaactca attgactgtt ttgggacaac caaaggctgc tccatccgtt 360

actttgtttc caccatcttc agaagaattg caagctaaca aggctacttt ggtttgtttg 420

atttccgatt tttacccagg agctgttact gttgcttgga aggctgattc ctcaccagtt 480

aaggctggtg ttgaaactac tactccatct aagcaatcaa acaacaagta cgctgcttca 540

tcatacttgt ccttgactcc agaacaatgg aagtcacata gatcatactc atgtcaagtt 600

actcatgaag gaagtactgt tgaaaagact gttgctccaa ctgaatgttc t 651

<210> 108

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

ccgctcgaga aaagagaggc tgaagctcaa gctgttttga ctcaacc 47

<210> 109

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

aaggaaaaaa gcggccgctt aagaacattc agttggagca ac 42

<210> 110

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 110

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Thr Ser Gly Gly Arg Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Phe Arg Gln Tyr Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Val Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 111

<211> 1344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

gaggtccagt tgttggagtc tggtggtggt ttggttcagc caggtggatc tttgagattg 60

tcttgtgcag catctggatt cactttttct tcttttccaa tggcttgggt tagacaggca 120

ccaggtaaag gtttggagtg ggtttctact atttctactt ctggtggtag aacttactac 180

agagattctg tcaagggtag attcactatt tctagagata actctaaaaa cactttgtac 240

ttgcaaatga actctttgag agctgaagat acagccgttt actattgcgc taagtttaga 300

caatactctg gtggttttga ttactgggga caaggtactt tggttactgt ttcttctgct 360

tctactaagg gaccatctgt ctttccattg gcaccatctt ctaaatctac ttctggagga 420

actgccgcct tgggatgttt ggtcaaggat tatttccctg agcctgtcac agtttcttgg 480

aactctggtg cattgacatc tggtgttcac acattcccag cagtcttgca atcttctggt 540

ttgtactctt tgtcttctgt cgttacagtc ccttcttctt ctttgggtac tcaaacctac 600

atctgtaatg ttaatcataa gccttctaac acaaaagttg ataagaaagt cgagccaaaa 660

tctgttgaca aaacacatac ctgcccacca tgtccagctc ctgagttgtt gggaggtcct 720

tctgtttttt tgttcccacc taagccaaag gatacattga tgatttccag aactcctgaa 780

gttacatgtg tcgttgtcga tgtttctcat gaagatcctg aggttaaatt caattggtac 840

gttgatggtg tcgaagttca taacgctaag actaaaccaa gagaagaaca gtacaattct 900

acttatagag tcgtctctgt tttgaccgtt ttgcatcaag attggttgaa cggaaaggag 960

tataagtgca aagtttctaa taaggctttg cctgccccta ttgagaagac catttctaag 1020

gctaaaggtc agcctagaga acctcaagtt tacactttgc caccttccag agaggaaatg 1080

actaagaacc aggtctcttt gtggtgcttg gttaagggtt tttacccttc tgacatcgct 1140

gttgagtggg aatctaacgg acagcctgaa aataactata aaacaacacc tccagttttg 1200

gattctgacg gttctttctt tttgtattct aagttgacag tcgataagtc cagatggcaa 1260

caaggaaacg tcttttcttg ttctgttatg catgaagcct tgcataatca ttacacccag 1320

aaatctttgt ctttgtctcc aggt 1344

<210> 112

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

agagaggctg aagctgaggt ccagttgttg gagtctg 37

<210> 113

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

tcaatgatga tgatgatgat gtcaacctgg agacaaagac aaagatttc 49

<210> 114

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

gacagatggt cccttagtag aagc 24

<210> 115

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

ctaagggacc atctgtctgt ccattg 26

<210> 116

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

ctaagggacc atctgtcttt ccatgtgcac catcttc 37

<210> 117

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

ctaagggacc atctgtcttt ccattgtgtc catcttctaa atc 43

<210> 118

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

ctaagggacc atctgtcttt ccattggcat gttcttctaa atctacttc 49

<210> 119

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

ctaagggacc atctgtcttt ccattggcac catgttctaa atctact 47

<210> 120

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

ctaagggacc atctgtcttt ccattggcac catcttgtaa atctact 47

<210> 121

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

ctaagggacc atctgtcttt ccattggcac catcttcttg ttctacttct ggag 54

<210> 122

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

ctaagggacc atctgtcttt ccattggcac catcttctaa atgtacttct ggag 54

<210> 123

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

ctaagggacc atctgtcttt ccattggcac catcttctaa atcttgttct ggaggaact 59

<210> 124

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

ctaagggacc atctgtcttt ccattggcac catcttctaa atctacttgt ggaggaact 59

<210> 125

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

agatgtcaat gcaccagagt tcc 23

<210> 126

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

ggtgcattga catcttgtgt tcacacattc cca 33

<210> 127

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

ggtgcattga catctggtgt ttgtacattc ccagc 35

<210> 128

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

ggtgcattga catctggtgt tcacacatgt ccagcagtct tgc 43

<210> 129

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

ggtgcattga catctggtgt tcacacattc tgtgcagtct tgcaa 45

<210> 130

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

ggtgcattga catctggtgt tcacacattc ccagcatgtt tgcaatcttc t 51

<210> 131

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

ggtgcattga catctggtgt tcacacattc ccagcagtct tgtgttcttc tggtttg 57

<210> 132

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

ttgcaagact gctgggaatg tgtg 24

<210> 133

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

ccagcagtct tgcaatgttc tggtttg 27

<210> 134

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

ccagcagtct tgcaatcttg tggtttgtac tctttg 36

<210> 135

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

ccagcagtct tgcaatcttc ttgtttgtac tctttgtc 38

<210> 136

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

ccagcagtct tgcaatcttc tggttgttac tctttgtctt c 41

<210> 137

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

gtacaaacca gaagattgca agac 24

<210> 138

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

caatcttctg gtttgtactg tttgtcttct gtc 33

<210> 139

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

caatcttctg gtttgtactc tttgtgttct gtcgttacag 40

<210> 140

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

caatcttctg gtttgtactc tttgtcttct tgtgttacag tcccttc 47

<210> 141

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

caatcttctg gtttgtactc tttgtcttct gtcgtttgtg tcccttcttc ttc 53

<210> 142

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

tttcttatca acttttgtgt tag 23

<210> 143

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

caaaagttga taagaaatgt gagccaaaat ctg 33

<210> 144

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

caaaagttga taagaaagtc tgtccaaaat ctgttg 36

<210> 145

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

caaaagttga taagaaagtc gagtgtaaat ctgttgac 38

<210> 146

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

aagaaagtcg agccatgttc tgttgacaaa aca 33

<210> 147

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

aagaaagtcg agccaaaatg tgttgacaaa aca 33

<210> 148

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

aagaaagtcg agccaaaatc ttgcgacaaa acacata 37

<210> 149

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 149

Asp Ile Gln Leu Thr Gln Pro Asn Ser Val Ser Thr Ser Leu Gly Ser

1 5 10 15

Thr Val Lys Leu Ser Cys Thr Leu Ser Ser Gly Asn Ile Glu Asn Asn

20 25 30

Tyr Val His Trp Tyr Gln Leu Tyr Glu Gly Arg Ser Pro Thr Thr Met

35 40 45

Ile Tyr Asp Asp Asp Lys Arg Pro Asp Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Phe Leu Thr Ile His Asn

65 70 75 80

Val Ala Ile Glu Asp Glu Ala Ile Tyr Phe Cys His Ser Tyr Val Ser

85 90 95

Ser Phe Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Val Ser

210 215

<210> 150

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

gatattcaat tgactcaacc taactcagtt tccacttcct tgggttcgac tgttaagttg 60

tcatgtactt tgtcctctgg taacattgaa aacaactacg ttcattggta tcaattgtac 120

gaaggtagat cccctactac tatgatttac gatgatgata agagacctga tggagttcct 180

gatagatttt ccggttctat tgatagaagc tctaactccg cttttttgac tattcataac 240

gttgctattg aagatgaagc tatttacttt tgtcattctt acgtttcttc ctttaacgtt 300

tttggaggtg gaactaagtt gactgttttg agacaaccaa aggctgctcc atccgttact 360

ttgtttccac catcttcaga agaattgcaa gctaacaagg ctactttggt ttgtttgatt 420

tccgattttt acccaggagc tgttactgtt gcttggaagg ctgattcctc accagttaag 480

gctggtgttg aaactactac tccatctaag caatcaaaca acaagtacgc tgcttcatca 540

tacttgtcct tgactccaga acaatggaag tcacatagat catactcatg tcaagttact 600

catgaaggaa gtactgttga aaagactgtt gctccaactg aagtttct 648

<210> 151

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

agagaggctg aagctgatat tcaattgact caac 34

<210> 152

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

tcaatgatga tgatgatgat gtcaagaaac ttcagttgga gcaac 45

<210> 153

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

tggagcagcc tttggttgtc tc 22

<210> 154

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

ccaaaggctg ctccatgtgt tactttgttt ccacc 35

<210> 155

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

ccaaaggctg ctccatccgt ttgtttgttt ccaccatctt c 41

<210> 156

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

ccaaaggctg ctccatccgt tactttgtgt ccaccatctt cag 43

<210> 157

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

ccaaaggctg ctccatccgt tactttgttt tgtccatctt cagaagaatt g 51

<210> 158

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

ccaaaggctg ctccatccgt tactttgttt ccaccatgtt cagaagaatt g 51

<210> 159

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

agccttaact ggtgaggaat cagc 24

<210> 160

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

tcaccagtta aggcttgtgt tgaaactact actc 34

<210> 161

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

tcaccagtta aggctggtgt ttgtactact actccatcta ag 42

<210> 162

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

tcaccagtta aggctggtgt tgaaacttgt actccatcta agcaatc 47

<210> 163

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

tcaccagtta aggctggtgt tgaaactact tgtccatcta agcaatca 48

<210> 164

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

tcaccagtta aggctggtgt tgaaactact acttgttcta agcaatcaaa c 51

<210> 165

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

tcaccagtta aggctggtgt tgaaactact actccatgta agcaatc 47

<210> 166

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 166

tcaccagtta aggctggtgt tgaaactact actccatcta agtgttcaaa caacaagta 59

<210> 167

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 167

gttgtttgat tgcttagatg gag 23

<210> 168

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 168

taagcaatca aacaactgtt acgctgcttc atcatac 37

<210> 169

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 169

taagcaatca aacaacaagt actgtgcttc atcatacttg tc 42

<210> 170

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 170

taagcaatca aacaacaagt acgctgcttg ttcatacttg tccttgac 48

<210> 171

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 171

taagcaatca aacaacaagt acgctgcttc atcatgtttg tccttgactc cag 53

<210> 172

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 172

taagcaatca aacaacaagt acgctgcttc atcatacttg tgtttgactc cagaacaat 59

<210> 173

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 173

caatgatgat gatgatgatg tcaagaaact tcagttggag cacaagtctt ttcaacag 58

<210> 174

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 174

caatgatgat gatgatgatg tcaagaaact tcagttggac aaacagtctt ttcaacag 58

<210> 175

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 175

tcaatgatga tgatgatgat gtcaagaaac ttcagtacaa gcaacagtct tttcaac 57

<210> 176

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 176

tcaatgatga tgatgatgat gtcaagaaac ttcacatgga gcaacagtct tttc 54

<210> 177

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 177

tcaatgatga tgatgatgat gtcaagaaac acaagttgga gcaacagtc 49

<210> 178

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 178

tcaatgatga tgatgatgat gtcaagaaca ttcagttgga gcaac 45

<210> 179

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 179

tcaatgatga tgatgatgat gtcaacaaac ttcagttgga gcaac 45

<210> 180

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 180

atcaactttt gtgttagaag gc 22

<210> 181

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 181

taacacaaaa gttgatgata aagtcgagcc aaaatctg 38

<210> 182

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 182

taacacaaaa gttgatgata aatgtgagcc aaaatctg 38

<210> 183

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 183

taacacaaaa gttgatgata aagtctgtcc aaaatctg 38

<210> 184

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 184

taacacaaaa gttgatgata aagtcgagtg taaatctg 38

<210> 185

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 185

taacacaaaa gttgatgata aagtcgagcc atgttctg 38

<210> 186

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 186

taacacaaaa gttgatgata aagtcgagcc aaaatgtg 38

<210> 187

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 187

atcaactttt gtgttagaag gc 22

<210> 188

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 188

taacacaaaa gttgatgaaa aagtcgagcc aaaatctg 38

<210> 189

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 189

taacacaaaa gttgatgaaa aatgtgagcc aaaatctg 38

<210> 190

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 190

taacacaaaa gttgatgaaa aagtctgtcc aaaatctg 38

<210> 191

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 191

taacacaaaa gttgatgaaa aagtcgagtg taaatctg 38

<210> 192

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 192

taacacaaaa gttgatgaaa aagtcgagcc atgttctg 38

<210> 193

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 193

taacacaaaa gttgatgaaa aagtcgagcc aaaatgtg 38

<210> 194

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 194

tgaagatggt ggaaacaaag taac 24

<210> 195

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 195

gtttccacca tcttcaaagg aattgcaagc taacaaggct ac 42

<210> 196

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 196

tgaagatggt ggaaacaaag taacaca 27

<210> 197

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 197

tgaagatggt ggaaacaaac aaac 24

<210> 198

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 198

gttagcttgc aattcctttg aacatggtgg aaacaaag 38

<210> 199

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 199

gaattgcaag ctaacaaggc tac 23

<210> 200

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 200

gttagcttgc aattcctttg aagatggaca aaacaaag 38

<210> 201

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 201

gttagcttgc aattcctttg aagatggtgg acacaaag 38

<210> 202

<211> 451

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 202

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Met Tyr Gly Thr Thr Asp Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Tyr Phe Thr Gly Thr Gly Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly His His His

435 440 445

His His His

450

<210> 203

<211> 1353

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 203

caagttcaat tggttcaatc cggagctgaa gttaagaagc ctgggtcatc cgttaaggtt 60

tcatgtaagg cttcaggata ctcttttact gattaccata ttcattgggt tagacaagct 120

ccaggtcaag gtttggaatg gatgggagtt attaacccta tgtacggaac tactgattac 180

aaccaaagat ttaagggtag agttactatt actgctgatg aatccacttc cactgcttac 240

atggaattgt cctctttgag atcagaagat actgctgttt actactgtgc tagatacgat 300

tactttactg gaactggtgt ttactgggga caaggaactt tggttactgt ttcttccgct 360

tccactaagg gaccatccgt ttttccattg gctccatgtt ctagatccac ttccgaatca 420

actgctgctt tgggatgttt ggttaaggat tactttccag aaccagttac tgtttcatgg 480

aactcaggag ctttgacttc cggagttcat acttttccag ctgttttgca atcctcagga 540

ttgtactctt tgtcttcagt tgttactgtt ccatcttctt ccttgggtac taagacttac 600

acttgtaacg ttgatcataa gccatctaac actaaggttg ataagagagt tgaatctaag 660

tacggacctc catgtccacc atgtccagct cctgaatttt tgggtggacc atccgttttt 720

ttgtttccac caaagccaaa ggatactttg atgatttcta gaactcctga agttacttgt 780

gttgttgttg atgtttcaca agaagatcct gaagttcaat ttaactggta cgttgatgga 840

gttgaagttc ataacgctaa gactaagcct agagaagaac aatttaactc cacttacaga 900

gttgtttccg ttttgactgt tttgcatcaa gattggttga acggtaagga atacaagtgt 960

aaggtttcta acaagggatt gccatcctct attgaaaaga ctatttcaaa ggctaagggt 1020

caacctagag aacctcaagt ttacactttg ccaccttcac aagaagaaat gactaagaac 1080

caagtttcct tgtcttgtgc tgttaaggga ttttacccat ccgatattgc tgttgaatgg 1140

gaatcaaacg gtcaacctga aaacaactac aagactactc caccagtttt ggattctgat 1200

ggatcatttt ttttggtttc tagattgact gttgataagt ctagatggca agaaggtaac 1260

gttttttcat gttccgttat gcatgaagct ttgcataaca gatttactca aaagtccttg 1320

tccttgtcat tgggacatca tcatcatcat cat 1353

<210> 204

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 204

ccgctcgaga aaagagaggc tgaagctcaa gttcaattgg ttcaatccg 49

<210> 205

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 205

aaggaaaaaa gcggccgctt aatgatgatg atgatgatgt cccaatgaca aggacaag 58

<210> 206

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 206

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Val His Ser

20 25 30

Arg Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ile Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Leu Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 207

<211> 657

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 207

gatattgtta tgactcaaac tccattgtcc ttgtccgtta ctccaggtca acctgcttct 60

atttcatgta gatcatctag gtccttggtt cattccagag gtaacactta cttgcattgg 120

tacttgcaaa agccaggaca atcaccacaa ttgttgattt acaaggtttc taacagattt 180

attggtgttc ctgatagatt ttccggatct ggatcaggaa ctgattttac tttgaagatt 240

tcgcgggttg aagctgaaga tgttggagtt tactactgtt cccaatccac tcatttgcct 300

tttacttttg gtcaaggaac taagttggaa attaagagaa ctgttgctgc accttctgtt 360

ttcatttttc ctccatctga cgagcagttg aagtctggta cagcatctgt tgtctgtttg 420

ttgaacaact tctacccaag agaagcaaag gttcaatgga aggttgacaa cgccttgcaa 480

tctggtaact ctcaggaatc tgttactgaa caagattcta aggattctac ttactctttg 540

tcttctacat tgacattgtc taaggctgat tacgaaaagc ataaggttta cgcttgtgaa 600

gttactcatc aaggattgtc ttctcctgtc acaaaatctt ttaacagagg tgagtgt 657

<210> 208

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 208

ccgctcgaga aaagagaggc tgaagctgat attgttatga ctcaaactc 49

<210> 209

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 209

aaggaaaaaa gcggccgctt aacactcacc tctgttaaaa g 41

<210> 210

<211> 446

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 210

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Met Arg Asn Lys Asn Tyr Gln Tyr Gly Thr Tyr Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Ser Tyr Tyr Gly Phe Thr Ser Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Cys Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 211

<211> 1146

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 211

gaagttcaat tggttgaatc tggaggtgga ttggttcaac ctggtggatc attgagattg 60

tcatgtgctg cttctggatt taactttaac gattacttta tgaactgggt tagacaagct 120

ccaggaaagg gattggaatg ggttgctcaa atgagaaaca agaactacca atacggtact 180

tactacgctg aatccttgga aggtagattt actatttcta gagatgattc aaagaactcc 240

ttgtacttgc aaatgaactc attgaagact gaagatactg ctgtttacta ctgtgctaga 300

gaatcatact acggatttac ttcttactgg ggacaaggaa ctttggttac tgtttcttcc 360

gcttccacta agggaccatc cgtttttcca ttggctccat cttctagatc cacttccgaa 420

tcaactgctg ctttgggatg tttggttaag gattactttc cagaaccagt tactgtttca 480

tggaactcag gagctttgac ttccggagtt catacttttc cagctgtttt gcaatcctca 540

ggattgtact ctttgtcttc agttgttact gttccatctt cttccttggg tactaagact 600

tacacttgta acgttgatca taagccatct aacactaagg ttgataagag agttgaatct 660

aagtacggac ctccatgtcc accatgtcca gctcctgaat ttttgggtgg accatccgtt 720

tttttgtttc caccaaagcc aaaggatact ttgatgattt ctagaactcc tgaagttact 780

tgtgttgttg ttgatgtttc acaagaagat cctgaagttc aatttaactg gtacgttgat 840

ggagttgaag ttcataacgc taagactaag cctagagaag aacaatttaa ctccacttac 900

agagttgttt ccgttttgac tgttttgcat caagattggt tgaacggtaa ggaatacaag 960

tgtaaggttt ctaacaaggg attgccatcc tctattgaaa agactatttc aaaggctaag 1020

ggtcaaccta gagaacctca agtttacact ttgccacctt cacaagaaga aatgactaag 1080

aaccaagttt ccttgtgttg tttggttaag ggattttacc catccgatat tgctgttgaa 1140

tgggaa 1146

<210> 212

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 212

agagaggctg aagctgaagt tcaattggtt gaatctg 37

<210> 213

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 213

tcaatgatga tgatgatgat gttatcccaa tgacaaggac 40

<210> 214

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 214

aacggatggt cccttagtgg aagc 24

<210> 215

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 215

taagggacca tccgtttgtc cattggctcc a 31

<210> 216

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 216

taagggacca tccgtttttc catgtgctcc atcttctag 39

<210> 217

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 217

taagggacca tccgtttttc cattgtgtcc atcttctaga tc 42

<210> 218

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 218

taagggacca tccgtttttc cattggcttg ttcttctaga tccac 45

<210> 219

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 219

taagggacca tccgtttttc cattggctcc atgttctaga tccac 45

<210> 220

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 220

taagggacca tccgtttttc cattggctcc atcttgtaga tccacttc 48

<210> 221

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 221

agaagatgga gccaatggaa aaac 24

<210> 222

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 222

ttggctccat cttcttgttc cacttccgaa tc 32

<210> 223

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 223

ttggctccat cttctagatg tacttccgaa tc 32

<210> 224

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 224

ttggctccat cttctagatc ctgttccgaa tcaactgc 38

<210> 225

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 225

ttggctccat cttctagatc cacttgtgaa tcaactgctg c 41

<210> 226

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 226

ttggctccat cttctagatc cacttcctgt tcaactgctg ctttg 45

<210> 227

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 227

ggaagtcaaa gctcctgagt tcca 24

<210> 228

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 228

ggagctttga cttcctgtgt tcatactttt c 31

<210> 229

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 229

ggagctttga cttccggagt ttgtactttt ccagctg 37

<210> 230

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 230

ggagctttga cttccggagt tcatacttgt ccagctgttt tg 42

<210> 231

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 231

ggagctttga cttccggagt tcatactttt tgtgctgttt tgcaatcctc 50

<210> 232

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 232

ggagctttga cttccggagt tcatactttt ccagcttgtt tgcaatcctc agg 53

<210> 233

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 233

aacagctgga aaagtatgaa ctc 23

<210> 234

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 234

acttttccag ctgtttgtca atcctcagga ttg 33

<210> 235

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 235

acttttccag ctgttttgtg ttcctcagga ttgt 34

<210> 236

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 236

acttttccag ctgttttgca atgttcagga ttgtactc 38

<210> 237

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 237

acttttccag ctgttttgca atcctgtgga ttgtactctt tg 42

<210> 238

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 238

acttttccag ctgttttgca atcctcatgt ttgtactctt tgtc 44

<210> 239

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 239

acttttccag ctgttttgca atcctcagga tgttactctt tgtcttc 47

<210> 240

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 240

gtacaatcct gaggattgca aaac 24

<210> 241

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 241

tcctcaggat tgtactgttt gtcttcag 28

<210> 242

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 242

tcctcaggat tgtactcttt gtgttcagtt gttac 35

<210> 243

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 243

tcctcaggat tgtactcttt gtcttcatgt gttactgttc catcttc 47

<210> 244

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 244

tcctcaggat tgtactcttt gtcttcagtt gtttgtgttc catcttcttc 50

<210> 245

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 245

tcctcaggat tgtactcttt gtcttcagtt gttactgttt gttcttcttc cttgggtac 59

<210> 246

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 246

aaccaaacat cccaaagcag cag 23

<210> 247

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 247

ctttgggatg tttggttgat gattactttc cagaa 35

<210> 248

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 248

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Gly Ile Ser

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Asn Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Val

210

<210> 249

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 249

gatattcaaa tgactcaatc accatcctcc ttgtctgctt ccgttggaga tagagttact 60

attacttgtc aagcttccca agatattgga atttcattgt catggtatca acaaaagcca 120

ggtaaggctc ctaagttgtt gatttacaac gctaacaact tggctgatgg tgttccatct 180

agattttctg ggagtggttc tggaactgat tttactttga ctatttcttc attgcaacct 240

gaagattttg ctacttacta ctgtttgcaa cataactcag ctccatacac ttttggtcaa 300

ggaactaagt tggagattaa gagaactgtc gctgcacctt ctgttttcat ttttcctcca 360

tctgacgagc agttgaagtc tggtacagca tctgttgtct gtttgttgaa caacttctac 420

ccaagagaag caaaggttca atggaaggtt gacaacgcct tgcaatctgg taactctcag 480

gaatctgtta ctgaacaaga ttctaaggat tctacttact ctttgtcttc tacattgaca 540

ttgtctaagg ctgattacga aaagcataag gtttacgctt gtgaagttac tcatcaagga 600

ttgtcttctc ctgtcacaaa atcttttaac agaggtgagg tt 642

<210> 250

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 250

agagaggctg aagctgatat tcaaatgact caatc 35

<210> 251

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 251

tcaatgatga tgatgatgat gttaaacctc acctctgtta aaagattttg 50

<210> 252

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 252

aggtgcagcg acagttctct taatc 25

<210> 253

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 253

gaactgtcgc tgcaccttgt gttttc 26

<210> 254

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 254

gaactgtcgc tgcaccttct gtttgtattt ttcctccatc 40

<210> 255

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 255

gaactgtcgc tgcaccttct gttttcattt gtcctccatc tgac 44

<210> 256

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 256

gaactgtcgc tgcaccttct gttttcattt tttgtccatc tgacgagcag 50

<210> 257

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 257

gaactgtcgc tgcaccttct gttttcattt ttccttgttc tgacgagcag ttg 53

<210> 258

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 258

gaactgtcgc tgcaccttct gttttcattt ttcctccatg tgacgagcag ttg 53

<210> 259

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 259

accagattgc aaggcgttgt caac 24

<210> 260

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 260

gccttgcaat ctggttgttc tcaggaatct g 31

<210> 261

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 261

gccttgcaat ctggtaactc ttgtgaatct gttactg 37

<210> 262

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 262

gccttgcaat ctggtaactc tcaggaatgt gttactgaac 40

<210> 263

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 263

gccttgcaat ctggtaactc tcaggaatct tgtactgaac aagattc 47

<210> 264

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 264

gccttgcaat ctggtaactc tcaggaatct gtttgtgaac aagattctaa g 51

<210> 265

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 265

gccttgcaat ctggtaactc tcaggaatct gttacttgtc aagattctaa g 51

<210> 266

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 266

agaatcctta gaatcttgtt cag 23

<210> 267

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 267

gattctaagg attcttgtta ctctttgtc 29

<210> 268

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 268

gattctaagg attctactta ctgtttgtct tc 32

<210> 269

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 269

gattctaagg attctactta ctctttgtgt tctaca 36

<210> 270

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 270

gattctaagg attctactta ctctttgtct tcttgtttga ca 42

<210> 271

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 271

gattctaagg attctactta ctctttgtct tctacattgt gtttgtctaa ggctg 55

<210> 272

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 272

gattctaagg attctactta ctctttgtct tctacattga cattgtgtaa ggctg 55

<210> 273

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 273

tcaatgatga tgatgatgat gtcaaacctc acctctgtta caagattttg t 51

<210> 274

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 274

tcaatgatga tgatgatgat gtcaaacctc acctctacaa aaagattttg t 51

<210> 275

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 275

tcaatgatga tgatgatgat gtcaaacctc accacagtta aaagattttg t 51

<210> 276

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 276

tcaatgatga tgatgatgat gtcaaacctc acatctgtta aaag 44

<210> 277

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 277

tcaatgatga tgatgatgat gtcaaacaca acctctgtta aaag 44

<210> 278

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 278

tcaatgatga tgatgatgat gtcaacactc acctctgtta aaag 44

<210> 279

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 279

accagacttc aactgctcgt caga 24

<210> 280

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 280

gcagttgaag tctggtagag catctgttgt ctg 33

Claims

1. A method for increasing the correct pairing ratio of heavy and light chains during the manufacture of a bispecific antibody, said method comprising the steps of:

2. The method of claim 1, wherein the amino acids in the CH1-CL domain of IgG1 that form the engineered interchain disulfide bond are selected from the following table:

3. the method of claim 2, wherein the amino acids in the CH1-CL domain of IgG1 that form the engineered interchain disulfide bond are selected from the group consisting of: mutation of selected amino acids [ EU numbering ] in the heavy chain to cysteine/selected amino acids [ EU numbering ] in the kappa chain to cysteine:

F126C/F118C、L128C/P120C、A129C/P120C、P130C/F118C、S132C/F118C、K133C/S121C、S134C/F118C、G166C/T172C、G166C/S174C、G166C/T180C、H168C/T180C、F170C/Q160C、F170C/T164C、F170C/T172C、F170C/S174C、F170C/S176C、F170C/T180C、P171C/T172C、P171C/S174C、P171C/T180C、V173C/S174C、Q175C/S174C、Q175C/S176C、Q175C/T180C、S176C/S162C、S181C/T172C、S181C/S176C、S181C/T180C、S183C/S114C、S183C/F118C、S183C/Q160C、S183C/S174C、S183C/T178C、S183C/T180C、V185C/T172C、V185C/S174C、V185C/T178C、V185C/T180C、T187C/S114C、T187C/T172C、T187C/S174C、T187C/T178C、K218C/F118C、S219C/F116C、S219C/F118C、S219C/P120C。

4. the method of claim 1, wherein the amino acids in the CH1-CL domain of IgG1 that form the engineered interchain disulfide bond are selected from the following table:

5. the method of claim 4, wherein the amino acids in the CH1-CL domain of IgG1 that form the engineered interchain disulfide bond are selected from the group consisting of: mutation of selected amino acids [ EU numbering ] in the heavy chain to cysteine/selected amino acids [ Kabat numbering ] in the lambda chain to cysteine

6. The method of claim 1, wherein the amino acids in the CH1-CL domain of IgG4 that form the engineered interchain disulfide bond are selected from the following table:

7. the method of claim 6, wherein the amino acids in the CH1-CL domain of IgG4 that form the engineered interchain disulfide bond are selected from the group consisting of: mutation of selected amino acids [ EU numbering ] in the heavy chain to cysteine/selected amino acids [ EU numbering ] in the kappa chain to cysteine;

A129C/F209C、A129C/N210C、P130C/F116C、P130C/F118C、P130C/P119C、P130C/N210C、P130C/R211C、S132C/S114C、S132C/F116C、S132C/F118C、S132C/P120C、S132C/R211C、S132C/E213C、R133C/P119C、R133C/R211C、R133C/E213C、T135C/F116C、T135C/P120C、G166C/T178C、H168C/N158C、F170C/F182C、V173C/Q160C、V173C/S162C、Q175C/S162C、Q175C/T180C、S181C/T172C、S181C/S176C、S183C/N158C、S183C/S176C、V185C/E165C、V185C/T178C。

8. the method of claim 1, wherein the amino acids in the CH1-CL domain of IgG4 that form the engineered interchain disulfide bond are selected from the following table:

9. the method of any one of claims 1-8, further comprising the steps of: the charge of a pair of amino acids of the CH1-CL domain of the Fab arm is reversed by amino acid substitution.

10. The method of claim 9, wherein the wild-type positively charged lysine at position 213 of the heavy chain is substituted with a negatively charged amino acid (e.g., K213E, K213D); the wild-type negatively charged glutamic acid at position 123 of the light chain is substituted with a positively charged amino acid (e.g., E123K, E123R).

11. The method of claim 10, wherein the formation of the engineered interchain disulfide bond by amino acid substitution and the charge reversal of a pair of amino acids of the CH1-CL domain of the Fab arm by amino acid substitution can occur on the same Fab arm or on different Fab arms.

12. A method for increasing the correct pairing rate of heavy and light chains during the production of a bispecific antibody, said method comprising the steps of: charge-reversing a pair of amino acids of the CH1-CL domain of the Fab arm by amino acid substitution, wherein the wild-type positively charged lysine at position 213 of the heavy chain is substituted with a negatively charged amino acid (e.g., K213E, K213D); the wild-type negatively charged glutamic acid at position 123 of the light chain is substituted with a positively charged amino acid (e.g., E123K, E123R).

13. A method of making a bispecific antibody comprising the step of employing the method of any one of claims 1-12 during the manufacture of a bispecific antibody so as to increase the correct heavy and light chain pairing rate in the bispecific antibody.

14. A bispecific antibody prepared by the method of claim 13 or having an increased rate of correct heavy and light chain pairing in said bispecific antibody by the method of any one of claims 1-12.