Modified antibodies

Abstract

This specification provides multispecific antibodies containing lambda charge pairs introduced at the heavy-chain-light-chain interface to reduce chain mispairing.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 494,610, filed on April 6, 2023, which is incorporated herein by reference in its entirety.

[0002] (Reference to electronically submitted sequence listings) The contents of the electronically submitted sequence listing (name: IOTS-100-WO Sequence Listing.xml, size: 16,123 bytes, creation date: March 29, 2024) filed with this application are incorporated herein by reference in their entirety.

[0003] (Field of invention) This disclosure relates to multispecific antibodies containing lambda charge pairs introduced at the heavy-chain-light-chain interface as a strategy to reduce chain mispairing. This disclosure also relates to methods for producing these multispecific antibodies and their therapeutic uses. [Background technology]

[0004] Multispecific antibodies that recognize two or more epitopes are of increasing interest in diagnostic and therapeutic applications, as they may support novel mechanisms of action not available with monospecific antibodies. However, their generation presents challenges. Indiscriminate pairing of the heavy and light chains of two antibodies expressed in a single cell can result in the production of 10 different molecules, with only one being bispecific and the remaining pairings yielding non-functional or monospecific molecules.

[0005] Various strategies have been developed to overcome this problem and promote the correct assembly of desired multispecific antibodies. One such strategy to promote heterodimerization of two different heavy chains is the "knobs-into-hole" technique (Ridgway 1990). Strategies to avoid mispairing of light chains include the use of a common light chain (Merchant 1998), domain swapping (Schaefer 2011), and substitution of natural disulfide bonds with interchain disulfide bonds (Mazor 2015).

[0006] An example of a bispecific antibody form incorporating some of these modifications to improve the efficient production of these molecules is "DuetMab," described in PCT International Publication No. 2013 / 096291. The DuetMab antibody uses knob-into-hole technology for heterodimerization of two different heavy chains and increases the effectiveness of homologous heavy- and light-chain pairing by replacing the native disulfide bond at one of the CH1-CL interfaces with a modified disulfide bond.

[0007] While the strategies described above have provided some methods for reducing mispairing of chains, there remains a need to further improve polypeptide chain pairing in multispecific antibodies and facilitate their efficient production. This invention has been made in view of the above circumstances. [Overview of the project]

[0008] We identified amino acid residues at the interface between lambda LC and HC where charge pairs can be introduced, and demonstrated that the introduction of these lambda charge pairs can favorably improve chain pairing beyond what was achieved in previous antibody formats.

[0009] Therefore, in one embodiment, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to a first heavy chain constant region 1 (CH1), wherein the first light chain comprises a constant light chain lambda region (CLλ), and (b) A multispecific antibody is provided which includes a second antigen-binding arm, which includes a second light chain disulfide-bonded to a second CH1, The first antigen-binding arm is a pair at the following positions: (i) Position 117 of CLλ, and position 141 of the first CH1, (ii) Position 117 of CLλ, and position 185 of the first CH1, (iii) Position 119 of CLλ, and position 128 of the first CH1, (iv) Position 134 of CLλ, and position 128 of the first CH1, (v) Position 134 of CLλ, and position 145 of the first CH1, (vi) Position 134 of CLλ, and position 183 of the first CH1, (vii) Position 136 of CLλ, and position 185 of the first CH1, (viii) Position 178 of CLλ, and position 173 of the first CH1, (ix) Including one or more lambda charge pairs located at position 117 of CLλ and position 187 of the first CH1, The lambda charge pair comprises a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the charge pair, and The numbering follows the EU index.

[0010] In some embodiments, the lambda charge pair is located at position 117 of CLλ and position 141 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. a. Arginine at position 117 of CLλ, and aspartic acid at position 141 of the first CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 141 of the first CH1, c. Arginine at position 117 of CLλ, and serine at position 141 of the first CH1, d. Arginine at position 117 of CLλ, and threonine at position 141 of the first CH1, Lysine at position 117 of e.CLλ, and aspartic acid at position 141 of the first CH1, Lysine at position 117 of f.CLλ, and glutamic acid at position 141 of the first CH1, Lysine at position 117 of g.CLλ, and serine at position 141 of the first CH1, and Lysine at position 117 of h.CLλ, and threonine at position 141 of the first CH1.

[0011] In some embodiments, the lambda charge pair is selected from a. to e. of the above list. In some embodiments, the lambda charge pair is selected from any one of a., b., and e. of the above list. In some embodiments, the lambda charge pair is selected from a. and b. of the above list. In some embodiments, the lambda charge pair is arginine at position 117 of CLλ and aspartic acid at position 141 of the first CH1.

[0012] In some embodiments, the lambda charge pair is located at position 117 of CLλ and position 185 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. a. Arginine at position 117 of CLλ, and aspartic acid at position 185 of the first CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 185 of the first CH1, c. Arginine at position 117 of CLλ, and serine at position 185 of the first CH1, d. Arginine at position 117 of CLλ, and threonine at position 185 of the first CH1, Lysine at position 117 of e.CLλ, and aspartic acid at position 185 of the first CH1, Lysine at position 117 of f.CLλ and Glutamic acid at position 185 of the first CH1, Lysine at position 117 of g.CLλ, Serine at position 185 of the first CH1, and Lysine at position 117 of h.CLλ and Threonine at position 185 of the first CH1.

[0013] In some embodiments, the lambda charge pair is located at positions 119 of CLλ and 128 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. Arginine at position 119 of a.CLλ and Aspartic acid at position 128 of the first CH1, Arginine at position 119 of b.CLλ and Glutamic acid at position 128 of the first CH1, Arginine at position 119 of c.CLλ and Serine at position 128 of the first CH1, Arginine at position 119 of d.CLλ and Threonine at position 128 of the first CH1, Lysine at position 119 of e.CLλ and Aspartic acid at position 128 of the first CH1, Lysine at position 119 of f.CLλ and Glutamic acid at position 128 of the first CH1, Lysine at position 119 of g.CLλ, Serine at position 128 of the first CH1, and Lysine at position 119 of h.CLλ and Threonine at position 128 of the first CH1.

[0014] In some embodiments, the lambda charge pair is located at positions 134 of CLλ and 128 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. Arginine at position 134 of a.CLλ and Aspartic acid at position 128 of the first CH1, Arginine at position 134 of b.CLλ and Glutamic acid at position 128 of the first CH1, Arginine at position 134 of c.CLλ and Serine at position 128 of the first CH1, d. Arginine at position 134 of CLλ, and threonine at position 128 of the first CH1, Lysine at position 134 of e.CLλ, and aspartic acid at position 128 of the first CH1, Lysine at position 134 of f.CLλ, and glutamic acid at position 128 of the first CH1, Lysine at position 134 of g.CLλ, and serine at position 128 of the first CH1, and Lysine at position 134 of h.CLλ, and threonine at position 128 of the first CH1.

[0015] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 145 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 145 of the first CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 145 of the first CH1, c. Arginine at position 134 of CLλ, and serine at position 145 of the first CH1, d. Arginine at position 134 of CLλ, and threonine at position 145 of the first CH1, Lysine at position 134 of e.CLλ, and aspartic acid at position 145 of the first CH1, Lysine at position 134 of f.CLλ, and glutamic acid at position 145 of the first CH1, Lysine at position 134 of g.CLλ, and serine at position 145 of the first CH1, and Lysine at position 134 of h.CLλ, and threonine at position 145 of the first CH1.

[0016] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 183 of the first CH1. In some embodiments, the lambda charge pair is lysine at position 134 of CLλ and aspartic acid or serine at position 183 of the first CH1.

[0017] In some embodiments, the lambda charge pair is located at position 136 of CLλ and position 185 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. a. Arginine at position 136 of CLλ, and aspartic acid at position 185 of the first CH1, b. Arginine at position 136 of CLλ, and glutamic acid at position 185 of the first CH1, c. Arginine at position 136 of CLλ, and serine at position 185 of the first CH1, d. Arginine at position 136 of CLλ, and threonine at position 185 of the first CH1, Lysine at position 136 of e.CLλ, and aspartic acid at position 185 of the first CH1, Lysine at position 136 of f.CLλ, and glutamic acid at position 185 of the first CH1, Lysine at position 136 of g.CLλ, and serine at position 185 of the first CH1, and Lysine at position 136 of h.CLλ, and threonine at position 185 of the first CH1.

[0018] In some embodiments, the lambda charge pair is located at position 178 of CLλ and position 173 of the first CH1. In some embodiments, the lambda charge pair is selected from the following list. a. Arginine at position 178 of CLλ, and aspartic acid at position 173 of the first CH1, b. Arginine at position 178 of CLλ, and glutamic acid at position 173 of the first CH1, c. Arginine at position 178 of CLλ, and serine at position 173 of the first CH1, d. Arginine at position 178 of CLλ, and threonine at position 173 of the first CH1, Lysine at position 178 of e.CLλ, and aspartic acid at position 173 of the first CH1, Lysine at position 178 of f.CLλ, and glutamic acid at position 173 of the first CH1, Lysine at position 178 of g.CLλ, and serine at position 173 of the first CH1, and Lysine at position 178 of h.CLλ, and threonine at position 173 of the first CH1.

[0019] As further described herein, lambda charge pairs can be combined with other approaches to promote light chain pairing, for example, to further increase the correct assembly of desired multispecific antibodies.

[0020] In some embodiments, the multispecific antibody has a native interchain disulfide bond at one of the CH1-CL interfaces that has been replaced by a modified interchain disulfide bond. This was one of the approaches taken in the formation of the DuetMab format described by Mazer 2015. In some embodiments, (i) The disulfide bond between the first light chain and the first CH1 is formed between the first light chain and a pair of modified cysteines of the first CH1, and the disulfide bond between the second light chain and the second CH1 is formed between a pair of native cysteines, or (ii) A disulfide bond is formed between the second light chain and the second CH1 between the second light chain and the pair of modified cysteines of the second CH1, and a disulfide bond is formed between the first light chain and the first CH1 between the pair of native cysteines.

[0021] In some embodiments, the modified pair of cysteines in the light chain and CH1 are located at position 122 of the light chain and position 126 of CH1, with the light chain containing a non-cysteine ​​residue at position 212 and CH1 containing a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0022] In some embodiments, the CLλ of the first light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 provides an exemplary wild-type (natural) CLλ, while SEQ ID NO: 2 provides an exemplary CLλ in which the cysteine ​​involved in the natural interchain disulfide bond is replaced with a modified cysteine ​​to form the modified disulfide bond.

[0023] In some embodiments, the first CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4 or SEQ ID NO: 5. SEQ ID NO: 4 provides an exemplary wild-type (natural) CH1, while SEQ ID NO: 5 provides an exemplary CH1 in which the cysteine ​​involved in the natural interchain disulfide bond is replaced with a modified cysteine ​​to form the modified disulfide bond.

[0024] In some embodiments, the second light chain includes a constant light chain kappa region (CLκ). As described herein, the use of different light chains (lambda and kappa) is advantageous because it allows for the selective purification of multispecific antibodies containing the correct light chain using methods such as light chain affinity chromatography. Including a kappa light chain in a multispecific antibody also makes it possible to include a kappa charge pair, which can facilitate pairing of the second CH1:CLκ polypeptide.

[0025] In some embodiments, the second antigen-binding arm comprises a kappa charge pair located at CLκ of the second light chain and the second CH1, the kappa charge pair of the second antigen-binding arm comprising a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position in the kappa charge pair of the second antigen-binding arm, and an uncharged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position in the kappa charge pair of the second antigen-binding arm.

[0026] In some embodiments, the load-charged amino acid residue in the kappa charge pair of the second antigen-binding arm is located at position 133 of CLκ, and the positively charged amino acid residue in the kappa charge pair is located at position 183 of the second CH1. In some embodiments, the load-charged amino acid residue at position 133 of CLκ is glutamic acid, and the positively charged amino acid residue at position 183 of the second CH1 is lysine.

[0027] In some embodiments, the CLκ of the second light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3.

[0028] In some embodiments, the second CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO: 1 or SEQ ID NO: 2.

[0029] In some embodiments, the first antigen-binding arm and / or the second antigen-binding arm include an Fc region. In some embodiments, the first antigen-binding arm includes a first Fc region, and the second antigen-binding arm includes a second Fc region. Various strategies can be used to facilitate the heterodimerization of the two heavy chains (i.e., heterodimerization of a first heavy chain containing a first CH1 and a first Fc region, and a second heavy chain containing a second CH1 and a second Fc region).

[0030] In some embodiments, the first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region. In some embodiments, these modifications are located at CH3 in the Fc region.

[0031] In some embodiments, the modification of the CH3 in one of the first and second Fc regions is a substitution of an amino acid residue with a larger side chain, thereby generating a bump (knob) on the surface of the CH3 domain, while the modification of the CH3 in the other Fc region is a substitution of an amino acid residue with a smaller side chain, thereby generating a cavity (hole) on the surface of the CH3 domain. Optionally, the CH3 domain containing the bump (knob) is part of the first heavy chain polypeptide, and the CH3 domain containing the cavity (hole) is part of the second heavy chain.

[0032] In some embodiments, the substitution for generating a knob is a substitution for tryptophan at position 366, and the substitution for generating a hole is a substitution that includes one or more of the following: i) Substitution of valine at position 407, ii) Substitution of serine at position 366, and iii) Substitution with alanine at position 368.

[0033] In some embodiments, the CH3 domain containing a knob contains cysteine ​​at position 354, and the CH3 domain containing a hole contains cysteine ​​at position 349.

[0034] In some embodiments, the multispecific antibody includes a lambda charge pair in combination with any one or more of the modified disulfide, kappa charge pair, and Fc modification to promote the heterodimerization described herein. For example, in some embodiments, the multispecific antibody includes a lambda charge pair in combination with the modified disulfide described herein. In some embodiments, the multispecific antibody includes a lambda charge pair in combination with the kappa charge pair described herein. In some embodiments, the multispecific antibody includes a lambda charge pair in combination with the Fc modification to promote the heterodimerization described herein. In some embodiments, the multispecific antibody includes a lambda charge pair in combination with the modified disulfide and Fc modification to promote the heterodimerization described herein. In some embodiments, the multispecific antibody includes a lambda charge pair in combination with the kappa charge pair and Fc modification to promote the heterodimerization described herein. In some embodiments, the multispecific antibody includes a lambda charge pair in combination with the modified disulfide and Fc modification to promote the heterodimerization described herein. In some embodiments, the multispecific antibody comprises a lambda charge pair in combination with a modified disulfide, a kappa charge pair, and an Fc modification to facilitate the heterodimerization described herein.

[0035] Methods for producing multispecific antibodies described herein are also provided herein. In some embodiments, the method is a) Expressing the first light chain and the second light chain, as well as the first CH1 and the second CH1 in host cells, b) Pairing the first light chain with the first CH1 to form the first binding arm, pairing the second light chain with the second CH1 to form the second binding arm, and pairing the first binding arm with the second binding arm to form a multispecific antibody, c) Purification of multispecific antibodies from host cells, including

[0036] In some embodiments, the method comprises producing a multispecific antibody, the method comprising expressing a first light chain, a second light chain, and a third light chain, as well as a first CH1, a second CH1, and a third CH1 in host cells, and expressing a first CH1, a second CH1, and a third CH1 in host cells, wherein the first light chain pairs with the first CH1 to form a first binding arm, the second light chain pairs with the second CH1 to form a second binding arm, the third light chain pairs with the third CH1, and the first binding arm pairs with the second and third binding arms to form a multispecific antibody, and the multispecific antibody is purified from host cells.

[0037] In some embodiments, the purification of multispecific antibodies includes affinity chromatography. In some embodiments, the purification of multispecific antibodies includes light chain affinity chromatography.

[0038] In some embodiments, less than 25%, less than 20%, less than 15%, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the light chain is mispaired after purification of the multispecific antibody. In some embodiments, less than 10% of the light chain is mispaired. In some embodiments, less than 5% of the light chain is mispaired. Preferred methods for determining the percentage of mispairing are described herein.

[0039] One or more nucleic acids encoding the first light chain and / or first CH1 of the multispecific antibodies described herein are also provided herein. In some embodiments, one or more nucleic acids further encode a second light chain and / or a second CH1. In some embodiments, one or more nucleic acids are part of a vector. Isolated host cells containing nucleic acids or vectors are also provided herein.

[0040] This specification also provides pharmaceutical compositions and therapeutic methods comprising pharmaceutical compositions or multispecific antibodies, as further described below. [Brief explanation of the drawing]

[0041] Next, embodiments and experiments demonstrating the principle of this disclosure will be described with reference to the attached drawings. [Figure 1A] This shows the interface between the CH1 group of the kappa light chain (LC) and heavy chain (HC) in the antibody. V133 of κLC and S183 of HC are labeled. [Figure 1B] This shows the interface between lambda LC and CH1 in the antibody. V134 and Y178 of lambda LC and S183K of HC are labeled. [Figure 2] The diagram includes schematic representations of DuetMab antibodies containing charge pairs. The “hole” HC on the left is disulfide-bonded to kappa LC via native cysteine ​​and contains a kappa charge pair (e.g., S183K / V133E) indicated by a minus ("-") sign on the kappa LC and a plus ("+") sign on the “hole” HC. The “knob” HC on the right is disulfide-bonded to lambda LC via modified cysteine ​​and contains a lambda charge pair indicated by a plus ("+") sign on the lambda LC and a minus ("-") sign on the “knob” HC. [Figure 3] This shows the interface between the lambda LC and CH1 of HC in an antibody containing exemplary lambda charge pairs (T117R and A141S). The T117R of the lambda LC and the A141S of the HC are labeled. [Figure 4] The corrected LC ratio % data from Table 1 were plotted on a scattering XY chart. Charge pair mutants #33, #34, #35, #36, and #41 were selected for further analysis based on their accurate LC ratio %. [Figure 5] The response signals and fitting curves for control sample #1 and mutant #33 are shown, with the latter representing the mutant tested. Reaction kinetics for the soluble monomeric form of antigen 2 were obtained using an Ocet384 instrument. The dissociation constant KD was calculated as the koff / kon ratio from the nonlinear fit of the data. [Figure 6]The transitions captured by DSC thermal stability measurements for the Fab, CH2, and CH3 domains, designated TM1, TM2, TM3, and TM4, are shown. [Figure 7] The UV chromatograms obtained from subunit LC / MS analysis of each sample are shown. No subunits corresponding to mismatched species were identified. [Figure 8] Mutants with different charge pairs were assayed for cytotoxic activity. Each point represents the mean of a triple well, and the mean ± standard error (SEM) is represented by an error bar. R347 is the isotype control. [Figure 9] Table 9 provides a representation of the corrected LC ratio % data plotted in a grouped box chart. [Figure 10] This specification provides a schematic diagram of the CH1-CL domain interface having the T117R mutation in the CL of the lambda light chain and the A141D mutation in the CH1 of the heavy chain, based on data generated from the crystallographic studies described herein. A strong hydrogen bond of approximately 2.4 Å appears to be formed between the OD1 atom of aspartic acid at position 141 of the CH1 domain and the NH1 atom of arginine at position 117 of the lambda light chain. [Figure 11] This specification provides a schematic diagram of the CH1-CL domain interface having the T117R mutation in the CL of the lambda light chain and the A141E mutation in the CH1 of the heavy chain, based on data generated from the crystallographic studies described herein. A hydrogen bond of approximately 3.0 Å appears to be formed between the OE1 atom of aspartic acid at position 141 of the CH1 domain and the NH1 atom of arginine at position 117 of the lambda light chain. [Modes for carrying out the invention]

[0042] Next, aspects and embodiments of this disclosure will be described with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0043] This specification provides multispecific antibodies (e.g., bispecific antibodies) that include a constant light chain lambda region (CLλ) in one binding arm of the multispecific antibody and a lambda charge pair located at a specific position in the heavy chain constant region 1 (CH1), as will be discussed in more detail below.

[0044] Methods for generating bispecific antibodies are well known. However, such methods are often limited by a multitude of possible antibody formations, which may involve several combinations of incorrect pairings of heavy and light chains. Such mispairings can reduce production efficiency. The use of lambda and kappa charge variants, as described herein, overcomes these limitations by preferentially pairing the lambda light chain with the correct CH1 in one binding arm, thereby generating a preferred bispecific antibody assembly. In particular, these lambda charge variants can be combined with well-known approaches used to promote accurate pairing of heavy and light chains, such as knob-into-hole (KiH), modified disulfide, and kappa charge pairs, as described in more detail below, to further improve the formation of a preferred bispecific antibody.

[0045] antibody The terms “antibody” or “antibody molecule” refer to immunoglobulins, whether naturally occurring or partially or entirely synthetically produced. Antibodies can be human or humanized. In some embodiments, antibodies are monoclonal antibody molecules. Examples of antibodies include immunoglobulin isotypes such as immunoglobulin G (IgG), and their isotype subclasses such as IgG1, IgG2, IgG3, and IgG4, as well as fragments thereof.

[0046] Antibodies are composed of two different types of polypeptide chains: one is called the heavy chain, and the other is called the light chain. Natural monospecific antibodies consist of two identical heavy chains and two identical light chains. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. The disulfide bonds linking the light and heavy chains are sometimes called "intra-chain" disulfide bonds to distinguish them from "inter-chain" disulfide bonds present within individual heavy and light chain polypeptides.

[0047] The light chains in natural antibodies are either "lambda (λ)" or "kappa (κ)" light chains, which differ in their amino acid sequences. The light chain consists of a single constant light chain region (CL) and a single light chain variable region (VL). An example of the amino acid sequence of the constant light chain lambda region (CLλ) is provided as SEQ ID NO: 1, and an example of the amino acid sequence of the constant light chain kappa region (CLκ) is provided as SEQ ID NO: 3. The light chains used in the multispecific antibodies described herein may be chimeric light chains, for example, containing CLλ and VLκ.

[0048] The IgG heavy chain consists of a heavy variable (VH) region and three heavy chain constant regions (CH1, CH2, and CH3), with a further "hinge region" between CH1 and CH2. An example of the IgG1 CH1 region amino acid sequence is provided as SEQ ID NO: 4. An example of the IgG1 CH2 amino acid sequence is provided as SEQ ID NO: 6. An example of the IgG1 CH3 amino acid sequence is provided as SEQ ID NO: 7. An example of the heavy chain amino acid sequence including CH1, the hinge, CH2, and CH3 is provided as SEQ ID NO: 8.

[0049] Unless otherwise specified, amino acid residue positions in constant domains, including amino acid sequences, substitutions, deletions, and insertions, described herein are numbered according to EU numbering (Edelman, 2007).

[0050] The light chain associates with VH and CH1 to form an "antigen binding arm," and the variable domains within the antigen binding arm interact to form an "antigen binding domain."

[0051] The "antigen-binding domain" represents a part of a molecule that binds to all or part of a target antigen and generally contains six complementarity-determining regions (CDRs), three of which are in the VH region: HCDR1, HCDR2, and HCDR3, and three in the VL region: LCDR1, LCDR2, and LCDR3. Together, the six CDRs define a paratope of the antigen-binding domain, which is part of the antigen-binding domain that binds to the target antigen. A monoclonal monospecific IgG antibody molecule contains two antigen-binding domains, each of which can bind to the same target (i.e., it is bivalent for a single target).

[0052] The VH and VL regions each contain a framework region (FR) on one side of each CDR, which provides a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region has the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0053] multispecific antibodies This disclosure provides multispecific antibodies. The multispecific antibodies according to this disclosure may be provided in an isolated form, meaning that they do not contain contaminants such as antibodies that can bind to other polypeptides and / or serum components.

[0054] The formation of disulfide bonds between cysteine ​​residues occurs during the folding of many proteins entering the secretory pathway. When a polypeptide chain breaks down, adjacent cysteines can form a covalent bond during a process catalyzed by members of the protein disulfide isomerase family. As used herein, the terms “disulfide link” or “disulfide linked” refer to a single covalent bond formed from the coupling of thiol groups, particularly cysteine ​​residues. In some embodiments, the covalent bond between two cysteines is located between the two sulfur atoms of each residue. However, depending on the environment, not all protein species can always have a disulfide present, for example, in the event of disulfide reduction. Therefore, the terms “disulfide link” or “disulfide linked” (whether natural or modified) also refer, in some embodiments, to the presence of two cysteine ​​residues that can form a disulfide bond, regardless of whether they are actually linked at individual points in time.

[0055] The multispecific antibodies of this disclosure can bind to at least two epitopes on the same antigen or on different antigens and include at least two antigen-binding arms, referred to herein as “first antigen-binding arm” and “second antigen-binding arm”. According to this disclosure, an “antigen-binding arm” includes a light chain, VH and CH1 (i.e., at least one constant domain and one variable domain from the heavy chain and light chain, respectively), the light chain being disulfide-bonded to CH1. Each antigen-binding arm may further include additional heavy chain regions, i.e., one or more of the hinge, CH2 and CH3. In some embodiments, the antigen-binding arm further includes an Fc region (i.e., the remaining portion of the heavy chain including the hinge, CH2 and CH3). In some embodiments, the multispecific antibody includes the complete heavy chain (i.e., VH, CH1, hinge, CH2 and CH3).

[0056] The first antigen-binding arm and the second antigen-binding arm differ from each other at least in their light chain amino acid sequences and CH1 amino acid sequences (i.e., the first and second light chains have different amino acid sequences, and the first and second CH1s have different amino acid sequences). In some embodiments, the heavy chain of the first antigen-binding arm can form a disulfide bond with the heavy chain of the second antigen-binding arm (for example, via interchain disulfide bonds between cysteine ​​molecules present in the Fc domain).

[0057] Examples of multispecific antibodies include bispecific antibodies that can bind to two epitopes, and triplicate antibodies that can bind to three epitopes. In some cases, a multispecific antibody is a bispecific antibody.

[0058] The bispecific antibody molecules may be provided in any preferred form. For preferred formats of the bispecific antibody molecules described herein and methods for producing them, see Kontermann, MAbs 2012, 4(2):182-197 and Kontermann and Brinkmann 2015, 20(7):838-847. In particular, see Figure 2 in Kontermann MAbs 2012, 4(2):182-19. Specific examples of bispecific antibody formats include DuetMab, kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair, and SEED-body. In certain embodiments, the bispecific antigen is DuetMab.

[0059] A specific exemplary format of an asymmetric IgG-like bispecific molecule is referred to as “DuetMab.” DuetMab antibodies utilize a knob-into-hole (KIH) technique for heterodimerization of two distinct heavy chains, increasing the effectiveness of congeneral heavy- and light-chain pairing by replacing a native disulfide bond at one of the CH1-CL interfaces with a modified disulfide bond. Disclosures relating to DuetMab can be found, for example, in U.S. Patent No. 9,527,927 and Mazor, 2015, which are incorporated herein by reference in their entirety, as further described below.

[0060] Lambda charge pair The terms “charge pair” and “charge mutation” are used interchangeably throughout this specification and refer to a positively charged amino acid residue and a negatively charged amino acid residue, one located in the light chain region of the antigen-binding arm (e.g., the constant light chain region) and the other in the heavy chain region (e.g., the constant heavy chain region 1 (CH1)), in a position intended to facilitate the association of the light and heavy chains. A “lambda charge pair” means an introduced or substituted charge pair in which a positively charged or uncharged amino acid residue is located in the lambda light chain (e.g., CLλ) and the constant heavy chain region (e.g., CH1). A “kappa charge pair” means an introduced or substituted charge pair in which a positively charged or uncharged amino acid residue in the light chain is located in the kappa light chain (e.g., CLκ) and the constant heavy chain region (e.g., CH1).

[0061] While we do not wish to be bound by theory, it is thought that the oppositely charged amino acid residues in the charge pair increase the attractive force of the heavy chain to the light chain in the antigen-binding arm, thereby promoting the formation of an antigen-binding arm with the correct heavy and light chains.

[0062] At least one of the amino acid residues in a charge pair is modified into an antigen-binding arm (i.e., at least one amino acid residue in the pair is not a wild-type amino acid residue). In some embodiments, both amino acid residues in a charge pair are modified into antigen-binding arms (i.e., neither amino acid residue in the pair is a wild-type amino acid residue).

[0063] Charged amino acid residues are typically found in nature. Examples of naturally occurring positively charged amino acid residues according to this disclosure include arginine, lysine, and histidine. Examples of naturally occurring negatively charged amino acid residues according to this disclosure include glutamic acid, serine, threonine, and aspartic acid. Although serine and threonine are often described as "uncharged" in the art, they have isoelectric points less than 6 and are therefore partially negatively charged at neutral pH. For the purposes of the charged pairs disclosed herein, serine and threonine are examples of negatively charged amino acid residues (along with glutamic acid and aspartic acid).

[0064] Therefore, a charge pair may include a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the charge pair. For example, a charge pair may include any one of the following amino acid residue pairs. Arginine and aspartic acid, Arginine and glutamic acid, Arginine and serine, Arginine and threonine, Lysine and aspartic acid, Lysine and glutamic acid, Lysine and serine, Lysine and threonine, Histidine and aspartic acid, Histidine and glutamic acid, Histidine and serine, and Histidine and threonine.

[0065] In some embodiments, the positively charged amino acid residue in the charge pair is located on the light chain, and the uncharged amino acid residue in the charge pair is located on the heavy chain. In other embodiments, the uncharged amino acid residue is located on the light chain, and the positively charged amino acid residue in the charge pair is located on the heavy chain.

[0066] The multispecific antibodies described herein contain a lambda charge pair in one of the antigen-binding arms (also referred to herein as the “first antigen-binding arm”). As illustrated herein, the lambda charge pair can be introduced at several positions to improve the correct pairing of light and heavy chains in the antigen-binding arm.

[0067] In some embodiments, the lambda charge pair includes a positively charged amino acid residue or an uncharged amino acid residue at positions 117, 119, 134, 136, or 178 of the steady-state light chain lambda region (CLλ). In some embodiments, the lambda charge pair includes a positively charged amino acid residue or an uncharged amino acid residue at positions 141, 185, 128, 145, 183, 185, 173, or 187 of CH1. As mentioned elsewhere, the numbering follows EU numbering. Positions 117, 119, 134, 136, and 178 of CLλ, according to EU numbering, correspond to amino acids 10, 12, 27, 29, and 71 of SEQ ID NO: 1 and SEQ ID NO: 2. Positions 141, 185, 128, 145, 183, 185, 173, and 187 of CH1, according to EU numbering, correspond to amino acids 24, 68, 11, 28, 66, 68, 56, and 70 of SEQ ID NO: 4 and SEQ ID NO: 5.

[0068] In some embodiments, the lambda charge pair is as follows: (i) Position 117 of CLλ and position 141 of CH1, (ii) Position 117 of CLλ and position 185 of CH1, (iii) Position 119 of CLλ and position 128 of CH1, (iv) Position 134 of CLλ and position 128 of CH1, (v) Position 134 of CLλ and position 145 of CH1, (vi) Position 134 of CLλ and position 183 of CH1, (vii) Position 136 of CLλ and position 185 of CH1, (viii) Position 178 of CLλ and position 173 of CH1, (ix) Located at one or more of the pair of positions at 117th position of CLλ and 187th position of CH1.

[0069] In some embodiments, the lambda charge pair is located at position 117 of CLλ and position 141 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 117 of CLλ, and aspartic acid at position 141 of CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 141 of CH1, c. Arginine at position 117 of CLλ, and serine at position 141 of CH1, d. Arginine at position 117 of CLλ, and threonine at position 141 of CH1, Lysine at position 117 of e.CLλ, and aspartic acid at position 141 of CH1, Lysine at position 117 of f.CLλ, and glutamic acid at position 141 of CH1, Lysine at position 117 of g.CLλ, and serine at position 141 of CH1, Lysine at position 117 of h.CLλ, and threonine at position 141 of CH1.

[0070] In some embodiments, the lambda charge pair is selected from any one of a. to f. in the above list. In some embodiments, the lambda charge pair is selected from any one of a. to e. in the above list. In some embodiments, the lambda charge pair is selected from any one of a, b, and e in the above list. In some embodiments, the lambda charge pair is a.

[0071] In some embodiments, the lambda charge pair is located at position 117 of CLλ and position 185 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 117 of CLλ, and aspartic acid at position 185 of CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 185 of CH1, c. Arginine at position 117 of CLλ, and serine at position 185 of CH1, d. Arginine at position 117 of CLλ, and threonine at position 185 of CH1, Lysine at position 117 of e.CLλ, and aspartic acid at position 185 of CH1, Lysine at position 117 of f.CLλ, and glutamic acid at position 185 of CH1, Lysine at position 117 of g.CLλ, and serine at position 185 of CH1, Lysine at position 117 of h.CLλ, and threonine at position 185 of CH1.

[0072] In some embodiments, the lambda charge pair is located at position 119 of CLλ and position 128 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 119 of CLλ, and aspartic acid at position 128 of CH1, b. Arginine at position 119 of CLλ, and glutamic acid at position 128 of CH1, c. Arginine at position 119 of CLλ, and serine at position 128 of CH1, d. Arginine at position 119 of CLλ, and threonine at position 128 of CH1, Lysine at position 119 of e.CLλ, and aspartic acid at position 128 of CH1, Lysine at position 119 of f.CLλ, and glutamic acid at position 128 of CH1, Lysine at position 119 of g.CLλ, and serine at position 128 of CH1, Lysine at position 119 of h.CLλ, and threonine at position 128 of CH1.

[0073] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 128 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 128 of CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 128 of CH1, c. Arginine at position 134 of CLλ, and serine at position 128 of CH1, d. Arginine at position 134 of CLλ, and threonine at position 128 of CH1, Lysine at position 134 of e.CLλ, and aspartic acid at position 128 of CH1, Lysine at position 134 of f.CLλ, and glutamic acid at position 128 of CH1, Lysine at position 134 of g.CLλ, and serine at position 128 of CH1, Lysine at position 134 of h.CLλ, and threonine at position 128 of CH1.

[0074] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 145 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 145 of CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 145 of CH1, c. Arginine at position 134 of CLλ, and serine at position 145 of CH1, d. Arginine at position 134 of CLλ, and threonine at position 145 of CH1, Lysine at position 134 of e.CLλ, and aspartic acid at position 145 of CH1, Lysine at position 134 of f.CLλ, and glutamic acid at position 145 of CH1, Lysine at position 134 of g.CLλ, and serine at position 145 of CH1, Lysine at position 134 of h.CLλ, and threonine at position 145 of CH1.

[0075] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 183 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 183 of CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 183 of CH1, c. Arginine at position 134 of CLλ, and serine at position 183 of CH1, d. Arginine at position 134 of CLλ, and threonine at position 183 of CH1, Lysine at position 134 of e.CLλ, and aspartic acid at position 183 of CH1, Lysine at position 134 of f.CLλ, and glutamic acid at position 183 of CH1, Lysine at position 134 of g.CLλ, and serine at position 183 of CH1, Lysine at position 134 of h.CLλ, and threonine at position 183 of CH1.

[0076] In some embodiments, the lambda charge pair is aspartic acid or serine at lysine at position 134 of CLλ and at position 183 of CH1. In the CH1 sequence provided as SEQ ID NO: 4 or SEQ ID NO: 5, position EU183 is serine, and therefore, it is not necessary to introduce a modification to CH1 in SEQ ID NO: 4 or SEQ ID NO: 5 in order to generate a charge pair with the positively charged amino acid at position 134 of CLλ.

[0077] In some embodiments, the lambda charge pair is located at position 136 of CLλ and position 185 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 136 of CLλ, and aspartic acid at position 185 of CH1, b. Arginine at position 136 of CLλ, and glutamic acid at position 185 of CH1, c. Arginine at position 136 of CLλ, and serine at position 185 of CH1, d. Arginine at position 136 of CLλ, and threonine at position 185 of CH1, Lysine at position 136 of e.CLλ, and aspartic acid at position 185 of CH1, Lysine at position 136 of f.CLλ, and glutamic acid at position 185 of CH1, Lysine at position 136 of g.CLλ, and serine at position 185 of CH1, Lysine at position 136 of h.CLλ, and threonine at position 185 of CH1.

[0078] In some embodiments, the lambda charge pair is located at position 178 of CLλ and position 173 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 178 of CLλ, and aspartic acid at position 173 of CH1, b. Arginine at position 178 of CLλ, and glutamic acid at position 173 of CH1, c. Arginine at position 178 of CLλ, and serine at position 173 of CH1, d. Arginine at position 178 of CLλ, and threonine at position 173 of CH1, Lysine at position 178 of e.CLλ, and aspartic acid at position 173 of CH1, Lysine at position 178 of f.CLλ, and glutamic acid at position 173 of CH1, Lysine at position 178 of g.CLλ, and serine at position 173 of CH1, Lysine at position 178 of h.CLλ, and threonine at position 173 of CH1.

[0079] In some embodiments, the first antigen-binding arm includes two or more lambda charge pairs. For example, the first antigen-binding arm may include 2, 3, 4, 5, 6, 7, 8, or 9 lambda charge pairs at positions (i) to (ix) above.

[0080] In the multispecific antibodies described herein, one antigen-binding arm (e.g., a first antigen-binding arm) includes one of the lambda charge pairs described above, and another antigen-binding arm (e.g., a second antigen-binding arm) includes either a different lambda charge pair described above or does not include a lambda charge pair.

[0081] For example, both the first and second antigen-binding arms of a multispecific antibody may contain the lambda charge pairs described above, and the lambda charge pair of the first antigen-binding arm may be different from the lambda charge pair of the second antigen-binding arm. That is, the lambda charge pair of the first antigen-binding arm may be located at any one of the pairs at positions (i) to (ix) above, and the lambda charge pair of the second antigen-binding arm may be located at different pairs within positions (i) to (ix) above. For example, the first antigen-binding arm may contain lambda charge pairs at position 117 of CLλ and position 141 of the first CH1, and the second antigen-binding arm may contain different lambda charge pairs, for example, at position 134 of CLλ and position 145 of the second CH1.

[0082] In another example, both the first and second antigen-binding arms in a multispecific antibody contain a lambda charge pair at the same position (i.e., one of (i) to (ix) above), but the positively charged amino acid residue and the uncharged amino acid residue are located on different polypeptide chains in each antigen-binding arm. That is, the first antigen-binding arm may contain a positively charged amino acid residue at one of the CLλ positions and an uncharged amino acid residue at the first CH1, and the second antigen-binding arm may contain an uncharged amino acid residue at the same position of CLλ and a positively charged amino acid residue at the same position of the second CH1, or vice versa. For example, the first antigen-binding arm may include a lambda charge pair consisting of arginine at position 117 of CLλ of the first antigen-binding arm and aspartic acid at position 141 of the first CH1, and the second antigen-binding arm may include a lambda charge pair consisting of aspartic acid at position 117 of CLλ of the second antigen-binding arm and arginine at position 141 of the second CH1.

[0083] In further examples, the first antigen-binding arm includes one or more of the lambda charge pairs described above, and the second antigen-binding arm includes a constant light chain kappa region (CLκ) (optionally having a kappa charge pair), as described in more detail below.

[0084] As demonstrated herein, multispecific antibodies containing lambda charge pairs exhibit improved correct light chain pairing compared to multispecific antibodies lacking lambda charge pairs. Specifically, when producing multispecific antibodies containing lambda charge pairs in the first antigen-binding arm, the proportion of multispecific antibodies containing the correct first light chain and first CH1 increases compared to the production of equivalent multispecific antibodies without lambda charge pairs.

[0085] As described in the examples, several methods are known that can be used to determine accurate light chain pairing. These include mass spectrometry-based approaches that can be used to establish the correct heavy / light chain association. If the second binding arm contains a kappa light chain, the ratio of kappa light chains to lambda light chains in the assembled multispecific antibody can be determined using microfluidic electrophoresis as a readout for the correct light chain ratio.

[0086] Therefore, in some embodiments, multispecific antibodies containing lambda charge pairs exhibit improved correct light chain pairing compared to equivalent multispecific antibodies lacking lambda charge pairs. In some embodiments, multispecific antibodies containing lambda charge pairs optionally exhibit correct light chain ratios of 90%, 95%, 96%, 97%, 98%, or greater than 99% (determined, for example, using microfluidic electrophoresis) after purification of the multispecific antibody using light chain affinity purification.

[0087] Combination with other counter-approach approaches The lambda charge pairs described herein may be combined with other strategies to promote heterodimerization in order to further increase the correct pairing of heavy-chain and light-chain polypeptides.

[0088] Non-limiting examples of strategies for promoting heterodimerization are described in more detail below and include disulfide modifications at the CH1 / CL interface, introduction of additional charge pairs (e.g., kappa charge pairs), and Fc region modifications such as knob-into-hole, as well as the use of strategies that enable fractionation and purification.

[0089] Modified disulfide In some embodiments, multispecific antibodies contain an engineered disulfide in addition to the lambda charge pair. "Engineered disulfide" means that the native interchain disulfide bond at the CH1-CL interface of one of the antigen-binding arms (e.g., 220 of CH1 and 212 of LC) is replaced by an engineered (non-native) interchain disulfide, while the other antigen-binding arm contains a native interchain disulfide bond at the CH1-CL interface. Engineered disulfides are typically formed by modifying cysteine ​​at the CL of the light chain and the corresponding CH1 of the heavy chain, replacing the cysteine ​​that normally forms an interchain disulfide. Disclosures relating to the introduction of engineered disulfides into multispecific antibodies for the purpose of promoting heterodimerization can be found, for example, U.S. Patent No. 9,527,927 and Mazor, 2015, both of which are incorporated herein by reference.

[0090] Therefore, in some embodiments, (i) The disulfide bond between the first light chain and the first CH1 is formed between the first light chain and a pair of modified cysteines of the first CH1, and the disulfide bond between the second light chain and the second CH1 is formed between a pair of native cysteines, or (ii) A disulfide bond is formed between the second light chain and the second CH1 between the second light chain and the pair of modified cysteines of the second CH1, and a disulfide bond is formed between the first light chain and the first CH1 between the pair of native cysteines.

[0091] In some embodiments, the modified pair of cysteines in the light chain and CH1 are located at position 122 of the light chain and position 126 of CH1, the same light chain contains a non-cysteine ​​residue at position 212, and the same CH1 contains a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0092] An exemplary amino acid sequence of CLλ containing the modified cysteine ​​is provided as SEQ ID NO: 2, and an exemplary amino acid sequence of CH1 containing the corresponding modified cysteine ​​for forming the modified disulfide is provided as SEQ ID NO: 5.

[0093] In the multispecific antibodies exemplified herein, the modified disulfide resides on the “first” antigen-binding arm containing a lambda charge pair, while the native disulfide resides on the “second” antigen-binding arm that does not contain a lambda charge pair. However, the reverse configuration, i.e., the native disulfide residing on the first antigen-binding arm and the modified disulfide on the second antigen-binding arm, is also specifically intended.

[0094] In some embodiments, a pair of modified cysteines in the constant light chain kappa region (CLκ) and CH1 are located at position 121 of CLκ and position 126 of CH1, the same CLκ containing a non-cysteine ​​residue at position 214, and the same CH1 containing a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0095] κ chain and charge pair In some embodiments, the second antigen-binding arm includes a constant light chain kappa region (CLκ). That is, in a multispecific antibody, one antigen-binding arm contains CLλ and the other antigen-binding arm contains CLκ. As described herein, techniques such as light chain affinity chromatography utilizing affinity resins specific to either CLκ or CLλ may be used to selectively purify an antibody based on its light chain. Examples of such affinity resins include LambdaFabSelect and KappaSelect resins available from GE Healthcare. Such methods may be used to selectively purify multispecific antibodies containing both CLκ and CLλ, and therefore may be used to improve the production of bispecific antibodies in this form.

[0096] An example of the CLκ amino acid sequence is provided as SEQ ID NO: 3.

[0097] In some embodiments, the second antigen-binding arm includes a kappa charge pair. As described above, the kappa charge pair refers to a positively charged amino acid residue and an uncharged amino acid residue, one of which is located on the kappa light chain (e.g., CLκ) and the other on the heavy chain of the antigen-binding arm (e.g., CH1), in a position intended to facilitate association between the light chain and CH1 of the second antigen-binding arm.

[0098] In some embodiments, the second antigen-binding arm includes a kappa charge pair located at position 133 of CLκ and position 183 of the second CH1. In some embodiments, the loaded amino acid residue in the kappa charge pair is at position 133 of CLκ, and the positively charged amino acid residue in the kappa charge pair is at position 183 of the second CH1. In other embodiments, the positively charged amino acid residue in the kappa charge pair is at position 133 of CLκ, and the loaded amino acid residue in the kappa charge pair is at position 183 of the second CH1. In some embodiments, the loaded amino acid residue (e.g., position 133 of CLκ) is glutamic acid, and the positively charged amino acid residue (e.g., position 183 of the second CH1) is lysine. As mentioned elsewhere, this numbering follows EU numbering.

[0099] Position 133 of CLκ, according to EU numbering, corresponds to amino acid position 26 of SEQ ID NO: 3. Position 183 of CH1, according to EU numbering, corresponds to amino acid 66 of SEQ ID NOs: 4 and 5.

[0100] In certain embodiments, the multispecific antibody comprises a first antigen-binding arm having a lambda charge pair as described above, and a second antigen-binding arm having a kappa charge pair as described above, wherein the multispecific antibody comprises a modified disulfide.

[0101] For example, in one embodiment, the first antigen-binding arm includes a lambda charge pair (e.g., position 117 of CLλ and position 141 of the first CH1), and the disulfide bond between the first light chain and the first CH1 is formed between a pair of modified cysteines in CLλ and the first CH1; and the second antigen-binding arm includes a kappa charge pair (e.g., position 133 of CLκ and position 183 of the second CH1), and the disulfide bond between the second light chain and the second CH1 is formed between a pair of native cysteines in CLκ of the second light chain and the second CH1.

[0102] In another embodiment, the first antigen-binding arm comprises a lambda charge pair (e.g., position 117 of CLλ and position 141 of the first CH1), and the disulfide bond between the first light chain and the first CH1 is formed between a pair of native cysteines in CLλ of the first light chain and the first CH1; the second antigen-binding arm comprises a kappa charge pair (e.g., position 133 of CLκ and position 183 of the second CH1), and the disulfide bond between the second light chain and the second CH1 is formed between a pair of modified cysteines in CLκ of the second light chain and the second CH1.

[0103] Fc region modification As described above, in some embodiments, the first antigen-binding arm and the second antigen-binding arm further include a first Fc region and a second Fc region (i.e., further include CH2 and CH3 regions of the heavy chain).

[0104] In some embodiments, the antibody molecule includes one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of a heterodimeric antibody molecule by promoting the formation of a first Fc region and a second Fc region. This may include a Knobs into Holes (KiH) strategy based on a single amino acid substitution in the CH3 domain that promotes heavy chain heterodimerization, as described by Ridgway, 1996. The knob mutant heavy chain CH3 has a small amino acid substituted with a larger amino acid, thereby creating a bump (knob) on the surface of the CH3 domain, while the hole mutant has a large amino acid substituted with a smaller amino acid, thereby creating a cavity (hole) on the surface of the CH3 domain. Further modifications may also be introduced to stabilize the association between the heavy chains.

[0105] Examples of CH3 modifications to enhance heterodimerization include the "hole" mutation Y407V / T366S / L368A on one Fc region and the "knob" mutation T366W on the other Fc region. These may further include stabilizing the cystine mutation Y349C (e.g., on the Fc region containing the "hole" mutation) and stabilizing the S354C mutation on the other Fc region (e.g., on the Fc region containing the "knob" mutation). Exemplary amino acid sequences of CH3 domains modified to include the "hole" mutation are provided as SEQ ID NOs: 9 and 10. Exemplary amino acid sequences of CH3 domains modified to include the "knob" mutation are provided as SEQ ID NOs: 11 and 12.

[0106] Therefore, in one embodiment, the substitution for generating a knob is a substitution for tryptophan at position 366, and the substitution for generating a hole is one or more of the following: i) Substitution of valine at position 407, ii) Substitution of serine at position 366, and iii) Substitution with alanine at position 368.

[0107] In the multispecific antibodies illustrated herein, the “knob” is located on the “first” antigen-binding arm containing a lambda charge pair, and the “hole” is located on the “second” antigen-binding arm that does not contain a lambda charge pair. However, the reverse configuration, i.e., the “hole” being located on the CH3 of the first antigen-binding arm and the “knob” being located on the CH3 of the second antigen-binding arm, is also specifically intended.

[0108] For example, one Fc region may include modifications to enable fractional elution by protein A chromatography. In short, one of the Fc regions may include protein A(Fc * This modification includes removing the bond to the heterodimer FcFc (called * This enables the selective purification of bispecific products. Fc * Suitable modifications for generating the region include substitution of H435 with arginine and substitution of Y436 with phenylalanine.

[0109] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, and The second antigen-binding arm includes the second Fc region, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0110] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, and It includes a second antigen-binding arm containing a second Fc region, The first and second Fc regions include modifications to promote heterodimerization of the first and second Fc regions, and the multispecific antibody contains the modified disulfide.

[0111] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, and It includes the kappa charge pair and a second antigen-binding arm containing a second Fc region, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0112] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, and It includes the kappa charge pair and a second antigen-binding arm containing a second Fc region, The first and second Fc regions include modifications to promote heterodimerization of the first and second Fc regions, and the multispecific antibody contains the modified disulfide.

[0113] Non-limiting examples of bispecific antibodies, including lambda charge pairs, kappa charge pairs, modified disulfides, and modifications to promote heterodimerization of the first and second Fc regions, are provided in the examples.

[0114] Other Fc modifications intended herein reduce or disable the binding of antibody molecules to one or more Fcγ receptors and / or complement, such as FcγRI, FcγRIIa, FcγRIIb, and FcγRIII. Such mutations reduce or suppress Fc effector function. Mutations that reduce or suppress the binding of antibody molecules to one or more Fcγ receptors and complement are well known, including, for example, the L234F / L235E / P331S “triple mutation” or “TM” (according to the European Union numbering rules) described by Organosesyan et al., Acta Crystallogr D Biol Crystallogr 64(6):700-704, 2008.

[0115] In some embodiments, the CH2 domain of one or both of the constant domains of the immunoglobulin heavy chain contains the following substitution: E233P / L234V / L235A / G236del / S267K. This combination of mutations may be referred to herein as an "Fc effector null mutation."

[0116] Other suitable Fc region amino acid substitutions or modifications are well known in the art and include, for example, triple substitutions (M252Y / S254T / T256E, "YTE" or "YTE" mutations) numbered according to the EU index, such as in Kabat, of methionine (M) to tyrosine (Y) substitution at position 252, serine (S) to threonine (T) substitution at position 254, and threonine (T) to glutamic acid (E) substitution at position 256 (M252Y / S254T / T256E, "YTE" or "YTE" mutations) (e.g., U.S. Patent No. 7,658,921, U.S. Patent Application Publication No. 2014 / 0302058, and Yu et al., Antimicrob. Agents Chemother., 61(1):e01020-16 (2017), the full contents of each of these are incorporated herein by reference). This combination of mutations may extend the half-life of the antibody.

[0117] Triple mutations, Fc effector null mutations, and YTE mutations, if present, may be located in one or both of the heavy chain constant domains. Typically, if present, they are located in both of the heavy chain constant domains.

[0118] CD3 target and T cell engager In some embodiments, one of the antigen-binding arms can bind to CD3.

[0119] CD3 (differentiation antigen group 3) is a protein complex composed of four subunits: the CD3γ chain, CD3δ chain, and two CD3ε chains. CD3 associates with the T cell receptor and ζ chain to generate an activation signal in T lymphocytes. Bispecific antibodies targeting CD3 and target cell antigens are used to force transient interactions between target cells and T cells, leading to crosslinking, T cell activation, and subsequent antigen-dependent T cell death of target cells.

[0120] Sequence identity and mutation As described herein, the first antigen-binding arm comprises a first light chain capable of forming a disulfide bond to a first CH1, the first light chain comprising a constant light chain lambda region (CLλ). Also as described herein, the second antigen-binding arm comprises a second light chain capable of forming a disulfide bond to a second CH1. In some embodiments, the second light chain comprises a constant light chain kappa region (CLκ).

[0121] In some embodiments, the CLλ of the first light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the CLλ of the first light chain comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0122] In some embodiments, the CLκ of the second light chain (if present) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 3. In some embodiments, the CLκ of the second light chain (if present) comprises an amino acid sequence of SEQ ID NO: 3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0123] In some embodiments, the first CH1 or the second CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, CH1 comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0124] One, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications may be added to the above modifications for introducing the charge pair, modified disulfide, and / or Fc region modification. For example, compared to the wild-type CLλ described in SEQ ID NO: 1, the CLλ used in the multispecific antibody may contain a lambda charge pair mutation, a modified disulfide (e.g., S122C and C212V), and one, two, three, four, five, six, seven, eight, nine, or ten further amino acid modifications. As another example, compared to the wild-type CH1 provided in SEQ ID NO: 4, the first CH1 used in the multispecific antibody may contain a lambda charge mutation, a modified disulfide (e.g., F126C, C220V), and one, two, three, four, five, six, seven, eight, nine, or ten further amino acid modifications.

[0125] Amino acid modifications may be insertions, substitutions, or deletions. In some embodiments, an amino acid modification is the substitution of an amino acid residue with any other naturally occurring or non-naturally occurring amino acid residue.

[0126] Naturally occurring residues may be classified into classes based on common side-chain properties. 1) Nonpolar aliphatic compounds: Glycine (G), methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I), 2) Polarity: Cysteine ​​(C), asparagine (N), glutamine (Q), proline (P), 3) Polarity, partial load: serine (S), threonine (T), 4) Acidic (loaded electricity): Aspartic acid (D), Glutamic acid (E), 5) Basic (positively charged): Histidine (H), Lysine (K), Arginine I, 6) Aromatic compounds: Tryptophan (W), tyrosine (Y), phenylalanine (F).

[0127] As described above, serine (S) and threonine (T) have isoelectric points less than 6 and are partially negatively charged at neutral pH; therefore, they are classified as “polar, partially negatively charged” in this specification.

[0128] Amino acid substitutions may be conservative amino acid substitutions. Conservative amino acid substitutions may include the exchange of one member of these classes with another member of the same class. For example, a conservative amino acid substitution may be a substitution using the acidic amino acid glutamic acid (E) instead of the acidic amino acid aspartic acid (D).

[0129] Nucleic acids, vectors, and host cells One or more nucleic acids encoding the multispecific antibodies described herein are also provided herein. In some embodiments, the nucleic acids are purified or isolated, for example, from other nucleic acids or naturally occurring biological materials. Those skilled in the art will have no difficulty preparing such nucleic acid molecules using methods well known in the art.

[0130] In some embodiments, one or more nucleic acids encode the light chain and / or CH1 as described herein. One or more nucleic acids encoding the first CH1 or the second CH1 may further encode other heavy chain domains, e.g., hinges, CH2 and CH3, or they may encode the complete heavy chain.

[0131] This disclosure also provides one or more vectors comprising nucleic acids encoding the multispecific antibodies described herein. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including a promoter sequence, a terminator fragment, a polyadenylation sequence, an enhancer sequence, a marker gene, and optionally other sequences. In some embodiments, the vector contains appropriate regulatory sequences for driving nucleic acid expression in host cells. The vector may optionally be a plasmid, a virus, such as a phage, or a phagemid.

[0132] Multispecific antibodies may be produced from light chain vectors and heavy chain vectors. The light chain vector may contain nucleic acids encoding a first light chain and nucleic acids encoding a second light chain, which may be present on the vector as separate cassettes (e.g., each operably connected to a different promoter). Similarly, the heavy chain vector may be used to encode both the first CH1 (and the first Fc region, if present) and the second CH1 (and the second Fc region, if present), which may be present on the vector as separate cassettes. Alternatively, separate vectors may be used to encode the first light chain, the second light chain, the first CH1 (and the first Fc region, if present), and the second CH1 (and the second Fc region, if present), respectively.

[0133] The nucleic acid molecules or vectors described herein may be introduced into host cells. Techniques for introducing nucleic acids or vectors into host cells are well established in the art, and any suitable technique may be used. Various host cells suitable for the production of recombinant antibody molecules are well known in the art and include bacterial, yeast, insect, or mammalian host cells. In some embodiments, the host cell is a mammalian cell such as a CHO, NS0, or HEK cell, e.g., a HEK293 cell. In some embodiments, the host cell is a CHO cell.

[0134] Method for producing multispecific antibodies Methods for producing multispecific antibodies as described herein are also provided herein. In some embodiments, the method includes: a) expressing a first light chain and a second light chain, and a first CH1 and a second CH1 in a host cell; b) pairing the first light chain with the first CH1 to form a first binding arm, pairing the second light chain with the second CH1 to form a second binding arm, and pairing the first binding arm with the second binding arm to form a multispecific antibody; and c) generating a multispecific antibody from a host cell.

[0135] Expressing the first and second light chains and the first and second CH1 in host cells may involve introducing nucleic acids or vectors into host cells (e.g., CHO cells) using preferred techniques as described above. The host cells may then be cultured using preferred techniques so that the light and heavy chain polypeptides pair up to form the first and second binding arms. During normal bispecific antibody development, the various light and heavy chain polypeptides associate with each other (e.g., via interchain disulfide bonds formed between native cysteine ​​and / or via cysteine ​​modified into bispecific antibodies as described herein), and the heavy chains associate with each other (e.g., via interchain disulfide bonds formed between cysteine ​​in two Fc domains). As described herein, the presence of lambda charge pairs ensures that the correct heavy / light chain pairs are formed in the bispecific antibody.

[0136] Techniques for purifying recombinant antibody molecules are well known in the art and include, for example, high-performance liquid chromatography, high-performance protein liquid chromatography, ion-exchange chromatography, and affinity chromatography, for example, using protein A or protein L or by conjugating them to affinity tags. In some embodiments, purification is carried out using affinity chromatography (e.g., protein A affinity chromatography). In some embodiments, purification further includes light chain affinity chromatography (in addition to, for example, protein A chromatography). As described herein, light chain affinity chromatography may be used to selectively purify multispecific antibodies containing both CLκ and CLλ, and therefore may be used to improve the production of bispecific antibodies in this format.

[0137] In some embodiments, less than 25%, 20%, 15%, or 10%, 5%, 4%, 3%, 2%, or 1% of the light chains in a multispecific antibody mispair (i.e., pair with CH1 from a different antigen-binding arm) after purification (e.g., by protein A affinity chromatography, or after protein A affinity chromatography and light chain affinity chromatography). Methods for determining accurate light chain pairing are well known in the art and include mass spectrometry and microfluidic electrophoresis, as described in more detail herein. In some cases, the method includes measuring the correct light chain pairing.

[0138] The method may also include formulating an antibody molecule into a pharmaceutical composition with optionally pharmaceutically acceptable excipients or other substances listed below.

[0139] treatment Therefore, the multispecific antibodies described herein may be useful for therapeutic applications such as cancer treatment.

[0140] The multispecific antibodies described herein may be used in methods for treating the human or animal body. Relevant aspects of this disclosure are: (i) A multispecific antibody as described herein for use as a pharmaceutical product, (ii) A multispecific antibody as described herein for use in a method of treating a disease or disorder, (iii) In the manufacture of a pharmaceutical product for use in the treatment of a disease or disorder, the multispecific antibodies described herein, (iv) A method for treating a disease or disorder in an individual, comprising administering to the individual a therapeutically effective amount of a multispecific antibody described herein.

[0141] The treatment may be any treatment or therapy that achieves any desired therapeutic effect, such as inhibiting or delaying the progression of the condition, and may include a reduction in the rate of progression, cessation of the rate of progression, improvement of the condition, cure or remission of the condition (either partially or completely), prevention, improvement, delay, reduction or cessation of one or more symptoms and / or signs of the condition, or extension of the survival of the individual or patient beyond what would be expected in the absence of treatment.

[0142] Preventive measures (i.e., treatments as prophylaxis) are also included. For example, individuals that are susceptible to or at risk of developing or recurring a disease such as cancer may be treated as described herein. Such treatments may prevent or delay the development or recurrence of the disease in the individual.

[0143] The treatment methods described may include administering at least one further treatment to the individual in addition to the multispecific antibody. Therefore, the multispecific antibody described herein may be administered to the individual alone or in combination with one or more other treatments. When the multispecific antibody is administered to the individual in combination with another treatment, the additional treatment may be administered to the individual simultaneously with, following, or separately from, the administration of the multispecific antibody. When the additional treatment is administered simultaneously with the multispecific antibody, the multispecific antibody and the additional treatment may be administered to the individual as a combination preparation. For example, the additional therapy may be a well-known therapy or treatment for the disease being treated.

[0144] While multispecific antibodies may be administered alone, they are typically administered in the form of a pharmaceutical composition that may contain at least one additional component in addition to the multispecific antibody. Therefore, another aspect of this disclosure provides a pharmaceutical composition comprising the multispecific antibody described herein. Methods for formulating a multispecific antibody into a pharmaceutical composition are also provided.

[0145] The pharmaceutical composition may include, in addition to the multispecific antibody, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. As used herein, “pharmaceutically acceptable” refers to a compound, material, composition, and / or dosage form that is suitable for use in contact with the tissue of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio, within the bounds of reasonable medical judgment. Each carrier, excipient, etc., must also be “acceptable” in the sense of being compatible with the other components of the formulation. The exact nature of the carrier or other material depends on the route of administration, which may be by infusion, injection, or any other preferred route, as discussed below.

[0146] The dose may be a "therapeutically effective amount," which is sufficient to provide a benefit to the individual. The actual amount administered, as well as the rate and time course of administration, depends on the nature and severity of what is being treated, the specific individual being treated, the individual's clinical condition, the cause of the disorder, the delivery site of the composition, the type of antibody molecule, the method of administration, the scheduling of administration, and other factors known to healthcare professionals. The prescription of treatment, such as the determination of the dosage, is the responsibility of the general practitioner and other physicians and may depend on the severity of the symptoms and / or progression of the disease being treated. ***

[0147] Features disclosed in the foregoing description, the following claims, or the accompanying drawings, expressed in a particular form or relating to means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, may be used, as necessary, separately or in any combination of such features, to implement the present disclosure in a variety of forms.

[0148] While this disclosure is described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Therefore, the exemplary embodiments of this disclosure described above are illustrative and not limiting. Various modifications to the embodiments of this disclosure may be made without departing from the spirit and scope of this disclosure.

[0149] Throughout this Spec., including the claims, it will be understood that unless the context requires otherwise, variations of the words “comprise” and “include,” as well as “comprises,” “comprising,” and “including,” mean the inclusion of the integer or step or group of integers or steps described, but not the exclusion of any other integer or step or group of integers or steps.

[0150] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, the alternative aspects include from one particular value and / or the other particular value. Similarly, where a value is expressed as an approximation by the use of the antecedent “about,” it will be understood that a particular value forms an alternative aspect. The term “about” with respect to numbers is optional and means, for example, ±10%. [Examples]

[0151] Example 1 - Design of charge pair mutants at the lambda LC-HC interface To improve correct chain pairing beyond what alternative disulfides can achieve in the DuetMab setting (see PCT International Publication No. 2013 / 096291 incorporated herein by reference), charge pairs were designed using amino acids involved in the lambda light chain (LC)-heavy chain (HC) interface. Previous strategies for improving HC / LC chain pairing involved modifying the interface between kappa LC and CH1 with a reverse-charged amino acid residue. It was recognized that charge pairs modified at the κLC-HC interface were unlikely to behave similarly if modified at equivalent positions at the λ / CH1 interface. For example, the presence of Y178 in lambda LC is expected to disrupt charge pairs modified to V134 of lambda LC (equivalent to V133 of kappa LC) and S138 of CH1 (see Figure 1B).

[0152] The following positions were evaluated as lambda light chain amino acids involved in interface formation with the CH1 domain: T117, F119, S122, E124, E125, K130, T132, V134, L136, S138, D139, E161, T163, S166, Q168, A174, S176, Y178, S180. In relation to this, the following heavy chain CH1 domains... The amino acids involved in interface formation with the lambda light chain CL domain are: S124, F126, L128, A129, S131, S132, K133, S134, A141, G143, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S181, S183, V185, T187, V211, and K213.

[0153] These amino acids were investigated individually or in pairs, in combination, or alone, using alternative interchain disulfides or leaving the disulfides in their natural state. The introduction of positively or partially positively charged amino acids means substituting the amino acid present at that position with lysine and arginine, and possibly asparagine, glutamine, or histidine. The introduction of negatively or partially negatively charged amino acids means substituting the amino acid present at that position with aspartic acid, glutamic acid, serine, threonine, and possibly asparagine or glutamine. Adding histidine residues to some of these positions makes it possible to introduce pH-dependent CH1-CL interactions.

[0154] Nine pair combinations at the lambda LC-HC interface that meet the above criteria are provided in Table 1 as a non-exhaustive list of examples, and improved pair formation was tested herein.

[0155] Table 1. All mutations presented here are specific to lambda light chain-containing molecules and are expected to function in wild-type and V12 formats (see below). Furthermore, the opposite charge pair [i.e., V134(D,E,S,T)-L128(R,K,H)] is also expected to provide a preferred pairing. Net uncharged side chains containing amino acids such as asparagine and glutamine have been found to be involved in the formation of hydrogen bonds with both positively and negatively charged amino acids, as well as with each other, and can therefore be used for substitutions with either positive or negative partial charges.

[0156] [Table 1]

[0157] Example 2 - Materials and Methods The experiments described in the subsequent examples were carried out using the materials and methods described herein. All reagents were obtained from Thermo Fisher Scientific, Waltham, MA, unless otherwise noted. As stated elsewhere, the terms “charge pair” and “charge mutation” are used interchangeably throughout this specification, and amino acid numbering is based on the EU numbering system unless otherwise noted.

[0158] Construction of pDuet-heavy chain and pDuet-light chain mammalian expression vectors for DuetMab with charge pairs. To construct a DuetMab antibody with a charge pair mutation at the heavy-light chain interface, the pDuet-heavy-chain and pDuet-light-chain plasmids described in PCT International Publication No. 2013 / 096291 and Mazor et al., 2015, were used as the backbone vector. Briefly, the pDuet-Heavy vector contained two human gamma monoheavy-chain (HC) cassettes to support HC heterodimerization. The former heavy chain possessed a "hole" set mutation (T366S / L368A / Y407V) and a stabilizing mutation (Y349C) in the CH3 domain, while the latter possessed a complementary "knob" mutation (T366W) and a stabilizing mutation (S354C) in CH3. However, the order of the cassettes could be easily reversed. The pDuet-Light vector contains two human light chain (LC) cassettes, the former containing a kappa constant domain (Cκ) and the latter containing a lambda constant domain (Cλ). The pDuet-Heavy and pDuet-Light vectors also contain mutations to remove the native interchain disulfide bond in CH1 / Cλ and provide an alternative disulfide bond indicated herein as "V12 DS" or "V12". Mutation F126C / C220V was introduced into the CH1 domain of the "knob" heavy chain, and mutation S122C / C212V was introduced into the lambda constant domain. The amino acid sequences of the constant domains in the exemplary DuetMab antigen backbone (before introduction of charge mutations) are provided below.

[0159] [Table 2]

[0160] The “knob-and-hole” sets of mutations and stabilizing / alternative disulfide bonds used herein are provided merely as examples. Those skilled in the art may use any other combination of “knob-and-hole” techniques and / or mutations for stabilizing / alternative disulfide bonds known in the art to support HC heterodimerization.

[0161] To construct a pDuet-Heavy vector with a charge mutation, the "Hole" heavy chain was cloned into the pDuet-Heavy vector using restriction cloning techniques with BssHII / HindIII by a synthetic DNA fragment of the VH-CH1-CH2-CH3 domain containing the above mutation for the "Hole" heavy chain. Optionally, the "Hole" heavy chain contained the charge mutation S183K in the CH1 domain. The "Knob" heavy chain was cloned into the vector using restriction cloning techniques with BsrGI / EcoRI by a synthetic DNA fragment of the VH-CH1-CH2-CH3 domain containing the above mutation for the "Knob" heavy chain. The "knob" heavy chains were optionally selected to contain one of the following charge mutations in the CH1 domain: L128D, L128E, L128S, L128T, A141D, A141E, A141S, A141T, L145D, L145E, L145S, L145T, S183D, V185D, V185E, V185S, V185T, V173D, V173E, V173S, and V173T.

[0162] To construct pDuet-Light with charge mutations, the κ light chain was cloned into the pDuet-Light vector using a synthetic DNA fragment of the VL-Cκ domain with BssHII / NheI restriction cloning technology. Optionally, the stationary kappa (Cκ) domain contained the charge mutation V133E. The lambda light chain was cloned into the pDuet-Light vector using a synthetic DNA fragment of the VL-Cλ domain containing the above-mentioned S122C / C212V mutation for the lambda light chain with BsrGI / EcoRI restriction cloning technology. Optionally, the stationary lambda (Cλ) domain contained one of the charge mutations V117R, V117K, F119R, F119K, V134R, V134K, L136R, L136K, Y178R, and Y178K. The light chain variable domain (VL) can be either a variable kappa domain (Vκ) or a variable lambda domain (Vλ).

[0163] Expression, affinity purification, and protein quantification. All constructs were transiently expressed in CHO cells in suspension using PEI-MAX (Polysciences, Inc., Warrington, PA) as the transfection reagent and grown in in-house prepared CHO medium. Vectors containing the following charge pair combinations were used for antibody expression in these studies. A schematic diagram of the constructed DuetMabs containing the charge pairs is shown in Figure 2. Bispecific antibodies were prepared against several different antigens expressed on the cell surface (referred to herein as antigens 1, 2, 3, 4, 5, and 6). Antigen 3 is CD3. The prepared bispecific antibodies were referred to as "Target 1 / Target 2-DuetMab" or simply "Target 1 / Target 2".

[0164] [Table 3-1]

[0165] [Table 3-2]

[0166] [Table 3-3]

[0167] The culture medium was collected 7–13 days after transfection and filtered through a 0.22 μm sterile filter. The antibody concentration in the culture supernatant was measured using an Octet 384 instrument with a protein A sensor (Sartorius, Gottingen, Germany) according to the manufacturer's protocol. The antibodies were purified by either protein A magnetic bead affinity purification (Genscript, Piscataway, NJ) or standard protein A affinity chromatography (Cytiva, Marlborough, MA), followed by light chain affinity chromatography if necessary, according to the manufacturer's protocol, and then buffer exchange in PBS (pH 7.2). The purity and oligomeric state of the purified molecules were determined by microfluidic electrophoresis and analytical size exclusion chromatography (see method below). Protein aggregates were removed by preparative SEC. The concentration of the purified antibodies was determined by reading the absorbance at 280 nm using the theoretically determined extinction coefficient.

[0168] Size exclusion chromatography (SEC) Analytical SEC-HPLC (Agilent 1260 Infinity HPLC system) was performed using a TSK-gel G3000SWxL column (Tosoh Biosciences, King of Prussia, PA) to determine the oligomeric state of the purified molecules. Preparative SEC-HPLC was performed using a Superdex 200 column (Cytiva) to remove protein aggregates.

[0169] Microfluidic-based electrophoresis To evaluate the ratio of kappa light chains to lambda light chains in the antibody, microfluidic electrophoresis was performed using a Bioanalyzer according to the manufacturer's protocol (Agilent, Santa Clara, CA), and the percentage of the correct light chain ratio was calculated based on this.

[0170] Binding kinetics assay The binding kinetics were measured by biolayer interferometry using an Octet384 instrument. A streptavidin (SA) biosensor was immobilized with biotinylated protein antigen (ACRO Biosystems, Newark, DE) in PBS pH 7.2, 1 mg / mL BSA, and 0.05% (v / v) TWEEN (Kinetic buffer). After washing the loaded biosensor in the same buffer, association and dissociation measurements were performed over the indicated time using various antibodies. The kinetic parameters (K) were then measured. on and K off ) and affinity (K D This was calculated from a nonlinear fit of the data using Octet384 software v.12.2.1.24.

[0171] Accelerated stability test Protein test samples were diluted to 1 mg / mL in PBS (pH 7.2) and divided into three equal aliquots to be used as control, thermal, and photostress samples. The control sample was incubated at 4°C for 14 days, the thermal stress sample at 45°C for 14 days, and the photostress sample was incubated at 25°C for 7 days in a glass vial in an ICH-compliant photostable chamber exposed to 3000 lux of cold white light. The samples were then analyzed by HP-SEC to determine the levels of aggregates, monomers, and fragments.

[0172] Differential scanning fluorescence (DSF) Samples were prepared in a 96-well PCR plate by combining 20 μL of 1 mg / mL protein sample in PBS (pH 7.2) with 5 μL of SYPRO Orange dye diluted 40-fold in PBS (pH 7.2) in a double-strand configuration. The plates were sealed and measured using a QuantStudio 7 Flex Real-Time PCR System. The samples were subjected to an initial equilibrium step at 25°C for 2 minutes, followed by a temperature gradient to 99°C in increments of 0.05°C / second. Fluorescence emission was monitored using a FAM filter set. The Tm value for each sample was calculated using the Boltzmann method with Protein Thermal Shift® software.

[0173] Subunit LC-MS analysis Subunit LC / MS analysis was performed to characterize the mispaired species. 50 μg of sample was dried and further reconstituted in 50 μL of 100 mM sodium phosphate buffer, pH 7.0. Digestion was performed by adding 60 units of FabALACTICA enzyme (IgdE) (Genovis AB, Lund, Sweden) to each sample and incubating at 37°C for 16–18 hours. A Waters ACQUITY UPLC system (Waters, Milford, MA) connected to a Waters Xevo G2-XS QTI mass spectrometer was used for subunit separation and mass determination. 2 μg of digested subunits were injected into a Waters BioResolve RP mAb polyphenyl column (2.1 × 150 mm, 2.7 mm, 450 Å) for separation. Mobile phase A contained 0.1% formic acid (FA) and 0.01% trifluoroacetic acid (TFA) in water, and mobile phase B contained 0.1% FA and 0.01% TFA in ACN water. A gradient from 25%B to 45%B was performed at a flow rate of 0.2 mL / min for 40 minutes. The column temperature was set to 75°C. The UV profile of the eluted subunits was acquired at a wavelength of 280 nm.

[0174] Differential scanning calorimetry analysis (DSC) The DSC experiments were carried out using a MICROCAL VP-DSC scanning microcalorimeter (Malvern, Northampton, MA). Before DSC analysis, all samples were diluted to approximately 0.6 mg / mL in phosphate buffered saline (PBS, pH 7.2). The exact concentration was determined from duplicate measurements using a UV-VIS spectrophotometer (NanoDrop 2000C). 400 μL of each sample and the corresponding buffer (PBS, pH 7.2) were immobilized in a 96-well plate and stored at 10 °C in an autosampler chamber until analysis. All DSC measurements were performed using a temperature window of 20 °C to 100 °C at a scan rate of 60 °C / hour. Before sample measurements, baseline measurements (buffer versus buffer) were obtained for subtraction from the sample measurements. Data analysis, baseline correction, and deconvolution were performed using Origin™ DSC software provided by Microcal. Baseline correction was carried out using a linear connecting function within the software. Deconvolution analysis was performed using a non-two-state model, and the best fit was obtained using 1 and 200 iteration cycles until the chi-square value was minimized. The interpretation of the DSC deconvolution results was based on the fact that different domains in the antibody format unfold independently. T 開始値 is defined as the temperature at which the thermogram begins to increase significantly from the baseline. T m values are defined as the temperature values corresponding to the maximum of each peak on the thermogram or the deconvolved thermogram.

[0175] Cell viability assay Cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). This assay quantifies the presence of ATP, which indicates the presence of metabolically active cells. Luminescence generated by the luciferase-catalyzed reaction of luciferin and ATP was measured using a luminescence plate reader. Briefly, target cells (NCI H358) were seeded at approximately 1 × 10 4Cells were seeded in 96-well plates at a cell / well density. Various concentrations of antibody were added to triplicate samples, and the cells were incubated in a humidified incubator at 37°C and 5% CO2 for 72 hours. After treatment, cells were exposed to CellTiter-Glo® reagent (Promega) for approximately 15 minutes, and OD409 was measured using an EnVision 2104 Multilabel plate reader (PerkinElmer). Cell viability was determined by measuring ATP levels compared to an antibody-free control.

[0176] Example 3 - Expression and characteristics of charge pair mutants Table 3 and Figure 4 summarize the expression and biochemical profiles of Antigen 1 / Antigen 2 DuetMabs with the proposed charge pair set produced in small cell cultures (3 mL). Figure 4 shows the accurate LC ratio data from Table 1 plotted on a scattered XY chart. Compared to controls #1 and #2, charge pair mutants #33, #34, #35, #36, and #41 showed improved accurate LC ratios and were selected for further analysis.

[0177] [Table 4-1]

[0178] [Table 4-2]

[0179] Table 4 summarizes the expression (Table 4A) and biochemical profiles (Table 4B) of Antigen 1 / Antigen 2 DuetMabs containing selected charge pair mutants #1, #2, #33, #34, #35, #36, and #41 produced in large-scale cell cultures (100 mL). The biochemical profiles of the selected cultured DuetMabs were consistent regardless of production scale. For further analysis, DuetMabs were further purified by light chain affinity chromatography to remove mispaired byproducts, and aggregates were removed by preparative SEC.

[0180] [Table 5]

[0181] [Table 6]

[0182] Figure 5 and Table 5 show the binding dynamics of Antigen 1 / Antigen 2 DuetMabs with selected charge-pair variants. Figure 5 shows the response signals and fitting curves for control sample #1 and variant #33, which are representative of the tested variants. The binding affinities of variants #33, #34, #35, #36, and #41 to antigen 2 were comparable to those of controls #1 and #2.

[0183] [Table 7]

[0184] Table 6 summarizes the thermal stability and accelerated stability profiles of Antigen 1 / Antigen 2 DuetMabs with selected charge-pair mutants, as determined by differential scanning fluorescence (DSF). NIP228 served as an IgG1 control. Antigen 1 / Antigen 2 DuetMab mutants did not show any concerns regarding aggregation or fragmentation after thermal stress. HP-SEC retention times for Antigen 1 / Antigen 2 DuetMab mutants were consistent with those of the NIP228 IgG1 control (ΔRT < 0.2 m from NIP228). DSF values ​​did not show significant differences between charge-pair mutants and were consistent with those of the NIP228 IgG1 control.

[0185] [Table 8]

[0186] Figures 6 and Table 7 show the thermal stability tests of Antigen 1 / Antigen 2 DuetMabs with selected charge-pair mutants using differential scanning calorimetry (DSC) analysis. Figure 3 shows the stacked thermograms for Antigen 1 / Antigen 2 DuetMab mutants. Deconvolution of the thermograms revealed transitions for the Fab, CH2, and CH3 domains, with some transitions overlapping and located under the same TM peak. Table 7 shows the deconvoluted TM and approximated T for Antigen 1 / Antigen 2 DuetMab charge-pair mutants. 開始値 List them. All mutants are similar to the approximate T 開始値 The study demonstrated that the selected charge pair did not significantly affect thermal stability.

[0187] [Table 9]

[0188] Figures 7 and Table 8 show the subunit mass spectrometry data of Antigen 1 / Antigen 2 DuetMabs with selected charge pair variants. The molecular integrity and LC / HC congruence of each variant were confirmed by alignment of the theoretical and measured masses.

[0189]

Table 10

[0190] Figure 8 shows the cytotoxicity characteristics of Antigen 1 / Antigen 2 DuetMabs with selected charge pair variants, determined by quantification of ATP indicating the presence of metabolically active cells. Variants #33, #34, #35, #36, and #41 showed cytotoxicity comparable to Control #1 and Control #2, suggesting that the charge pair variants did not affect the biological function of Antigen 1 / Antigen 2 of the DuetMab.

[0191] Figures 9 and Table 9 summarize the expression and biochemical profiles of selected charge pair variants in diverse Fv-cultured DuetMabs. Figure 9 shows the exact LC ratio data of Table 9 plotted in a grouped box chart. Charge pair variant #33, charge pair variant #34, charge pair variant #35, charge pair variant #36, and charge pair variant #41 showed improved exact LC ratios compared to Control #1 and #2 among different Fv DuetMabs.

[0192]

Table 11

[0193] Example 4 - Crystallographic Investigation of Proposed Mutations at the CH1 - CL (lambda) Interface To further investigate the lambda charge variants at the light chain:CH1 interface, X-ray crystallographic analysis was performed.

[0194] Fab Cloning and Expression (i) the variable domain of the light chain derived from the anti-antigen 2 antibody and the constant domain of the human lambda light chain containing the T117R, S122C, and C212V mutations, and (ii) the coding sequences of the variable domain of the heavy chain of the anti-antigen 2 antibody and the CH1 domain containing the A141D or A141E and F126C and C220V mutations were ordered as synthetic DNA gBlocks from Integrated DNA Technologies (Coralville, IA). The coding sequences of the light chains were flanked by the N-terminal BssHII and C-terminal NheI restriction sites, and the coding sequences of the heavy chains were flanked by the N-terminal BsrGI and C-terminal EcoRI restriction sites to facilitate cloning. The gBlocks were digested and inserted into a mammalian expression vector (pOE, AstraZeneca, Gaithersburg, MD). One Shot Top10 chemically competent E. coli cells (Invitrogen, Carlsbad, CA) were used as the host for gene cloning.

[0195] Both Fabs were transiently expressed in a suspension of human embryonic kidney (HEK) 293 cells using 293 fectin Transfection Reagent (Life Technologies, Carlsbad, CA) and a standard protocol. Cells were grown in FreeStyle 293-F Expression Medium (Life Technologies) for 10 days, supplied with proprietary cell supply solution (AstraZeneca), then the suspension was spun down and the supernatant was filtered through a 0.2 μM filter. The Fabs were purified from the supernatant using a 5 mL CaptureSelect CH1-XL column (Thermo Fisher Scientific, Waltham, MA), dialyzed against 25 mM Hepes pH 7, and further polished on a 5 mL HiTrap SP HP cation exchange column (Cytiva, Marlborough, MA) in a NaCl gradient to improve sample homogeneity.

[0196] Crystallization, crystal collection, and X-ray diffraction data acquisition Before setting up the crystallization screening, each Fab sample was individually electrophoresed on a Superdex 200 Increase 10 / 300 GL column (Cytiva) pre-equilibrated with 25 mM HEPES, pH 7.5, and 100 mM NaCl to ensure sample homogeneity. The initial crystallization tests for both proteins were performed by sitting-drop vapor diffusion at 20°C. Crystallization droplets were dispensed into a 96-well crystallization plate (Intelli-Plate 102-0001-20, Millipore). Crystallization was performed using a Phoenix crystallization robot (Art Robbins Instruments) and a commercially available crystallization screen (XCArt Robbins Instruments, Sunnyvale, CA). Droplets consisted of equal volumes of protein and reservoir buffer.

[0197] result Diffraction-quality crystals were collected directly from the original sitting drop plates using the following crystallization solutions: A141E: 0.1 M BIS-TRIS pH 6.5, 25% w / v PEG 3350 with a protein concentration of 18.4 mg / mL; A141D: 200 mM sodium chloride, 0.1 M BIS-TRIS pH 5.5, 25% w / v PEG 3350 with a protein concentration of 9 mg / mL. All crystals recovered for X-ray analysis were flash-cooled in liquid nitrogen, and diffraction experiments were performed at 100 K on beamline B14-1 of the Stanford Synchrotron Radiation Lightsource (Menlo Park, CA). Diffraction data collected from single crystals for each Fab were processed, integrated, and scaled using XDS software (Kabsch, 2010).

[0198] The structures of both Fab molecules were determined using molecular substitution with the MolRep program (Vagin, 1997) from the crystallographic software suite CCP4 (Winn, 2011). Model construction was performed using Coot (Emsley, 2004), and refinement was carried out using the Refmac5 program (Kovalevskiy, 2018).

[0199] The crystal of T117R / A141D Fab was diffracted to 2.1 Å. Upon completion of refinement, the inventors found, consistent with their predictions, that the side chains of the mutant amino acids actually established very strong hydrogen bonds (Figure 1).

[0200] The crystal of T117R / A141E Fab was diffracted to 2.0 Å. Upon completion of the refinement, the inventors found that the side chains of the mutant amino acids actually established hydrogen bonds, consistent with their predictions.

[0201] Consideration A comparison of these two Fab molecules shows that the mutant T117R / A141D establishes stronger (shorter) hydrogen bonds than T117R / A141E. This result was confirmed by a higher percentage of molecules with exact pairs for molecules containing the 117R / 141D pair.

[0202] array 1. Amino acid sequence of the WT CLλ constant region (SEQ ID NO: 1) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0203] 2. Amino acid sequence of the "V12" LC-lambda constant (CLλ) region modified to form a modified disulfide bridge (SEQ ID NO: 2) The following substitutions are underlined. Modified disulfides: S122C, C212V

[0204] TIFF2026515663000015.tif12166

[0205] 3. Amino acid sequence of the WT LC kappa constant (Cκ) region (SEQ ID NO: 3) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0206] 4. Amino acid sequence of IgG1 CH1 (SEQ ID NO: 4) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

[0207] 5. Amino acid sequence of "V12" CH1 modified to form modified disulfide bridges (SEQ ID NO: 5) The following substitutions are underlined. Modified disulfide: F126C, C220V

[0208] TIFF2026515663000016.tif12166

[0209] 6. Amino acid sequence of IgG1 CH2 (SEQ ID NO: 6) LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0210] 7. Amino acid sequence of IgG1 CH3 (SEQ ID NO: 7) GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0211] 8. Amino acid sequence of IgG1 heavy chain polypeptide (SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0212] 9. Amino acid sequence of IgG1 CH3 modified to include the "whole" mutation (SEQ ID NO: 9) The following substitutions are underlined. "Hole" mutations (T366S, L368A, and Y407V).

[0213] TIFF2026515663000017.tif11167

[0214] 10. Amino acid sequence of IgG1 CH3 modified to include stabilized cysteine ​​and "hole" mutation (SEQ ID NO: 10) The following substitutions are underlined. To stabilize "hole" mutations (T366S, L368A, and Y407V) and cysteine ​​mutations (Y349C).

[0215] TIFF2026515663000018.tif11165

[0216] 11. Amino acid sequence of IgG1 CH3 modified to include the "knob" mutation (SEQ ID NO: 11) The following substitutions are underlined. "Nobu" mutation (T366W). TIFF2026515663000019.tif11165

[0217] 12. Amino acid sequence of IgG1 CH3 modified to include stabilized cysteine ​​and "knob" mutation (SEQ ID NO: 12) The following substitutions are underlined. To stabilize the "knob" mutation (T366W) and the cysteine ​​mutation (S354C).

[0218] TIFF2026515663000020.tif11165

[0219] 13. Amino acid sequence of IgG1 heavy-chain polypeptide modified to include stabilized cysteine ​​and "hole" mutation (SEQ ID NO: 13) The following substitutions are underlined. To stabilize "hole" mutations (T366S, L368A, and Y407V) and cysteine ​​mutations (Y349C).

[0220] TIFF2026515663000021.tif30167

[0221] 14. Amino acid sequence of IgG1 heavy chain polypeptide modified to include stabilized cysteine ​​and "knob" mutation (SEQ ID NO: 14) The following substitutions are underlined. To stabilize the "knob" mutation (T366W) and the cysteine ​​mutation (S354C).

[0222] TIFF2026515663000022.tif29167

[0223] 15. Amino acid sequence of the "V12" IgG1 heavy chain polypeptide modified to include stabilizing cysteine, interchain cysteine ​​mutations, and "knob" mutations (SEQ ID NO: 15) The following substitutions are underlined. To stabilize the "knob" mutation (T366W), interchain cysteine ​​mutations (F126C and C220V), and cysteine ​​mutation (S354C).

[0224] TIFF2026515663000023.tif29166

Claims

1. A multispecific antibody, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to a first heavy chain constant region 1 (CH1), wherein the first light chain comprises a constant light chain lambda region (CLλ), and (b) comprising a second antigen-binding arm which includes a second light chain disulfide-bonded to a second CH1, The first antigen-binding arm is positioned in the following pairs: (i) Position 117 of CLλ and position 141 of the first CH1, (ii) Position 117 of CLλ and position 185 of the first CH1, (iii) Position 119 of CLλ and position 128 of the first CH1, (iv) Position 134 of CLλ and position 128 of the first CH1, (v) Position 134 of CLλ and position 145 of the first CH1, (vi) Position 134 of CLλ and position 183 of the first CH1, (vii) Position 136 of CLλ and position 185 of the first CH1, (viiii) Position 178 of CLλ and position 173 of the first CH1, (ix) Includes a lambda charge pair located at one or more positions of the CLλ (position 117) and the first CH1 (position 187), The lambda charge pair comprises a positively charged amino acid residue optionally selected from arginine, lysine, and histidine located at one position of the lambda charge pair, and a negatively charged amino acid residue optionally selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the lambda charge pair, and A multispecific antibody whose numbering follows the EU index.

2. The multispecific antibody according to claim 1, wherein the lambda charge pair is located at position 117 of CLλ and position 141 of the first CH1.

3. The lambda charge pairs are listed below: a. Arginine at position 117 of the CLλ, and aspartic acid at position 141 of the first CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 141 of the first CH1, c. Arginine at position 117 of CLλ, and serine at position 141 of the first CH1, d. Arginine at position 117 of CLλ, and threonine at position 141 of the first CH1, e. Lysine at position 117 of the Clλ, and aspartic acid at position 141 of the first CH1, f. Lysine at position 117 of CLλ, and glutamic acid at position 141 of the first CH1, g. Lysine at position 117 of the CLλ, and serine at position 141 of the first CH1, and h. The multispecific antibody according to claim 2, selected from lysine at position 117 of CLλ and threonine at position 141 of the first CH1.

4. The lambda charge pairs are listed below: a. Arginine at position 117 of the CLλ, and aspartic acid at position 141 of the first CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 141 of the first CH1, c. Arginine at position 117 of CLλ, and serine at position 141 of the first CH1, d. Arginine at position 117 of CLλ, and threonine at position 141 of the first CH1, and e. A multispecific antibody according to claim 2 or 3, selected from lysine at position 117 of CLλ and aspartic acid at position 141 of the first CH1.

5. The lambda charge pair is located at position 117 of CLλ and position 185 of the first CH1, and optionally the lambda charge pair is from the following list: a. Arginine at position 117 of the CLλ, and aspartic acid at position 185 of the first CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 185 of the first CH1, c. Arginine at position 117 of CLλ, and serine at position 185 of the first CH1, d. Arginine at position 117 of CLλ, and threonine at position 185 of the first CH1, e. Lysine at position 117 of the Clλ, and aspartic acid at position 185 of the first CH1, f. Lysine at position 117 of the CLλ, and glutamic acid at position 185 of the first CH1, g. Lysine at position 117 of the CLλ, and serine at position 185 of the first CH1, h. The multispecific antibody according to claim 1, selected from lysine at position 117 of CLλ and threonine at position 185 of the first CH1.

6. The lambda charge pair is located at position 119 of CLλ and position 128 of the first CH1, and optionally the lambda charge pair is from the following list: a. Arginine at position 119 of the CLλ, and aspartic acid at position 128 of the first CH1, b. Arginine at position 119 of CLλ, and glutamic acid at position 128 of the first CH1, c. Arginine at position 119 of CLλ, and serine at position 128 of the first CH1, d. Arginine at position 119 of CLλ, and threonine at position 128 of the first CH1, e. Lysine at position 119 of the Clλ, and aspartic acid at position 128 of the first CH1, f. Lysine at position 119 of the CLλ, and glutamic acid at position 128 of the first CH1, g. Lysine at position 119 of the CLλ, and serine at position 128 of the first CH1, and h. The multispecific antibody according to claim 1, selected from lysine at position 119 of CLλ and threonine at position 128 of the first CH1.

7. The lambda charge pair is located at position 134 of CLλ and position 128 of the first CH1, and optionally the lambda charge pair is from the following list: a. Arginine at position 134 of the CLλ, and aspartic acid at position 128 of the first CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 128 of the first CH1, c. Arginine at position 134 of CLλ, and serine at position 128 of the first CH1, d. Arginine at position 134 of CLλ, and threonine at position 128 of the first CH1, e. Lysine at position 134 of the Clλ, and aspartic acid at position 128 of the first CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 128 of the first CH1, g. Lysine at position 134 of the CLλ, and serine at position 128 of the first CH1, and h. A multispecific antibody according to claim 1, selected from lysine at position 134 of CLλ and threonine at position 128 of the first CH1.

8. The lambda charge pair is located at position 134 of CLλ and position 145 of the first CH1, and optionally the lambda charge pair is from the following list: a. Arginine at position 134 of the CLλ, and aspartic acid at position 145 of the first CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 145 of the first CH1, c. Arginine at position 134 of CLλ, and serine at position 145 of the first CH1, d. Arginine at position 134 of CLλ, and threonine at position 145 of the first CH1, e. Lysine at position 134 of the Clλ, and aspartic acid at position 145 of the first CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 145 of the first CH1, g. Lysine at position 134 of the CLλ, and serine at position 145 of the first CH1, and h. A multispecific antibody according to claim 1, selected from lysine at position 134 of CLλ and threonine at position 145 of the first CH1.

9. The multispecific antibody according to claim 1, wherein the lambda charge pair is located at position 134 of CLλ and position 183 of the first CH1, and optionally the lambda charge pair is lysine at position 134 of CLλ and aspartic acid or serine at position 183 of the first CH1.

10. The lambda charge pair is located at position 136 of CLλ and position 185 of the first CH1, and optionally the lambda charge pair is from the following list: a. Arginine at position 136 of the CLλ, and aspartic acid at position 185 of the first CH1, b. Arginine at position 136 of CLλ, and glutamic acid at position 185 of the first CH1, c. Arginine at position 136 of CLλ, and serine at position 185 of the first CH1, d. Arginine at position 136 of CLλ, and threonine at position 185 of the first CH1, e. Lysine at position 136 of the Clλ, and aspartic acid at position 185 of the first CH1, f. Lysine at position 136 of the CLλ, and glutamic acid at position 185 of the first CH1, g. Lysine at position 136 of the CLλ, and serine at position 185 of the first CH1, h. The multispecific antibody according to claim 1, selected from lysine at position 136 of CLλ and threonine at position 185 of the first CH1.

11. The lambda charge pair is located at position 178 of CLλ and position 173 of the first CH1, and optionally the lambda charge pair is from the following list: a. Arginine at position 178 of the CLλ, and aspartic acid at position 173 of the first CH1, b. Arginine at position 178 of CLλ, and glutamic acid at position 173 of the first CH1, c. Arginine at position 178 of CLλ, and serine at position 173 of the first CH1, d. Arginine at position 178 of CLλ, and threonine at position 173 of the first CH1, e. Lysine at position 178 of the Clλ, and aspartic acid at position 173 of the first CH1, f. Lysine at position 178 of CLλ, and glutamic acid at position 173 of the first CH1, g. Lysine at position 178 of the Clλ, and serine at position 173 of the first CH1, and h. The multispecific antibody according to claim 1, selected from lysine at position 178 of CLλ and threonine at position 173 of the first CH1.

12. (i) The disulfide bond between the first light chain and the first CH1 is formed between the first light chain and a pair of modified cysteines of the first CH1, and the disulfide bond between the second light chain and the second CH1 is formed between a pair of natural cysteines, or (ii) The multispecific antibody according to any one of claims 1 to 11, wherein the disulfide bond between the second light chain and the second CH1 is formed between the second light chain and a pair of modified cysteines of the second CH1, and the disulfide bond between the first light chain and the first CH1 is formed between a pair of native cysteines.

13. The multispecific antibody according to claim 12, wherein the pair of modified cysteines of the first light chain and the first CH1 are located at position 122 of the first light chain and position 126 of the first CH1, the first light chain contains a non-cysteine ​​residue at position 212, and the first CH1 contains a non-cysteine ​​residue at position 220, and optionally the non-cysteine ​​residue is valine.

14. The multispecific antibody according to any one of claims 1 to 13, wherein the CLλ of the first light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1 or SEQ ID NO:

2.

15. The multispecific antibody according to any one of claims 1 to 14, wherein the first CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4 or SEQ ID NO:

5.

16. The multispecific antibody according to any one of claims 1 to 15, wherein the second light chain comprises a constant light chain kappa region (CLκ).

17. The multispecific antibody according to claim 16, wherein the second antigen-binding arm comprises a kappa charge pair located at the CLκ and the second CH1 of the second light chain, and the kappa charge pair of the second antigen-binding arm comprises a positively charged amino acid residue selected from arginine, lysine, and histidine located at one position of the kappa charge pair of the second antigen-binding arm, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, and threonine located at the other position of the kappa charge pair of the second antigen-binding arm.

18. The charged amino acid residue in the kappa charge pair of the second antigen-binding arm is located at position 133 of CLκ, and the positively charged amino acid residue in the kappa charge pair is located at position 183 of the second CH1, The multispecific antibody according to claim 17, wherein the charged amino acid residue at position 133 of CLκ is glutamic acid, and the positively charged amino acid residue at position 183 of the second CH1 is lysine.

19. The multispecific antibody according to claim 17 or 18, wherein the CLκ of the second light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:

3.

20. The multispecific antibody according to any one of claims 1 to 19, wherein the second CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO: 1 or SEQ ID NO:

2.

21. A multispecific antibody, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to a first heavy chain constant region 1 (CH1), wherein the first light chain comprises a constant light chain lambda region (CLλ), (i) The first antigen-binding arm includes a lambda charge pair located at position 117 of CLλ and position 141 of the first CH1, wherein the lambda charge pair includes a positively charged amino acid residue selected from arginine, lysine, and histidine located at one position of the lambda charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, and threonine located at the other position of the lambda charge pair, and (ii) The disulfide bond between the first light chain and the first CH1 forms a first antigen-binding arm between the pair of modified cysteines of CLλ and the first CH1, and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to CH1, wherein the second light chain comprises a constant light chain kappa region (CLκ), (i) The second antigen-binding arm comprises a kappa charge pair located at CLκ and the second CH1, wherein the kappa charge pair comprises a positively charged amino acid residue selected from arginine, lysine, and histidine located at one position of the kappa charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, and threonine located at the other position of the kappa charge pair, and (ii) The disulfide bond between the second light chain and the second CH1 comprises a second antigen-binding arm formed between the CLκ and a pair of native cysteines of the second CH1, A multispecific antibody in which the numbering follows the EU index.

22. The multispecific antibody according to any one of claims 1 to 21, wherein the first antigen-binding arm further comprises a first Fc region, and the second antigen-binding arm further comprises a second Fc region.

23. The multispecific antibody according to claim 22, comprising modifications in the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region.

24. The multispecific antibody according to claim 23, wherein the modification is located at CH3 in the Fc region.

25. The multispecific antibody according to claim 24, wherein the modification of the CH3 in one of the first Fc region and the second Fc region is a substitution of an amino acid residue with a larger side chain, thereby generating a bump (knob) on the surface of the CH3 domain, and the modification of the CH3 in the other Fc region is a substitution of an amino acid residue with a smaller side chain, thereby generating a cavity (hole) on the surface of the CH3 domain, and optionally, the CH3 domain containing the bump (knob) is part of the first heavy chain polypeptide, and the CH3 domain containing the cavity (hole) is part of the second heavy chain.

26. The substitution for generating the knob is the substitution of tryptophan at position 366, and the substitution for generating the hole is the following: i) Substitution with valine at position 407, ii) Substitution with serine at position 366, and The multispecific antibody according to claim 25, wherein one or more substitutions are: iii) substitution to alanine at position 368.

27. The multispecific antibody according to claim 25 or 26, wherein the CH3 domain containing the knob contains cysteine ​​at position 354, and the CH3 domain containing the hole contains cysteine ​​at position 349.

28. At least one of the Fc regions is subjected to the following amino acid substitutions: (a) L234F / L235E / P331S, (b) E233P / L234V / L235A / G236del / S267K, and / or (c) A multispecific antibody according to any one of claims 22 to 27, comprising M252Y / S254T / T256E.

29. A method for producing a multispecific antibody according to any one of claims 1 to 28, a) Expressing the first light chain and the second light chain, and the first CH1 and the second CH1 in host cells, b) Pairing the first light chain with the first CH1 to form the first binding arm, pairing the second light chain with the second CH1 to form the second binding arm, and pairing the first binding arm with the second binding arm to form the multispecific antibody, c) A method comprising purifying the multispecific antibody from the host cells.

30. The method according to claim 29, wherein the purification of the multispecific antibody comprises affinity chromatography, and optionally the further purification of the antibody comprises light chain affinity chromatography.

31. The method according to claim 29 or 30, wherein less than 25%, less than 20%, less than 15%, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the light chain in the multispecific antibody is mispaired.

32. One or more nucleic acids encoding the first light chain and / or the first CH1 of the multispecific antibody according to any one of claims 1 to 28, Optionally, one or more nucleic acids further encode one or more of the following: the second light chain and the second CH1.

33. A vector comprising the nucleic acid described in claim 32.

34. An isolated host cell comprising the nucleic acid described in claim 32 or the vector described in claim 33.

35. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 28 and a pharmaceutically acceptable carrier.

36. A method for treating a disease in a patient requiring treatment for the disease, comprising administering to the patient an effective amount of a multispecific antibody according to any one of claims 1 to 28, or a pharmaceutical composition according to claim 35.

37. The method according to claim 36, wherein the disease is cancer.

38. A multispecific antibody according to any one of claims 1 to 28, or a pharmaceutical composition according to claim 35, for use as a pharmaceutical.

39. A multispecific antibody according to any one of claims 1 to 28, or a pharmaceutical composition according to claim 35, for use in the treatment of the aforementioned cancer.

40. Use of a multispecific antibody according to any one of claims 1 to 28, or a pharmaceutical composition according to claim 35, for the manufacture of a pharmaceutical for the treatment of the aforementioned cancer.

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