Dual-specificity modified antibodies

Abstract

A multispecific antibody is provided herein, comprising two antigen-binding arms capable of binding to a first target and at least one antigen-binding arm capable of binding to a second target. The multispecific antibody includes a lambda charge pair introduced at the interface between the heavy and light chains to reduce mispairing of the chains.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 494,905, filed on April 7, 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-101-WO Sequence Listing.xml, size: 18,719 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 a multispecific antibody comprising two antigen-binding arms capable of binding to a first target and one antigen-binding arm capable of binding to a second target, wherein the multispecific antibody includes a lambda charge pair introduced at the interface between the heavy and light chains 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] Bispecific antibodies that recognize two or different 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 several different molecules, with only one being bispecific and the remaining pairings resulting in non-functional or monospecific molecules.

[0005] Various strategies have been developed to overcome this problem and promote the correct assembly of the desired bispecificity. Such strategies to promote heterodimerization of two different heavy chains include techniques such as "knobs-into-hole" (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 bivalent bispecific format is "DuetMab," described in PCT International Publication Patent WO2013 / 096291. DuetMab antibodies use knob-into-hole technology for heterodimerization of two different heavy chains, increasing the effectiveness of congeneral heavy-light chain pairing by replacing the innate disulfide bond at one of the CH1-CL interfaces with a modified disulfide bond. An example of an asymmetric trivalent bispecific format is the "2+1" format, in which the bispecific antibody contains three antigen-binding arms, two of which bind to the same epitope and the third binds to a different epitope.

[0007] While the strategies described above have provided some methods to reduce mispairing of chains, additional mechanisms are still needed to improve polypeptide chain pairing in bispecific antibodies and facilitate their efficient production. [Overview of the Initiative]

[0008] In some embodiments, the disclosure relates to an improved pairing of chains in a bispecific antibody in a “2+1” format, the bispecific antibody comprising a first antigen-binding arm capable of binding to a first epitope, and two antigen-binding arms (a second antigen-binding arm and a third antigen-binding arm) capable of binding to a second epitope distinct from the first epitope. In this “2+1” format, the third antigen-binding arm is fused to either the first or second antigen-binding arm (for example, via a peptide linker between domains in each heavy chain domain).

[0009] Introducing charge pairs at the interface between lambda LC and lambda HC can improve the pairing of heavy chains (HC) and light chains (LC). However, charge pairs designed for κLC and CH1 interfaces were less likely to succeed when applied to λLC and CH1 interfaces. Amino acid residues at the interface between lambda LC and HC into which charge pairs can be introduced were identified, and it was demonstrated that the introduction of these lambda charge pairs can favorably improve chain pairing beyond what was achieved in previous DuetMab bispecificity formats.

[0010] It has been further established that the identified lambda charge pair can be used to improve pairing in the "2+1" format of bispecificity. As demonstrated herein, 2+1 bispecific antibodies containing the lambda charge pair were well expressed, showed high levels (>90%) of correct chain pairing, and possessed good molecular stability. Furthermore, 2+1 bispecific antibodies containing the lambda charge pair and an antigen-binding arm that monovalently targets CD3 on T cells and bivalently targets another (non-T cell) target were demonstrated to induce stronger activity in T cell cytotoxicity assays than that achieved by bispecific antibodies that monovalently bind both of its targets. Thus, in some embodiments, only a single antigen-binding domain binds to CD3 in a 2+1 bispecific antibody, which is referred to as a bispecific T cell engager DuetMab ("TED2").

[0011] Therefore, in one embodiment, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to the first heavy chain constant region 1 (CH1), and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, (c) comprising a third antigen-binding arm, which includes a third light chain disulfide-bonded to a third CH1, The first antigen-binding arm binds to the first epitope, and the second and third antigen-binding arms bind to the second epitope. The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm. The first antigen-binding arm, or the second and third antigen-binding arms, are located in the constant light chain lambda region (CLλ) and CH1 at the following positions: (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) Including one or more lambda charge pairs located at position 117 of CLλ and position 187 of CH1, The lambda charge pair comprises a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the lambda 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 lambda charge pair, and Bispecific antibodies, numbered according to the EU index, are provided herein.

[0012] In some embodiments, the lambda charge pair is located at position 117 of CLλ and position 141 of 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 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, e. Lysine at position 117 of CLλ and aspartic acid at position 141 of CH1, f. Lysine at position 117 of CLλ and glutamic acid at position 141 of CH1, g. Lysine at position 117 of CLλ and serine at position 141 of CH1, and h. Lysine at position 117 of CLλ and threonine at position 141 of CH1.

[0013] 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 CH1.

[0014] In some embodiments, the lambda charge pair is located at position 117 of CLλ and position 185 of 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 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, e. Lysine at position 117 of CLλ and aspartic acid at position 185 of CH1, f. Lysine at position 117 of CLλ and glutamic acid at position 185 of CH1, g. Lysine at position 117 of CLλ, serine at position 185 of CH1, and h. Lysine at position 117 of CLλ and threonine at position 185 of CH1.

[0015] In some embodiments, the lambda charge pair is located at position 119 in CLλ and position 128 in CH1. In some embodiments, the lambda charge pair is 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, e. Lysine at position 119 of CLλ and aspartic acid at position 128 of CH1, f. Lysine at position 119 of CLλ and glutamic acid at position 128 of CH1, g. Lysine at position 119 of CLλ, serine at position 128 of CH1, and h. Lysine at position 119 of CLλ and threonine at position 128 of CH1.

[0016] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 128 of 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 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.

[0017] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 145 of 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 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.

[0018] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 183 of CH1. In some embodiments, the lambda charge pair is lysine at position 134 of CLλ and aspartic acid or serine at position 183 of CH1.

[0019] In some embodiments, the lambda charge pair is located at position 136 of CLλ and position 185 of 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 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.

[0020] In some embodiments, the lambda charge pair is located at position 178 of CLλ and position 173 of 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 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.

[0021] In some embodiments, the first antigen-binding arm includes a lambda charge pair, while the second and third antigen-binding arms do not include the same lambda charge pair. In other embodiments, both the second and third antigen-binding arms include the same lambda charge pair, while the first antigen-binding arm does not include the same lambda charge pair.

[0022] As further described herein, charge pairing can be combined with other approaches to promote light chain pairing, for example, to further increase the correct assembly of the desired trispecific antibody.

[0023] 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. (i) The disulfide bond between the first light chain and the first CH1 is formed between the modified pair of cysteines of the first light chain and the first CH1, and both the disulfide bonds between the second light chain and the second CH1, and between the third light chain and the third CH1 are formed between the pair of native cysteines, or (ii) Both disulfide bonds between the second light chain and the second CH1, and between the third light chain and the third CH1, are formed between the second and third light chains and between the second and third CH1, and the disulfide bond between the first light chain and the first CH1 is formed between the pair of native cysteines.

[0024] 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.

[0025] In some embodiments, CLλ 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.

[0026] In some embodiments, 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.

[0027] In some embodiments, the light chain(s) in the antigen-binding arm(s) lacking a lambda charge pair include 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 a second CH1:CLκ polypeptide.

[0028] In some embodiments, an antigen-binding arm comprising a light chain having CLκ comprises a kappa charge pair located at CLκ and CH1 of the antigen-binding arm, the kappa charge pair comprising a positively charged amino acid residue selected from arginine, lysine, or histidine located at one of the positions of the kappa charge pair, and an uncharged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the kappa charge pair.

[0029] In some embodiments, the load amino acid residue in the kappa charge pair 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 corresponding CH1 in its antigen-binding arm. In some embodiments, the load amino acid residue at position 133 of CLκ is glutamic acid, and the positively charged amino acid residue at position 183 of the corresponding CH1 in its antigen-binding arm is lysine.

[0030] In some embodiments, CLκ 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.

[0031] In some embodiments, the bispecific antibody is provided in a "2+2" format, comprising a fourth antigen-binding arm containing a fourth light chain disulfide-bonded to a fourth CH1. The fourth antigen-binding arm binds to the same epitope as the first antigen-binding arm; therefore, the first and fourth antigen-binding arms bind to the first epitope, and the second and third antigen-binding arms bind to the second epitope. Thus, in the 2+2 format, the bispecific antibody comprises two binding arms that bind to one epitope and two binding arms that bind to different epitopes. Similar to the first antigen-binding arm, the fourth antigen-binding arm contains a lambda charge pair; i.e., both the first and fourth antigen-binding arms contain a lambda charge pair.

[0032] In some embodiments, the second and third light chains are identical (i.e., they have the same amino acid sequence).

[0033] 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).

[0034] 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.

[0035] 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.

[0036] In some embodiments, if the substitution for generating a knob is a substitution for tryptophan at position 366, and the substitution for generating a hole is a substitution for generating a hole, then one or more of the following are possible: i) Substitution of valine at position 407, ii) Substitution with serine at position 366, and iii) Substitution with alanine at position 368.

[0037] 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.

[0038] 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.

[0039] In some embodiments, the first antigen-binding arm binds to an epitope on CD3.

[0040] In some embodiments, the multispecific antibody further includes an additional antigen-binding domain, optionally being VHH. Including an additional antigen-binding domain in addition to the "2+1" bispecificity enables the production of a triplicate antibody. Furthermore, the use of VHH as one of the antigen-binding domains may be advantageous from a manufacturing standpoint compared to including an additional antigen-binding arm containing further VH, VL, and CH1, as it reduces the number of heavy and light chains present during the production of the triplicate antibody.

[0041] In some embodiments, an additional antigen-binding domain (e.g., VHH) can bind to an epitope on CD8. In some embodiments, the first antigen-binding arm can bind to CD3, and the additional antigen-binding domain (e.g., VHH) can bind to an epitope on CD8. While we do not wish to be constrained by theory, including an antigen-binding domain (e.g., VHH) that can bind to CD8 in a multispecific antibody is possible. + This is thought to enable preferential activation of T cells, which may improve therapeutic efficacy.

[0042] Therefore, this format may be referred to as the quadruple-specific T-cell engager DuetMab ("TED4"), and in some embodiments, comprises two antigen-binding domains that bind to the same target. In some embodiments, the TED4 format comprises two antigen-binding domains that bind to the same or different antigen targets, a CD3-binding domain, and a CD8-binding domain.

[0043] In some embodiments, one of the antigen-binding arms of an antigen1 / antigen2 / CD3 TriMab can bind to an epitope on antigen1 or antigen2 that does not induce cytotoxicity. In some embodiments, the antigen1-binding arm of an antigen1 / antigen2 / CD3 TriMab induces cytotoxicity, while the antigen2-binding arm acts as an anchoring arm that does not have the ability to induce cytotoxicity in cells expressing only antigen2. In some embodiments, the antigen2-binding arm of an antigen1 / antigen2 / CD3 TriMab induces cytotoxicity, while the antigen1-binding arm acts as an anchoring arm that does not have the ability to induce cytotoxicity in cells expressing only antigen1. The inability of the anchoring arm to induce cytotoxicity may be due, for example, to binding to a distal membrane epitope that interferes with the ability to form an active immunological synapse.

[0044] Methods for producing multispecific antibodies described herein are also provided herein. In some embodiments, the method is a) Expressing the first light chain, the second light chain, and the third light chain, as well as the first CH1, the second CH1, and the third 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, pairing the third light chain with the third CH1, 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

[0045] 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, 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 the host cell.

[0046] In some embodiments, the purification of multispecific antibodies includes affinity chromatography. In some embodiments, the purification of multispecific antibodies includes light chain affinity chromatography.

[0047] 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.

[0048] One or more nucleic acids encoding the multispecific antibodies described herein are also provided herein. Isolated host cells containing nucleic acids or vectors are also provided herein.

[0049] This specification also provides pharmaceutical compositions and therapeutic methods comprising pharmaceutical compositions or multispecific antibodies, as further described below.

[0050] This disclosure includes the described aspects and combinations of features unless such combinations are clearly impossible or expressly avoided. [Brief explanation of the drawing]

[0051] 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 triplicate 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. [Figure 12]The diagram includes a schematic representation of the DuetMab "2+1" antibody containing charge pairs. The "knob" HC on the right is disulfide-bonded to the lambda LC via a 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. The CH1 and VH regions of this knob HC and lambda LC form the "first antigen binding arm". The "hole" HC on the left is disulfide-bonded to the kappa LC via a 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 CH1 and VH regions of this "hole" HC and kappa LC form the "second antigen binding arm". The third antigen-binding arm is fused by a peptide linker from the N-terminus of its CH1 to the C-terminus of the "knob" HC. The CH1 of the third antigen-binding arm is disulfide-bonded to kappa LC via native cysteine ​​and contains a kappa charge pair. As indicated by the different shadings, the first antigen-binding arm binds to a first epitope (e.g., CD3), while the second and third binding arms bind to a second different epitope. [Figure 13] Representative images are provided showing the kinetics of antigen 1 in its soluble monomer form (top image) and CD3 epsilon / delta heterodimer form (bottom image) obtained using the Ocet384 instrument. [Figure 14] UV chromatograms of subunit LC / MS analysis of the antigen 1 / CD3 2+1 bispecific antibody (upper chromatogram) and the NIP228 / CD3 2+1 bispecific antibody (lower chromatogram) are provided. No subunits corresponding to mispaired species were identified. [Figure 15] This provides representative DSC thermal stability measurement plots for antigen 1 / CD3 2+1 bispecific antibody (solid line) and NIP228 / CD3 2+1 bispecific antibody (dotted line). [Figure 16]This report provides data on cell death assays and T cell activation (CD8 and CD4) for DuetMabs and Duet 2(2+1) bispecific antibodies. Each cytotoxic point represents the mean of a triplicate well, and the mean ± standard error (SEM) is represented by error bars. [Figure 17] This includes a schematic diagram of a DuetMab "2+2" antibody containing charge pairs. The antibody corresponds to the antibody in Figure 12, which has an additional antigen-binding arm. Antigen-binding arms of matching colors bind to the same epitope. A shows a symmetrical configuration of the "2+2" format, with an antigen-binding arm for a first epitope located at the N-terminus of the heavy chain and an antigen-binding arm for a second epitope immediately to its C-terminus. B shows an asymmetrical configuration of the "2+2" format, where one heavy chain contains an antigen-binding arm for a first epitope at its N-terminus and the other contains an antigen-binding arm for a second epitope at its N-terminus. [Modes for carrying out the invention]

[0052] 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.

[0053] This specification provides multispecific antibodies in a "2+1" format (e.g., bispecific antibodies) that include a constant light chain lambda region (CLλ) in one or more binding arms of the multispecific antibody and a lambda charge pair located at a specific position in the heavy chain constant region 1 (CH1). Multispecific antibodies that include an additional antigen-binding domain (e.g., VHH) in addition to the "2+1" bispecific antigen-binding arms are also provided herein.

[0054] Methods for generating multispecific antibodies are well known. However, such methods are often limited by a multitude of possible antibody formations, which may include 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 preferred multispecific antibody assemblies. In particular, these lambda charge variants, when 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, can further improve the formation of preferred multispecific antibodies and reduce the formation of mispaired variants.

[0055] 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.

[0056] 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.

[0057] 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 a constant light chain lambda region (CLλ) amino acid sequence is provided as SEQ ID NO: 1, and an example of a constant light chain kappa region (CLκ) amino acid sequence 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.

[0058] 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.

[0059] 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).

[0060] The light chain associates with VH and CH1 in the heavy chain to form an "antigen binding arm," and the variable domains within the antigen binding arm interact to form an "antigen binding domain."

[0061] The “antigen-binding domain” refers to a portion of a molecule that binds to all or part of a target epitope, and generally includes six complementarity-determining regions (CDRs), three of which are in the VH region: HCDR1, HCDR2, and HCDR3, and three of which are in the VL region: LCDR1, LCDR2, and LCDR3. Together, the six CDRs define a paratope of the antigen-binding domain, which is a portion of the antigen-binding domain that binds to a target epitope. As used herein, the term “epitope” refers to the portion of the antigen to which the antigen-binding domain binds. A monoclonal monospecific IgG antibody molecule contains two antigen-binding domains, each of which can bind to the same epitope (i.e., it is bivalent for a single epitope). As used herein, the term “valent” refers to the presence of a specific number of antigen-binding domains in the antibody that binds to the epitope.

[0062] 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.

[0063] Bispecific “2+1” antibodies This disclosure provides multispecific (e.g., bispecific) antibodies in a "2+1" format. The multispecific antibodies according to this disclosure may be provided in an isolated form, meaning they do not contain contaminants such as antibodies that can bind to other polypeptides and / or serum components.

[0064] The multispecific antibodies of this disclosure can bind to two different epitopes on the same antigen or on different antigens, each comprising three antigen-binding arms referred to herein as the “first antigen-binding arm,” the “second antigen-binding arm,” and the “third antigen-binding arm.” According to this disclosure, the “antigen-binding arm” comprises 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. References herein to the first domain, the second domain, and the third domain (CH1, VH, VL, etc.) refer to the described domains of the first antigen-binding arm, the second antigen-binding arm, and the third antigen-binding arm, respectively.

[0065] 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.

[0066] In the "2+1" format of this multispecific antibody, the first antigen-binding arm is monovalently bound to the first epitope, while the second and third antigen-binding arms are divalently bound to the second epitope, which is distinct from the first epitope. The third antigen-binding arm is typically fused to either the first or second antigen-binding arm via a peptide linker between heavy chain domains. Suitable peptide linkers are well known in the art and may consist of 5-100 amino acids, 5-50 amino acids, 5-25 amino acids, or 5-15 amino acids. The peptide linker is mainly formed from glycine and serine amino acid residues and may contain the amino acid sequence GGGGS (SEQ ID NO: 16) or SGGGGS (SEQ ID NO: 17). In one embodiment, the peptide linker contains or consists of (GGGGS)2 (SEQ ID NO: 18).

[0067] The third antigen-binding arm is fused to one of the two heavy chains present in the multispecific antibody. In some embodiments, the third antigen-binding arm is fused at its N-terminus of the CH1 domain to the C-terminus of the VL domain of either the first or second antigen-binding arm. Thus, in one embodiment, the N-terminus of the CH1 domain of the third antigen-binding arm is fused to the C-terminus of the VL domain of the first antigen-binding arm. A schematic diagram of this exemplary embodiment is shown in Figure 12. In another embodiment, the N-terminus of the CH1 domain of the third antigen-binding arm is fused to the C-terminus of the VL domain of the second antigen-binding arm.

[0068] The first antigen-binding arm and the second 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 first antigen-binding arm and the second antigen-binding arm further include an Fc region (i.e., the remaining portion of the heavy chain including the hinge, CH2, and CH3). In some embodiments, the first antigen-binding arm and the second antigen-binding arm include a complete heavy chain (i.e., VH, CH1, hinge, CH2, and CH3). In some embodiments, the heavy chain of the first antigen-binding arm is disulfide-bonded to the heavy chain of the second antigen-binding arm (e.g., via interchain disulfide bonds between cysteine ​​present in the Fc domain, or the ability to form such bonds).

[0069] The first antigen-binding arm differs from the second and third binding arms in at least one of the CDR and CH1 amino acid sequences in the VL of the light chain, reflecting the fact that the first antigen-binding arm binds to a different epitope than those bound by the second and third antigen-binding arms.

[0070] In some embodiments, the second antigen-binding arm and the third antigen-binding arm have the same CDR sequence. In other embodiments, they have the same VH and VL sequences. In some embodiments, the second light chain and the third light chain are the same (i.e., they have the same amino acid sequence).

[0071] Bispecific 2+2 antibody Multiple specific (e.g., bispecific) antibodies in the "2+2" format, a variation of the "2+1" format, are also provided.

[0072] Essentially, the "2+2" format multispecific antibody includes an additional fourth antigen-binding arm in addition to the features of the "2+1" format described above. Similar to the first to third antigen-binding arms described above, the fourth antigen-binding arm includes a fourth light chain disulfide-bonded to a fourth CH1. The fourth antigen-binding arm binds to the same epitope as the first antigen-binding arm (referred to herein as the "first epitope"). Thus, while the "2+1" format antibody binds monovalently to the first epitope and divalently to the second epitope, the "2+2" format antibody binds divalently to each epitope.

[0073] Similar to "2+1" format antibodies, antigen-binding arms that bind to the same epitope may be located on the same heavy chain of the multispecific antibody or on different heavy chains. Typically, the four antigen-binding arms are arranged such that each heavy chain of the antibody includes one antigen-binding arm that binds to a first epitope and one antigen-binding arm that binds to a second epitope. In these embodiments, a third antigen-binding arm is fused to the first antigen-binding arm, and a fourth antigen-binding arm is fused to the second antigen-binding arm.

[0074] Alternatively, the antigen-binding arms may be arranged such that each heavy chain of the antibody specifically binds to one epitope, i.e., each heavy chain of the antibody binds to the same epitope, by including two antigen-binding arms. Thus, in some embodiments, a third antigen-binding arm is fused to a second antigen-binding arm, and a fourth antigen-binding arm is fused to a first antigen-binding arm.

[0075] When the 2+2 format is arranged such that each heavy chain contains one antigen-binding arm bound to a first epitope and one antigen-binding arm bound to a second epitope, the antibody may have a symmetric or asymmetric configuration. The reference to symmetry / asymmetry refers to the arrangement of antigen-binding arms within the two heavy chains of the antibody. Schematic diagrams of the symmetric and asymmetric configurations are shown in Figures 17A and 17B, respectively.

[0076] In a symmetrical configuration, both antigen-binding arms that recognize the first epitope are located either at the N-terminus or C-terminus of the antigen-binding arm that recognizes the second epitope. Thus, in some embodiments, the 2+2 format is symmetrically configured, with the third antigen-binding arm fused to the N-terminus of the first antigen-binding arm and the second antigen-binding arm fused to the N-terminus of the fourth antigen-binding arm. In other embodiments, the 2+2 format is symmetrically configured, with the third antigen-binding arm fused to the C-terminus of the first antigen-binding arm and the second antigen-binding arm fused to the C-terminus of the fourth antigen-binding arm.

[0077] In an asymmetric configuration, one antigen-binding arm that binds to the first epitope is located on the N-terminal side of the antigen-binding arm that binds to the second epitope, and the other antigen-binding arm that binds to the first epitope is located on the C-terminal side of the antigen-binding arm that binds to the second epitope. Thus, in some embodiments, the 2+2 format is asymmetrically configured such that a third antigen-binding arm is fused to the N-terminus of the first antigen-binding arm and a second antigen-binding arm is fused to the C-terminus of a fourth antigen-binding arm (or vice versa).

[0078] As described above, the first and second antigen-binding arms may further include additional heavy chain regions, such as Fc regions. From the above consideration of possible arrangements of the "2+2" antibody format, it will be understood that, in this context, if additional heavy chain regions are present, the additional binding arms may be located between the first and / or second antigen-binding arms and their respective additional heavy chain regions. For example, in some embodiments, a third antigen-binding arm may be located between the first antigen-binding arm and its additional heavy chain region (e.g., Fc domain). In some embodiments, a fourth antigen-binding arm may be located between the second antigen-binding arm and its additional heavy chain region.

[0079] As shown above with respect to the second and third antigen-binding arms, the first and fourth antigen-binding arms may have the same CDR sequence. In some embodiments, they have the same VH and VL sequences. In some embodiments, the first and fourth light chains are the same.

[0080] 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 an uncharged 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 located in the heavy chain region (e.g., constant heavy chain region 1 (CH1)) and positioned to facilitate the association of the light and heavy chains. A “lambda charge pair” means a charge pair in which a positively charged amino acid residue or an uncharged amino acid residue is located in the lambda light chain (e.g., CLλ). A “kappa charge pair” means a charge pair in which a positively charged amino acid residue or an uncharged amino acid residue in the light chain is located in the kappa light chain (e.g., CLκ).

[0081] 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.

[0082] 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).

[0083] 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).

[0084] Therefore, the 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, the 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.

[0085] 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.

[0086] As illustrated herein, lambda charge pairs can be introduced at several positions to improve the correct pairing of light and heavy chains in the antigen-binding arm.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In some embodiments, the antigen-binding arm containing lambda charge pairs 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.

[0100] In the multispecific antibodies described herein, the lambda charge pairs defined above are located either i) within the first antigen-binding arm, or ii) within the second and third antigen-binding arms, but the same lambda charge pair is not located within all three antigen-binding arms. In the context of the "2+2" format antibodies described herein, the lambda charge pairs defined above are located either i) within the first and fourth antigen-binding arms, or ii) within the second and third antigen-binding arms, but the same lambda charge pair is not located within all four antigen-binding arms.

[0101] In this specification, a singular (e.g., "a" or "the") reference to a charge pair or domain also includes multiple charge pairs or multiple domains unless the context clearly indicates otherwise. For example, as described herein, a first antigen-binding arm, or both of the second and third antigen-binding arms, may contain a lambda charge pair. In embodiments where both the second and third antigen-binding arms contain a lambda charge pair, it is understood that CH1 and CLλ of the second antigen-binding arm contain the lambda charge pair, and CH1 and CLλ of the third antigen-binding arm contain the same lambda charge pair.

[0102] In some embodiments, the lambda charge pair is located within the first antigen-binding arm. That is, the first light chain contains CLλ, and the lambda charge pair is located between the position in CLλ of the first light chain and the first CH1. In these embodiments, the second and third antigen-binding arms contain a different lambda charge pair than that present in the first antigen-binding arm, or do not contain a lambda charge pair (e.g., they contain wild-type CLλ, or they contain the constant light chain kappa region (CLκ)). In the context of a "2+2" format antibody, in these embodiments, the lambda charge pair present in the first antigen-binding arm is also present in the fourth antigen-binding arm.

[0103] In some embodiments, the lambda charge pair is located within the second and third antigen-binding arms. That is, the second and third light chains contain CLλ, and the lambda charge pair is located between the position in the CLλ of the second and third light chains and the second and third CH1. In these embodiments, the first antigen-binding arm (and, if present, the fourth antigen-binding arm) contains a different lambda charge pair than that present in the second and third antigen-binding arms, or does not contain a lambda charge pair (e.g., it contains wild-type CLλ or a constant light chain kappa region (CLκ)).

[0104] For example, the first, second, and third (and fourth, if present) antigen-binding arms of a multispecific antibody may all include the lambda charge pairs described above, and the lambda charge pair of the first (and fourth, if present) antigen-binding arm is different from the lambda charge pairs of the second and third antigen-binding arms. That is, the lambda charge pair of the first (and fourth, if present) antigen-binding arm is located in any one of the pairs at positions (i) to (ix) above, and the lambda charge pairs of the second and third antigen-binding arms may be located in different pairs within positions (i) to (ix) above. For example, the first antigen-binding arm may include a lambda charge pair at position 117 in CLλ of the first antigen-binding arm and position 141 in the first CH1, and the second and third antigen-binding arms may include different lambda charge pairs at position 134 in CLλ of the second and third antigen-binding arms and at position 145 in the second and third CH1s.

[0105] In another example, the first, second, and third (and, if present, the fourth) antigen-binding arms of a multispecific antibody may all 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 of each antigen-binding arm. That is, the first antigen-binding arm (and, if present, the fourth) may contain a positively charged amino acid residue at one of the CLλ positions and an uncharged amino acid residue at the first CH1, the second and third antigen-binding arms may contain an uncharged amino acid residue at the same CLλ position of the second and third antigen-binding arms and a positively charged amino acid residue at the same position of the second and third 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 and third antigen-binding arms may include a lambda charge pair consisting of aspartic acid at position 117 of CLλ of the second and third antigen-binding arms and arginine at position 141 of the second and third CH1, respectively.

[0106] In further examples, the first antigen-binding arm (and, if present, the fourth antigen-binding arm) includes the lambda charge pair described above, and both the second and third antigen-binding arms optionally include a constant light chain kappa region (CLκ) having a kappa charge pair, as described in more detail below. Or, the second and third antigen-binding arms include the lambda charge pair described above, and the first antigen-binding arm (and, if present, the fourth antigen-binding arm) optionally includes a constant light chain kappa region (CLκ) having a kappa charge pair, as described in more detail below.

[0107] 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.

[0108] 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 a multispecific antibody contains a mixture of kappa and lambda light chains, the ratio of kappa to lambda light chains in the assembled multispecific antibody can be determined using microfluidic electrophoresis as a readout for the correct light chain ratio.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Modified disulfide In some embodiments, multispecific antibodies contain modified disulfides in addition to lambda charge pairs. "Engineered disulfide" means that the natural interchain disulfide bond at the CH1-CL interface (e.g., 220 of CH1 and 212 of LC) of the first antigen-binding arm, or the second antibody-binding arm, and the third antibody-binding arm, is replaced by a modified (non-natural) interchain disulfide, while the other antigen-binding arms contain a natural interchain disulfide bond at the CH1-CL interface. Modified 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 would normally form an interchain disulfide. Disclosures relating to the introduction of modified disulfides into multispecific antibodies for the purpose of promoting heterodimerization can be found, for example, in U.S. Patent No. 9,527,927 and Mazor, 2015, both of which are incorporated herein by reference.

[0113] Therefore, in some embodiments, (i) Disulfide bonds are formed between the first light chain and the first CH1 between the modified pair of cysteines of the first light chain and the first CH1, and between the second light chain and the second CH1, and between the third light chain and the third CH1 between the pair of native cysteines. A schematic diagram of this embodiment is shown in Figure 12. (ii) Disulfide bonds between the second light chain and the second CH1, and between the third light chain and the third CH1 are formed between the modified pair of cysteines of the second light chain and the third light chain and the second CH and the third CH1, and disulfide bonds between the first light chain and the first CH1 are formed between the pair of native cysteines, or (iii) All three antigen-binding arms contain a modified disulfide, but the modified cysteine ​​in the first antigen-binding arm is located in a different position (e.g., in the light chain) than the modified cysteines in the second and third antigen-binding arms.

[0114] In any of these three embodiments, in the "2+2" format, the fourth antigen-binding arm has the same type of disulfide bond as the first antigen-binding arm.

[0115] In some embodiments, the pair of modified cysteines CLλ and CH1 are located at position 122 of CLλ and position 126 of CH1, with the same CLλ containing a non-cysteine ​​residue at position 212 and the same CH1 containing a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0116] 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.

[0117] In the bispecific antibodies illustrated herein, the modified disulfide is located on a first (monovalent) antigen-binding arm containing a lambda charge pair, while the native disulfide is located on a second (divalent) antigen-binding arm and a third antigen-binding arm that do not contain a lambda charge pair. However, other configurations are also specifically envisioned, for example, in which the native disulfide is located on an antigen-binding arm containing a lambda charge pair, and the modified disulfide is located on an arm lacking a lambda charge pair.

[0118] 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.

[0119] κ chain and charge pair In some embodiments, at least one of the antigen-binding arms contains a light chain having a constant light chain kappa region (CLκ). That is, in a multispecific antibody, one antigen-binding arm contains CLλ and a different 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 multispecific antibodies in this form.

[0120] In one embodiment, the first antigen-binding arm (and, if present, the fourth antigen-binding arm) includes a lambda charge pair, and the second and third antigen-binding arms include CLκ as part of the second and third light chains. In another embodiment, the second and third antigen-binding arms include a lambda charge pair, and the first antigen-binding arm (and, if present, the fourth antigen-binding arm) includes CLκ as part of the first light chain.

[0121] An example of the CLκ amino acid sequence is provided as SEQ ID NO: 3.

[0122] In some embodiments, the antigen-binding arm containing CLκ 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 the CH1 of the second antigen-binding arm.

[0123] In some embodiments, the antigen-binding arm containing CLκ 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.

[0124] 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.

[0125] In certain exemplary embodiments, the multispecific antibody comprises a first antigen-binding arm (and optionally a fourth antigen-binding arm) having the lambda charge pair described above, and a second and third antigen-binding arm having the kappa charge pair described above, wherein the multispecific antibody contains a modified disulfide. Such exemplary embodiments are shown in schematic diagrams provided in Figures 12 and 17.

[0126] For example, in one exemplary embodiment, the first antigen-binding arm (and optionally the fourth 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 (and optionally between the fourth light chain and the fourth CH1) is formed between the modified pair of cysteines of CLλ and the first CH1; the second and third antigen-binding arms comprises kappa charge pairs (e.g., position 133 of two CLκ and position 183 of the second and third CH1), and the disulfide bonds between the second light chain and the second CH1, and between the third light chain and the third CH1 are formed between the pair of native cysteines of CLκ of the second and third light chains and the second and third CH1.

[0127] Other combinations of kappa charge pairs and modified disulfides are also specifically considered. In one such embodiment, the first antigen-binding arm (and optionally a fourth antigen-binding arm) comprises a lambda charge pair and a native disulfide, while the second and third antigen-binding arms comprise a kappa charge pair and a modified disulfide. In another embodiment, the first antigen-binding arm (and optionally a fourth antigen-binding arm) comprises a kappa charge pair and a modified disulfide, while the second and third antigen-binding arms comprise a lambda charge pair and a native disulfide. In yet another embodiment, the first antigen-binding arm (and optionally a fourth antigen-binding arm) comprises a kappa charge pair and a native disulfide, while the second and third antigen-binding arms comprise a lambda charge pair and a modified disulfide.

[0128] 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).

[0129] In some embodiments, multispecific antibodies include one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote pairing of heterodimeric antibody molecules by promoting pairing 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 heavy chains.

[0130] 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.

[0131] 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 with serine at position 366, and iii) Substitution with alanine at position 368.

[0132] In the multispecific antibodies exemplified herein, the “knob” is located on the first (monovalent) antigen-binding arm, and the “hole” is located on the second antigen-binding arm (which, together with the third antigen-binding arm, binds bivalently to the second epitope). This configuration is shown in the schematic diagram provided as Figure 12. However, the reverse configuration, i.e., where the “hole” is located on the CH3 of the first antigen-binding arm and the “knob” is located on the CH3 of the second antigen-binding arm, is also specifically intended.

[0133] Other examples of CH3 modifications for enhancing heterodimerization are, for example, Table 1 of Brinkmann and Kontermann, MABS 9(2), 182-212, 2017, which are specifically incorporated herein by reference.

[0134] For example, one Fc region may include modifications to enable fractional elution by protein A chromatography, as described by Tustian (Barton), 2016. Briefly, one of the Fc regions may include modifications to remove binding to protein A (called Fc*), which can enable selective purification of the heterodimer FcFc* multispecific product. Examples of suitable modifications for generating the Fc* region include substitution of H435 with arginine and substitution of Y436 with phenylalanine.

[0135] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, The second antigen-binding arm, which includes the second Fc region, and Including the above-mentioned third antigen-binding arm, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0136] In some embodiments, multispecific antibodies are The first antigen-binding arm, including the first Fc region, A second antigen-binding arm including the lambda charge pair described above, which includes a second Fc region, and A third antigen-binding arm containing the same lambda charge pair as the second antigen-binding arm described above, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0137] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, The second antigen-binding arm, which includes the second Fc region, and Including the above-mentioned third antigen-binding arm, 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.

[0138] In some embodiments, multispecific antibodies are The first antigen-binding arm, including the first Fc region, A second antigen-binding arm including the lambda charge pair described above, which includes a second Fc region, and A third antigen-binding arm containing the same lambda charge pair as the second antigen-binding arm described above, 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.

[0139] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, The kappa charge pair and the second antigen-binding arm including the second Fc region, It includes a third antigen-binding arm containing the same kappa charge pair as the second antigen-binding arm described above, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0140] In some embodiments, multispecific antibodies are The above kappa charge pair and the first antigen-binding arm including the first Fc region, The lambda charge pair and the second antigen-binding arm including the second Fc region, A third antigen-binding arm containing the same lambda charge pair as the second antigen-binding arm described above, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0141] In some embodiments, multispecific antibodies are The above-mentioned lambda charge pair and the first antigen-binding arm including the first Fc region, The kappa charge pair and the second antigen-binding arm including the second Fc region, and It includes a third antigen-binding arm containing the same kappa charge pair as the second antigen-binding arm described above, 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.

[0142] In some embodiments, multispecific antibodies are The above kappa charge pair and the first antigen-binding arm including the first Fc region, The above-mentioned lambda charge pair and a second antigen-binding arm including a second Fc region, and A third antigen-binding arm containing the same lambda charge pair as the second antigen-binding arm described above, 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.

[0143] Non-limiting examples of multispecific 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.

[0144] 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 / or complement are well known, such as 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.

[0145] 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."

[0146] Other suitable Fc region amino acid substitutions or modifications are well known in the art and include, for example, a triple substitution (M252Y / S254T / T256E, referred to as the "YTE" or "YTE" mutation) 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) (see, 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) (each of these is incorporated herein by reference in whole)). This combination of mutations may extend the half-life of the antibody.

[0147] 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.

[0148] In some embodiments, the Fc region includes YTE mutations and triple mutations. In other embodiments, the Fc region includes YTE mutations and Fc effector null mutations.

[0149] CD3 target and T cell engager In some embodiments, the first antigen-binding arm can bind to CD3.

[0150] 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 the target cell. The 2+1 format of bispecific antibodies is well-suited for CD3 binding because the goal is to bind to the CD3 protein only in a monovalent state, so that the T cell receptor is crosslinked and activated only upon binding to the target cell.

[0151] Additional antigen-binding domains Also described herein are multispecific antibodies further comprising an additional antigen-binding domain capable of binding to a third epitope distinct from the first and second epitopes (e.g., a third epitope on a third antigen). Such an antibody may be a trispecific tetravalent antibody: the first antigen-binding arm binds monovalently to the first epitope, the second and third antigen-binding arms bind bivalently to the second epitope, while the additional antigen-binding domain binds monovalently to the third epitope. Alternatively, such an antibody may be a trispecific pentavalent antibody: the first and fourth antigen-binding arms bind bivalently to the first epitope, the second and third antigen-binding arms bind bivalently to the second epitope, and the additional antigen-binding domain binds monovalently to the third epitope.

[0152] In some embodiments, the additional antigen-binding domain is a single-domain antibody, such as a heavy-chain variable (VH) domain lacking CH1 and a light chain. Heavy-chain variable domains derived from naturally occurring light-chain-lacking heavy-chain antibodies are referred to herein as VHH to distinguish them from the VH of conventional quadruple-chain immunoglobulins. These VHH molecules may originate from antibodies produced in camelid species such as camels, alpacas, dromedaries, llamas, and guanacos. Non-camelid species can also produce naturally occurring light-chain-lacking heavy-chain antibodies, and such VHHs are also included.

[0153] Camelid heavy chain antibodies (VHH) can be obtained through genetic engineering processes. See U.S. Patent No. 5,759,808. As with other non-human antibody fragments, the amino acid sequence of Camelid VHH can be modified by recombination to obtain a sequence that more closely mimics the human sequence, i.e., "humanized," thereby reducing the antigenicity of Camelid VHH to humans. Furthermore, key elements derived from Camelid VHH can be transferred to the human VH domain to obtain a camelid human VH domain.

[0154] VHH has a molecular weight one-tenth that of human IgG molecules and a physical diameter of only a few nanometers. VHH itself possesses extremely high thermal stability, stability against extreme pH and proteolytic digestion, and low antigenicity. Using VHH as one of the antigen-binding domains can reduce the number of heavy and light chains present during the production of trispecific antibodies and may therefore be advantageous from a manufacturing standpoint compared to including additional antigen-binding arms containing VH, VL, and CH1.

[0155] The camelid antigen-binding domain (e.g., VHH) may be fused to one of the first, second, third, or fourth antigen-binding arms, typically via a peptide linker. Suitable peptide linkers are well known in the art and may consist of 5-100 amino acids, 5-50 amino acids, 5-25 amino acids, or 5-15 amino acids. The peptide linker is mainly formed from glycine and serine amino acid residues and may contain the amino acid sequence GGGGS or SGGGGS. In one embodiment, the peptide linker contains or consists of (GGGGS)2.

[0156] In some embodiments in which a third antigen-binding arm is fused to the first antigen-binding arm, an additional antigen-binding domain (e.g., VHH) is fused to the second antigen-binding arm. Alternatively, if the third antigen-binding arm is fused to the second antigen-binding arm, an additional antigen-binding domain (e.g., VHH) is fused to the first antigen-binding arm.

[0157] In some embodiments, an additional antigen-binding domain (e.g., Fab or VHH) can bind to an epitope on CD8. In some embodiments, the first antigen-binding arm can bind to CD3, and the additional antigen-binding domain (e.g., VHH) can bind to an epitope on CD8.

[0158] CD8 (differentiation antigen group 8) is a dimer consisting of a pair of CD8 chains. The most common form of CD8 is composed of a CD8-α chain and a CD8-β chain. CD8 acts as a co-receptor on MHC-I-restricted T cells, binding to a nearly invariant region of MHC-I at a site different from where the T cell receptor binds. + It acts to enhance the antigen sensitivity of T cells. While we do not wish to be bound by theory, including an antigen-binding domain (e.g., VHH) that can bind to CD8 in multispecific antibodies is a way to enhance the sensitivity of CD8. + This is thought to enable preferential activation of T cells, which may offer superior therapeutic efficacy.

[0159] Sequence identity and mutation As described herein, a "2+1" format bispecific antibody comprises a first antigen-binding arm, a second antigen-binding arm, and a third antigen-binding arm, at least one of which comprises a constant light chain lambda region (CLλ) and a lambda charge pair between CLλ corresponding to CH1. Antigen-binding arms comprising a constant light chain kappa region (CLκ) are also described herein. Furthermore, multispecific antibodies comprising an additional antigen-binding region (e.g., VHH) in addition to the binding arms of a "2+1" bispecific antibody are also described herein.

[0160] In some embodiments, the CLλ of the 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.

[0161] In some embodiments, the light chain CLκ (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 (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.

[0162] In some embodiments, the first CH1, the second CH1, and / or the third CH1 include 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 includes 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.

[0163] 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 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.

[0164] 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.

[0165] 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).

[0166] 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.

[0167] 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).

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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). As described above, the third light chain may be the same as the second light chain and therefore may be encoded by the same nucleic acid. Alternatively, the third light chain may be a different nucleic acid.

[0172] The heavy chain vector may be used to encode both the first CH1 and the first VH (and the first Fc region, if present) and the second CH1 and the second VH (and the second Fc region, if present), which may exist on the vector as separate cassettes. As described above, in the "2+1" format, the CH1 of the third antigen-binding arm is fused to the VH of either the first or second antigen-binding arm. Thus, when the third antigen-binding arm is fused to the first antigen-binding arm, the third CH1 and the third VH are encoded by the nucleic acid encoding the first CH1 and the first VH, and when the third antigen-binding arm is fused to the second antigen-binding arm, the third CH1 and the third VH are encoded by the nucleic acid encoding the second CH1 and the second VH. The same applies, if extended, to the nucleic acid encoding the antibody in the "2+2" format described above. Similarly, if an additional antigen-binding domain (e.g., VHH) is present, it is fused to either the VH of the first antigen-binding arm or the second antigen-binding arm, and is therefore encoded by a nucleic acid encoding the first CH1 and the first VH or the nucleic acid encoding the second CH1 and the second VH.

[0173] 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.

[0174] Method for producing multispecific antibodies Methods for producing the multispecific antibodies described herein are also provided herein.

[0175] In some embodiments, the method is a) Expressing the first light chain, the second light chain, and the third light chain, as well as the first CH1, the second CH1, and the third 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, pairing the third light chain with the third CH1, and pairing the first binding arm with the second and third binding arms to form a multispecific antibody. c) Purification of multispecific antibodies from host cells, including

[0176] In some embodiments, part (a) further comprises expressing a fourth light chain and a fourth CH1 in a host cell. In such cases, part (b) further comprises pairing the fourth light chain with the fourth CH1 to form a fourth binding arm, and pairing the first to fourth binding arms to form a multispecific antibody.

[0177] Expressing the first light chain, the second light chain, and the third light chain, and optionally the fourth light chain, and the first CH1, the second CH1, and the third CH1, and optionally the fourth CH1, in host cells may involve introducing nucleic acids or vectors into host cells (e.g., CHO cells) using the preferred techniques described above. The host cells may then be cultured using preferred techniques so that the light chain and heavy chain polypeptides pair up to form the first and second binding arms. During normal multispecific antibody development, the various light chain and heavy chain polypeptides associate with each other (e.g., via interchain disulfide bonds formed between native cysteine ​​and / or via cysteine ​​modified into multispecific 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 multispecific antibodies.

[0178] 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 multispecific antibodies in this format.

[0179] 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.

[0180] The method may also include formulating an antibody molecule into a pharmaceutical composition with optionally pharmaceutically acceptable excipients or other substances listed below.

[0181] treatment Therefore, the multispecific antibodies described herein may be useful for therapeutic applications such as cancer treatment.

[0182] 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.

[0183] The individual may be a patient, or more specifically, a human patient.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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, the term “pharmaceutically acceptable” refers to a compound, material, composition, and / or dosage form that, within reasonable medical judgment, is suitable for use in contact with the tissue of a subject (e.g., human) without causing excessive toxicity, irritation, allergic response, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc., must also be “acceptable” in the sense of being compatible with the other components of the formulation.

[0189] 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, and the scheduling of administration. ***

[0190] 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.

[0191] 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.

[0192] To avoid any doubt, any theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0193] Any section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0194] Throughout this Spec., including the claims, unless the context requires otherwise, the terms “comprise” and “include,” as well as variations such as “comprises,” “comprising,” and “including,” will be understood to 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.

[0195] 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]

[0196] 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. Suitable pairs of amino acids at the lambda LC-HC interface for substitution were identified. The following four criteria were applied: the Cb atom must point toward the interface (for glycine amino acids, the carbonyl must point away from the interface), there must be available space beyond the Cb to introduce a longer amino acid, and there must be at least one acceptable combination of Chi angles that positions the side chain of the new amino acid in a position that does not collide with existing amino acids.

[0197] The following positions were evaluated as λ 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 The domains involved in interface formation with λ light chain CL domains 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.

[0198] These amino acids were investigated one pair at a time, or in combination, in pairs or alone, using alternative interchain disulfides or leaving the disulfides intact. The introduction of an amino acid having a positive charge or a partially positive charge means substituting the amino acid present at that position with lysine and arginine, and optionally asparagine or glutamine or histidine. The introduction of an amino acid having a negative charge or a partially negative charge means substituting the amino acid present at that position with aspartic acid, glutamic acid, serine, threonine, and optionally asparagine or glutamine. By adding histidine residues to some of these positions, it becomes possible to introduce pH-dependent CH1-CL interactions.

[0199] Nine pairs of combinations at the lambda LC-HC interface that meet the above criteria are provided in Table 1 as a non-exhaustive example and tested for improved pairing.

[0200]

Table 1

[0201] Example 2 - Materials and Methods Using the materials and methods described herein, the experiments described in the subsequent examples were conducted. All reagents were obtained from Thermo Fisher Scientific, Waltham, MA, unless otherwise noted. As described elsewhere, the terms "charge pair" and "charge variant" are used interchangeably throughout this specification, and the amino acid numbering is based on the EU numbering system, unless otherwise noted.

[0202] Construction of pDuet-heavy chain and pDuet-light chain mammalian expression vectors for DuetMab with charge pairs To construct DuetMab antibodies with charge pair mutations at the heavy-light chain interface, the pDuet-heavy-chain and pDuet-light-chain plasmids described in PCT International Publication No. WO2013 / 096291 and Mazor et al., 2015, were used as the backbone vectors. 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.

[0203] [Table 2]

[0204] 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.

[0205] 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.

[0206] 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).

[0207] 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".

[0208] [Table 3-1]

[0209] [Table 3-2]

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] Differential scanning fluorimetry (DSF) In a 96-well PCR plate, 20 μL of a 1 mg / mL protein sample in PBS (pH 7.2) was combined in duplicate with 5 μL of SYPRO Orange dye diluted 40-fold in PBS (pH 7.2) to prepare the samples. The plates were sealed and measured on a QuantStudio 7 Flex Real-Time PCR System. The samples were subjected to an initial equilibration step at 25 °C for 2 minutes, followed by a temperature gradient up to 99 °C in 0.05 °C / second increments. Fluorescence emission was monitored using a FAM filter set. The Tm value of each sample was calculated using Protein Thermal Shift™ software with the Boltzmann method.

[0216] Subunit LC-MS analysis Subunit LC / MS analysis was performed to characterize the mispaired species. 50 μg of the sample was dried and reconstituted further in 50 μL of 100 mM sodium phosphate buffer, pH 7.0. Digestion was carried out 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) coupled to a Waters Xevo G2-XS QTOF mass spectrometer was used for subunit separation and mass determination. 2 μg of the digested subunits were injected onto a Waters BioResolve RP mAb phenyl column (2.1 × 150 mm, 2.7 mm, 450 Å) for separation. Mobile phase A contained 0.1% formic acid (FA), 0.01% trifluoroacetic acid (TFA) in water, and mobile phase B contained 0.1% FA, 0.01% TFA in water in ACN. A gradient from 25% B to 45% B was run at a flow rate of 0.2 mL / min for 40 minutes. The column temperature was set at 75 °C. The UV profile of the eluted subunits was acquired at a wavelength of 280 nm.

[0217] Differential scanning calorimetry analysis (DSC) The DSC experiments were performed 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 performed using the linear connection 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 The T value is defined as the temperature at which the thermogram begins to increase significantly from the baseline. T m The T value is defined as the temperature value corresponding to the maximum of each peak on the thermogram or the deconvolved thermogram.

[0218] 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 resulting from the luciferase-catalyzed reaction of luciferin and ATP was measured using a luminometer plate reader. Briefly, target cells expressing antigen 1 were cultured in RPMI 1640 medium supplemented with 0.1% BSA and 0.2 ng / mL human recombinant EGF 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.

[0219] 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.

[0220] [Table 4-1]

[0221] [Table 4-2]

[0222] 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.

[0223] [Table 5]

[0224] [Table 6]

[0225] 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.

[0226] [Table 7]

[0227] Table 6 summarizes the thermal stability and accelerated stability profiles of antigen 1 / antigen 2 DuetMabs with selected charge-pair variants, as measured by differential scanning fluorescence (DSF). NIP228 served as the IgG1 control. Antigen 1 / antigen 2 DuetMab variants did not show any concerns regarding aggregation or fragmentation after thermal stress. HP-SEC retention times for antigen 1 / antigen 2 DuetMab variants were consistent with those of the NIP228 IgG1 control (ΔRT < 0.2 m from NIP228). DSF values ​​did not differ significantly between charge-pair variants and were consistent with those of the NIP228 IgG1 control.

[0228] [Table 8]

[0229] Figures 6 and Table 7 show the thermal stability tests of antigen 1 / antigen 2 DuetMabs with selected charge-pair variants using differential scanning calorimetry (DSC) analysis. Figure 3 shows the stacked thermograms for antigen 1 / antigen 2 DuetMab variants. 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 variants. 開始 List the values. All mutants have similar approximate T values. 開始 The values ​​were found, indicating that the selected charge pairs did not significantly affect thermal stability.

[0230] [Table 9]

[0231] Figure 7 and Table 8 show the subunit mass spectral data of antigen 1 / antigen 2 DuetMabs with selected charge-pair variants. Molecular integrity and LC / HC association identity of each variant were confirmed by alignment of theoretical and measured masses.

[0232] [Table 10]

[0233] Figure 8 shows the cytotoxic properties of antigen-1 / antigen-2 DuetMabs with selected charge-pair variants, as determined by ATP quantification, which indicates the presence of metabolically active cells. Variants #33, #34, #35, #36, and #41 showed cytotoxicity comparable to controls #1 and #2, suggesting that the charge-pair variants did not affect the biological function of antigen-1 / antigen-2 in DuetMab.

[0234] Figure 9 and Table 9 summarize the expression and biochemical profiles of selected charge-pair mutants in diverse Fv cultured DuetMabs. Figure 9 shows the accurate LC ratio data from Table 9 plotted in grouped box charts. Charge-pair mutants #33, #34, #35, #36, and #41 showed improved accurate LC ratios among different Fv DuetMabs compared to controls #1 and #2.

[0235] [Table 11]

[0236] Example 4 - Crystallographic investigation of the proposed mutation at the CH1-CL (lambda) interface To further investigate lambda charge variants at the light chain:CH1 interface, X-ray crystallography was performed.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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).

[0241] 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).

[0242] 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 10).

[0243] The crystal of T117R / A141E Fab was diffracted to 2.0 Å. Upon completion of refinement, the inventors found that the side chains of the mutant amino acids actually established hydrogen bonds, consistent with their predictions (Figure 11).

[0244] Example 5 - Generation of a 2+1 bispecific antibody containing a lambda charge pair 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.

[0245] Construction of a p2+1 heavy chain mammalian expression vector for Duet2(2+1) bispecific antibodies with charge mutations. The p2+1-heavy chain vector was constructed on the pDuet-heavy chain skeleton described in Example 2. To construct the p2+1-heavy chain vector with charge mutations, the "hole" heavy chain was cloned into the vector by BssHII / HindIII as previously described. The "knob" heavy chain was cloned into the vector using restriction cloning techniques with BsrGI / EcoRI with synthetic DNA fragments of the VH-CH1-VH-CH1-CH2-CH3 domain, where the preceding VH-CH1 segment corresponds to the sequence found on the "hole" heavy chain, and the subsequent VH-CH1 segment contained charge mutations A141D and V12 DS in VH and CH1 for different targets. The pDuet-Light vector is common to both the Duet2(2+1) bispecific construct and the DuetMab construct.

[0246] Expression, affinity purification, and protein quantification. All Duet2(2+1) bispecific constructs were transiently expressed and purified for the DuetMab molecule as described above.

[0247] Binding kinetic assays, accelerated stability tests, subunit LC-MS analysis, and differential scanning calorimetry (DSC) were performed for the DuetMab protein essentially as described above.

[0248] xCELLigence Cell Death Assay T cell-mediated cytotoxicity was evaluated using xCELLigence Real-Time Cell Analyzer (ACEA Biosciences). A total of 1 × 10⁶ cells were resuspended in RPMI1640 medium supplied with 10% thermally inactivated FBS and 50 μM 2-mercaptoethanol per well. 4 Target cells were seeded into E-plates. After cell adhesion overnight at 37°C and 5% CO2, effector cells (PBMCs derived from healthy donors) were added at an E:T ratio of 10:1, and various concentrations of antibodies were added. Cell index (i.e., relative cell impedance) values ​​were monitored every 10 minutes, and the time point closest to 48 hours (upper or lower) was used for data analysis. Cytotoxicity was normalized to the maximum cell index value without antibody treatment and plotted using GraphPad Prism v 9.0.0.

[0249] CD69 and CD25 expression assays Human T cell activation was characterized in a T cell-mediated cytotoxicity assay using flow cytometry. After a two-day incubation, T cells were collected from the cytotoxicity assay and analyzed. T cells from PBMCs were measured by staining for surface expression of CD2 and CD4 (both obtained from BioLegend, San Diego, CA, USA). T cell activation was determined by staining for both T cell activation markers (CD69, CD25) (both obtained from BioLegend, San Diego, CA, USA) and analyzed using a FACSymhony A3 flow cytometer from BD. Data were analyzed with FlowJo v 10.6.1 and plotted using GraphPad Prism v 9.0.0.

[0250] result Table 10 summarizes the expression and biochemical profiles of antigen 1 / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules possessing selected charge pair sets, produced in 500 mL cell cultures. For further analysis, DuetMabs were further purified by light chain affinity chromatography to remove mispaired byproducts, and aggregates were removed by preparative SEC.

[0251] [Table 12]

[0252] Figure 13 shows the binding dynamics of the antigen 1 / CD3 Duet2(2+1) bispecific molecule to antigen 1 and CD3 antigen. Table 11 shows the response signals and fitting curves.

[0253] [Table 13]

[0254] Table 12 summarizes the thermal stability and accelerated stability profiles of antigen 1 / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules as determined by differential scanning fluorescence (DSF). The Duet2(2+1) bispecific molecules did not show any concerns regarding aggregation or fragmentation after thermal stress.

[0255] [Table 14]

[0256] Figure 14 and Table 13 show the subunit mass spectral data of antigen 1 / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules. Molecular integrity and LC / HC association identity of each variant were confirmed by alignment of theoretical and measured masses.

[0257] [Table 15]

[0258] Figure 15 and Table 14 show the thermal stability tests of antigen 1 / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules using differential scanning calorimetry (DSC) analysis. Figure 15 shows the stacked thermograms for antigen 1 / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules. 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 14 lists the deconvoluted TM and approximate T onset values ​​for the Duet2(2+1) bispecific molecules.

[0259] [Table 16]

[0260] Figure 16 shows the cytotoxic properties of DuetMab and Duet2(2+1) bispecific molecules as determined by the xCELLigence cytotoxicity assay. Antigen 1 / CD3 Duet2 bispecificity, with two anti-antigen 1 Fab arms, showed superior efficacy in eliminating antigen 1-expressing target cells compared to the corresponding 1+1 antigen 1 / CD3 DuetMab. Wells treated with antigen 1 / CD3 Duet2 bispecificity showed increased CD8 and CD4 T cell activation compared to wells treated with antigen 1 / CD3 DuetMab. NIP228 / CD3 DuetMab and NIP228 / CD3 Duet2(2+1) bispecific antibodies were included in this assay as isotype controls. The control molecules showed low cytotoxicity and induced limited CD8 and CD4 T cell activation.

[0261] array 1. Amino acid sequence of the WT CLλ constant region (SEQ ID NO: 1) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0262] 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

[0263] [Table 17]

[0264] 3. Amino acid sequence of the WT LC kappa constant (Cκ) region (SEQ ID NO: 3) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0265] 4. Amino acid sequence of IgG1 CH1 (SEQ ID NO: 4) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

[0266] 5. Amino acid sequence of "V12" CH1 modified to form a modified disulfide bridge (SEQ ID NO: 5) The following substitutions are underlined. Modified disulfides: F126C, C220V

[0267] [Table 18]

[0268] 6. Amino acid sequence of IgG1 CH2 (SEQ ID NO: 6) LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0269] 7. Amino acid sequence of IgG1 CH3 (SEQ ID NO: 7) GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] 8. Amino acid sequence of IgG1 heavy chain polypeptide (SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0271] 9. Amino acid sequence of IgG1 CH3 modified to include the "hole" mutation (SEQ ID NO: 9) The following substitutions are underlined. "Hole" mutations (T366S, L368A, and Y407V).

[0272] [Table 19]

[0273] 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).

[0274] [Table 20]

[0275] 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).

[0276] [Table 21]

[0277] 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).

[0278] [Table 22]

[0279] 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).

[0280] [Table 23]

[0281] 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).

[0282] [Table 24]

[0283] 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).

[0284] [Table 25]

[0285] 16. Linker (Sequence ID 16), GGGGS

[0286] 17. Linker (SEQ ID NO: 17) SGGGGS

[0287] 18. Linker (Sequence ID 18), GGGGSGGGGS

Claims

1. It is 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), and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, The first antigen-binding arm binds to the first epitope, and the second antigen-binding arm and the third antigen-binding arm bind to the second epitope. The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm. The first antigen-binding arm, or both the second antigen-binding arm and the third antigen-binding arm, are located in the steady-state light chain lambda region (CLλ) of the light chain and in the CH1 at the following positions: (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, (viiii) Position 178 of CLλ and position 173 of CH1, (ix) Located at one or more of the pairs of positions 117 of CLλ and 187 of CH1, The lambda charge pair comprises a positively charged amino acid residue optionally selected from arginine, lysine, or 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 CH1.

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

4. The lambda charge pairs are listed below: 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, e. The multispecific antibody according to claim 2, selected from lysine at position 117 of CLλ and aspartic acid at position 141 of CH1.

5. The lambda charge pair is located at position 117 of CLλ and position 185 of CH1, and optionally the lambda charge pair is 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, e. Lysine at position 117 of CLλ, and aspartic acid at position 185 of CH1, f. Lysine at position 117 of CLλ, and glutamic acid at position 185 of CH1, g. Lysine at position 117 of CLλ, and serine at position 185 of CH1, h. The multispecific antibody according to claim 1, selected from lysine at position 117 of CLλ and threonine at position 185 of CH1.

6. The lambda charge pair is located at position 119 of CLλ and position 128 of CH1, and optionally the lambda charge pair is 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, e. Lysine at position 119 of CLλ, and aspartic acid at position 128 of CH1, f. Lysine at position 119 of CLλ, and glutamic acid at position 128 of CH1, g. Lysine at position 119 of CLλ, and serine at position 128 of CH1, h. A multispecific antibody according to claim 1, selected from lysine at position 119 of CLλ and threonine at position 128 of CH1.

7. The lambda charge pair is located at position 134 of CLλ and position 128 of CH1, and optionally the lambda charge pair is 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, e. Lysine at position 134 of CLλ, and aspartic acid at position 128 of CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 128 of CH1, g. Lysine at position 134 of CLλ, and serine at position 128 of CH1, h. A multispecific antibody according to claim 1, selected from lysine at position 134 of CLλ and threonine at position 128 of CH1.

8. The lambda charge pair is located at position 134 of CLλ and position 145 of CH1, and optionally the lambda charge pair is 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, e. Lysine at position 134 of CLλ, and aspartic acid at position 145 of CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 145 of CH1, g. Lysine at position 134 of CLλ, and serine at position 145 of CH1, h. A multispecific antibody according to claim 1, selected from lysine at position 134 of CLλ and threonine at position 145 of 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 CH1, and optionally the lambda charge pair is lysine at position 134 of CLλ and aspartic acid or serine at position 183 of CH1.

10. The lambda charge pair is located at position 136 of CLλ and position 185 of CH1, and optionally the lambda charge pair is 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, e. Lysine at position 136 of CLλ, and aspartic acid at position 185 of CH1, f. Lysine at position 136 of CLλ, and glutamic acid at position 185 of CH1, g. Lysine at position 136 of CLλ, and serine at position 185 of CH1, h. The multispecific antibody according to claim 1, selected from lysine at position 136 of CLλ and threonine at position 185 of CH1.

11. The lambda charge pair is located at position 178 of CLλ and position 173 of CH1, and optionally the lambda charge pair is 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, e. Lysine at position 178 of CLλ, and aspartic acid at position 173 of CH1, f. Lysine at position 178 of CLλ, and glutamic acid at position 173 of CH1, g. Lysine at position 178 of CLλ, and serine at position 173 of CH1, h. The multispecific antibody according to claim 1, selected from lysine at position 178 of CLλ and threonine at position 173 of CH1.

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

13. The multispecific antibody according to claim 12, wherein the pair of modified cysteines of the light chain and CH1 are located at position 122 of the light chain and position 126 of the CH1, the light chain contains a non-cysteine ​​residue at position 212, the 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 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, the second CH1, and / or the third 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 light chain in the antigen-binding arm lacking the lambda charge pair comprises a constant light chain kappa region (CLκ).

17. The multispecific antibody according to claim 16, wherein the antigen-binding arm containing CLκ includes a kappa charge pair located at CLκ and CH1 of the antigen-binding arm, and the kappa charge pair comprises a positively charged amino acid residue selected from arginine, lysine, or histidine located at one of the positions of the kappa 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 kappa charge pair.

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

19. The multispecific antibody according to claim 17 or 18, wherein the CLκ 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.

20. It is 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, or histidine located at one position of the lambda 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 lambda charge pair, and (ii) The disulfide bond between the first light chain and the first CH1 is formed between the CLλ and a modified pair of cysteines of the first CH1, and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, (i) The second light chain and the third light chain include a steady light chain kappa region (CLκ), and (ii) Both the second antigen-binding arm and the third antigen-binding arm include a kappa charge pair located at CLκ and the corresponding CH1, wherein the kappa charge pair includes a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the kappa 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 kappa charge pair, and (iii) The disulfide bonds between the second light chain and the second CH1, and between the third light chain and the third CH1, include a third antigen-binding arm formed between the CLκ and a pair of native cysteine ​​in the second CH1, The first antigen-binding arm binds to the first epitope, and the second antigen-binding arm and the third antigen-binding arm bind to the second epitope. The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm, and A multispecific antibody whose numbering follows the EU index.

21. The fourth antigen-binding arm further comprises a fourth light chain disulfide-bonded to a fourth CH1, wherein the fourth antigen-binding arm is bound to the first epitope. The first antigen-binding arm and the fourth antigen-binding arm both include the lambda charge pair, and (a) The third antigen-binding arm is fused to the first antigen-binding arm, the fourth antigen-binding arm is fused to the second antigen-binding arm, or (b) The multispecific antibody according to any one of claims 1 to 20, wherein the third antigen-binding arm is fused to the second antigen-binding arm and the fourth antigen-binding arm is fused to the first antigen-binding arm.

22. (a) The third antigen-binding arm is fused to the N-terminus of the first antigen-binding arm, (b) The multispecific antibody according to claim 21, wherein the second antigen-binding arm is fused to the C-terminus of the fourth antigen-binding arm.

23. (a) The third antigen-binding arm is fused to the N-terminus of the first antigen-binding arm, and the second antigen-binding arm is fused to the N-terminus of the fourth antigen-binding arm, or (b) The multispecific antibody according to claim 21, wherein the third antigen-binding arm is fused to the C-terminus of the first antigen-binding arm, and the second antigen-binding arm is fused to the C-terminus of the fourth antigen-binding arm.

24. (i) The second light chain and the third light chain are the same, and / or (ii) The multispecific antibody according to any one of claims 1 to 23, wherein the first light chain and the fourth light chain are the same.

25. The multispecific antibody according to any one of claims 1 to 24, wherein the first antigen-binding arm further comprises a first Fc region, and the second antigen-binding arm further comprises a second Fc region.

26. The multispecific antibody according to claim 25, 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.

27. The multispecific antibody according to claim 26, wherein the modification is located at CH3 in the Fc region.

28. The multispecific antibody according to claim 27, 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.

29. 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 iii) One or more substitutions of alanine at position 368, according to claim 28.

30. The multispecific antibody according to claim 28 or 29, wherein the CH3 domain containing the knob contains cysteine ​​at position 354, and the CH3 domain containing the hole contains cysteine ​​at position 349.

31. 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 25 to 30, comprising M252Y / S254T / T256E.

32. The multispecific antibody according to any one of claims 1 to 31, wherein the first antigen-binding arm binds to an epitope on CD3.

33. A multispecific antibody according to any one of claims 1 to 32, further comprising an additional antigen-binding domain, wherein the additional antigen-binding domain is VHH, and optionally the VHH binds to an epitope on CD8.

34. A method comprising expressing the first light chain, the second light chain, and the third light chain, as well as the first CH1, the second CH1, and the third CH1 in a host cell, wherein the first light chain is paired with the first CH1 to form a first binding arm, the second light chain is paired with the second CH1 to form a second binding arm, the third light chain is paired with the third CH1 to form a third binding arm, and (a) The first coupling arm is in opposition to the second coupling arm and the third coupling arm, or (b) The first coupling arm and the third coupling arm face the second coupling arm, A method for producing a multispecific antibody according to any one of claims 1 to 33, wherein the multispecific antibody is formed thereby, and the multispecific antibody is purified from the host cell.

35. The method according to claim 34, further comprising expressing the fourth light chain and the fourth CH1 in the host cell, wherein the fourth light chain pairs with the fourth CH1 to form the fourth binding arm, and the first binding arm and the third binding arm pair with the second binding arm and the fourth binding arm to form the multispecific antibody.

36. The method according to claim 35, wherein the purification of the multispecific antibody comprises affinity chromatography, and optionally further comprises purifying the antibody using light chain affinity chromatography.

37. The method according to claim 35 or 36, 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.

38. One or more nucleic acids encoding a multispecific antibody according to any one of claims 1 to 33.

39. A vector comprising the nucleic acid described in claim 38.

40. An isolated host cell comprising the nucleic acid described in claim 38 or the vector described in claim 39.

41. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 33 and a pharmaceutically acceptable carrier.

42. 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 33, or a pharmaceutical composition according to claim 41.

43. The method according to claim 42, wherein the disease is cancer.

44. A multispecific antibody according to any one of claims 1 to 33, or a pharmaceutical composition according to claim 41, for use as a pharmaceutical.

45. A multispecific antibody according to any one of claims 1 to 33, or a pharmaceutical composition according to claim 41, for use in the treatment of cancer.

46. Use of a multispecific antibody according to any one of claims 1 to 33, or a pharmaceutical composition according to claim 41, for the manufacture of a pharmaceutical for the treatment of the aforementioned cancer.

Images