Trispecific modified antibody

Abstract

This specification provides a tripspecific antibody comprising three antigen-binding arms, each capable of binding to a different target, wherein each antigen-binding arm contains a different lambda or kappa charge pair introduced at the interface of the respective 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 / 494,929, 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-102-WO Sequence Listing.xml, size: 18,720 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 triplicate antibody containing three antigen-binding arms, each capable of binding to a different target, wherein each antigen-binding arm contains a different lambda or kappa charge pair introduced at the interface of its respective heavy and light chains as a strategy to reduce chain mispairing. In some cases, the triplicate antibody also contains a modified disulfide. This disclosure also relates to methods for producing these triplicate antibodies and their therapeutic use. [Background technology]

[0004] Multispecific 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 bispecific antibody form incorporating some of these modifications to improve the efficient production of these molecules is "DuetMab," described in Mazor 2015 and PCT International Publication No. 2013 / 096291. DuetMab antibodies use knob-into-hole technology for heterodimerization of two different heavy chains and increase the effectiveness of homologous heavy- and light-chain pairing by replacing the native disulfide bond at one of the CH1-CL interfaces with a modified disulfide bond.

[0007] The natural evolution of bispecific antibodies involved the introduction of triplicate antibodies capable of interacting with three distinct epitopes. However, the introduction of further antigen-binding arms often increases the number of heavy and light chains that need to be precisely paired, presenting further challenges for the efficient construction of these molecules.

[0008] Therefore, additional mechanisms are still needed to improve polypeptide chain pairing in triplicate antibodies and promote their efficient production. The present invention has been made in view of the above circumstances. [Overview of the project]

[0009] Described herein is the pairing of heavy chains (HC) and light chains (LC) achieved by introducing charge pairs at the interface between lambda LC and lambda HC. Also described are amino acid residues at the interface between lambda LC and HC in which charge pairs can be introduced to favorably improve chain pairing beyond what was achieved with previous DuetMab bispecificity formats. It has been further established that triplicate antibodies containing three different antigen-binding arms can be produced using the newly identified lambda charge pairs. In particular, the following was observed: - Efficient pairing of the first antigen-binding arm can be achieved by using the lambda charge pair between CH1 and CLλ of the first antigen-binding arm. -Efficient pairing of the second antigen-binding arm can be achieved by using the kappa charge pair between CH1 and CLκ of the second antigen-binding arm, and - Efficient pairing of the third antigen-binding arm can be achieved by using the kappa charge pair between CH1 and CLκ of the third antigen-binding arm, where the charged amino acid residue is in the opposite arrangement to that of the second antigen-binding arm.

[0010] Having a kappa charge pair in the opposite configuration to the kappa charge pair of the second antigen-binding arm means that if the kappa charge pair of the second antigen-binding arm contains a positively charged amino acid residue on CH1 and a negatively charged amino acid residue on CLκ, then the kappa charge pair of the third antigen-binding arm contains a negatively charged amino acid residue on CH1 and a positively charged amino acid residue on CLκ. As demonstrated herein, triplicate antibodies containing this lambda and kappa charge pair combination were efficiently produced with a high degree (>90%) of correct chair pairing.

[0011] Therefore, in one embodiment, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to a first heavy chain constant region 1 (CH1), wherein the first light chain comprises a constant light chain lambda region (CLλ), and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain kappa region (CLκ), and (c) A triplicate antibody is provided herein that comprises a third antigen-binding arm, the third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises CLκ. The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm. The first antigen-binding arm includes one or more lambda charge pairs, each containing a positively charged amino acid residue and an uncharged amino acid residue, located at the interface between the first CH1 and CLλ. Optionally, the second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and the CLκ of the second light chain, and the third antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and the CLκ of the third light chain, and the charged amino acid residues of the kappa charge pair located on the third CH1 and the CLκ of the third light chain have the opposite charge to those of the kappa charge pair located on the second CH1 and the CLκ of the second light chain, and The positively charged amino acid residue is arbitrarily selected from arginine, lysine, or histidine, and the uncharged amino acid residue is arbitrarily selected from aspartic acid, glutamic acid, serine, or threonine.

[0012] Furthermore, it was recognized that triplicate antibodies with correct chain pairing can also be produced when the lambda and kappa chains are swapped. Therefore, in another embodiment, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to a first heavy chain constant region 1 (CH1), wherein the first light chain comprises a constant light chain kappa region (CLκ), and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain lambda region (CLλ), and (c) A triplicate antibody is provided herein that comprises a third antigen-binding arm, the third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises CLλ. The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm. The first antigen-binding arm optionally includes a kappa charge pair containing a positively charged amino acid residue and an uncharged amino acid residue located at the interface between the first CH1 and CLκ. The second antigen-binding arm includes one or more lambda charge pairs, which include a positively charged amino acid residue and a loaded amino acid residue located at the interface between the second CH1 and the CLλ of the second light chain, and The third antigen-binding arm optionally includes one or more lambda charge pairs, each containing a positively charged amino acid residue and an uncharged amino acid residue, located at the interface between the third CH1 and the CLλ of the third light chain, and the charged amino acid residues of the one or more lambda charge pairs located on the third CH1 and the CLλ of the third light chain are optionally opposite in charge to the charged amino acid residues of the one or more lambda charge pairs located on the second CH1 and the CLλ of the second light chain, and The positively charged amino acid residue is arbitrarily selected from arginine, lysine, or histidine, and the uncharged amino acid residue is arbitrarily selected from aspartic acid, glutamic acid, serine, or threonine.

[0013] In some embodiments, a trispecific antibody is (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), (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain kappa region (CLκ), and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises CLκ, The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm, The first antigen-binding arm comprises a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the first CH1 and CLλ, The second antigen-binding arm comprises a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and CLκ of the second light chain, and The third antigen-binding arm comprises a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and CLκ of the third light chain, and the charged amino acid residues located on the third CH1 and CLκ of the third light chain have an opposite charge to the charged amino acid residues located on the second CH1 and CLκ of the second light chain, and The positively charged amino acid residue is selected from arginine, lysine, or histidine, and the negatively charged amino acid residue is selected from aspartic acid, glutamic acid, serine, or threonine.

[0014] In another aspect, (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 kappa region (CLκ), the first antigen-binding arm, (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain lambda region (CLλ), the second antigen-binding arm, and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises CLλ, a trispecific antibody comprising the third antigen-binding arm is provided herein, The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm, The first antigen-binding arm comprises a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the first CH1 and CLκ, The second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and the CLλ of the second light chain, and The third antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and the CLκ of the third light chain, and the charged amino acid residue located on the third CH1 and the CLκ of the third light chain has the opposite charge to the charged amino acid residue located on the second CH1 and the CLκ of the second light chain, and The positively charged amino acid residue is selected from arginine, lysine, or histidine, and the uncharged amino acid residue is selected from aspartic acid, glutamic acid, serine, or threonine.

[0015] 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 pairs of positions at 117th position of CLλ and 187th position of CH1, The numbering follows the EU index.

[0016] In some embodiments, the lambda charge pair is located at position 117 in CLλ and position 141 in 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, 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.

[0017] 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.

[0018] 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, 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.

[0019] 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, 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] In some embodiments, the kappa charge pair is located at position 133 of CLκ and position 183 of the corresponding CH1 in its antigen-binding arm.

[0026] In some embodiments, a kappa charge pair located at the interface between a second CH1 and the CLκ of the second light chain comprises a charged amino acid residue on the CLκ of the second light chain and a charged amino acid residue on the second CH1, and a kappa charge pair located at the interface between a third CH1 and the CLκ of the third light chain comprises a charged amino acid residue on the CLκ of the third light chain and a charged amino acid residue on the third CH1.

[0027] In some embodiments, the kappa charge pair located at the interface between the second CH1 and the CLκ of the second light chain comprises a charged amino acid residue at position 133 of the CLκ of the second light chain and a charged amino acid residue at position 183 of the second CH1, and the kappa charge pair located at the interface between the third CH1 and the CLκ of the third light chain comprises a charged amino acid residue at position 133 of the CLκ of the third light chain and a charged amino acid residue at position 183 of the third CH1.

[0028] In some embodiments, the negatively charged amino acid residue in the kappa charge pair is glutamic acid. In some embodiments, the positively charged amino acid residue in the kappa charge pair is lysine.

[0029] 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.

[0030] In some embodiments, the triplicate antibody has a native interchain disulfide bond at one of the CH1-CL interfaces, which is replaced by a modified interchain disulfide bond. In some embodiments, the disulfide bond between the first light chain and the first CH1 is formed between the first light chain and a pair of modified cysteines of the first CH1, and 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 a pair of native cysteines.

[0031] Furthermore, it has been recognized that the introduction of modified disulfides to additional antigen-binding arms (in addition to the first antigen-binding arm) may be used to further improve correct pairing. For example, in embodiments in which both the second and third antigen-binding arms contain kappa charge pairs, the introduction of a modified disulfide to one of the arms (e.g., the third antigen-binding arm) may limit any potential mispairing between the second and third antigen-binding arms. If both the first and third antigen-binding arms contain modified disulfides, it is desirable that the modified disulfide introduced to the third antigen-binding arm is different from the modified disulfide introduced to the first antigen-binding arm.

[0032] Accordingly, in some embodiments, the disulfide bond between the first light chain and the first CH1 is formed between the first light chain and a pair of modified cysteines of the first CH1, the disulfide bond formed between the second light chain and the second CH1 is formed between a pair of native cysteines, and the disulfide bond formed between the third light chain and the third CH1 is formed between the third light chain and a pair of modified cysteines of the third CH1, and the pair of inserted cysteines of the third light chain and the third CH1 are at different amino acid residue positions than the pair of inserted cysteines of the first light chain and the first CH1.

[0033] In some embodiments, a modified pair of cysteines in the light chain (e.g., CLλ) are located at position 122 of the light chain and position 126 of CH1, the light chain (e.g., CLλ) contains a non-cysteine ​​residue at position 212, and CH1 contains a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0034] In some embodiments, a modified pair of cysteines in the light chain (e.g., CLκ) are located at position 121 of the light chain and position 126 of CH1, the light chain (e.g., CLκ) contains a non-cysteine ​​residue at position 214, and CH1 contains a non-cysteine ​​residue at position 220. In some embodiments, the non-cysteine ​​residue is valine.

[0035] In some embodiments, the disulfide bond between the first light chain and the first CH1 is formed between a pair of modified cysteines at position 122 of CLλ and position 126 of the first CH1, wherein CLλ contains a non-cysteine ​​residue at position 212, and the first CH1 contains a non-cysteine ​​residue at position 220. The disulfide bond formed between the second light chain and the second CH1 is formed between a pair of natural cysteine, and The disulfide bond formed between the third light chain and the third CH1 is formed between a pair of modified cysteines at position 121 of CLκ and position 126 of the first CH1, with CLκ containing a non-cysteine ​​residue at position 214 and the first CH1 containing a non-cysteine ​​residue at position 220.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] In some embodiments, CLκ comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO: 1 or SEQ ID NO: 2.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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 of serine at position 366, and iii) Substitution with alanine at position 368.

[0044] 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.

[0045] In some embodiments, the triplicate antibody comprises the above-mentioned combination of lambda and kappa charge pairs, the above-mentioned modified disulfide, and the above-mentioned Fc modification to promote heterodimerization.

[0046] In some embodiments, one of the antigen-binding arms binds to an epitope on CD3.

[0047] Methods for producing the triplicate 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 triple-specific antibody. c) Purification of a triplicate antibody from host cells, and the following:

[0048] In some embodiments, the method comprises producing a multispecific antibody, the method comprising expressing a first light chain, a second light chain, and a third light chain, as well as a first CH1, a second CH1, and a third CH1 in host cells, and expressing a first CH1, a second CH1, and a third CH1 in host cells, wherein the first light chain pairs with the first CH1 to form a first binding arm, the second light chain pairs with the second CH1 to form a second binding arm, the third light chain pairs with the third CH1, and the first binding arm pairs with the second and third binding arms to form a multispecific antibody, and the multispecific antibody is purified from host cells.

[0049] In some embodiments, the purification of a triplicate antibody includes affinity chromatography. In some embodiments, the purification of a triplicate antibody includes light chain affinity chromatography.

[0050] 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 trispecific 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. A preferred method for determining the percentage of mispairing is described herein.

[0051] One or more nucleic acids encoding the triplicate antibodies described herein are also provided herein. Isolated host cells containing nucleic acids or vectors are also provided herein.

[0052] This specification also provides pharmaceutical compositions and therapeutic methods comprising pharmaceutical compositions or trispecific antibodies, as further described below.

[0053] This disclosure includes the described aspects and combinations of features unless such combinations are clearly impossible or expressly avoided. [Brief explanation of the drawing]

[0054] Next, embodiments and experiments demonstrating the principle of this disclosure will be described with reference to the attached drawings. [Figure 1A] This shows the interface between the CH1 group of the kappa light chain (LC) and heavy chain (HC) in the antibody. V133 of κLC and S183 of HC are labeled. [Figure 1B] This shows the interface between lambda LC and CH1 in the antibody. V134 and Y178 of lambda LC and S183K of HC are labeled. [Figure 2] The diagram includes schematic representations of DuetMab antibodies containing charge pairs. The “hole” HC on the left is disulfide-bonded to kappa LC via native cysteine ​​and contains a kappa charge pair (e.g., S183K / V133E) indicated by a minus ("-") sign on the kappa LC and a plus ("+") sign on the “hole” HC. The “knob” HC on the right is disulfide-bonded to lambda LC via modified cysteine ​​and contains a lambda charge pair indicated by a plus ("+") sign on the lambda LC and a minus ("-") sign on the “knob” HC. [Figure 3] This shows the interface between the lambda LC and CH1 of HC in an antibody containing exemplary lambda charge pairs (T117R and A141S). The T117R of the lambda LC and the A141S of the HC are labeled. [Figure 4]The corrected LC ratio % data from Table 1 were plotted on a scattering XY chart. Charge pair mutants #33, #34, #35, #36, and #41 were selected for further analysis based on their accurate LC ratio %. [Figure 5] The response signals and fitting curves for control sample #1 and mutant #33 are shown, with the latter representing the mutant tested. Reaction kinetics for the soluble monomeric form of antigen 2 were obtained using an Ocet384 instrument. The dissociation constant KD was calculated as the koff / kon ratio from the nonlinear fit of the data. [Figure 6] The transitions captured by DSC thermal stability measurements for the Fab, CH2, and CH3 domains, designated TM1, TM2, TM3, and TM4, are shown. [Figure 7] The UV chromatograms obtained from subunit LC / MS analysis of each sample are shown. No subunits corresponding to mismatched species were identified. [Figure 8] Mutants with different charge pairs were assayed for cytotoxic activity. Each point represents the mean of a triple well, and the mean ± standard error (SEM) is represented by an error bar. R347 is the isotype control. [Figure 9] Table 9 provides a representation of the corrected LC ratio % data plotted in a grouped box chart. [Figure 10] This specification provides a schematic diagram of the CH1-CL domain interface having the T117R mutation in the CL of the lambda light chain and the A141D mutation in the CH1 of the heavy chain, based on data generated from the crystallographic studies described herein. A strong hydrogen bond of approximately 2.4 Å appears to be formed between the OD1 atom of aspartic acid at position 141 of the CH1 domain and the NH1 atom of arginine at position 117 of the lambda light chain. [Figure 11]This specification provides a schematic diagram of the CH1-CL domain interface having the T117R mutation in the CL of the lambda light chain and the A141E mutation in the CH1 of the heavy chain, based on data generated from the crystallographic studies described herein. A hydrogen bond of approximately 3.0 Å appears to be formed between the OE1 atom of aspartic acid at position 141 of the CH1 domain and the NH1 atom of arginine at position 117 of the lambda light chain. [Figure 12] This diagram contains a schematic representation of the "TrmAb" tripspecific 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 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 from the N-terminus of its CH1 to the C-terminus of the "knob" HC by a peptide linker. The CH1 of the third antigen-binding arm is disulfide-bonded to kappa LC via a modified cysteine ​​and contains a kappa charge pair having the opposite charge to that of the second antigen-binding arm, this kappa charge pair consisting of the negatively charged amino acid residue ("-") of CH1 and the positively charged amino acid residue ("+") in kappa LC. As indicated by the different shading, the first antigen-binding arm (dark shading) binds to the first epitope, the second antigen-binding arm (light shading) binds to the second epitope, and the third binding arm (shaded shading) binds to the third epitope. [Figure 13] The UV chromatograms of subunit LC / MS analysis of HER2 / EFGR / CD3 TriMab are shown. No subunits corresponding to mismatched species were identified. [Figure 14] The transitions captured by DSC thermal stability measurements for the Fab, CH1, CH2, and CH3 domains, referred to as Tm1, Tm2, ​​and Tm3, are shown. [Figure 15] Representative images showing the continuous binding of TriMab to soluble monomeric forms of HER2 and EGFR, and to the heterodimer form of CD3 epsilon / δ, obtained using the Ocet384 instrument are provided. A: HER2 / EGFR / CD3 TriMab. B: HER2 / VκS93A+VHP97A / CD3 TriMab. [Figure 16-1] This paper provides cytotoxic activity and T cell activation data for HER2 / EGFR / CD3 TriMab and affinity-modulated HER2 / VκS93A+VHP97A EGFR / CD3. (A) Double and single-positive cells tested to simulate target tumors and normal tissues, respectively. (B) Selective cytotoxic activity (top) and CD8+ T cell activation (middle) and CD4+ T cell activation (bottom). Each point on the graph represents the mean of the two wells, and the mean ± standard error (SEM) is represented by error bars. [Figure 16-2] This paper provides cytotoxic activity and T cell activation data for HER2 / EGFR / CD3 TriMab and affinity-modulated HER2 / VκS93A+VHP97A EGFR / CD3. (A) Double and single-positive cells tested to simulate target tumors and normal tissues, respectively. (B) Selective cytotoxic activity (top) and CD8+ T cell activation (middle) and CD4+ T cell activation (bottom). Each point on the graph represents the mean of the two wells, and the mean ± standard error (SEM) is represented by error bars. [Modes for carrying out the invention]

[0055] 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.

[0056] This specification provides a triply specific antibody containing lambda and kappa charge pairs in its three antigen-binding arms, as will be discussed in more detail below.

[0057] Methods for producing bispecific and triplicate antibodies are well known. However, such methods are often limited by a multitude of possible antibody formations, which may involve several combinations of incorrect pairings of heavy and light chains. Such mispairings can reduce production efficiency. The combined use of lambda and kappa charge variants, as described herein, overcomes these limitations by preferentially pairing the correct light chain with the correct CH1 in one binding arm, thereby generating triplicate antibody assemblies. Furthermore, these charge pairs can be combined with well-known approaches used to promote accurate heavy and light chain pairing, such as knob-into-hole (KiH) and modified disulfides, as described in more detail below, to further improve the formation of triplicate antibodies.

[0058] 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.

[0059] 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.

[0060] The light chains in natural antibodies are either "lambda (λ)" or "kappa (κ)" light chains, which differ in their amino acid sequences. The light chain consists of a single constant light chain region (CL) and a single light chain variable region (VL). An example of the amino acid sequence of the constant light chain lambda region (CLλ) is provided as SEQ ID NO: 1, and an example of the amino acid sequence of the constant light chain kappa region (CLκ) is provided as SEQ ID NO: 3. The light chains used in the trispecific antibodies described herein may be chimeric light chains, for example, containing CLλ and VLκ.

[0061] 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.

[0062] 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).

[0063] 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."

[0064] 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.

[0065] 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.

[0066] Three chroma format This disclosure provides a triplicate antibody. The triplicate antibody according to this disclosure may be provided in an isolated form, meaning that it is free from contaminants such as antibodies that can bind to other polypeptides and / or serum components.

[0067] 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.

[0068] The triplicate antibodies of this disclosure can bind to three different epitopes on the same antigen, or in some embodiments, on different antigens, and include 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” includes a light chain, VH, and CH1 (i.e., at least one constant domain and one variable domain from the heavy chain and light chain, respectively), the light chain being disulfide-bonded to CH1. 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.

[0069] The first and second antigen-binding arms in the trispecific antibody may further include additional heavy chain regions, i.e., one or more of the hinge, CH2, and CH3. In some embodiments, the first and second antigen-binding arms 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 and second antigen-binding arms 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 present in natural (e.g., IgG) antibodies).

[0070] The third antigen-binding arm is typically fused to the heavy chain of either the first or second antigen-binding arm via a peptide linker between the 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, in some embodiments, includes the amino acid sequence GGGGS (SEQ ID NO: 16) or SGGGGS (SEQ ID NO: 17). In one embodiment, the peptide linker includes or consists of (GGGGS)2 (SEQ ID NO: 18).

[0071] 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 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.

[0072] Charge pair The terms “charge pair” and “charge mutation” are used interchangeably throughout this specification and refer to a positively charged amino acid residue and a negatively charged amino acid residue, one located in the light chain region of the antigen-binding arm (e.g., the constant light chain region) and the other in the heavy chain region (e.g., the constant heavy chain region 1 (CH1)), in a position intended to facilitate the association of the light and heavy chains. A “lambda charge pair” means an introduced or substituted charge pair in which a positively charged or uncharged amino acid residue is located in the lambda light chain (e.g., CLλ) and the constant heavy chain region (e.g., CH1). A “kappa charge pair” means an introduced or substituted charge pair in which a positively charged or uncharged amino acid residue in the light chain is located in the kappa light chain (e.g., CLκ) and the constant heavy chain region (e.g., CH1).

[0073] While we do not wish to be constrained 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.

[0074] 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).

[0075] In some embodiments, the positively charged amino acid residue in a charge pair is located on the light chain, and the uncharged amino acid residue in a charge pair is located on the corresponding heavy chain. In other embodiments, in a charge pair, the uncharged amino acid residue is located on the light chain, and the positively charged amino acid residue is located on the corresponding heavy chain.

[0076] 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).

[0077] Therefore, a charge pair may include a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the charge pair. For example, a charge pair may include any one of the following amino acid residue pairs. Arginine and aspartic acid, Arginine and glutamic acid, Arginine and serine, Arginine and threonine, Lysine and aspartic acid, Lysine and glutamic acid, Lysine and serine, Lysine and threonine, Histidine and aspartic acid, Histidine and glutamic acid, Histidine and serine, and Histidine and threonine.

[0078] Lambda charge pair 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In some exemplary embodiments, the first antigen-binding arm includes a lambda charge pair, as shown in Figure 12. In other embodiments, the first antigen-binding arm includes a kappa charge pair, and both the second and third antigen-binding arms include a lambda charge pair, with the third CH1 and the charged amino acid residue located on the CLλ of the third light chain having the opposite charge to the second CH1 and the charged amino acid residue located on the CLλ of the second light chain.

[0093] In some exemplary embodiments, as shown in Figure 12, the positively charged amino acid residue in the lambda charge pair is located on the light chain, and the uncharged amino acid residue in the lambda 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 lambda charge pair is located on the heavy chain.

[0094] Kappa charge pair In the tripspecific antibodies described herein, at least one antigen-binding arm comprises 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.

[0095] In some embodiments, the antigen-binding arm (or more) 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., at position 133 of CLκ) is glutamic acid, and the positively charged amino acid residue (e.g., at position 183 of the second CH1) is lysine. As mentioned elsewhere, this numbering follows EU numbering.

[0096] 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.

[0097] In some exemplary embodiments, both the second and third antigen-binding arms include a kappa charge pair, as shown in Figure 12. In other embodiments, the first antigen-binding arm includes a kappa charge pair, while the second and third antigen-binding arms include a lambda charge pair.

[0098] In some exemplary embodiments, as shown in Figure 12, the positively charged amino acid residue of the kappa charge pair in the second antigen-binding arm is located on the second CH1 and the uncharged amino acid residue is located on the second light chain, and the positively charged amino acid residue of the kappa charge pair in the third antigen-binding arm is located on the third light chain and the uncharged amino acid residue is located on the third CH1. In some embodiments, the positively charged amino acid residue of the kappa charge pair in the second antigen-binding arm is located on the second light chain and the uncharged amino acid residue is located on the second CH1, and the positively charged amino acid residue of the kappa charge pair in the third antigen-binding arm is located on the third CH1 and the uncharged amino acid residue is located on the third light chain.

[0099] Combinations of charge pairs As described herein, the exemplary triplicate antibody contains a lambda charge pair on the first antigen-binding arm, a kappa charge pair on the second antigen-binding arm, and a kappa charge pair on the third antigen-binding arm, where the charged amino acid residue located on the CLκ of the third CH1 and third light chain is opposite in charge to the charged amino acid residue located on the CLκ of the second CH1 and second light chain. While we do not wish to be bound by theory, for example, the third CLκ is not thought to preferentially bind to the second CH1 because both chains contain the same (e.g., positive) charge at the HC:LC interface. As demonstrated herein, the combination of these lambda pairs and oppositely charged kappa pairs resulted in a triplicate antibody with a high degree (>90%) of correct chair pairing.

[0100] Therefore, in some embodiments, The first antigen-binding arm includes a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the first CH1 and CLλ. The second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and the CLκ of the second light chain, and The third antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and the CLκ of the third light chain, and the charged amino acid residue located on the third CH1 and the CLκ of the third light chain has the opposite charge to the charged amino acid residue located on the second CH1 and the CLκ of the second light chain.

[0101] In other embodiments, the first antigen-binding arm contains a kappa charge pair, while the second and third antigen-binding arms contain a reversed lambda charge pair. Therefore, in some embodiments, The first antigen-binding arm includes a kappa charge pair containing a positively charged amino acid residue and an uncharged amino acid residue located at the interface between the first CH1 and CLκ. The second antigen-binding arm includes a lambda charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and the CLλ of the second light chain, and The third antigen-binding arm includes a lambda charge pair comprising a positively charged amino acid residue and an uncharged amino acid residue located at the interface between the third CH1 and the CLλ of the third light chain, and the charged amino acid residue located on the third CH1 and the CLλ of the third light chain has the opposite charge to that of the charged amino acid residue located on the second CH1 and the CLλ of the second light chain.

[0102] For example, the charge pair in the second antigen-binding arm may be formed from a positively charged amino acid residue in the second CH1 and a negatively charged amino acid residue in the second light chain, and the kappa charge pair in the third antigen-binding arm may be formed from a negatively charged amino acid residue in the third CH1 and a positively charged amino acid residue in the third light chain.

[0103] Alternatively, the charge pair in the second antigen-binding arm may be formed from a charged amino acid residue in the second CH1 and a positively charged amino acid residue in the second light chain, and the kappa charge pair in the third antigen-binding arm may be formed from a positively charged amino acid residue in the third CH1 and a charged amino acid residue in the third light chain.

[0104] As described in the examples, several methods are known that can be used to determine accurate light chain pairing. These include mass spectrometry-based approaches that can be used to establish the correct heavy / light chain association. If the triplicate antibody contains a mixture of kappa and lambda light chains, the ratio of kappa to lambda light chains in the assembled triplicate antibody can be determined using microfluidic electrophoresis as a readout for the correct light chain ratio.

[0105] Therefore, in some embodiments, triplicate antibodies containing lambda charge pairs exhibit improved correct light chain pairing compared to equivalent triplicate antibodies lacking lambda charge pairs. In some embodiments, triplicate 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 triplicate antibody using light chain affinity purification.

[0106] 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 antibodies based on their light chains. Examples of such affinity resins include LambdaFabSelect and KappaSelect resins available from GE Healthcare. Such methods may be used to selectively purify trispecific antibodies containing both CLκ and CLλ, and therefore may be used to improve the production of trispecific antibodies in this form.

[0107] Combination with other counter-approach approaches The 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.

[0108] Non-limiting examples of strategies for promoting heterodimerization are described in more detail below and include the use of disulfide modifications at the CH1 / CL interface, Fc region modifications such as knob-into-hole, and those that enable fractionation and purification strategies.

[0109] Modified disulfide In some embodiments, triplicate antibodies contain an engineered disulfide in addition to 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 at least one of the first, second, and third antigen-binding arms is replaced by an engineered (non-natural) interchain disulfide, while the other antigen-binding arms contain a natural interchain disulfide bond at the CH1-CL interface. Engineered disulfides are typically formed by modifying cysteine ​​in the CL of the light chain and the corresponding CH1 of the heavy chain, replacing the cysteine ​​that normally forms an interchain disulfide. Disclosures relating to the introduction of engineered disulfides into antibodies for the purpose of promoting heterodimerization can be found, for example, in U.S. Patent No. 9,527,927 (which is incorporated herein by reference in its entirety).

[0110] In some embodiments, a disulfide bond is formed between the light chain and at least one CH1 of the antigen-binding arms between the pair of modified cysteines of the light chain and the CH1 of the antigen-binding arms. In some embodiments, a disulfide bond is formed between the light chain and CH1 in two of the antigen-binding arms (e.g., a first antigen-binding arm and a third antigen-binding arm, a first antigen-binding arm and a second antigen-binding arm, a first antigen-binding arm and a third antigen-binding arm, or a second antigen-binding arm and a third antigen-binding arm) between the pair of modified cysteines of the light chain and the CH1 of the two antigen-binding arms.

[0111] In some embodiments, the disulfide bond between the first light chain and the first CH1 is formed between a pair of modified cysteines of the first light chain and the first CH1. In some embodiments, the disulfide bond between the third light chain and the third CH1 is formed between a pair of modified cysteines of the third light chain and the third CH1. As described above, the light chain may contain CLλ or CLκ. In some embodiments, the pair of modified cysteines of CLλ and CH1 are located at position 122 of CLλ and position 126 of CH1, 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.

[0112] 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.

[0113] 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.

[0114] In some embodiments, the disulfide bond between the first light chain and the first CH1 is formed between a pair of modified cysteines of the first light chain and the first CH1, the disulfide bond formed between the second light chain and the second CH1 is formed between a pair of native cysteines, and the disulfide bond formed between the third light chain and the third CH1 is Formed between a pair of natural cysteine, or A pair of modified cysteines are formed between the third light chain polypeptide and the third heavy chain polypeptide, and the pair of inserted cysteines in the third light chain polypeptide and the third heavy chain polypeptide are located at different amino acid residue positions than the pair of inserted cysteines in the first light chain polypeptide and the first heavy chain polypeptide.

[0115] In some embodiments, the disulfide bond between the first light chain and the first CH1 is formed between a pair of modified cysteines at position 122 of CLλ and position 126 of the first CH1, wherein CLλ contains a non-cysteine ​​residue at position 212, and the first CH1 contains a non-cysteine ​​residue at position 220. The disulfide bond formed between the second light chain and the second CH1 is formed between a pair of natural cysteine, and The disulfide bond formed between the third light chain and the third CH1 is formed between a pair of modified cysteines at position 121 of CLκ and position 126 of the first CH1, with CLκ containing a non-cysteine ​​residue at position 214 and the first CH1 containing a non-cysteine ​​residue at position 220.

[0116] In one exemplary embodiment, the triplicate antibody comprises a first antigen-binding arm having the lambda charge pair and modified disulfide described above, a second antigen-binding arm having the kappa charge pair and native disulfide described above, and a third antigen-binding arm having the kappa charge pair and modified disulfide described above. Such embodiments are shown in the schematic diagram provided in Figure 12.

[0117] Other combinations of charge pairs and modified disulfides are also specifically considered. In one such embodiment, the first 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 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 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.

[0118] 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).

[0119] In some embodiments, the triplicate antibody includes one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of a heterodimeric antibody molecule by promoting the pairing of the first Fc region and the second Fc region. This may include a Knobs into Holes (KiH) strategy based on a single amino acid substitution in the CH3 domain that promotes heavy chain heterodimerization, as described by Ridgway, 1996. The knob mutant heavy chain CH3 has a small amino acid substituted with a larger amino acid, thereby creating a bump (knob) on the surface of the CH3 domain, while the hole mutant has a large amino acid substituted with a smaller amino acid, thereby creating a cavity (hole) on the surface of the CH3 domain. Further modifications may also be introduced to stabilize the association between the heavy chains.

[0120] 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.

[0121] Therefore, in one embodiment, the substitution for generating a knob is a substitution for tryptophan at position 366, and the substitution for generating a hole is one or more of the following: i) Substitution of valine at position 407, ii) Substitution of serine at position 366, and iii) Substitution with alanine at position 368.

[0122] In the triplicate antibodies illustrated herein, the “knob” is located on the first antigen-binding arm containing a lambda charge pair, and the “hole” is located on the second antigen-binding arm containing one of the kappa charge pairs. This arrangement is shown in the schematic diagram provided as Figure 12. However, the reverse arrangement, i.e., the arrangement in which 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.

[0123] For example, one Fc region may include modifications to enable fractional elution by protein A chromatography, as described by Tustian (Barton), 2016. In short, one of the Fc regions may include modifications to enable fractional elution by protein A chromatography. * This modification includes removing the bond to the heterodimer FcFc (called *This enables the selective purification of bispecific products. Fc * Suitable modifications for generating the region include substitution of H435 with arginine and substitution of Y436 with phenylalanine.

[0124] Other Fc modifications that may be used in addition to those used to enhance heterodimerization include those that reduce or inhibit 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 inhibit Fc effector function. Mutations that reduce or inhibit 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.

[0125] 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."

[0126] 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 "YTE" or "YTE mutation") numbered according to the EU index, such as in Kabat, consisting of a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256 (referred to as M252Y / S254T / T256E, "YTE" or "YTE mutation") (e.g., U.S. Patent No. 7,658,921, U.S. Patent Application Publication No. 2014 / 0302058, and Yu et al., Antimicrob. Agents Chemother., 61(1):e01020-16 (2017), the full contents of each of these are incorporated herein by reference). This combination of mutations may extend the half-life of the antibody.

[0127] 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.

[0128] 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.

[0129] In some cases, The first antigen-binding arm comprises a lambda charge pair and a first Fc region, the disulfide bond between the first light chain and the first CH1 is formed between the first light chain and a pair of modified cysteines of the first CH1, and the first Fc region comprises a "knob" mutation. The second antigen-binding arm comprises a kappa charge pair and a second Fc region, the disulfide bond formed between the second light chain and the second CH1 is formed between a pair of native cysteine ​​molecules, and the second Fc region contains a "hole" mutation, The third antigen-binding arm contains a kappa charge pair, and the charged amino acid residue located on the CLκ of the third CH1 and third light chain has the opposite charge to the charged amino acid residue located on the CLκ of the second CH1 and second light chain, and the disulfide bond formed between the third light chain and the third CH1 is Formed between a pair of natural cysteine, or A pair of modified cysteines are formed between the third light chain polypeptide and the third heavy chain polypeptide, and the pair of inserted cysteines in the third light chain polypeptide and the third heavy chain polypeptide are located at different amino acid residue positions than the pair of inserted cysteines in the first light chain polypeptide and the first heavy chain polypeptide.

[0130] Non-limiting examples of triplicate 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.

[0131] Quadruple Specific Format Also described herein are tetraspecific antibodies having the characteristics of the triplicate antibodies described herein, further comprising an additional antigen-binding domain capable of binding to a fourth epitope (e.g., a fourth epitope on a fourth antigen) that is typically distinct from the first, second, and third epitopes. Thus, this format may be referred to as a tetraspecific T-cell engager DuetMab ("TED4"). In some embodiments, the TED4 format comprises two antigen-binding domains capable of binding to the same target.

[0132] 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.

[0133] 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.

[0134] VHH has a molecular weight one-tenth that of human IgG molecules and a physical diameter of only a few nanometers. VHH itself has extremely high thermal stability, stability against extreme pH and proteolytic digestion, and low antigenicity.

[0135] An additional antigen-binding domain (e.g., VHH) may be fused to one of the first, second, or third antigen-binding arm heavy chains, 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, in some embodiments, includes the amino acid sequence GGGGS (SEQ ID NO: 16) or SGGGGS (SEQ ID NO: 17). In one embodiment, the peptide linker includes or consists of (GGGGS)2 (SEQ ID NO: 18).

[0136] 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.

[0137] 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.

[0138] 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 MCHI-restricted T cells, binding to a nearly invariant region of MHCI 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.

[0139] target In some embodiments, one of the antigen-binding arms can bind to CD3.

[0140] 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 and triplicate antibodies targeting CD3 and target cell antigens (or multiple antigens) are used to force transient interactions between target cells (or multiple cells) and T cells, leading to crosslinking, T cell activation, and subsequent antigen-dependent T cell death of the target cell. Often, the goal is simply monovalent binding to the CD3 protein so that the T cell receptor is crosslinked and activated only upon binding to the target cell. Thus, in some embodiments, only a single antigen-binding domain binds to CD3 and is referred to as a triplicate T cell activator DuetMab ("TED3").

[0141] In some embodiments, one of the antigen-binding arms can bind to CD8.

[0142] 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.

[0143] In some embodiments, one antigen-binding arm can bind to CD3, and the other antigen-binding arm can bind to CD8.

[0144] 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.

[0145] Sequence identity and mutation As described herein, the triplicate antibodies described herein comprise 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 CH1, and at least one of which comprises a constant light chain kappa region (CLκ) and a kappa charge pair between CLκ-corresponding CH1.

[0146] 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, one or more CLλ of the 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.

[0147] In some embodiments, the light chain 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. In some embodiments, one or more light chain CLκ 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.

[0148] 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, the first CH1, the second CH1, and / or the third CH1 include an 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.

[0149] 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 modifications. For example, compared to the wild-type CLλ described in SEQ ID NO: 1, the CLλ used in the tripspecific 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 tripspecific 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.

[0150] 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.

[0151] 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).

[0152] 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.

[0153] 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).

[0154] Nucleic acids, vectors, and host cells One or more nucleic acids encoding the triplicate 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 not have difficulty preparing such nucleic acid molecules using methods well known in the art.

[0155] In some embodiments, one or more nucleic acids encode the first light chain, the second light chain, and / or the third light chain as described herein, and / or the first CH1, the second CH1, and / or the third CH1 as described herein. One or more nucleic acids encoding the first CH1 or the second CH1 may further encode other heavy chain domains, such as hinges, CH2 and CH3, or they may encode a complete heavy chain.

[0156] This disclosure also provides one or more vectors comprising nucleic acids encoding the triplicate 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.

[0157] Triple-specific antibodies may be produced from one or more light chain vectors and one or more heavy chain vectors. The light chain vector may contain nucleic acids encoding a first light chain, a second light chain, and a third light chain, which may be present on the vector as separate cassettes (e.g., each operably connected to a different promoter). Alternatively, separate vectors may be used, i.e., one containing the nucleic acid encoding the first light chain, one containing the nucleic acid encoding the second light chain, and one containing the nucleic acid encoding the third light chain.

[0158] 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 be present on the vector as separate cassettes. Alternatively, separate vectors may be used, namely one containing the nucleic acid encoding the first CH1 and the first VH (and the first Fc region, if present), and another containing the nucleic acid encoding the second CH1 and the second VH (and the second Fc region, if present). As described above, in the triplicity format described herein, the CH1 of the third antigen-binding arm is fused to the heavy chain of either the first antigen-binding arm or the second antigen-binding arm. Therefore, when the third antigen-binding arm fuses with the first antigen-binding arm, the third CH1 and third VH are encoded by the nucleic acid encoding the first CH1 and first VH, and when the third antigen-binding arm fuses with the second antigen-binding arm, the third CH1 and third VH are encoded by the nucleic acid encoding the second CH1 and second VH.

[0159] 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.

[0160] Method for producing triple-specific antibodies A method for producing the triplicate antibodies described herein is also provided herein.

[0161] 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 (for example, via the Fc region), thereby forming a triply specific antibody. c) Purification of a triplicate antibody from host cells, and the following:

[0162] Expressing the first light chain, second light chain, and third light chain, as well as the first CH1, second CH1, and third CH1 in host cells may involve introducing nucleic acids or vectors into host cells (e.g., CHO cells) using preferred techniques as described above. The host cells may then be cultured using preferred techniques so that the light chain and heavy chain polypeptides pair up to form the first and second binding arms. During normal 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 in the triplicate antibody 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 and kappa charge pairs, and (if present) modified disulfides, facilitates the formation of correct heavy / light chain pairs in the triplicate antibody.

[0163] 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, by 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 trispecific antibodies containing both CLκ and CLλ, and therefore may be used to improve the production of trispecific antibodies in this format.

[0164] In some embodiments, less than 25%, 20%, 15%, or 10%, 5%, 4%, 3%, 2%, or 1% of the light chains in the trispecific 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.

[0165] The method may also include formulating an antibody molecule into a pharmaceutical composition with optionally pharmaceutically acceptable excipients or other substances listed below.

[0166] treatment Therefore, the triplicate antibodies described herein may be useful for therapeutic applications such as cancer treatment.

[0167] The triplicate antibodies described herein may be used in methods for treating the human or animal body. Relevant aspects of this disclosure are: (i) A triplicate antibody as described herein for use as a pharmaceutical product, (ii) A triplicate antibody described herein for use in a method of treating a disease or disorder, (iii) The trispecific antibodies described herein in the manufacture of a pharmaceutical product for use in the treatment of a disease or disorder, and (iv) A method for treating a disease or disorder in an individual, comprising administering to the individual a therapeutically effective amount of a triplicate antibody described herein.

[0168] The individual may be a patient, and in some embodiments, may be a human patient.

[0169] 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.

[0170] 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.

[0171] The treatment methods described may include administering at least one additional treatment to the individual in addition to the triplicate antibody. Therefore, the triplicate antibody described herein may be administered to the individual alone or in combination with one or more other treatments. When the triplicate 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 triplicate antibody. When the additional treatment is administered simultaneously with the triplicate antibody, the triplicate 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.

[0172] Triple-specific antibodies may be administered alone, but they are typically administered in the form of a pharmaceutical composition that may contain at least one additional component in addition to the triple-specific antibody. Therefore, another aspect of this disclosure provides a pharmaceutical composition comprising the triple-specific antibody described herein. Methods for formulating a triple-specific antibody into a pharmaceutical composition are also provided.

[0173] The pharmaceutical composition may include, in addition to the tripspecific antibody, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. As used herein, “pharmaceutically acceptable” refers to a compound, material, composition, and / or dosage form that, 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. The exact nature of the carrier or other material depends on the route of administration, which may be by infusion, injection, or any other preferred route, as discussed below.

[0174] 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. ***

[0175] 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.

[0176] 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.

[0177] To avoid any ambiguity, any theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. Any section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0178] Throughout this Spec., including the claims, unless the context requires otherwise, the words “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.

[0179] 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]

[0180] 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.

[0181] It was recognized that charge pairs modified at the κLC-HC interface are unlikely to behave similarly if modified at an equivalent position at the λ / CH1 interface. For example, the presence of Y178 in lambda LC is expected to destroy the charge pairs modified at V134 in lambda LC (equivalent to V133 in kappa LC) and S138 in CH1 (see Figure 1B).

[0182] 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.

[0183] These amino acids were investigated individually or in pairs, in combination, or alone, using alternative interchain disulfides or leaving the disulfides in their natural state. The introduction of positively or partially positively charged amino acids means substituting the amino acid present at that position with lysine and arginine, and possibly asparagine, glutamine, or histidine. The introduction of negatively or partially negatively charged amino acids means substituting the amino acid present at that position with aspartic acid, glutamic acid, serine, threonine, and possibly asparagine or glutamine. Adding histidine residues to some of these positions makes it possible to introduce pH-dependent CH1-CL interactions.

[0184] Nine pair combinations at the lambda LC-HC interface that meet the above criteria are provided in Table 1 as non-exhaustive examples, and the improved pair formation described herein was tested.

[0185] [Table 1]

[0186] Example 2 - Materials and Methods The experiments described in the subsequent examples were carried out using the materials and methods described herein. All reagents were obtained from Thermo Fisher Scientific, Waltham, MA, unless otherwise noted. As stated elsewhere, the terms “charge pair” and “charge mutation” are used interchangeably throughout this specification, and amino acid numbering is based on the EU numbering system unless otherwise noted.

[0187] Construction of pDuet-heavy chain and pDuet-light chain mammalian expression vectors for DuetMab with charge pairs. To construct a DuetMab antibody with a charge pair mutation at the heavy-light chain interface, the pDuet-heavy-chain and pDuet-light-chain plasmids described in PCT International Publication No. 2013 / 096291 and Mazor et al., 2015, were used as the backbone vector. Briefly, the pDuet-Heavy vector contained two human gamma monoheavy-chain (HC) cassettes to support HC heterodimerization. The former heavy chain possessed a "hole" set mutation (T366S / L368A / Y407V) and a stabilizing mutation (Y349C) in the CH3 domain, while the latter possessed a complementary "knob" mutation (T366W) and a stabilizing mutation (S354C) in CH3. However, the order of the cassettes could be easily reversed. The pDuet-Light vector contains two human light chain (LC) cassettes, the former containing a kappa constant domain (Cκ) and the latter containing a lambda constant domain (Cλ). The pDuet-Heavy and pDuet-Light vectors also contain mutations to remove the native interchain disulfide bond in CH1 / Cλ and provide an alternative disulfide bond indicated herein as "V12 DS" or "V12". Mutation F126C / C220V was introduced into the CH1 domain of the "knob" heavy chain, and mutation S122C / C212V was introduced into the lambda constant domain. The amino acid sequences of the constant domains in the exemplary DuetMab antigen backbone (before introduction of charge mutations) are provided below.

[0188] [Table 2]

[0189] 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.

[0190] 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.

[0191] For the construction of pDuet-Light with charge variants, the κ light chain was cloned into the pDuet-Light vector using restriction cloning technology with BssHII / NheI with a synthetic DNA fragment of the VL-Cκ domain. Optionally, the constant kappa (Cκ) domain contained the charge variant V133E. Using restriction cloning technology with BsrGI / EcoRI, the λ light chain was cloned into the pDuet-Light vector with a synthetic DNA fragment of the VL-Cλ domain containing the above S122C / C212V mutation for the λ light chain. Optionally, the constant lambda (Cλ) domain contained one of the charge variants V117R, V117K, F119R, F119K, V134R, V134K, L136R, L136K, Y178R, and Y178K. The light chain variable domain (VL) can be either the variable kappa domain (Vκ) or the variable lambda domain (Vλ).

[0192] 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 CHO medium prepared in-house. Vectors containing the following charge pair combinations were used for the expression of antibodies in these studies. A schematic diagram of the constructed DuetMabs containing the charge pairs is shown in Figure 2. Bispecific antibodies were made against several different antigens (referred to herein as Antigen 1, 2, 3, 4, 5, and 6) expressed on the surface of cells. Antigen 3 is CD3. The bispecific antibodies made were referred to as "Target 1 / Target 2-DuetMab" or simply "Target 1 / Target 2".

[0193] [Table 3-1]

[0194] [Table 3-2]

[0195] 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.

[0196] 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.

[0197] 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.

[0198] Binding kinetic assay The binding kinetics were measured by biolayer interferometry on an Octet 384 instrument. A streptavidin (SA) biosensor was immobilized with biotinylated protein antigen (ACRO Biosystems, Newark, DE) in PBS pH 7.2, 1 mg / mL BSA, 0.05% (v / v) TWEEN (Kinetic buffer). After washing the loaded biosensor in the same buffer, various antibodies were used to perform association and dissociation measurements over the indicated times. The kinetic parameters (K on and K off ) and affinity (K D ) were calculated from the non-linear fitting of the data using Octet 384 software v.12.2.1.24.

[0199] Accelerated stability test The protein test samples were diluted to 1 mg / mL in PBS (pH 7.2) and divided into three equal aliquots for use as control, heat, and light stress samples. The control samples were incubated at 4 °C for 14 days, the heat stress samples were incubated at 45 °C for 14 days, and the light stress samples were incubated at 25 °C for 7 days in glass vials in an ICH-compliant photostability chamber exposed to 3000 lux of cool white light. The samples were then analyzed by HP-SEC to determine the levels of aggregates, monomers, and fragments.

[0200] Differential scanning fluorimetry (DSF) Samples were prepared in a 96-well PCR plate by combining 20 μL of 1 mg / mL protein sample in PBS (pH 7.2) with 5 μL of SYPRO Orange dye diluted 40-fold in PBS (pH 7.2) in a double-strand configuration. The plates were sealed and measured using a QuantStudio 7 Flex Real-Time PCR System. The samples were subjected to an initial equilibrium step at 25°C for 2 minutes, followed by a temperature gradient to 99°C in increments of 0.05°C / second. Fluorescence emission was monitored using a FAM filter set. The Tm value for each sample was calculated using the Boltzmann method with Protein Thermal Shift® software.

[0201] Subunit LC-MS analysis Subunit LC / MS analysis was performed to characterize the mispaired species. 50 μg of sample was dried and further reconstituted in 50 μL of 100 mM sodium phosphate buffer, pH 7.0. Digestion was performed by adding 60 units of FabALACTICA enzyme (IgdE) (Genovis AB, Lund, Sweden) to each sample and incubating at 37°C for 16–18 hours. A Waters ACQUITY UPLC system (Waters, Milford, MA) connected to a Waters Xevo G2-XS QTI mass spectrometer was used for subunit separation and mass determination. 2 μg of digested subunits were injected into a Waters BioResolve RP mAb polyphenyl column (2.1 × 150 mm, 2.7 mm, 450 Å) for separation. Mobile phase A contained 0.1% formic acid (FA) and 0.01% trifluoroacetic acid (TFA) in water, and mobile phase B contained 0.1% FA and 0.01% TFA in ACN water. A gradient from 25%B to 45%B was performed at a flow rate of 0.2 mL / min for 40 minutes. The column temperature was set to 75°C. The UV profile of the eluted subunits was acquired at a wavelength of 280 nm.

[0202] Differential scanning calorimetry analysis (DSC) DSC experiments were performed using a MICROCAL VP-DSC scanning microcalorimeter (Malvern, Northampton, MA). Prior to DSC analysis, all samples were diluted to approximately 0.6 mg / mL in phosphate-buffered saline (PBS, pH 7.2). Accurate concentrations were determined by dual measurements using a UV-VIS spectrophotometer (NanoDrop 2000C). 400 μL of each sample and its 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 with a scanning speed of 60°C / hour. Before sample measurements, baseline measurements (buffer vs. buffer) were obtained for subtraction from 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-dual-state model, and the best fit was obtained using 1 and 200 repeat cycles until the chi-squared value was minimized. The interpretation of the DSC deconvolution results was based on the fact that different domains in the antibody format unfold independently. The T start value is defined as the temperature at which the thermogram begins to increase significantly from baseline. The Tm value is defined as the temperature value corresponding to the maximum value of each peak on the thermogram or the deconvoluted thermogram.

[0203] 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. The luminescence produced by the luciferase-catalyzed reaction between luciferin and ATP was measured using a luminescence plate reader. Briefly, target cells (NCI H358) were induced to fluoresce in RPMI 1640 medium supplemented with 0.1% BSA and 0.2 ng / mL human recombinant EGF, resulting in approximately 1 × 10⁶ cells. 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.

[0204] 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.

[0205] [Table 4-1]

[0206] [Table 4-2]

[0207] Table 4 summarizes the expression (Table 4A) and biochemical profiles (Table 4B) of Antigen 1 / Antigen 2 DuetMabs having selected charge pair variants #1, #2, #33, #34, #35, #36, and #41 produced in large-scale cell culture (100 mL). The biochemical profiles of the selected culture DuetMabs were consistent regardless of production scale. For further analysis, the DuetMabs were further purified by light chain affinity chromatography to remove mispaired by-products and aggregates were removed by preparative SEC.

[0208]

Table 5

[0209]

Table 6

[0210] Figure 5 and Table 5 show the binding kinetics of Antigen 1 / Antigen 2 DuetMabs having selected charge pair variants. Figure 5 shows the response signals and fitting curves of control sample #1 and variant #33, which are representative of the variants tested. The binding affinities of variants #33, #34, #35, #36, and #41 for Antigen 2 were equivalent to those of control #1 and control #2.

[0211]

Table 7

[0212] Table 6 summarizes the thermal stability and accelerated stability profiles of Antigen 1 / Antigen 2 DuetMabs with selected charge-pair mutants, as determined by differential scanning fluorescence (DSF). NIP228 served as an IgG1 control. Antigen 1 / Antigen 2 DuetMab mutants did not show any concerns regarding aggregation or fragmentation after thermal stress. HP-SEC retention times for Antigen 1 / Antigen 2 DuetMab mutants were consistent with those of the NIP228 IgG1 control (ΔRT < 0.2 m from NIP228). DSF values ​​did not show significant differences between charge-pair mutants and were consistent with those of the NIP228 IgG1 control.

[0213] [Table 8]

[0214] Figures 6 and Table 7 show the thermal stability tests of Antigen 1 / Antigen 2 DuetMabs with selected charge-pair mutants using differential scanning calorimetry (DSC) analysis. Figure 3 shows the stacked thermograms for Antigen 1 / Antigen 2 DuetMab mutants. Deconvolution of the thermograms revealed transitions for the Fab, CH2, and CH3 domains, with some transitions overlapping and located under the same TM peak. Table 7 shows the deconvoluted TM of Antigen 1 / Antigen 2 DuetMab charge-pair mutants. M and approximated T 開始 The values ​​are listed. All mutants had similar approximate T-start values, indicating that the selected charge pair did not significantly affect thermal stability.

[0215] [Table 9]

[0216] Figure 7 and Table 8 show subunit mass spectral data of Antigen 1 / Antigen 2 DuetMabs with selected charge-pair mutants. Molecular integrity and LC / HC association identity of each mutant were confirmed by alignment of theoretical and measured masses.

[0217] [Table 10]

[0218] Figure 8 shows the cytotoxic properties of selected charge-pair variants of Antigen 1 / Antigen 2 DuetMabs, as determined by ATP quantification, which indicates the presence of metabolically active cells. Variants #33, #34, #35, #36, and #41 exhibited 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 DuetMabs.

[0219] 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.

[0220] [Table 11]

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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).

[0226] 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).

[0227] Crystals of T117R / A141D Fab were 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). The formation of this bond is a desired outcome for this mutant pair, and the inventors consider it to be the basis for the favorable pairing of these two polypeptide chains.

[0228] Crystals of T117R / A141E Fab were diffracted to 2.0 Å. Upon completion of refinement, the inventors found, consistent with their predictions, that the side chains of the mutant amino acids actually established hydrogen bonds (Figure 11). The formation of this bond is considered to be the basis for the favorable pairing of these two polypeptide chains.

[0229] Consideration A comparison of these two Fab molecules shows that the mutant T117R / A141D establishes stronger (shorter) hydrogen bonds than T117R / A141E. This result was confirmed by a higher percentage of molecules with exact pairs for molecules containing the 117R / 141D pair.

[0230] Example 5 - Production of a triplicate antibody containing lambda and kappa charge pairs 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.

[0231] Construction of mammalian expression vectors for pTriMab heavy chain, pTriMab light chain 1, and pTriMab light chain 2 for Trimab with three charge pairs. The pTriMab-heavy chain vector was constructed on the pDuet-heavy chain skeleton described in Example 1 above. To construct a pTriMab-heavy chain vector with a charge mutation, the "hole" heavy chain was cloned into the vector by BssHII / HindIII as previously described, and the VH-CH1 segment in the "hole" heavy chain was defined as "Fab1" having the VH and CH1 charge mutation S183K for the first target. The "knob" heavy chain was cloned into the vector using restriction cloning techniques with BsrGI / EcoRI with a synthetic DNA fragment of the VH-CH1-VH-CH1-CH2-CH3 domain. The preceding VH-CH1 segment of the "knob" heavy chain was defined as "Fab2" having the VH and charge mutation S183E for the second target, and an optional V12 DS for CH1. The subsequent VH-CH1 segment of the "knob" heavy chain was defined as "Fab3," which has charge mutations A141D and V12 DS in VH and CH1 for a third target. To use terminology as used elsewhere in this specification, "Fab1" corresponds to the second antigen-binding arm, "Fab2" corresponds to the third antigen-binding arm fused to the first antigen-binding arm, and "Fab3" corresponds to the first antigen-binding arm.

[0232] The vectors pTriMab-light chain 1 and pTriMab-light chain 2 were constructed on the pDuet-light chain skeleton described in Example 1 above. To construct pTriMab-light chain 1 with a charge mutation, the κ light chain of Fab1 was cloned into the vector by BssHII / NheI as previously described, and the Cκ domain contained the charge mutation V133E. The second (lambda)LC cassette in pTriMab-light chain 1 was removed.

[0233] For the construction of pTriMab-light chain 2 with charge mutations, the kappa light chain of Fab2 was cloned into a vector by BssHII / NheI as previously described, and the Cκ domain contained the charge mutation V133K and, optionally, V12 DS (S121C / C214V for Cκ). The λ light chain of Fab3 was cloned into a vector by BsrGI / EcoRI as previously described, and the Cλ domain contained the charge mutation T117R and V12 DS.

[0234] Expression, affinity purification, and protein quantification. All TriMab constructs were transiently expressed and purified using the same method as described above for the DuetMab molecule.

[0235] 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.

[0236] Simultaneous joining using octets The simultaneous binding of recombinant soluble HER2, EGFR, and CD3 proteins was measured by biolayer interferometry using an Octet384 instrument (ForteBio, Fremont, CA). 3 mg / mL of BSA and 2 μg / mL of triplicate TriMab antibody in 0.05% (v / v) Tween-20 (assay buffer) were captured on an anti-human IgG Fc biosensor (ForteBio) in PBS pH 7.2. After a washing step to remove any unbound proteins, each immobilized biosensor was subjected to sequential association and dissociation interactions, first with HER2 (100 nM), followed by EGFR (200 nM) and CD3 (200 nM). Association and dissociation curves were calculated from nonlinear fitting of the data using Octet384 software v.9.0.

[0237] Cell viability and T cell activation FACS assay Cytotoxicity and T cell activation assays of redirected T cells were performed using a BD FACSymphony® A5 Cell Analyzer. To determine selective cytotoxicity, parental NCI-H358 HER2 KO cells expressing only EGFR ("single-positive") or parental NCI-H358 expressing both EGFR and HER2 ("double-positive") were first stained with CellTracker® Violet (Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were then combined with human PBMCs (peripheral blood mononuclear cells, effector cell:target cell ratio 10:1) and suspended in RPMI1640 supplemented with 10% thermally inactivated FBS and 50 μM 2-mercaptoethanol, and seeded in 96-well plates. Various concentrations of antibodies were added to triple samples, and the cells were incubated in a humidified incubator at 37°C and 5% CO2. To detect T cell activation, cells were stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD69 antibodies (all from BioLegend, San Diego, CA, USA). Data were analyzed using FlowJo v 10.6.1. The number of depleted target cells was determined using the following formula: % lysis = 100 - (surviving cells in the treated group / surviving cells in the untreated control group × 100), and plotted using GraphPad Prism v 9.0.0.

[0238] result Table 10 summarizes the expression and biochemical profiles of HER2 / EGFR / CD3 TriMab with a selected set of charge pairs produced in 500 mL cell cultures. For further analysis, TriMab was further purified by protein A affinity chromatography, and aggregates were removed by preparative CHT column (an incompressible mixed-mode chromatography medium using cation exchange and calcium-affinity interactions).

[0239] [Table 12]

[0240] Table 11 summarizes the thermal stability and accelerated stability profiles of HER2 / EGFR / CD3 TriMab molecules as determined by differential scanning fluorescence (DSF). HER2 / EGFR / CD3 TriMab molecules showed no signs of aggregation or fragmentation after thermal stress.

[0241] [Table 13]

[0242] Figure 13 and Table 12 show the subunit mass spectral data of HER2 / EGFR / CD3 TriMab. Molecular integrity and LC / HC association identity of each peak were confirmed by alignment of theoretical and measured masses.

[0243] [Table 14]

[0244] Figure 14 and Table 13 show the thermal stability study of HER2 / EGFR / CD3 TriMab using differential scanning calorimetry (DSC) analysis. Figure 14 shows the stacked thermogram of HER2 / EGFR / CD3 TriMab. Table 13 lists the deconvoluted TM and approximated T start values ​​for HER2 / EGFR / CD3 TriMab.

[0245] [Table 15]

[0246] Figure 15 shows the simultaneous binding of HER2 / EGFR / CD3 TriMab to HER2, EGFR, and CD3 antigens.

[0247] Figure 16 shows the EGFR / CD3 DuetMab, determined by cell viability and T cell activation FACS assay.

[0248] This study demonstrates the cytotoxic and T-cell activating properties of HER2 / EGFR / CD3 TriMab and HER2 / VκS93A+VHP97A EGFR / CD3 TriMab molecules. NCI-H358 Wt cancer cells positive for both HER2 and EGFR antigens simulated dual-positive target cells in this study, while NCI-H358 HER2 KO cells positive only for EGFR antigen simulated single-positive non-target normal tissue. EGFR affinity-modified HER2 / V κ S93A+V H The P97A EGFR / CD3 TriMab mutant mediated a greater degree of target selectivity compared to the EGFR-high affinity, EGFR / CD3 DuetMab, and HER2 / EGFR / CD3 TriMab molecules, as reflected by the preferential death of bipositive target cells over EGFR-single-positive non-target cells. The improved selectivity mediated by HER2 / VκS93A+VHP97A EGFR / CD3 TriMab for target cells was also reflected by significantly reduced levels of CD8 and CD4 T cell activation when incubated with EGFR-single-positive non-target cells.

[0249] array 1. Amino acid sequence of the WT CLλ constant region (SEQ ID NO: 1) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0250] 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

[0251] [Table 16]

[0252] 3. Amino acid sequence of the WT LC kappa constant (Cκ) region (SEQ ID NO: 3) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0253] 4. Amino acid sequence of IgG1 CH1 (SEQ ID NO: 4) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

[0254] 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

[0255] [Table 17]

[0256] 6. Amino acid sequence of IgG1 CH2 (SEQ ID NO: 6) LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0257] 7. Amino acid sequence of IgG1 CH3 (SEQ ID NO: 7) GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0258] 8. Amino acid sequence of IgG1 heavy chain polypeptide (SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0259] 9. Amino acid sequence of IgG1 CH3 modified to include the "whole" mutation (SEQ ID NO: 9) The following substitutions are underlined. "Hole" mutations (T366S, L368A, and Y407V).

[0260] [Table 18]

[0261] 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).

[0262] [Table 19]

[0263] 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).

[0264] [Table 20]

[0265] 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).

[0266] [Table 21]

[0267] 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).

[0268] [Table 22]

[0269] 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).

[0270] [Table 23]

[0271] 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).

[0272] [Table 24]

[0273] 16. Linker (Sequence ID 16) GGGGS

[0274] 17. Linker (Sequence ID 17) SGGGGS

[0275] 18. Linker (Sequence ID 18) GGGGSGGGGS

Claims

1. It is a triplicate antibody, (a) A first antigen-binding arm comprising a first light chain disulfide-bonded to a first heavy chain constant region 1 (CH1), wherein the first light chain comprises a constant light chain lambda region (CLλ), and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain kappa region (CLκ), and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises CLκ, The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm. The first antigen-binding arm includes one or more lambda charge pairs, each containing a positively charged amino acid residue and an uncharged amino acid residue located at the interface between the first CH1 and the CLλ. Optionally, the second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the second CH1 and the CLκ of the second light chain, and the third antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and a negatively charged amino acid residue located at the interface between the third CH1 and the CLκ of the third light chain, wherein the charged amino acid residue of the kappa charge pair located on the third CH1 and the CLκ of the third light chain has the opposite charge to that of the kappa charge pair located on the second CH1 and the CLκ of the second light chain, and A triple-specific antibody in which the positively charged amino acid residue is arbitrarily selected from arginine, lysine, or histidine, and the uncharged amino acid residue is arbitrarily selected from aspartic acid, glutamic acid, serine, or threonine.

2. It is a triplicate 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 kappa region (CLκ), and (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain lambda region (CLλ), and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises a third antigen-binding arm comprising a CLλ The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm. The first antigen-binding arm optionally includes a kappa charge pair comprising a positively charged amino acid residue and an uncharged amino acid residue located at the interface between the first CH1 and the CLκ, The second antigen-binding arm includes one or more lambda charge pairs, each comprising a positively charged amino acid residue and an uncharged amino acid residue located at the interface between the second CH1 and the CLλ of the second light chain, and The third antigen-binding arm optionally includes one or more lambda charge pairs, each containing a positively charged amino acid residue and an uncharged amino acid residue, located at the interface between the third CH1 and the CLλ of the third light chain, and the charged amino acid residues of the one or more lambda charge pairs located on the third CH1 and the CLλ of the third light chain optionally have the opposite charge to the charged amino acid residues of the one or more lambda charge pairs located on the second CH1 and the CLλ of the second light chain, and A triple-specific antibody in which the positively charged amino acid residue is arbitrarily selected from arginine, lysine, or histidine, and the uncharged amino acid residue is arbitrarily selected from aspartic acid, glutamic acid, serine, or threonine.

3. The one or more lambda charge pairs described above are 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, (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, A trispecific antibody according to claim 1 or 2, wherein the numbering follows the EU index.

4. The triplicate antibody according to any one of claims 1 to 3, wherein one or more lambda charge pairs are located at position 117 of CLλ and position 141 of CH1.

5. The one or more lambda charge pairs mentioned above 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. A triplicate antibody according to any one of claims 1 to 5, selected from lysine at position 117 of CLλ and threonine at position 141 of CH1.

6. The one or more lambda charge pairs mentioned above 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. A trispecific antibody according to any one of claims 1 to 6, selected from lysine at position 117 of CLλ and aspartic acid at position 141 of CH1.

7. The one or more lambda charge pairs are located at position 117 of CLλ and position 185 of CH1, and optionally, the one or more lambda charge pairs are 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. A triplicate antibody according to any one of claims 1 to 6, selected from lysine at position 117 of CLλ and threonine at position 185 of CH1.

8. The one or more lambda charge pairs are located at position 119 of CLλ and position 128 of CH1, and optionally, the one or more lambda charge pairs are 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 triplicate antibody according to any one of claims 1 to 7, selected from lysine at position 119 of CLλ and threonine at position 128 of CH1.

9. The one or more lambda charge pairs are located at position 134 of CLλ and position 128 of CH1, and optionally, the one or more lambda charge pairs are 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 triplicate antibody according to any one of claims 1 to 8, selected from lysine at position 134 of CLλ and threonine at position 128 of CH1.

10. The one or more lambda charge pairs are located at position 134 of CLλ and position 145 of CH1, and optionally, the one or more lambda charge pairs are 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 triplicate antibody according to any one of claims 1 to 9, selected from lysine at position 134 of CLλ and threonine at position 145 of CH1.

11. The triplicate antibody according to any one of claims 1 to 10, wherein the one or more lambda charge pairs are located at position 134 of CLλ and position 183 of CH1, and optionally, the one or more lambda charge pairs are lysine at position 134 of CLλ and aspartic acid or serine at position 183 of CH1.

12. The one or more lambda charge pairs are located at position 136 of CLλ and position 185 of CH1, and optionally, the one or more lambda charge pairs are 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. A triplicate antibody according to any one of claims 1 to 11, selected from lysine at position 136 of CLλ and threonine at position 185 of CH1.

13. The one or more lambda charge pairs are located at position 178 of CLλ and position 173 of CH1, and optionally, the one or more lambda charge pairs are from the following list: i. Arginine at position 178 of CLλ, and aspartic acid at position 173 of CH1, j. Arginine at position 178 of CLλ, and glutamic acid at position 173 of CH1, k. Arginine at position 178 of CLλ, and serine at position 173 of CH1, l. Arginine at position 178 of CLλ, and threonine at position 173 of CH1, m. Lysine at position 178 of CLλ, and aspartic acid at position 173 of CH1, n. Lysine at position 178 of CLλ, and glutamic acid at position 173 of CH1, o. Lysine at position 178 of CLλ, and serine at position 173 of CH1, h. A triplicate antibody according to any one of claims 1 to 12, selected from lysine at position 178 of CLλ and threonine at position 173 of CH1.

14. The kappa charge pair is located at position 133 of CLκ and position 183 of the corresponding CH1 in the antigen-binding arm. A triplicate antibody according to any one of claims 1 to 13, wherein, optionally, the loaded amino acid residue in the kappa pair is glutamic acid, and the positively charged amino acid residue in the kappa pair is lysine.

15. The triplicate antibody according to any one of claims 1 to 14, wherein the kappa charge pair located at the interface between the second CH1 and the CLκ of the second light chain comprises a charged amino acid residue on the CLκ of the second light chain and a charged amino acid residue on the second CH1, and the kappa charge pair located at the interface between the third CH1 and the CLκ of the third light chain comprises a charged amino acid residue on the CLκ of the third light chain and a charged amino acid residue on the third CH1.

16. The triplicate antibody according to any one of claims 1 to 15, wherein the disulfide bond between the light chain and the CH1 in at least one of the antigen-binding arms is formed between a pair of modified cysteine ​​in the light chain and the CH1 in the antigen-binding arm.

17. (i) The disulfide bond between the first light chain and the first CH1 is formed between the pair of modified cysteines of the first light chain and the first CH1, (ii) The disulfide bond between the second light chain and the second CH1 is formed between a pair of natural cysteine, and / or (iii) (a) The disulfide bond between the third light chain polypeptide and the third heavy chain polypeptide is formed between a pair of natural cysteine, or (b) The triplicate antibody according to any one of claims 1 to 16, wherein the disulfide bond between the third light chain and the third CH1 is formed between a pair of modified cysteines of the third light chain and the third CH1, and the pair of inserted cysteines of the third light chain and the third CH1 are at different amino acid residue positions than the pair of inserted cysteines of the first light chain and the first CH1.

18. The triplicate antibody according to claim 17, wherein the pair of modified cysteines of the first light chain and the first CH1 are located at position 122 of the first light chain and position 126 of the first CH1, the first light chain contains a non-cysteine ​​residue at position 212, and the first CH1 contains a non-cysteine ​​residue at position 220, and optionally the non-cysteine ​​residue is valine.

19. The triplicate antibody according to claim 17 or 18, wherein the pair of modified cysteines of the third light chain and the third CH1 are located at position 121 of the third light chain and position 126 of the third CH1, the third light chain contains a non-cysteine ​​residue at position 214, and the third CH1 contains a non-cysteine ​​residue at position 220, and optionally the non-cysteine ​​residue is valine.

20. The triplicate antibody according to any one of claims 1 to 19, 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.

21. The triplicate antibody according to any one of claims 1 to 20, 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.

22. The triplicate antibody according to any one of claims 1 to 21, 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.

23. It is a triplicate 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 comprising a positively charged amino acid residue and an uncharged amino acid residue located at position 117 of CLλ and position 141 of the first CH1, and (ii) The disulfide bond between the first light chain and the first CH1 is formed between the first antigen-binding arm and the modified pair of cysteines of the CLλ of the first light chain and the first CH1, (b) A second antigen-binding arm comprising a second light chain disulfide-bonded to a second CH1, wherein the second light chain comprises a constant light chain kappa region (CLκ), (i) The second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and an uncharged amino acid residue located at position 133 of the CLκ of the second light chain and position 183 of the second CH1, and (ii) The disulfide bond between the second light chain and the second CH1 forms a second antigen-binding arm between the pair of native cysteine ​​molecules of the CLκ of the second light chain and the second CH1, and (c) A third antigen-binding arm comprising a third light chain disulfide-bonded to a third CH1, wherein the third light chain comprises CLκ, (i) The second antigen-binding arm includes a kappa charge pair comprising a positively charged amino acid residue and an uncharged amino acid residue located at position 133 of the CLκ of the second light chain and position 183 of the second CH1, wherein the charged amino acid residue located on the CLκ of the third CH1 and the third light chain has the opposite charge to the charged amino acid residue located on the CLκ of the second CH1 and the second light chain, and (ii) The disulfide bond between the CLκ of the third light chain and the third CH1 is formed between a pair of modified cysteines of the CLκ of the third light chain and the third CH1, and the pair of inserted cysteines of the CLκ of the third light chain and the third CH1 are at different or the same amino acid residue positions as the pair of inserted cysteines of the CLλ of the first light chain and the first CH1, comprising a third antigen-binding arm, The third antigen-binding arm is fused to the first antigen-binding arm or the second antigen-binding arm, and A triple-specific antibody in which the positively charged amino acid residue is arbitrarily selected from arginine, lysine, or histidine, and the uncharged amino acid residue is arbitrarily selected from aspartic acid, glutamic acid, serine, or threonine.

24. The triplicate antibody according to claim 23, wherein the CLλ of the first light chain and the pair of modified cysteines of the first CH1 are located at position 122 of the first light chain and position 126 of the first CH1, the first light chain contains a non-cysteine ​​residue at position 212, the first CH1 contains a non-cysteine ​​residue at position 220, and optionally the non-cysteine ​​residue is valine.

25. The triplicate antibody according to claim 23 or 24, wherein the CLκ of the third light chain and the pair of modified cysteines of the third CH1 are located at position 121 of the third light chain and position 126 of the third CH1, the third light chain contains a non-cysteine ​​residue at position 214, the third CH1 contains a non-cysteine ​​residue at position 220, and optionally the non-cysteine ​​residue is valine.

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

27. The triplicate antibody according to claim 26, 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.

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

29. The triplicate antibody according to claim 28, 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.

30. The substitution to generate the knob is the substitution of tryptophan at position 366, and the substitution to generate the hole is as follows: i) Substitution with valine at position 407, ii) Substitution with serine at position 366, and iii) The triplicate antibody according to claim 29, wherein one or more of the substitutions for alanine at position 368 are present.

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

32. 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 triplicate antibody according to any one of claims 26 to 31, comprising M252Y / S254T / T256E

33. A triplicate antibody according to any one of claims 1 to 32, wherein one of the antigen-binding arms binds to an epitope on CD3.

34. A triplicate antibody according to any one of claims 1 to 33, wherein one of the antigen-binding arms binds to an epitope on CD8.

35. A method for producing a multispecific antibody according to any one of claims 1 to 34, 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 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 binding arm and the third binding arm to form a multispecific antibody, and purifying the multispecific antibody from the host cell.

36. The method according to claim 35, wherein the purification of the triple-specific 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 trispecific antibody is mispaired.

38. One or more nucleic acids encoding a trispecific antibody according to any one of claims 1 to 34.

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 triplicate antibody according to any one of claims 1 to 34 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 trispecific antibody according to any one of claims 1 to 34, or a pharmaceutical composition according to claim 41.

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

44. A triplicate antibody according to any one of claims 1 to 34, or a pharmaceutical composition according to claim 41, for use as a pharmaceutical.

45. A trispecific antibody according to any one of claims 1 to 34, or a pharmaceutical composition according to claim 41, for use in the treatment of cancer.

46. Use of a triplicate antibody according to any one of claims 1 to 34, or a pharmaceutical composition according to claim 41, for the manufacture of a pharmaceutical for the treatment of the aforementioned cancer.

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