JP2023116826A - antigen binding molecule - Google Patents

Abstract

To provide novel therapeutic agents or candidates thereof for treating or preventing diseases, including autoimmune diseases.SOLUTION: Provided is an antigen binding molecule that targets an immune synapse by specifically binding to a complex consisting of a co-inhibitory molecule and its ligand localized at the immune synapse. The antigen binding molecule preferably has agonist activity to the co-inhibitory molecule. The antigen binding molecule can be used not only as a drug having an immunosuppressive effect based on agonist activity toward the co-inhibitory molecule, but also for the detection of a complex consisting of a co-inhibitory molecule and its ligand.SELECTED DRAWING: None

Description

The present invention provides an antigen-binding molecule, a co-inhibitory molecule agonist, a co-inhibitory molecule signal activator, a pharmaceutical composition, a detection agent for a complex comprising a co-inhibitory molecule and its ligand, a method of suppressing immunity, and a co-inhibitory molecule and It relates to a method for detecting a complex consisting of its ligand.

Medicines that pharmacologically target molecules present in immune synapses have attracted attention (Non-Patent Document 1). Among such molecules, therapies targeting co-inhibitory molecules are aimed at cancer treatment. Antibodies targeting CTLA-4 and PD-1, for example, have been approved by the US Food and Drug Administration (FDA), and clinical trials are underway to block TIM-3, LAG3, and TIGIT.

Although the efficacy of agonists against co-inhibitory molecules has been demonstrated in mouse models of autoimmune diseases, no agonists have yet demonstrated efficacy in human diseases (Non-Patent Document 2).

Agonists for PD-1, one of the co-inhibitory molecules, have been tested.
For example, an antibody called PD-1 AB-6 has been disclosed to bind to a β-sheet with a PD-1 loop consisting of residues 100-105 of PD-1 (US Pat. The article discloses that PD-1 AB-6 is designed to provide an agonist through PD-1.
It has also been disclosed that a substance having specificity for human PD-1 is an excellent substance capable of transducing human PD-1 signals (Patent Document 2). However, it has not been experimentally shown whether the bispecific antibodies reactive against PD-1 and CD3 used in the examples of the article have agonistic activity against PD-1.

Phase 1 clinical trials for psoriasis are currently underway using a PD-1 agonistic antibody called CC-90006 (Non-Patent Document 3).
Recently, phase 1 clinical trials for autoimmune diseases have started using ONO-4685, a bispecific antibody that targets both PD-1 and CD3 (Non-Patent Documents 4 and 5). ). ONO-4685 cross-links PD-1 and CD3 expressed on the same T cells and acts as a PD-1 agonist, and cross-links cells expressing PD-1 and CD3 expressed on different cells Therefore, it is expected to have an effect of damaging T cells (Non-Patent Document 6).

WO2018/053405 WO2004/072286

The immunological synapse as a pharmacological target. Finetti F, Baldari CT., Pharmacol. Res. 2018 Vol. 134, pp.118-133. Immune Checkpoints as Therapeutic Targets in Autoimmunity. Christopher P. et al., Frontiers in Immunology 2018, Vol. 9, Article 2306. U.S. National Library of Medicine, ClinicalTrials.gov, "Study to Evaluate the Safety, Tolerability, Pharmacokinetics, Pharmacodynamics, and Immunogenicity of CC-90006 in Subjects With Mild to Moderate Plaque-type Psoriasis", ClinicalTrials.gov Identifier: NCT03337022, Recruiting Status : Recruiting, First Posted: November 8, 2017, Last Update Posted: May 28, 2019 (https://clinicaltrials.gov/ct2/show/NCT03337022) U.S. National Library of Medicine, ClinicalTrials.gov, "Single Ascending Dose Study to Assess the Safety, Tolerability, PK and PD of ONO-4685 in Japanese and Caucasian Healthy Adult Male Subjects", ClinicalTrials.gov Identifier: NCT04079062, Recruiting Status: Recruiting , First Posted: September 6, 2019, Last Update Posted: September 6, 2019 (https://clinicaltrials.gov/ct2/show/NCT04079062) ONO PHARMACEUTICAL CO., LTD. (4528) Summary of Financial Results for the First Quarter of the Fiscal Year Ending March 31, 2020, Supplementary Materials, August 1, 2019 (https://www.ono.co.jp/jpnw/ir/pdf/k_hosoku/ 2020_1h.pdf) ONO PHARMACEUTICAL CO., LTD. (4528) Financial Results for the First Quarter of the Fiscal Year Ending March 2020, Script, August 1, 2019 (https://www.ono.co.jp/jpnw/ir/pdf/k_setsumei/20190805_1. pdf)

There is an unsatisfactory supply of medicines for treating or preventing diseases, including autoimmune diseases. Accordingly, it is expected that additional therapeutic agents or candidates for treating or preventing diseases, including autoimmune diseases, will be created.

The present inventors have created and experimentally confirmed the concept of immune synapse-targeted antigen-binding molecules with potential efficacy against autoimmune diseases. Specifically, the antigen-binding molecule targets the immune synapse by specifically binding to a complex consisting of a co-inhibitory molecule localized at the immune synapse and its ligand. Said antigen-binding molecule preferably has agonist activity to said co-inhibitory molecule. The antigen-binding molecule can be provided for detection of a complex consisting of a co-inhibitory molecule and its ligand, as well as a drug having an immunosuppressive effect based on agonist activity to the co-inhibitory molecule.

The following invention is provided as one aspect in this specification.
[1] An antigen-binding molecule comprising a first antigen-binding domain,
An antigen-binding molecule, wherein said first antigen-binding domain is capable of specifically binding a first complex consisting of a first co-inhibitory molecule and a first co-inhibitory molecule ligand to said first co-inhibitory molecule.
[2] the specific binding of the first antigen-binding domain to the first complex is either the first co-inhibitory molecule in the first complex or the first co-inhibitory molecule ligand; The antigen-binding molecule of [1], which is bound by intermolecular force with one or both.
[3] The antigen-binding molecule of [1] or [2], which can activate the first intracellular signal downstream of the first co-inhibitory molecule in the immune synapse where the first complex is present.
[4] The antigen-binding molecule of [3], wherein activation of the first intracellular signal can suppress activation of T cells.
[5] The immune synapse is formed between a cell expressing MHC and the co-inhibitory molecule ligand and the T cell expressing the co-inhibitory molecule, [3] or [ 4] antigen-binding molecule.
[6] An antigen-binding molecule comprising a first antigen-binding domain,
said first antigen binding domain is capable of binding a first complex consisting of a first co-inhibitory molecule and a first co-inhibitory molecule ligand to said first co-inhibitory molecule, and said first antigen binding domain The binding activity to the first complex of the first co-inhibitory molecule that does not form the first complex and the first co-inhibitory molecule ligand that does not form the first complex An antigen-binding molecule that has higher binding activity to either or both.
[7] binding of the first antigen-binding domain to the first complex is either one of the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex, or The antigen-binding molecule of [6], which is bound by intermolecular forces with both.
[8] The antigen-binding molecule of [6] or [7], which can activate the first intracellular signal downstream of the first co-inhibitory molecule at the immune synapse where the first complex is present.
[9] The antigen-binding molecule of [8], wherein activation of the first intracellular signal can suppress activation of T cells.
[10] The immune synapse is formed between a cell expressing MHC and the co-inhibitory molecule ligand and the T cell expressing the co-inhibitory molecule, [8] or [ 9] antigen-binding molecule.
[11] The antigen-binding molecule of any one of [6] to [10], wherein the binding activity satisfies one or both of the following conditions (a) and (b):
(a) cells of the first co-inhibitory molecule ligand in flow cytometry using a first cell that forcibly expresses the first co-inhibitory molecule but does not forcibly express the first co-inhibitory molecule ligand; The binding activity of the first antigen-binding domain to the first cell in the presence of the first soluble polypeptide containing the ectodomain or a portion thereof is determined by the non-binding activity of the first soluble polypeptide higher than in presence;
(b) in flow cytometry using a second cell that forcibly expresses the first co-inhibitory molecule ligand but does not forcibly express the first co-inhibitory molecule, The binding activity of the first antigen-binding domain to the second cell in the presence of a second soluble polypeptide comprising the domain or a portion thereof is reduced by the absence of the second soluble polypeptide. higher than below.
[12] The antigen-binding molecule of [10], wherein both the first cell and the second cell are derived from Chinese hamster ovary cells.
[13] An antigen-binding molecule comprising a first antigen-binding domain,
an antigen, wherein said first antigen-binding domain is capable of specifically binding to an epitope present in a first complex consisting of a first co-inhibitory molecule and a first co-inhibitory molecule ligand to said first co-inhibitory molecule binding molecule.
[14] The epitope is present only in the first co-inhibitory molecule that does not form the first complex or only in the first co-inhibitory molecule ligand that does not form the first complex not, the antigen-binding molecule of [13].
[15] the specific binding of the first antigen-binding domain to the first complex is either the first co-inhibitory molecule in the first complex or the first co-inhibitory molecule ligand; The antigen-binding molecule of [13] or [14], which is bound by intermolecular forces with one or both.
[16] The antigen binding of any one of [13] to [15], which can activate a first intracellular signal downstream of the first co-inhibitory molecule at an immune synapse where the first complex resides. molecule.
[17] The antigen-binding molecule of [16], wherein activation of the first intracellular signal can suppress activation of T cells.
[18] said immune synapse is formed between cells expressing MHC and said co-inhibitory molecule ligand and said T cells expressing said co-inhibitory molecule, [16] or [ 17].
[19] a third cell expressing the first co-inhibitory molecule and capable of measuring the intensity of the first intracellular signal, an antigen-independent T cell receptor activator and the first co-inhibitory molecule and a fourth cell that expresses a ligand is used in a state in which they can contact each other, and the first intracellular signal in the third cell is partially inhibited by an inhibitory antibody against the first co-inhibitory molecule ligand In the system for measuring the intensity of the first intracellular signal, which is performed under conditions of suppression, the intensity of the first intracellular signal in the presence of the antigen-binding molecule is The antigen-binding molecule of any of [3] to [5], [8] to [10], and [16] to [18] that is elevated compared to its presence.
[20] The antigen-binding molecule of [19], wherein the third cell is derived from Jurkat cells and the fourth cell is derived from Chinese Hamster Ovary cells.
[21] said intensity of said first intracellular signal is directly between said first co-inhibitory molecule and said intracellular domain when said first co-inhibitory molecule is activated; or The antigen-binding molecule of [19] or [20], measured based on proximity to intracellular proteins with which it indirectly interacts.
[22] The antigen-binding molecule of [21], wherein the intracellular protein is a dephosphorylation enzyme.
[23] The T cell receptor and the first co-inhibitory molecule are expressed, the intensity of a second intracellular signal that can be activated downstream of the T cell receptor is measurable, and the first A fifth cell capable of suppressing the second intracellular signal by activation of the intracellular signal and a sixth cell expressing an antigen-independent T cell receptor activator and the first co-inhibitory molecule ligand are used under conditions that allow contact with each other, and
under conditions in which inhibition of the second intracellular signal in the fifth cell by the first co-inhibitory molecule ligand is partially inhibited by an inhibitory antibody to the first co-inhibitory molecule ligand; In the second intracellular signal intensity measurement system,
wherein the intensity of the second intracellular signal in the presence of the antigen-binding molecule is lower than in the absence of the antigen-binding molecule [3] to [5], [8] to [10], and The antigen-binding molecule of any one of [16] to [22].
[24] The antigen-binding molecule of [23], wherein the fifth cell is derived from Jurkat cells and the sixth cell is derived from Chinese hamster ovary cells.
[25] The antigen-binding molecule of [23] or [24], wherein the second intracellular signal is NFAT activity.
[26] the combination of said first co-inhibitory molecule and said first co-inhibitory molecule ligand is a combination of PD-1 and PD-L1, a combination of PD-1 and PD-L2, a combination of BTLA and HVEM, TIGIT and CD155 combination, TIGIT and CD112 combination, LAG-3 and MHC class II molecule combination, CTLA4 and CD80 combination, CTLA4 and CD86 combination, TIM-3 and galectin-9 combination, TIM-3 and phosphatidylserine combination The antigen-binding molecule of any one of [1] to [25], which is any one selected from the group consisting of a combination, a combination of TIM-3 and CEACAM-1, and a combination of TIM-3 and HMGB1.
[27] the antigen-binding molecule is a multiple antigen-binding molecule further comprising a second antigen-binding domain;
Any of [1] to [26], wherein the second antigen-binding domain is capable of specifically binding a second co-inhibitory molecule capable of forming a second complex with a second co-inhibitory molecule ligand antigen-binding molecule.
[28] The antigen-binding molecule of [27], which can activate a third intracellular signal downstream of the second co-inhibitory molecule at an immune synapse where the first complex is present.
[29] The antigen-binding molecule of [28], wherein activation of the third intracellular signal can suppress the second intracellular signal.
[30] binding of said second antigen binding domain to said second co-inhibitory molecule does not compete with binding of said second co-inhibitory molecule ligand to said second co-inhibitory molecule, from [27] Any antigen-binding molecule of [29].
[31] the combination of said second co-inhibitory molecule and said second co-inhibitory molecule ligand is a combination of PD-1 and PD-L1, a combination of PD-1 and PD-L2, a combination of BTLA and HVEM, TIGIT and CD155 combination, TIGIT and CD112 combination, LAG-3 and MHC class II molecule combination, CTLA4 and CD80 combination, CTLA4 and CD86 combination, TIM-3 and galectin-9 combination, TIM-3 and phosphatidylserine combination The antigen-binding molecule of any one of [27] to [31], which is any one combination selected from the group consisting of a combination, a combination of TIM-3 and CEACAM-1, and a combination of TIM-3 and HMGB1.
[32] The antigen binding of any of [27] to [31], wherein the first antigen-binding domain and the second antigen-binding domain are antibody variable regions or target antigen-binding fragments thereof molecule.
[33] The antigen-binding molecule of any one of [27] to [32], wherein said first co-inhibitory molecule is the same as or different from said second co-inhibitory molecule.
[34] When the first co-inhibitory molecule is the same as the second co-inhibitory molecule, the epitope in the first co-inhibitory molecule to which the first antigen-binding domain binds is the second antigen The antigen-binding molecule of [33], which is different from the epitope on said second co-inhibitory molecule bound by the binding domain.
[35] The antigen-binding molecule of any of [27] to [34], wherein said first co-inhibitory molecule ligand is the same as or different from said second co-inhibitory molecule ligand.
[36] The antigen-binding molecule of any of [28] or [29], wherein said first intracellular signal is the same as or different from said third intracellular signal.
[37] The antigen-binding molecule of any one of [1] to [36], further comprising an antibody constant region.
[38] The antigen-binding molecule of [31], wherein at least one residue in the constant region is different from the corresponding amino acid residue in the constant region of the native antibody.
[39] A co-inhibitory molecule agonist comprising the antigen-binding molecule of any one of [1] to [38].
[40] A co-inhibitory molecule signal activator comprising the antigen-binding molecule of any one of [1] to [38].
[41] A pharmaceutical composition comprising the antigen-binding molecule of any one of [1] to [38].
[42] The pharmaceutical composition of [41] for treating or preventing a disease caused by hyperimmunity.
[43] A first complex comprising the antigen-binding molecule of any one of [1] to [38], comprising a first co-inhibitory molecule and a first co-inhibitory molecule ligand to the first co-inhibitory molecule detection agent.
[44] A method of suppressing immunity, which comprises administering the antigen-binding molecule of any one of [1] to [38] to a subject.
[45] from a first co-inhibitory molecule and a first co-inhibitory molecule ligand to said first co-inhibitory molecule, comprising contacting an immune cell with the antigen-binding molecule of any one of [1] to [38] A first method for detecting a complex.
[46] A method for producing an antigen-binding molecule, which comprises culturing a cell containing a nucleic acid encoding the antigen-binding molecule of any one of [1] to [38].
[47] from a non-human animal immunized with a third complex comprising a first co-inhibitory molecule or a partial polypeptide thereof and a first co-inhibitory molecule ligand for said first co-inhibitory molecule or a partial polypeptide thereof, A method for producing an antigen-binding molecule, which comprises collecting plasma or blood containing the antigen-binding molecule of any one of [1] to [38].
[48] The third complex binds the first co-inhibitory molecule or partial polypeptide thereof to the first co-inhibitory molecule ligand or partial polypeptide thereof via a linker or directly by a chemical bond. [47].
[49] The production method of [47] or [48], further comprising obtaining a composition containing the antigen-binding molecule with increased purity from the plasma or the blood.
[50] The production method of any one of [47] to [49], wherein the antigen-binding molecule is a polyclonal antibody.
[51] from a non-human animal immunized with a third complex comprising a first co-inhibitory molecule or a partial polypeptide thereof and a first co-inhibitory molecule ligand for said first co-inhibitory molecule or a partial polypeptide thereof, A method for producing an antigen-binding molecule, which comprises isolating a cell clone expressing the antigen-binding molecule of any one of [1] to [38].
[52] the third complex binds the first co-inhibitory molecule or a partial polypeptide thereof to the first co-inhibitory molecule ligand or a partial polypeptide thereof via a linker or directly by a chemical bond; The production method of [51], which is one molecule obtained by
[53] The production method of [51] or [52], further comprising extracting and isolating the nucleic acid encoding the antigen-binding molecule from the cell clone.
[54] The production method of [53], further comprising recombination of the nucleic acid.
[55] The production method of [54], further comprising introducing the recombinant nucleic acid into another cell to express the antigen-binding molecule in the other cell.
[56] The production method of [55], further comprising obtaining a composition containing the expressed antigen-binding molecule.
[57] The production method of [56], further comprising increasing the purity of the expressed antigen-binding molecule in the composition.

The antigen-binding molecule of the present invention targets the immune synapse by specifically binding to a complex consisting of a co-inhibitory molecule localized at the immune synapse and its ligand, and has agonist activity against the co-inhibitory molecule. It can be a therapeutic agent or a candidate thereof for treating or preventing diseases including autoimmune diseases.

Figure 1 shows the PD-1 signal induction of a bispecific antibody containing an anti-PD-1 arm and an anti-CD3 arm (SHP-2 It is a figure which shows the evaluation result of recruitment) activity. "OKT3//949" is a bispecific antibody containing an OKT3-derived arm and a clone 949-derived arm, and "CE115TR//949" is a bispecific antibody containing a CE115TR-derived arm and a clone 949-derived arm. means FIG. 2 shows the results of evaluating the effect of a bispecific antibody containing an anti-PD-1 arm and an anti-human CD3 arm on the proliferation of human CD4-positive T cells using a T cell proliferation assay. "OKT3/IC17" is a bispecific antibody comprising an OKT3-derived arm and an anti-KLH antibody-derived arm, and "OKT3/PD1-17 (7J13)" comprises an OKT3-derived arm and an anti-KLH antibody-derived arm "OKT3/clone10" is a bispecific antibody comprising an OKT3-derived arm and a clone10-derived arm, "OKT3/clone949" is a bispecific antibody comprising an OKT3-derived arm and a clone949-derived arm A specific antibody is meant. Of these, OKT3/IC17 was used as a negative control. FIG. 3 shows the design of the produced linker-fused human PD-L1/human PD-1 complex protein. The left figure shows the design of Linker complex #1 with a linker inserted between N131 of human PD-L1 and N33 of human PD-1, and the right figure shows the design of E146 of human PD-1 and F19 of human PD-L1. Linker complex#2 is shown with the linker inserted in between. FIG. 4 shows the design of the produced disulfide bond-introduced human PD-L1/human PD-1 complex protein. S-S complex#1 that combines the Y56C variant of human PD-L1 and the A132C variant of human PD-1, and the S-S complex that combines the A18C variant of human PD-L1 and the G90C variant of human PD-1 #2 was produced. FIG. 5 shows the results of specificity evaluation of the anti-human PD-L1/human PD-1 complex antibody. 30 clones (LPB0006, LPB0010, LPB0017, LPB0036, LPB0038, LPC0001, LPC0002, LPC0006, LPC0007, LPC0008, LPC0011, LPC0012, LPC0017, LPC0019, LPC0020, LPC0021, LPC0021, LPC0001) PC0039, LPC0063, LPD0061, LPD0067, LPD0073, LPD0074, LPD0075, LPD0079 , LPE0015, LPE0017, LPE0024, LPE0027, LPE0065, LPE0076, LPB0006, LPB0010, LPB0017, LPB0036, LPB0038, LPC0001, LPC0002, LPC0006, LPC0007, LPC0008, LPC0 011, LPC0012, LPC0017, LPC0019, LPC0020, LPC0021, LPC0039, LPC0063, LPD0061 , LPD0067, LPD0073, LPD0074, LPD0075, LPD0079, LPE0015, LPE0017, LPE0024, LPE0027, LPE0065, LPE0076) bind to the human PD-L1-bound complex. FIG. 5-1 is a continuation of FIG. 5-1; Figure 6 shows PD-1 signal induction (SHP-2 recruitment) activity of anti-human PD-1 antibodies (clone949 and PDA0129) measured by the SHP-2 recruitment assay system using NanoBRET®. It is a figure which shows an evaluation result. “129 homo” is the anti-human PD-1 antibody PDA0129 produced in Example 9, and “949 homo” is the anti-human PD-1 antibody clone 949 produced in Example 1. Figure 7 shows PD-1 signal induction of anti-human PD-L1/human PD-1 complex antibodies (LPC0039 and LPB0006) (SHP -2 recruitment) activity evaluation results. “LPC0039 homo” and “LPB0006 homo” are anti-human PD-L1/human PD-1 complex antibodies produced in Example 7. FIG. 8 shows the results of evaluating the inhibitory activity of anti-human PD-1 antibodies (clone 949, PDA0129, clone 10, PD1-17) on binding between human PD-1 and human PD-L1. PD-1 inhibitory antibodies 5C4 and pembrolizumab were used as positive controls. Anti-KLH (anti-KLH antibody) that does not bind to either PD-1 or PD-L1 was used as a negative control. Figure 9 shows the PD of bispecific antibodies comprising an anti-PD-L1/PD-1 complex arm and an anti-PD-1 arm measured by the SHP-2 recruitment assay system using NanoBRET®. FIG. 3 shows the evaluation results of -1 signal induction (SHP-2 recruitment) activity. "LPE0024", "LPB0017", "LPC0039", "LPC0020", "LPC0017" refer to arms from the anti-human PD-L1/human PD-1 complex antibody generated in Example 7, and "949" and "129 ' refers to the arms from the anti-human PD-1 antibodies clone 949 and PDA0129, respectively. FIG. 9B is a continuation of FIG. 9A; FIG. 10 shows the results of evaluating suppression of NFAT activity by a bispecific antibody containing an anti-PD-L1/PD-1 complex arm and an anti-PD-1 arm. "LPB0010", "LPB0017", "LPC0039", "LPE0024", "LPC0011", "LPC0020", "LPC0001", and "LPB0010" are arms from the anti-human PD-L1 / human PD-1 complex antibody prepared in Example 7. "Clone 949" "PDA0129" refers to the arms from the anti-human PD-1 antibody. FIG. 11 is a diagram explaining the design of each molecular type concept and the function of each arm examined in this example. FIG. 12 is a diagram explaining the mechanism of action of each molecular type concept examined in this example.

A. Definition As used herein, the term "co-inhibitory molecule" refers to a membrane protein expressed in T cells, which suppresses the activity or activation of T cells by specific binding of co-inhibitory molecule ligands expressed in antigen-presenting cells. It is the molecule that produces the signal.
As used herein, the term "co-inhibitory molecule ligand" refers to a membrane protein expressed in antigen-presenting cells, and by specific binding to the co-inhibitory molecule expressed in T cells, the co-inhibitory molecule is expressed in T cells. It is a molecule that produces an intracellular signal through which the activity or activation of the T cell is suppressed.
As used herein, the term "a complex consisting of a co-inhibitory molecule and a co-inhibitory molecule ligand" means that when the co-inhibitory molecule is a combination capable of binding to the co-inhibitory molecule ligand, the co-inhibitory molecule ligand It refers to a complex formed by molecules that are bound to each other.
As used herein, "intermolecular force" is mainly an electromagnetic force acting between a molecule and another molecule. Intermolecular forces are exemplified by ionic interactions, hydrogen bonding, dipole interactions, and van der Waals forces. Intermolecular forces do not include covalent bonds. In macromolecules, intermolecular forces, rather than covalent bonds, can occur between different moieties within the molecule.
As used herein, the term "binding activity" is used to indicate the strength of intermolecular force. The binding activity of a molecule to another molecule is determined by the sum of various intermolecular forces generated between those molecules. Flow cytometry (FCM), surface plasmon resonance (SPR), biolayer interference (BLI), enzyme-linked immunosorbent assay (ELISA) can be employed as common techniques to measure binding activity between molecules. .
As used herein, “specific binding” refers to binding to a target antigen when, for example, the binding activity to the target antigen is higher than that to other antigens. In the present specification, "binding activity" is defined in "i) binding activity" of "(b-2) Second aspect" below.
As used herein, the term “intramolecular conformational change” refers to any one or both conformational changes caused by binding of a co-inhibitory molecule ligand to a co-inhibitory molecule (Structure, 2015, Vol.23, pp. 2341-2348. and J Biol Chem, 2013, Vol.288, pp.11771-11785.). A protein that has formed a higher-order structure so as to have a physiological function may have a plastic region on the surface of the protein that causes a change in its three-dimensional structure through intermolecular interactions with other proteins. A change in protein conformation means a change in the relative positional relationship of a specific amino acid residue with other amino acid residues in a protein that has formed a higher-order structure so as to have a physiological function. Specifically contemplated herein are intramolecular conformational changes that occur upon binding of a co-inhibitory molecule ligand to a co-inhibitory molecule.
As used herein, an "arm" of an antibody means a portion that constitutes a single valence in a bivalent antibody similar to naturally occurring antibodies. Specifically, the arm refers to a portion of an antibody in which one heavy chain Fab and one light chain Fab are linked via disulfide bonds. Antibodies comprising arms are not limited to bivalent antibodies and can be monovalent or multivalent antibodies.

As used herein, the term "antibody" is used in the broadest sense, and as long as it exhibits the desired antigen-binding activity, it is not limited to, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., It encompasses a variety of antibody structures, including bispecific antibodies) and antibody fragments.

An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv ); and multispecific antibodies formed from antibody fragments.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain, including at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Follows the EU Numbering System (also known as the EU Index), as described in MD 1991.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of variable domains usually consist of four FR domains: FR1, FR2, FR3 and FR4. Accordingly, HVR and FR sequences usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, antibodies are analyzed, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). Purified to greater than 95% or 99% purity as measured. For a review of methods for assessment of antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are mutated antibodies that may arise (e.g., mutated antibodies containing naturally occurring mutations, or mutated antibodies that arise during the manufacture of monoclonal antibody preparations. are identical and/or bind to the same epitope, except that they are present in the same amount. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention include, but are not limited to, hybridoma technology, recombinant DNA technology, phage display technology, transgenic animals containing all or part of the human immunoglobulin locus. Such and other exemplary methods for making monoclonal antibodies are described herein.

"Native antibodies" refer to immunoglobulin molecules with varying structures that occur in nature. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

The terms "variable region" or "variable domain" refer to the domains of the heavy or light chains of an antibody that are responsible for binding the antibody to the antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies are usually similar, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). have a structure. (See, eg, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind a particular antigen may be isolated using the VH or VL domains from the antibody that binds the antigen to screen a complementary library of VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as "expression vectors".

B. Antigen-Binding Molecules In one aspect of the invention, an antigen-binding molecule comprises a first antigen-binding domain.
The first antigen-binding domain may have any chemical structure as long as it can bind to the first complex, for example, it may be a low-molecular-weight compound or a high-molecular-weight compound. . The first complex consists of a first co-inhibitory molecule and a first co-inhibitory molecule ligand. The first co-inhibitory molecule ligand is a ligand to the first co-inhibitory molecule. Specific embodiments of the first antigen-binding domain are exemplified in the section "(b-10) Other embodiments" described below.

(b-1) First aspect In one aspect, the first antigen-binding domain can specifically bind to the first complex.

In this aspect, the specific binding of the first antigen-binding domain to the first complex can be binding by intermolecular forces to the first co-inhibitory molecule in the first complex, It may be binding by intermolecular forces to the first co-inhibitory molecule ligand in the complex, or intermolecular binding to both the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex. It can be force-bonded. Specific binding of the first antigen-binding domain to the first complex preferably results from intermolecular forces with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex. may be a bond by The existence of these various binding modalities is dependent on the presence of either the first co-inhibitory molecule or the first co-inhibitory molecule ligand as long as the first antigen-binding domain specifically binds to the first complex. or that intermolecular forces can form on both.

In this embodiment, when the first antigen-binding domain forms a bond with either the first co-inhibitory molecule or the first co-inhibitory molecule ligand by intermolecular forces, the first co-inhibitory molecule ligand is It is envisioned that there is an intramolecular conformational change in either or both the first co-inhibitory molecule and the first co-inhibitory molecule ligand when bound to the first co-inhibitory molecule. In this case, the first antigen-binding domain specifically recognizes the conformation changed by the binding between these two molecules.
For example, if there is a conformational change in one molecule of the first co-inhibitory molecule and the first co-inhibitory molecule ligand when the first co-inhibitory molecule ligand binds to the first co-inhibitory molecule, The first antigen-binding domain recognizes the three-dimensional structure changed by the binding, and binds to the first co-inhibitory molecule or the first co-inhibitory molecule ligand that has undergone the change in three-dimensional structure by intermolecular force. can form.
On the other hand, if there is a conformational change in both the first co-inhibitory molecule ligand and the first co-inhibitory molecule ligand when the first co-inhibitory molecule ligand binds to the first co-inhibitory molecule ligand, The first antigen-binding domain recognizes the conformation of either the first co-inhibitory molecule or the first co-inhibitory molecule ligand changed by the binding, and the first co-inhibitory molecule or the first co-inhibitory molecule ligand with intermolecular forces.

In this embodiment, when the first antigen-binding domain forms an intermolecular bond with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand, the first co-inhibitory molecule ligand is the first There may or may not be a conformational change within the molecules of the first co-inhibitory molecule and the first co-inhibitory molecule ligand upon binding to the co-inhibitory molecule of . In this case, if a conformational change occurs, the conformation of either or both of the first co-inhibitory molecule and the first co-inhibitory molecule ligand can be changed.
In this case, the first antigen-binding domain recognizes an arrangement of atoms unique to the first complex and binds both the first co-inhibitory molecule and the first co-inhibitory molecule ligand by intermolecular forces. can form The arrangement of all atoms recognized by the first antigen-binding domain in this case is either the first co-inhibitory molecule that does not form the first complex, or the first co-inhibitory molecule that does not form the first complex. co-inhibitory molecular ligands alone.

(b-2) Second Aspect In an aspect different from (b-1) above, the first antigen-binding domain can bind to the first complex.

In this aspect, the first complex, wherein the binding activity of the first antigen-binding domain to the first complex is higher than the binding activity to the first co-inhibitory molecule that does not form the first complex Forming a first complex with binding activity to the first co-inhibitory molecule that is higher than the binding activity to the first co-inhibitory molecule ligand that does not form the body, or to the first co-inhibitory molecule that does not form the first complex higher than both binding activities to the first co-inhibitory molecule ligand that was not. The binding activity of the first antigen-binding domain to the first complex preferably includes the binding activity to the first co-inhibitory molecule that does not form the first complex and the activity to form the first complex. The binding activity to the first co-inhibitory molecule ligand is higher than both.

In this embodiment, the binding of the first antigen-binding domain to the first complex can be binding by intermolecular forces to the first co-inhibitory molecule in the first complex, wherein the first complex or by intermolecular forces with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex. can be a bond. The binding of the first antigen-binding domain to the first complex is preferably binding by intermolecular forces with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex. can be The existence of these various binding modalities is the first co-inhibitory molecule and the first co-inhibitory molecule, as long as the first antigen-binding domain binds to the first complex and has the binding activity described above. It means that intermolecular forces can be formed on either one or both of the ligands.

In this embodiment, when the first antigen-binding domain forms a bond with either the first co-inhibitory molecule or the first co-inhibitory molecule ligand by intermolecular forces, the first co-inhibitory molecule ligand is It is envisioned that there is an intramolecular conformational change in either or both the first co-inhibitory molecule and the first co-inhibitory molecule ligand when bound to the first co-inhibitory molecule. In this case, the first antigen-binding domain recognizes the conformation changed by binding between these two molecules more easily than the conformation before the change caused by the binding.
For example, if there is a conformational change in one molecule of the first co-inhibitory molecule and the first co-inhibitory molecule ligand when the first co-inhibitory molecule ligand binds to the first co-inhibitory molecule, The first antigen-binding domain recognizes the conformation changed by the binding more easily than the conformation before the change, and the first co-inhibitory molecule or the first co-inhibition molecule that has undergone the change in conformation It is easy to form a bond with a ligand by intermolecular force.
On the other hand, if there is a conformational change in both the first co-inhibitory molecule ligand and the first co-inhibitory molecule ligand when the first co-inhibitory molecule ligand binds to the first co-inhibitory molecule ligand, The first antigen-binding domain recognizes the conformation of either the first co-inhibitory molecule or the first co-inhibitory molecule ligand changed by the binding more easily than the conformation before the change, and It is likely to form a bond with the first co-inhibitory molecule or the first co-inhibitory molecule ligand that has undergone a conformational change due to intermolecular forces.

In this embodiment, when the first antigen-binding domain forms an intermolecular bond with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand, the first co-inhibitory molecule ligand is the first There may or may not be a conformational change within the molecules of the first co-inhibitory molecule and the first co-inhibitory molecule ligand upon binding to the co-inhibitory molecule of . In this case, if a conformational change occurs, the conformation of either or both of the first co-inhibitory molecule and the first co-inhibitory molecule ligand can be changed. In this case, the first antigen-binding domain has an atomic arrangement unique to the first complex and an atomic arrangement in the first co-inhibitory molecule that does not form the first complex and the first complex. and are more likely to form bonds with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand by intermolecular forces. The arrangement of all atoms recognized by the first antigen-binding domain in this case is either the first co-inhibitory molecule that does not form the first complex, or the first co-inhibitory molecule that does not form the first complex. co-inhibitory molecular ligands alone.

i) Binding Activity The binding activity in this embodiment may be measured by a generally used intermolecular binding activity measurement method. Flow cytometry (FCM), surface plasmon resonance (SPR), biolayer interference (BLI), enzyme-linked immunosorbent assay (ELISA) can be selected as methods for measuring binding activity between molecules. When the antigen-binding molecule contains only one of the first antigen-binding molecules, when the binding activity of the antigen-binding molecule to the first complex is measured by these assays, the first complex of the antigen-binding molecule Affinity to is measured. When the antigen-binding molecule contains a plurality of first antigen-binding molecules, when the binding activity of the antigen-binding molecule to the first complex is measured by these assays, the binding activity of the antigen-binding molecule to the first complex is Avidity is measured.

Binding activity in this embodiment is preferably measured by flow cytometry. Binding of the first co-inhibitory molecule to the first co-inhibitory molecule ligand in the first complex, as the first complex is formed on the surface of the cell when the avidity is measured by flow cytometry The embodiment more closely approximates the in vivo environment than other measurements. The binding activity of the antigen-binding molecule to the first complex measured by flow cytometry tends to reflect the in vivo environment.

An example of flow cytometry for measuring the binding activity in this embodiment is performed using a first cell that forces expression of the first co-inhibitory molecule and does not force expression of the first co-inhibitory molecule ligand. .
In this example, the binding activity of the first antigen-binding domain to the first cell is measured in the presence and absence of the first soluble polypeptide.
In the example, the binding activity of the first antigen-binding domain to the first cell in the presence of the first soluble polypeptide is higher than in the absence of the first soluble polypeptide. .

The first cell may be any animal-derived cell that forcibly expresses the first co-inhibitory molecule and does not forcibly express the first co-inhibitory molecule ligand. The first cell preferably does not substantially express the first co-inhibitory molecule ligand. The first cell more preferably does not express the first co-inhibitory molecule ligand.
The first cells are preferably cells that can be cultured in suspension. A first cell type includes cells derived from Chinese hamster ovary cells.

The first antigen-binding domain used for measurement in this example may be contained in the antigen-binding molecule or may be a part of the antigen-binding molecule. An amino acid sequence that is not contained in the antigen-binding molecule may be added to the first antigen-binding domain used for measurement.
The first soluble polypeptide used for the measurement in this example may be any polypeptide as long as it contains the extracellular domain of the first co-inhibitory molecule ligand or a portion thereof and is soluble. . The extracellular domain of the first co-inhibitory molecule ligand comprises an Ig-V-like domain and an Ig-C-like domain. The first soluble polypeptide used for measurement should contain at least the Ig-V-like domain of the first co-inhibitory molecule ligand. That is, the first soluble polypeptide used for measurement may be any polypeptide as long as it contains the Ig-V-like domain of the first co-inhibitory molecule ligand and is soluble. The first soluble polypeptide used for measurement preferably comprises the Ig-V-like domain of the first co-inhibitory molecule ligand and is in soluble form.

Another example of flow cytometry for measuring the binding activity in this embodiment uses a second cell that forces expression of the first co-inhibitory molecule ligand and does not force expression of the first co-inhibition molecule. is done.
In this example, the binding activity of the first antigen-binding domain to the second cell is measured in the presence and absence of the second soluble polypeptide.
In this example, the binding activity of the first antigen-binding domain to the second cell in the presence of the second soluble polypeptide is higher than in the absence of the second soluble polypeptide. .

The second cell may be any animal-derived cell that forcibly expresses the first co-inhibitory molecule ligand and does not forcibly express the first co-inhibitory molecule. The second cell preferably does not substantially express the first co-inhibitory molecule. The second cell more preferably does not express the first co-inhibitory molecule.
The second cells are preferably cells that can be cultured in suspension. A second cell type includes cells derived from Chinese hamster ovary cells.

The first antigen-binding domain used for measurement in this example may be contained in the antigen-binding molecule or may be a part of the antigen-binding molecule. An amino acid sequence that is not contained in the antigen-binding molecule may be added to the first antigen-binding domain used for measurement.
The second soluble polypeptide used for measurement in this example may be any polypeptide as long as it contains the extracellular domain of the first co-inhibitory molecule or a portion thereof and is soluble. The extracellular domain of the first co-inhibitory molecule contains an Ig-V-like domain. The second soluble polypeptide used for measurement should contain at least the Ig-V-like domain of the first co-inhibitory molecule. That is, the second soluble polypeptide used for measurement may be any polypeptide as long as it contains the Ig-V-like domain of the first co-inhibitory molecule and is soluble. The second soluble polypeptide used for measurement preferably contains the Ig-V-like domain of the first co-inhibitory molecule and is in soluble form.

Specific embodiments of the binding activity include embodiments that satisfy one or both of the following conditions (a) and (b).
(a) cells of the first co-inhibitory molecule ligand in flow cytometry using a first cell that forcibly expresses the first co-inhibitory molecule but does not forcibly express the first co-inhibitory molecule ligand; The binding activity of the first antigen-binding domain to the first cell in the presence of the first soluble polypeptide containing the ectodomain or a portion thereof is determined by the non-binding activity of the first soluble polypeptide higher than in existence.
(b) in flow cytometry using a second cell that forcibly expresses the first co-inhibitory molecule ligand but does not forcibly express the first co-inhibitory molecule, The binding activity of the first antigen-binding domain to the second cell in the presence of a second soluble polypeptide comprising the domain or a portion thereof is reduced by the absence of the second soluble polypeptide. higher than below.

In this specific embodiment, the first cell of (a) can be derived from Chinese Hamster Ovary cells. The second cell in (b) can be derived from Chinese Hamster Ovary cells. Both the first cell and the second cell can be derived from Chinese Hamster Ovary cells.
In this specific embodiment, "forced expression" is expression of a foreign protein by genetic recombination in cells.
Flow cytometry can be commonly used by those skilled in the art, and commercially available flow cytometry can be used.
The first soluble polypeptide may be any polypeptide comprising the extracellular domain of the first co-inhibitory molecule ligand or a portion thereof, and is preferably in soluble form. The extracellular domain of the first co-inhibitory molecule ligand or part thereof preferably comprises the Ig-V-like domain of the first co-inhibitory molecule ligand and is in soluble form.
The second soluble polypeptide may be any polypeptide comprising the extracellular domain of the first co-inhibitory molecule or a portion thereof, and is preferably in soluble form. The extracellular domain of the first co-inhibitory molecule or part thereof preferably comprises the Ig-V-like domain of the first co-inhibitory molecule and is in soluble form.

As a mechanism by which the binding activity is detected in the above embodiment (a), first, the first soluble polypeptide is forcibly expressed in the first cell in the presence of the first soluble polypeptide. A first complex is formed on the first cell surface by binding to the first co-inhibitory molecule. A polypeptide comprising the first antigen-binding domain (which may be an antigen-binding molecule) then binds to the first complex formed. This binding is detected by flow cytometry. Since the first antigen-binding domain specifically binds to the first complex, this binding is not detected in the absence of the first soluble polypeptide.
As a mechanism by which the binding activity is detected in the above embodiment (b), first, the second soluble polypeptide is forcibly expressed in the second cell in the presence of the second soluble polypeptide. A first complex is formed on the second cell surface by binding to the first co-inhibitory molecule ligand. A polypeptide comprising the first antigen-binding domain (which may be an antigen-binding molecule) then binds to the first complex formed. This binding is detected by flow cytometry. Since the first antigen binding domain specifically binds to the first complex, this binding is not detected in the absence of the second soluble polypeptide.

(b-3) Third embodiment In an embodiment different from the above (b-1) and (b-2), the first antigen-binding domain comprises the first co-inhibitory molecule and the first co-inhibitory molecule. It can specifically bind to an epitope present in a first complex consisting of a first co-inhibitory molecule ligand to the molecule.
The epitope is preferably absent from the first co-inhibitory molecule alone not forming the first complex or only the first co-inhibitory molecule ligand not forming the first complex. In this aspect, when both a portion of the first co-inhibitory molecule that does not form the first complex and a portion of the first co-inhibitory molecule ligand that does not form the first complex are used as epitopes In addition to encompassing Conformation present in the epitope and in the first co-inhibitory molecule ligand that does not form the first complex but not in the first co-inhibitory molecule ligand that does not form the first complex The epitope includes the case where the conformational change described below is used as the epitope, such as the case where the is used as the epitope.

In this embodiment, the specific binding of the first antigen-binding domain to the epitope can be binding by intermolecular forces to the first co-inhibitory molecule in the first complex, may be intermolecular binding to the first co-inhibitory molecule ligand, or in intermolecular binding to both the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex. could be. The specific binding of the first antigen-binding domain to the epitope is preferably by intermolecular binding of both the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex. could be. The existence of these different binding modalities can be attributed to either or both the first co-inhibitory molecule and the first co-inhibitory molecule ligand, so long as the first antigen binding domain specifically binds to the epitope. It means that intermolecular forces can be formed. The intermolecular force can be an intermolecular force that occurs between the first antigen binding domain and the epitope.

In this embodiment, when the first antigen-binding domain forms a bond with either the first co-inhibitory molecule or the first co-inhibitory molecule ligand by intermolecular forces, the first co-inhibitory molecule ligand is It is envisioned that there is an intramolecular conformational change in either or both the first co-inhibitory molecule and the first co-inhibitory molecule ligand when bound to the first co-inhibitory molecule. In this case, the first antigen-binding domain specifically recognizes, as an epitope, the conformation changed by the binding between these two molecules.
For example, if there is a conformational change in one molecule of the first co-inhibitory molecule and the first co-inhibitory molecule ligand when the first co-inhibitory molecule ligand binds to the first co-inhibitory molecule, The first antigen-binding domain recognizes the conformation changed by the binding as an epitope, and the first co-inhibitory molecule or the first co-inhibitory molecule ligand and the intermolecular force that caused the change in conformation can form a bond.
On the other hand, if there is a conformational change in both the first co-inhibitory molecule ligand and the first co-inhibitory molecule ligand when the first co-inhibitory molecule ligand binds to the first co-inhibitory molecule ligand, The first antigen-binding domain recognizes, as an epitope, the conformation of either the first co-inhibitory molecule or the first co-inhibitory molecule ligand that has changed due to its binding, and undergoes a conformational change. It can form an intermolecular bond with the first co-inhibitory molecule or the first co-inhibitory molecule ligand.

In this embodiment, when the first antigen-binding domain forms an intermolecular bond with both the first co-inhibitory molecule and the first co-inhibitory molecule ligand, the first co-inhibitory molecule ligand is the first There may or may not be a conformational change within the molecules of the first co-inhibitory molecule and the first co-inhibitory molecule ligand upon binding to the co-inhibitory molecule of . In this case, if a conformational change occurs, the conformation of either or both of the first co-inhibitory molecule and the first co-inhibitory molecule ligand can be changed.
In this case, the first antigen-binding domain recognizes an arrangement of atoms unique to the first complex as an epitope, and the epitope and molecule in both the first co-inhibitory molecule and the first co-inhibitory molecule ligand. Inter-force bonds can be formed. The arrangement of all atoms in the epitope recognized by the first antigen-binding domain in this case is either only the first co-inhibitory molecule not forming the first complex, or not forming the first complex. Absent only in the first co-inhibitory molecule ligand.

(b-4) Effect of antigen-binding molecule on immune synapse In the above-described embodiments (b-1), (b-2) and (b-3), the antigen-binding molecule At the synapse, a first intracellular signal downstream of the first co-inhibitory molecule may be activated.
The "first intracellular signal downstream of the first co-inhibitory molecule" means an intracellular signal that occurs when the first co-inhibitory molecule is activated in a cell that expresses the first co-inhibitory molecule. do. In T cells, activation of the first intracellular signal may have the function of suppressing the signal from the T cell receptor. As a result, T cell activation can be suppressed. Here, T cell activation refers to T cell phenotypes that appear when immunity is activated, such as T cell proliferation and differentiation into activated cells, as well as increased production of cytokines that stimulate immunity. is. T cells specifically include CD4(+)FoxP3(-) T cells, and CD8(+) T cells.

"Immune synapse" is a ring-shaped structure formed between cells expressing co-inhibitory molecule ligands and cells of the immune system expressing co-inhibitory molecules that are in close proximity and contact. (Front Immunol 2016 Vol.7 Article 255, Immunology 2011 Vol.133 pp.420-425). Synapses formed between cells expressing MHC and co-inhibitory molecule ligands and T cells expressing co-inhibitory molecules are exemplified as immune synapses.

(b-5) Measurement of the intensity of the first intracellular signal In one embodiment, the intensity of the first intracellular signal generated by the activation of the first co-inhibitory molecule can be measured by in vitro experiments. In this measurement system, when the intensity of the first intracellular signal in the presence of the antigen-binding molecule is higher than in the absence of the antigen-binding molecule, the first intracellular signal is activated by the antigen-binding molecule. It can be said that

In this embodiment, the third cell expressing the first co-inhibitory molecule and the fourth cell expressing the antigen-independent T cell receptor activator and the first co-inhibitory molecule ligand are contactable with each other. A measurement system used in such a state is exemplified as the first intracellular signal intensity measurement system. The third cell is a cell capable of measuring the intensity of the first intracellular signal, which is the downstream intracellular signal generated by activation of the first co-inhibitory molecule.

When the first intracellular signal in the third cell is maximally activated by the first co-inhibitory molecule ligand in the fourth cell in the measurement system, the antigen-binding molecule is the first activation of intracellular signals cannot be confirmed. Therefore, in said exemplification, the measurement of the strength of the first intracellular signal is performed to maximize the activation of the first intracellular signal in the third cell by the first co-inhibitory molecule ligand expressed by the fourth cell. performed under unmet conditions. To achieve such conditions, a method of partially suppressing the first intracellular signal in the third cell by using an inhibitory antibody against the first co-inhibitory molecule ligand in the fourth cell is applied. obtain. A known inhibitory antibody can be applied as the inhibitory antibody against the first co-inhibitory molecule ligand. For example, as an inhibitory antibody against PD-L1, YW243.55S70 (see US2016/0222117 A1; heavy chain variable region SEQ ID NO: 16 and light chain variable region SEQ ID NO: 17) can be used.

In the exemplification, the third cell can be derived from Jurkat cells. A fourth cell may be derived from Chinese Hamster Ovary cells. In a preferred embodiment, the third cell is derived from Jurkat cells and the fourth cell is derived from Chinese Hamster Ovary cells. The fourth cell is exemplified by Promega's PD-L1-expressing artificial antigen-presenting cells (#J109A).

In said exemplification, the strength of the first intracellular signal interacts directly or indirectly with the intracellular domain of the first co-inhibitory molecule and said intracellular domain that occurs when the first co-inhibitory molecule is activated. It can be measured based on proximity to working intracellular proteins. The proximity detection system may be any detection system as long as it can observe the proximity between molecules present in cells and quantify the intensity thereof. A specific example is NanoBRET (registered trademark).

In the exemplification, the intracellular protein that directly or indirectly interacts with the intracellular domain of the first co-inhibitory molecule may be known or newly discovered by an experimenter. good. Dephosphorylation enzymes are exemplified as the intracellular proteins. Specifically, when the co-inhibitory molecule is PD-1, the dephosphorylation enzyme is exemplified by SHP-2.

(b-6) Measurement of suppression of immune activation signal by first intracellular signal In one aspect, immune activation signal is suppressed by first intracellular signal generated by activation of first co-inhibitory molecule It can be confirmed by in vitro experiments. In the experiment, when the strength of the immune activation signal in the presence of the antigen-binding molecule is lower than in the absence of the antigen-binding molecule, it can be said that the antigen-binding molecule suppresses the immune activation signal. . The immune activation signal is preferably a second intracellular signal that can be activated downstream of the T cell receptor in T cells.

In this embodiment, a fifth cell expressing the T cell receptor and the first co-inhibitory molecule and a sixth cell expressing the antigen-independent T cell receptor activator and the first co-inhibitory molecule ligand A measurement system that is used under conditions that allow contact with each other is exemplified as the second intracellular signal intensity measurement system. The fifth cell is capable of measuring the strength of a second intracellular signal that is a downstream intracellular signal resulting from activation of the T cell receptor, wherein activation of the first intracellular signal results in Cells in which intracellular signals can be suppressed. The sixth cell can use the fourth cell used in (b-5) above.

In the fifth cell of the measurement system, the second intracellular signal of the first co-inhibitory molecule of the fifth cell is generated due to the binding of the first co-inhibitory molecule ligand of the sixth cell. When activation and subsequent activation of the first intracellular signal lead to a state of maximum suppression, it cannot be confirmed that the antigen-binding molecule suppresses the second intracellular signal. Therefore, the measurement of the intensity of the second intracellular signal in the exemplification indicates that the second intracellular signal in the fifth cell is maximally suppressed by the first co-inhibitory molecule ligand expressed by the sixth cell. under no conditions. In order to achieve such conditions, a method of partially suppressing suppression of the second intracellular signal in the fifth cell by using an inhibitory antibody against the first co-inhibitory molecule ligand in the sixth cell. can be applied. A known inhibitory antibody can be applied as the inhibitory antibody against the first co-inhibitory molecule ligand. For example, as an inhibitory antibody against PD-L1, YW243.55S70 (see US2016/0222117 A1; heavy chain variable region SEQ ID NO: 16 and light chain variable region SEQ ID NO: 17) can be used.

In the exemplification, the fifth cell can be derived from Jurkat cells. A sixth cell may be derived from Chinese Hamster Ovary cells. In a preferred embodiment, the fifth cell is derived from Jurkat cells and the sixth cell is derived from Chinese Hamster Ovary cells. The sixth cell is exemplified by Promega's PD-L1-expressing artificial antigen-presenting cells (#J109A).

In this exemplification, the strength of the second intracellular signal can be measured based on transcriptional activity by transcription factors acting downstream of the T cell receptor signal. The measurement system for transcriptional activity may be based on modification or binding of factors known to act downstream of T cell receptor signals, or based on changes in gene expression level. Said modification may be a change in the phosphorylation state of a particular protein. The binding may be an interaction between specific proteins. A reporter gene assay in which a gene encoding a protein having some enzymatic activity is fused to a promoter recognized by a specific transcription factor may be used to detect the change in gene expression level. Specifically, in an aspect in which the second intracellular signal is the activity of the transcription factor NFAT, a reporter gene fused downstream of the NFAT response element so that the expression of luciferase can be controlled is the first. 5 cells and reacted with luciferase, is exemplified as a reporter gene assay that can measure the intensity of the signal downstream of the T cell receptor signal.

(b-7) Combination of the first co-inhibitory molecule and the first co-inhibitory molecule ligand In one aspect, the first co-inhibitory in the first complex to which the first antigen-binding domain can specifically bind The molecule can be appropriately selected from co-inhibitory molecules that are known at the time by a person who intends to obtain an antigen-binding molecule. First co-inhibitory molecules typically include PD-1, BTLA, TIGIT, LAG-3, CTLA4, and TIM-3.

In one aspect, the first co-inhibitory molecule ligand in the first complex can be appropriately selected from the range of co-inhibitory molecule ligands known at the time by the person seeking to obtain the antigen-binding molecule. First co-inhibitory molecular ligands typically include PD-L1, PD-L2, HVEM, CD155, CD112, MHC class II molecules, CD80, CD86, galectin-9, phosphatidylserine, CEACAM-1, and HMGB1. be done.

In these embodiments, the combination of the first co-inhibitory molecule and the first co-inhibitory molecule ligand is preferably a combination of PD-1 and PD-L1, a combination of PD-1 and PD-L2, a combination of BTLA and HVEM , TIGIT and CD155 combination, TIGIT and CD112 combination, LAG-3 and MHC class II molecule combination, CTLA4 and CD80 combination, CTLA4 and CD86 combination, TIM-3 and galectin-9 combination, TIM-3 and Any one selected from the group consisting of a combination of phosphatidylserine, a combination of TIM-3 and CEACAM-1, and a combination of TIM-3 and HMGB1.

(b-8) Multispecific antigen-binding molecule In one aspect, the antigen-binding molecule may further comprise a second antigen-binding domain. In that case, the antigen-binding molecule is a multispecific antigen-binding molecule capable of binding to more than one antigen or epitope. The second antigen binding domain is capable of specifically binding a second co-inhibitory molecule capable of forming a second complex with a second co-inhibitory molecule ligand. The second antigen binding domain may or may not bind to the second complex. The first antigen-binding domain is characterized by binding to the first complex, whereas the second antigen-binding domain may or may not bind to the second complex The two domains differ in that respect. Preferably, the second antigen binding domain has agonist activity towards the second co-inhibitory molecule.
The second antigen-binding domain may have any chemical structure as long as it has the characteristics described above, and may be, for example, a low-molecular-weight compound or a high-molecular-weight compound. Specific embodiments of the second antigen-binding domain are exemplified in the section "(b-10) Other Embodiments" described below.

In this embodiment, the multispecific antigen binding molecule can activate a third intracellular signal downstream of the second co-inhibitory molecule at the immune synapse where the first complex resides. The intensity of the third intracellular signal can be measured in the same manner as "measurement of the intensity of the first intracellular signal" in (b-5) above. When measuring the intensity of the third intracellular signal, in the "measurement of the intensity of the first intracellular signal", the first co-inhibitory molecule is the second co-inhibitory molecule, the first co-inhibitory molecule Each ligand is replaced by a second co-inhibitory molecule ligand. The ability of the multispecific antigen-binding molecule to activate the third intracellular signal downstream of the second co-inhibitory molecule is the agonistic activity of the second antigen-binding domain to the second co-inhibitory molecule. preferable.

In this embodiment, activation of the third intracellular signal may suppress the second intracellular signal. The intensity of the second intracellular signal can be measured in the same manner as "measurement of suppression of immunostimulatory signal by first intracellular signal" in (b-6) above.

In this embodiment, binding of the second antigen binding domain to the second co-inhibitory molecule preferably does not compete with binding of the second co-inhibitory molecule ligand to the second co-inhibitory molecule.

(b-9) A combination of a second co-inhibitory molecule and a second co-inhibitory molecule ligand In one aspect, the second co-inhibitory molecule to which the second antigen-binding domain can specifically bind is a multispecific antigen The co-inhibitory molecule can be appropriately selected from the range known at the time by a person who intends to obtain the binding molecule. Second co-inhibitory molecules typically include PD-1, BTLA, TIGIT, LAG-3, CTLA4, and TIM-3.

In one embodiment, the second co-inhibitory molecule ligand capable of forming a second complex with the second co-inhibitory molecule is a co-inhibitory molecule within the range known at the time by a person seeking to obtain a multispecific antigen-binding molecule. It can be appropriately selected from inhibitory molecule ligands. Second co-inhibitory molecular ligands typically include PD-L1, PD-L2, HVEM, CD155, CD112, MHC class II molecules, CD80, CD86, galectin-9, phosphatidylserine, CEACAM-1, and HMGB1. be done.

In these embodiments, the combination of the second co-inhibitory molecule and the second co-inhibitory molecule ligand is preferably a combination of PD-1 and PD-L1, a combination of PD-1 and PD-L2, a combination of BTLA and HVEM , TIGIT and CD155 combination, TIGIT and CD112 combination, LAG-3 and MHC class II molecule combination, CTLA4 and CD80 combination, CTLA4 and CD86 combination, TIM-3 and galectin-9 combination, TIM-3 and Any one selected from the group consisting of a combination of phosphatidylserine, a combination of TIM-3 and CEACAM-1, and a combination of TIM-3 and HMGB1.

(b-10) Other Embodiments In another embodiment, one molecule of the first co-inhibitory molecule may be present in the first complex, and a plurality of molecules of the first co-inhibitory molecule are present in the first complex. may be There may be one molecule of the first co-inhibitory ligand in the first complex, or there may be multiple molecules of the first co-inhibitory ligand. The first complex may comprise molecules other than the first co-inhibitory molecule and the first co-inhibitory molecule ligand.

In other embodiments, the first antigen-binding domain can be the variable region of an antibody or fragment thereof that is capable of binding to the target antigen. The first antigen-binding domain is preferably the variable region of an antibody or a fragment thereof that is capable of binding the target antigen. The "variable region of an antibody or its target antigen-binding fragment" in this embodiment means an arm. The first antigen-binding domain can specifically bind the first complex with only one arm. By doing so, the antigen-binding molecule can localize to the immune synapse when the first complex is present at the immune synapse.

In other embodiments, the second antigen binding domain can be the variable region of an antibody or fragment thereof that is capable of binding to the target antigen. The second antigen binding domain is preferably the variable region of an antibody or a fragment thereof that is capable of binding the target antigen. The "variable region of an antibody or its target antigen-binding fragment" in this embodiment means an arm. The second antigen binding domain may specifically bind the second co-inhibitory molecule with only one arm. Upon such binding, the second antigen binding domain can activate a third intracellular signal.

Both the first antigen-binding domain and the second antigen-binding domain are preferably antibody variable regions or fragments thereof that are capable of binding to the target antigen.

In other embodiments, the first co-inhibitory molecule ligand may be the same as or different from the second co-inhibitory molecule ligand. The first co-inhibitory molecule may be the same as or different from the second co-inhibitory molecule. Similarly in their downstream signals, the first intracellular signal may be the same as or different from the third intracellular signal. When the first co-inhibitory molecule is the same as the second co-inhibitory molecule, preferably the epitopes in the co-inhibitory molecule bound by the first antigen-binding domain and the second antigen-binding domain are different from each other.

In other embodiments, the antigen-binding molecule can further comprise an antibody constant region. At least one residue in the constant region of the antibody may differ from the amino acid residue at the corresponding position in the constant region of the native antibody. In further embodiments, the antigen binding molecule can be an antibody or antibody fragment. The antibody is preferably an isolated antibody.

In other embodiments, the antigen-binding molecule may contain a polypeptide, sugar chain, or low-molecular-weight compound in addition to the first antigen-binding domain, the second antigen-binding domain, and the constant region of the antibody. These structures may be linked to each other by linkers or covalent bonds, or may exist as a complex due to intermolecular forces. Preferably, all constituents are covalently linked directly or via linkers. The type of linker is not particularly limited as long as it can be used for medicine.

(b-11) Manufacturing method In another aspect of the present invention, a method for manufacturing the antigen-binding molecule described above is provided.
In one aspect, a method for producing an antigen-binding molecule can comprise culturing a cell containing a nucleic acid encoding the antigen-binding molecule.

In another embodiment, the method for producing an antigen-binding molecule comprises a first co-inhibitory molecule or a partial polypeptide thereof and a third complex comprising a first co-inhibitory molecule ligand for the first co-inhibitory molecule or a partial polypeptide thereof It can involve immunizing the body against a non-human animal. The third complex is formed by directly binding the first co-inhibitory molecule or partial polypeptide thereof to the first co-inhibitory molecule ligand or partial polypeptide thereof via a linker or chemically. It can be a molecule.
The linker is not particularly limited as long as it can chemically bind the first co-inhibitory molecule or partial polypeptide thereof to the first co-inhibitory molecule ligand or partial polypeptide thereof. The chemical bond is not particularly limited as long as it allows the third complex to function as an antigen. Examples of such chemical bonds include covalent bonds. If the chemical bond is a covalent bond, the stability of the third complex is enhanced. The covalent bond includes a disulfide bond utilizing a thiol group present on the side chain of a cysteine residue.

The production method may comprise collecting plasma or blood containing the antigen-binding molecule from the non-human animal immunized with the third complex. The production method may further comprise obtaining a composition containing the antigen-binding molecule with increased purity from the plasma or the blood.
In this aspect, in the production method, the antigen-binding molecule can be a polyclonal antibody.

The production method can comprise isolating cell clones expressing the antigen-binding molecule from the non-human animal immunized with the third conjugate. The production method may further comprise extracting and isolating the nucleic acid encoding the antigen-binding molecule from the cell clone. The manufacturing method may further comprise recombining the nucleic acid. The production method may further comprise introducing the recombinant nucleic acid into another cell to express the antigen-binding molecule in the other cell. The manufacturing method can further comprise obtaining a composition containing the expressed antigen-binding molecule. The production method may further comprise increasing the purity of the expressed antigen-binding molecule in the composition.

C. Use of antigen-binding molecule (c-1) Co-inhibitory molecule agonist In another aspect of the present invention, a co-inhibitory molecule agonist is provided.
In one aspect, the co-inhibitory molecule agonist comprises an antigen binding molecule as described above. The co-inhibitory molecule agonist has been unexpectedly used as an agonist to the co-inhibitory molecule, in addition to being used with the expectation of acting as an agonist to the co-inhibitory molecule, but as a result of the use It also includes cases where it was or was found to have agonist activity against.

(c-2) Co-inhibitory molecule signal activator In another aspect of the present invention, a co-inhibitory molecule signal activator is provided.
In one aspect, the co-inhibitory molecule signaling activator comprises an antigen binding molecule as described above. The co-inhibitory molecule signal activator has been unexpectedly used as an agonist to a co-inhibitory molecule, in addition to the case where it is expected to act as an agonist to the co-inhibitory molecule, but as a result of the use Cases that have or are found to have agonistic activity to co-inhibitory molecules are also included.

(c-3) Pharmaceutical Composition In another aspect of the present invention, a pharmaceutical composition is provided.
In one aspect, the pharmaceutical composition may contain the antigen-binding molecules described above. The pharmaceutical composition can be used to treat or prevent diseases caused by immune hypersensitivity. Diseases caused by abnormal hyperimmunity include autoimmune diseases.

"Autoimmune disease" refers to a non-malignant disease or disorder arising from and directed against the individual's own tissues. As used herein, autoimmune disease specifically excludes malignant or cancerous diseases or conditions, in particular B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic Exclude chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia. Examples of autoimmune diseases or disorders include, but are not limited to: inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); scleroderma and sclerosis; inflammatory bowel disease-related reactions (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)); dermatitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T-cell infiltration and chronic inflammatory responses; atherosclerosis; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes (e.g., type I diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and by cytokines and T lymphocytes typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis mediated immune reactions associated with acute and delayed-type hypersensitivity; pernicious anemia (Addison's disease); diseases with extravasation of leukocytes; central nervous system (CNS) inflammatory disorders; anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; Nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

In this aspect, the pharmaceutical composition may contain other ingredients. The other ingredients include other active ingredients applied to target diseases and pharmaceutically acceptable carriers. The other active ingredient may be appropriately selected depending on the target disease and is not particularly limited. The pharmaceutically acceptable carrier is not particularly limited and includes buffers, excipients, stabilizers, or preservatives. One or more of the other ingredients may be used in the pharmaceutical composition.

(c-4) Complex detection agent and detection method In another aspect of the present invention, a first complex comprising a first co-inhibitory molecule and a first co-inhibitory molecule ligand for the first co-inhibitory molecule is provided.
In one aspect, the detection agent may contain an antigen-binding molecule as described above. The presence of the first complex is not visible to the naked eye, but is revealed by the detecting agent. The subject for which the detection agent is used is not particularly limited as long as it is suspected that the first complex exists. It can be a mixture or cells.

In another aspect of the invention, a method is provided for detecting a first complex consisting of a first co-inhibitory molecule and a first co-inhibitory molecule ligand to the first co-inhibitory molecule.
In one aspect, the detection method can comprise contacting an immune cell with an antigen-binding molecule as described above.
In the detection method, antigen-presenting cells may be in close proximity to the immune cells. The immune cell may express a first co-inhibitory molecule and the antigen-presenting cell may express a first co-inhibitory molecule ligand. The immune cells can be T cells. The detection method may be applied in vivo or ex vivo. When the detection method is applied in vitro, the immune cells may be brought into contact with the antigen-binding molecule while alive, or may be immobilized on a slide glass. In the detection method, the immune cells may be present in the tissue, or mixed with one or more cells other than the immune cells.

(c-5) Method for Suppressing Immunity In another aspect of the present invention, a method for suppressing immunity is provided.
In one aspect, the method can comprise administering an antigen-binding molecule as described above to a subject. When T cells are activated by antigen-presenting cells in the subject's body, or when T cells are expected to be activated by antigen-presenting cells, the antigen-binding molecule is administered to the subject. This suppresses the activation of the T cells and suppresses the subject's immunity.
When a T cell is activated by an antigen presenting cell, downstream signals of the T cell receptor on the T cell are activated. On the other hand, the first co-inhibitory molecule ligand expressed in antigen-presenting cells binds to the first co-inhibitory molecule expressed in T cells to form a first complex. The antigen-binding molecule binds to the first complex and activates the downstream signal of the first co-inhibitory molecule. In the method, the mechanism by which the immunity of the subject is suppressed by suppressing the downstream signal of the activated T cell receptor by the downstream signal of the activated first co-inhibitory molecule is is assumed.

The antigen-binding molecule of the present invention targets an immune synapse by specifically binding to a complex consisting of a co-inhibitory molecule localized at the immune synapse and its ligand, and has agonistic activity against the co-inhibitory molecule. Therefore, the antigen-binding molecule can be a therapeutic agent or a candidate thereof for treating or preventing diseases including autoimmune diseases. By containing at least the first antigen-binding domain, the antigen-binding molecule has agonistic activity against co-inhibitory molecules at the immune synapse. By forming the antigen-binding molecule into a multiple antigen-binding molecule containing a second antigen-binding domain in addition to the first antigen-binding domain, the antigen-binding molecule has a significant agonistic activity against co-inhibitory molecules in the immune synapse.
On the other hand, when it is intended to obtain an antigen-binding molecule having agonist activity to a co-stimulatory molecule, the first and a first costimulatory molecule ligand to said first costimulatory molecule can be replaced by a first costimulatory molecule and a first costimulatory molecule ligand to said first costimulatory molecule, respectively. The antigen-binding molecule obtained in this case is a third antigen capable of specifically binding to a fourth complex consisting of the first costimulatory molecule and the first costimulatory molecule ligand for the first costimulatory molecule. Contains the binding domain. Such antigen-binding molecules may have agonistic activity for co-stimulatory molecules and thus may be candidates for the treatment of cancer and infectious diseases. In this case, the antigen-binding molecule can specifically bind to a second costimulatory molecule capable of forming a fifth complex with a second costimulatory molecule ligand, in addition to the third antigen-binding domain. A multi-antigen-binding molecule containing four antigen-binding domains is expected to have significant agonistic activity against co-stimulatory molecules.

Example 1 Generation of Anti-Human PD-1 Antibodies, Anti-Human CD3 Antibodies, and Bispecific Antibodies Comprising Anti-Human PD-1 Antibody-Derived Arms and Anti-CD3 Antibody-Derived Arms
In order to create agonists that induce PD-1 immunosuppressive signals, it was thought necessary to co-localize the T-cell receptor with PD-1 (J. Exp. Med. Vol. 209 No. 6 1201-1217). The present inventors generated a bispecific antibody consisting of an anti-PD-1 arm and an anti-CD3 arm and evaluated its ability to induce PD-1 immunosuppressive signals.

Three anti-human PD-1 monoclonal antibodies PD1-17 (heavy chain variable region SEQ ID NO: 1 and light chain variable region sequence No: 2 (see WO2004/056875), and heavy chain constant region SEQ ID NO: 11 and light chain constant region SEQ ID NO: 12), antibody949 (clone 949) (heavy chain variable region SEQ ID NO: 3 and light chain variable region SEQ ID NO: 4 (see WO2011/110621), and heavy chain constant region SEQ ID NO: 11 and light chain constant region SEQ ID NO: 13), clone 10 (heavy chain variable region SEQ ID NO: 5 and light chain variable region SEQ ID NO: :6 (see WO2010/029435), heavy chain constant region SEQ ID NO: 11, light chain constant region SEQ ID NO: 13) were prepared by methods known to those skilled in the art.

As anti-human CD3 antibodies, two types of anti-human CD3 monoclonal antibody OKT3 (heavy chain variable region SEQ ID NO: 7 (NCBI Accession: 1SY6_H, GI: 49259178), light chain variable region SEQ ID NO: 8 (NCBI Accession: 1SY6_L, GI: 49259177), heavy chain constant region SEQ ID NO: 14, and light chain constant region SEQ ID NO: : 13), and CE115TR (heavy chain variable region SEQ ID NO: 9, light chain variable region SEQ ID NO: 10, heavy chain constant region SEQ ID NO: 14, light chain constant region SEQ ID NO: 15) by a method known to those skilled in the art. was prepared.
The present inventors used arms derived from the three anti-human PD-1 antibodies described above (also referred to herein as "anti-PD-1 arms"), and anti-human CD3 monoclonal antibodies derived from the two anti-human CD3 monoclonal antibodies described above. A bispecific antibody containing an anti-PD-1 arm and an anti-CD3 arm was prepared by a method known to those skilled in the art by combining arms (hereinafter also referred to as "anti-CD3 arm") according to the purpose of the experiment. In the following examples, a bispecific antibody in which two types of arms are combined is, for example, an anti-human PD-1 clone 949-derived arm and an anti-human CD3 antibody OKT3-derived arm. Such bispecific antibodies are referred to as "clone949/OKT3", "OKT3/clone949", "clone949//OKT3" or "OKT3//clone949".

(Example 2) Evaluation of PD-1 signal-inducing activity of bispecific antibodies containing anti-PD-1 arm and anti-CD3 arm (SHP-2 recruitment)
PD-1 signal-inducing activity of bispecific antibodies containing anti-CD3 and anti-PD-1 arms is a phosphatase that interacts with the intracellular domain of PD-1 upon PD-1 signal induction It was evaluated using the proximity of SHP2 (SHP-2 recruitment) as an index. The NanoBRET (registered trademark) PD-1/SHP2 Interaction Assay system (Promega) was used for the evaluation. The PD-1 signal-inducing activity was determined by the ratio of the fluorescence signal (618 nm) by BRET when PD-1 and SHP2 were in close proximity to the luminescence (460 nm) from the donor SHP2.
The day before the assay, PD-L1-expressing antigen-presenting cells (Promega, #J109A) were plated at 4.0 x ¹⁰⁴ cells/100 mL/well in F-12 medium (Gibco, 11765-054) containing 10% FBS in 96-well plates. (Costar, #3917) and cultured in a CO ² incubator at 37° C. for 16-24 hours. HaloTag® nanoBRET® 618 Ligand (Promega, #G980A) was diluted 250-fold with Opti-MEM (Gibco, #31985-062) on the day of assay. The medium of the cultured PD-L1-expressing antigen-presenting cells was removed, and 25 µL/well of diluted HaloTag (registered trademark) nanoBRET (registered trademark) 618 Ligand was added. PD-L1 inhibitory antibody (YW243.55S70) (see US2016/0222117 A1) (sometimes referred to as "anti-PD-L1" or "PDL1b" in this example and drawings) 0.08 μg/mL An evaluation sample (40, 8, 1.6 µg/mL) diluted with Opti-MEM containing was added at 25 µL/well. The amino acid sequence of the variable region of the PD-L1 inhibitory antibody (YW243.55S70) was described in US2016/0222117 A1 (heavy chain variable region SEQ ID NO: 16, light chain variable region SEQ ID NO: 17). In the PD-L1 inhibitory antibody, the heavy chain constant region is a human IgG1-based modified Fc body F1332 (SEQ ID NO: 18), and the light chain constant region is a human κ chain constant region k0MT (SEQ ID NO: 19 ) was used. Add PD-1/SHP2 Jurkat cells (Promega, #CS2009A01) to the above 96-well plate at 5 x 10 ⁴ cells/50 μL/well, suspend well, and incubate at 37°C for 2.5 hours in a 5% CO ² incubator. cultured. nanoBRET (registered trademark) Nano-Glo (registered trademark) substrate (Promega, #N157A) was diluted 100-fold with Opti-MEM, added to a 96-well plate cultured at 25 mL/well, and allowed to stand at room temperature for 30 minutes. Em460mM and Em618nm were then measured with Envision (PerkinElmer, 2104 EnVision).
The obtained values were applied to Equations 1 and 2 below to calculate BRET Ratio (mBU).

［式２］

[Formula 1]

[Formula 2]

The assay results are shown in FIG. As shown in the figure, the SHP2 recruitment of the bispecific antibody containing anti-PD-1 arm and anti-CD3 arm, ie PD-1 signal-inducing activity, was not clearly demonstrated.

(Example 3) Evaluation of bispecific antibody containing anti-PD-1 arm and anti-CD3 arm in T cell proliferation assay Bispecific antibody containing anti-PD-1 arm and anti-human CD3 arm is primary CD4 positive In order to evaluate whether it has suppressive activity against T cells, an anti-human CD3 arm and an anti-KLH antibody (herein referred to as "IC17") against CD4-positive T cells isolated from fresh blood collected from healthy subjects were tested. ) (heavy chain variable region SEQ ID NO: 112, light chain variable region SEQ ID NO: 113, heavy chain constant region SEQ ID NO: 11, and light chain constant region SEQ ID NO: 13) and bispecific A sex antibody (referred to as "OKT3/IC17" in Figure 2) was evaluated as a negative control. On the day of evaluation, blood collected from a healthy subject was mixed with an equal amount of RPMI1640 (Nakarai, #30264-56), and then gently layered on a centrifugation tube filled with Ficoll in an amount of 15/50 of the blood mixing solution. After centrifugation at 400 g for 35 minutes in a centrifuge set at 20° C., the leukocyte fraction on the upper layer of Ficoll was separated into a new centrifuge tube and diluted to 50 mL with RPMI1640 (Nakarai, #30264-56). After centrifugation at 500 g for 15 minutes in a centrifuge at 20° C., a cell (PBMC) pellet was recovered. Human CD4+ T Cell Isolation Kit (miltenyi, Cat#130-096-533) was used to isolate human CD4+ T cells from the collected PBMCs according to the procedure of the kit. After that, the obtained CD4-positive T cells were added to a 96-well round-bottom plate at 7.6 x 10 ⁵ cells/well, and 10 ng/mL IL-2 and evaluation antibodies (20, 5, 1.25, 0.31, 0.08 μg/ mL) were cultured in the presence of After 5 days of culture, cell proliferation was assessed by Cell Titer-Glo® Luminescent Cell Viability Assay (promega, G7570) according to the kit's protocol. The results are shown in FIG.

Bispecific antibodies comprising three anti-PD-1 and anti-human CD3 arms were tested, as shown in FIG. It rather promoted the proliferation of human CD4-positive T cells compared to bispecific antibodies containing anti-KLH antibody-derived arms. From this result, it is difficult to suppress the proliferation of CD4-positive T cells and suppress the immune response caused by CD4-positive T cells with a bispecific antibody containing an anti-PD-1 arm and an anti-human CD3 arm. was suggested.

(Example 4) Production of human PD-1 extracellular domain protein
Based on the gene sequence information (NM_005018) encoding human PD-1 obtained from GenBank, HMM+38 (nucleotide sequence is SEQ ID NO: 20, amino acid Sequence No. 21) was added to the N-terminus. A linker sequence, a histidine tag, a linker sequence, and a BAP tag (biotinylation tag, GLNDIFEAQKIEWHE) were added to the C-terminus to prepare an expression construct (nucleotide sequence: SEQ ID NO: 22, amino acid sequence: SEQ ID NO: 23). The gene designed above was synthesized and subcloned into an in-house constructed vector (pBEF-OriP) for mammalian cells to obtain an expression vector. Human PD-1 extracellular domain protein was transiently expressed by introducing this expression vector into Expi293 (registered trademark) F cells (Thermo Fisher) using the ExpiFectamine (registered trademark) 293 Transfection Kit. . The obtained culture supernatant was subjected to ion-exchange chromatography using an AKTA (registered trademark) Avant25 device (GE Healthcare) and HiTrap (registered trademark) Q HP column (GE Healthcare), and affinity chromatography using a HisTrap (registered trademark) HP column (GE Healthcare). Purified by chromatography. The fraction containing the human PD-1 extracellular domain protein was concentrated with an ultrafiltration membrane, followed by gel filtration chromatography (size exclusion chromatography) using a Superdex (registered trademark) 200 increase 10/300 column (GE Healthcare). A highly purified monomer fraction of the human PD-1 extracellular domain protein was obtained by further increasing the purification purity and removing multimers.

In order to obtain a human PD-1 ectodomain protein with a BAP tag attached with biotin, the expression vector for the human PD-1 ectodomain protein and the expression vector for the biotin ligase BirA were added to Expi293 (registered trademark) F cells. Biotin was added to the target protein by transfection with ExpiFectamine (registered trademark) 293 Transfection Kit. Purification was performed by ion exchange chromatography using a HiTrap (registered trademark) Q HP column (GE Healthcare) and affinity chromatography using a HisTrap (registered trademark) HP column (GE Healthcare) in the same procedure as above. Then, affinity chromatography using Streptavidin Mutein Matrix (Roche) was performed for the purpose of increasing the purity of the biotin adduct. Fractions containing human PD-1 extracellular domain protein were concentrated with an ultrafiltration membrane and then subjected to gel filtration chromatography (size exclusion chromatography) using a HiLoad 16/600 Superdex® 200 pg column (GE Healthcare). ) to further increase the purification purity and remove multimers to obtain a highly purified monomer fraction of the biotin-adducted human PD-1 extracellular domain protein.

(Example 5) Production of human PD-L1 extracellular domain protein
Based on the gene sequence information (NM_014143) encoding human PD-L1 obtained from GenBank, HMM+38 (nucleotide sequence is SEQ ID NO: 20, amino acid SEQ ID NO: 21) was added to the N-terminus, and a linker sequence, a histidine tag, a linker sequence, and a BAP tag (biotinylation tag, GLNDIFEAQKIEWHE) were added to the C-terminus to form an expression construct (the base sequence is SEQ ID NO: 24, the amino acid sequence is SEQ ID NO: 25). The gene designed above was synthesized and subcloned into a mammalian cell vector (pBEF-OriP) to obtain an expression vector. Human PD-L1 extracellular domain protein was transiently expressed by introducing this expression vector into FreeStyle (registered trademark) CHO cells (Thermo Fisher). The obtained culture supernatant was subjected to ion-exchange chromatography using an AKTA (registered trademark) Avant25 device (GE Healthcare) and HiTrap (registered trademark) Q HP column (GE Healthcare), and affinity chromatography using a HisTrap (registered trademark) HP column (GE Healthcare). Purified by chromatography. Fractions containing human PD-L1 extracellular domain protein were concentrated with an ultrafiltration membrane and then subjected to gel filtration chromatography (size exclusion chromatography) using a HiLoad 16/600 Superdex® 200 pg column (GE Healthcare). ) to further increase the purification purity and remove multimers to obtain a highly purified monomer fraction of the human PD-L1 extracellular domain protein.

The human PD-L1 ectodomain protein with biotin added to the BAP tag was prepared by mixing the above-mentioned purified human PD-L1 ectodomain protein with the purified protein of BirA enzyme (biotin ligase) and performing the biotin addition reaction. I did it by doing The final concentrations were 35 μM human PD-L1 extracellular domain protein, 10 mM ATP, 0.825 μM BirA enzyme, 50 mM Tris-HCl pH 8.3, 10 mM Mg(OAc) ₂ , 0.05 mM Biotin. The mixture was left to react at 4°C for about 16 hours. Unreacted biotin was removed by substitution with 20 mM NaPhospate, 600 mM NaCl, pH 7.2 by dialysis using Slide-A-Lyzer Dialysis Cassettes, 3.5K (Thermo Fisher). Then, affinity chromatography using Streptavidin Mutein Matrix (Roche) was performed for the purpose of increasing the purity of the biotin adduct. Fractions containing human PD-L1 extracellular domain protein were concentrated with an ultrafiltration membrane and then subjected to gel filtration chromatography (size exclusion chromatography) using a Superdex® 200 Increase 10/300 GL column (GE Healthcare). ) to further increase the purification purity and remove multimers to obtain a highly purified monomer fraction of the biotin-adducted human PD-L1 extracellular domain protein.

(Example 6) Production of human PD-L1 / human PD-1 complex protein
(1) Preparation of linker-fused human PD-L1 / human PD-1 complex protein By inserting a linker sequence between them, we tried to stabilize them as a complex by bringing them closer together. Figure 3 shows the design. For human PD-1, a C93S modification was introduced to remove free cysteines. A version in which a linker was inserted between N131 of human PD-L1 and N33 of human PD-1 was designated as Linker complex #1 (nucleotide sequence: SEQ ID NO: 26, amino acid sequence: SEQ ID NO: 27) (Fig. 3, left). ). A version in which a linker was inserted between E146 of human PD-1 and F19 of human PD-L1 was designated as Linker complex #2 (nucleotide sequence: SEQ ID NO: 28, amino acid sequence: SEQ ID NO: 29) (Fig. 3 right). For both complex proteins, a signal sequence (HMM+38) was added to the N-terminus, and a linker sequence, a histidine tag, a linker sequence and a BAP tag (GLNDIFEAQKIEWHE) were added to the C-terminus to construct expression constructs. The protein was transiently expressed by introducing this expression vector into FreeStyle (registered trademark) CHO cells (Thermo Fisher). The obtained culture supernatant was subjected to ion-exchange chromatography using an AKTA (registered trademark) Avant25 device (GE Healthcare) and HiTrap (registered trademark) Q HP column (GE Healthcare), and affinity chromatography using a HisTrap (registered trademark) HP column (GE Healthcare). Purified by chromatography. Fractions containing human PD-L1/human PD-1 complex protein were concentrated with an ultrafiltration membrane and then subjected to gel filtration chromatography using a HiLoad 26/600 Superdex® 200 pg column (GE Healthcare) ( A highly pure human PD-L1 / human PD-1 complex protein was obtained by further increasing the purification purity and removing multimers by size exclusion chromatography.

For the preparation of human PD-L1/human PD-1 complex protein with biotin added to the BAP tag, the purified human PD-L1/human PD-1 complex protein described above was subjected to purification of the BirA enzyme (biotin ligase). It was performed by mixing with protein and performing Biotin addition reaction. The final concentrations were 25 μM or 26 μM human PD-L1 / human PD-1 complex protein, 10 mM ATP, 0.825 μM BirA enzyme, 50 mM Tris-HCl pH8.3, 10 mM Mg(OAc)2, 0.05 mM Biotin. The mixture was mixed so as to obtain the desired temperature, and left to react at 4°C for about 16 hours. Unreacted biotin was removed by substitution with 20 mM NaPhospate, 600 mM NaCl, pH 7.2 by dialysis using Slide-A-Lyzer G2 Dialysis Cassettes, 10K (Thermo Fisher). Then, affinity chromatography using Streptavidin Mutein Matrix (Roche) was performed for the purpose of increasing the purity of the biotin adduct. Fractions containing human PD-L1/human PD-1 complex protein were concentrated with an ultrafiltration membrane and then subjected to gel filtration chromatography using a Superdex® 200 Increase 10/300 GL column (GE Healthcare) ( By further increasing the purification purity and removing multimers by size exclusion chromatography), a highly pure fraction of biotin adduct human PD-L1/human PD-1 complex protein was obtained.

(2) Production of disulfide bond-introduced human PD-L1 / human PD-1 complex protein By replacing the interacting amino acid residues of human PD-L1 and human PD-1 with cysteine residues, ectopic The formation of disulfide bonds was promoted to stabilize the complex.

Residues for cysteine substitution were selected from structural modeling based on reported co-crystal structural data (PDB:3BIK). As molecular design software, Design Protein tools of Discovery Studio (Dassault Systèmes, Inc.) and Protein Design functions of MOE (Chemical Computing Group) were used. These softwares select C atoms that are in close proximity between human PD-L1 and human PD-1, and select residues that can form disulfide bonds by replacing them with cysteines from modeling using rotamer. did. A version combining the Y56C variant of human PD-L1 and the A132C variant of human PD-1 was designated as S-S complex#1, and the A18C variant of human PD-L1 and the G90C variant of human PD-1 were combined. The version is S-S complex#2. FIG. 4 illustrates the design of a disulfide bond-introduced human PD-L1/human PD-1 complex protein. For expression purification, human PD-L1 and human PD-1 were transformed into heterodimers for the purpose of increasing the efficiency of heterodimer formation between human PD-L1 and human PD-1 and facilitating purification. We proceeded to express and purify by fusing with Fc containing modifications of Knobs into Hole, a technique that promotes formation.

Human PD-L1 immunoglobulin domain variable region F19-A132 linker sequence, BAP tag (GLNDIFEAQKIEWHE), linker sequence, PreScission protease recognition sequence (LEVLFQGP), linker sequence Human IgG1 type including hinge region It was fused to the N-terminus of the Fc domain. Modifications (L235R, G236R, S239K) to eliminate Fcγ receptor and complement C1q binding activity in the Fc domain and Hole modification (D356C) for Knobs into Hole technology to promote heterodimer formation , T366S, L368A, Y407V). A His tag for purification was added to the C-terminus of the fusion protein containing PD-L1 via a linker sequence.

On the other hand, for human PD-1 immunoglobulin domain variable region N33-E146 (including C93S modification) linked with linker sequence, BAP tag (GLNDIFEAQKIEWHE), linker sequence, PreScission protease recognition sequence (LEVLFQGP), linker sequence It was fused to the N-terminus of a human IgG1 type Fc domain including the hinge region. Modifications (L235R, G236R, S239K) and Knob modifications (Y349C, T366W) for lacking Fcγ receptor and complement C1q binding activity were introduced into the Fc domain. A FLAG tag for purification was added to the C-terminus of the fusion protein containing PD-1 via a linker sequence.

For each protein, an artificial secretory signal sequence, HMM+38, was added to the N-terminus to construct the expression construct.
The nucleotide sequence of hPDL1(Y56C)_BAP_PreSission_hole_His10 used for SS complex#1 is shown in SEQ ID NO: 30, the amino acid sequence is shown in SEQ ID NO: 31, and the nucleotide sequence of hPD1(A132C)_BAP_PreSission_knob_FLAG is shown in SEQ ID NO: 32, the amino acid sequence is shown in SEQ ID NO:33. The nucleotide sequence of hPDL1(A18C)_BAP_PreSission_hole_His10 used for SS complex#2 is shown in SEQ ID NO: 34, the amino acid sequence is shown in SEQ ID NO: 35, the nucleotide sequence of hPD1(G90C)_BAP_PreSission_knob_FLAG is shown in SEQ ID NO: 36, and the amino acid sequence is Shown in SEQ ID NO:37.

The gene designed above was synthesized and subcloned into a mammalian cell vector (pBEF-OriP) to obtain an expression vector. The protein was transiently expressed by introducing this expression vector into FreeStyle (registered trademark) CHO cells (Thermo Fisher).

The target protein was purified from the obtained culture supernatant by chromatography using an AKTA (registered trademark) 10S device (GE Healthcare) or an AKTA (registered trademark) Avant25 device (GE Healthcare). The heterodimer was isolated by first affinity chromatography using ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) followed by affinity chromatography using a HisTrap® HP column (GE Healthcare). A forming fraction was obtained. By reacting this heterodimeric protein with Turbo3C protease (Accelagen) that cleaves the PreScission recognition sequence, the human PD-L1/human PD-1 complex protein was cleaved from the Fc domain. 80 units of Turbo3C protease was mixed with 1 mg of human PD-L1/human PD-1 complex protein, and the mixture was filled in Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K (Thermo Scientific), and D Cleavage reaction was carried out at 4°C for about 16 hours while substituting with -PBS(-). Then, in order to remove unwanted substances such as cleaved Fc domains, uncleaved Fc fusions, and Turbo3C protease (His-tagged) contained in the sample, a HisTrap® HP column (GE Healthcare ) and a HiTrap® Protein A HP column (GE Healthcare) in series. The resulting fraction containing the cleaved human PD-L1/human PD-1 complex protein was concentrated with an ultrafiltration membrane, and then filtered using HiLoad 26/600 Superdex (registered trademark) 200 pg (GE Healthcare). A highly pure human PD-L1 / human PD-1 complex protein was obtained by further increasing the purification purity and removing multimers by gel filtration chromatography (size exclusion chromatography).

For the preparation of human PD-L1/human PD-1 complex protein with biotin added to the BAP tag, the purified human PD-L1/human PD-1 complex protein described above was subjected to purification of the BirA enzyme (biotin ligase). It was performed by mixing with protein and performing Biotin addition reaction. The final concentrations were 21 μM or 22 μM human PD-L1 / human PD-1 complex protein, 10 mM ATP, 0.825 μM BirA enzyme, 50 mM Tris-HCl pH8.3, 10 mM Mg(OAc)2, 0.05 mM Biotin. The mixture was mixed so as to obtain the desired temperature, and left to react at 4°C for about 16 hours. After concentrating the sample with an ultrafiltration membrane, the HisTrap® HP (GE Healthcare) and Superdex® 200 Increase 10/300 GL (GE Healthcare) are connected in series to allow the sample to flow to remove unwanted By adsorbing BirA and further increasing the purification purity by gel filtration chromatography (size exclusion chromatography) and removing multimers, we obtained a highly pure biotin adduct human PD-L1 / human PD-1 complex protein. Fractions were obtained.

(Example 7) Generation of anti-human PD-L1/human PD-1 complex protein antibodies from rabbits Each of the four human PD-L1/human PD-1 complex proteins prepared as described above, Linker Monoclonal antibodies were generated from rabbits using complex#1, Linker complex#2, SS complex#1, and SS complex#2 as antigens. 12-13 week old NZW rabbits were immunized with each of the four antigens. The antigen protein solution was mixed with an equal volume of TiterMax Gold adjuvant and administered intradermally to rabbits. Immunization was repeated four times at intervals of one week or more. Linker complex#1 and Linker complex#2 were administered at 200 µg/head for the first immunization, and 100 µg/head for subsequent immunizations. SS complex#1 and SS complex#2 were administered at 100 µg/head at all immunizations. One week after the final immunization, spleens and blood were collected from the immunized animals.

To enrich for antigen-specific B cells in spleen and blood cells, negative selection of cells binding to human PD-L1 ectodomain protein was performed by MACS using the autoMACS Pro Separator (miltenyi Biotec). A positive selection was then performed to enrich for B cells expressing rabbit IgG-type antibodies. For negative selection against PD-L1 ectodomain proteins, anti-biotin MicroBeads (Miltenyi Biotech, 130-090-485) were conjugated with human PD-L1 ectodomain proteins with biotin attached to the aforementioned BAP tag. there was. For positive selection of B cells expressing rabbit IgG antibodies, Anti-mouse IgG MicroBeads (Miltenyi Biotech, 130-048-401) conjugated with Mouse Anti-Rabbit IgG-PE (SouthernBiotech, 4090-9) were used. there was.

The MACS-enriched cells were sorted using a cell sorter (FACS aria III, BD) to further enrich for antibodies specific to the human PD-L1/human PD-1 complex protein. . Each of the four immunizing antigens (human PD-L1 / human PD-1 complex protein used for immunization with biotin added to the BAP tag) was bound to Streptavidin-APC (Miltenyi Biotech, 130-106- 791). At the same time, human PD-1 protein (R&D, 1086-PD) fused to human IgG1 type Fc was bound and stained with Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight 488 (ThermoFisher, SA5-10134). did B cells expressing IgG type antibodies were also sorted using Anti-Rabbit IgG-PE (SouthernBiotech, 4090-9). A cell sorter was used to collect B cells expressing IgG-type antibodies that bound to the human PD-L1/human PD-1 complex protein but not to the human PD-1 protein.

The resulting B cells were seeded at a density of 1 cell/well in 384-well plates with 12,500 cells/well of EL4 cells (European Collection of Cell Cultures) and 20-fold diluted activated rabbit T cell conditioned medium. Cultured for ˜12 days and subjected to antibody screening using secreted antibody in the B cell culture supernatant. The EL4 cells were cultured in advance by applying X-rays at a dose of 10 Gy using an X-ray irradiator MX-160Labo (mediXtec).
Activated rabbit T cell conditioned medium was prepared by mixing rabbit thymocytes in RPMI-1640 containing phytohaemagglutinin M (Roche, Cat. No. 11082132), phorbol 12-myristate 13-acetate (Sigma, Cat. No. P1585) and 2% FBS. It was prepared by culturing in culture medium. After culture, B cell culture supernatants were harvested for further evaluation and cell pellets were cryopreserved.

Using the secretory antibody contained in the B cell culture supernatant, screening was performed using the binding properties of the antibody as an index. MagPlex Microspheres immobilized with human PD-1 ectodomain protein, human PD-L1 ectodomain protein, the four human PD-L1/human PD-1 complex proteins previously described, and BSA as a negative control ( Luminex) and the binding to various proteins was confirmed with a flow cytometer (iQue Screener, Intellicyt). Binding analysis was performed by distinguishing different proteins by immobilizing them on MagPlex Microspheres with different fluorescence patterns. After streptoavidin was immobilized on MagPlex Microspheres, each biotinylated protein to be evaluated was immobilized and used for evaluation. Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific, A-11034) was used as the secondary antibody. Data analysis was performed using FlowJo software (Tomy Digital Biology). The results of this experiment show that it does not bind to the negative control BSA, human PD-1 ectodomain protein and human PD-L1 ectodomain protein, but only to each human PD-L1 / human PD-1 complex protein. Antibody-producing B cells were selected.

Selected B-cell RNA was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, Cat# R1053). Using the prepared RNA, the DNA encoding the antibody heavy chain variable region was amplified by reverse transcription PCR, and human IgG1 heavy chain constant region F1332 (SEQ ID NO: : 18) was recombined in-frame with the DNA encoding In addition, the DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR, and in the case of the rabbit-derived antibody light chain variable region, k0MTC (SEQ ID NO: 38), which is a modified human κ-type light chain constant region, was added. It was religated in-frame with the encoding DNA. Expression vectors containing the recombined antibody heavy and light chain coding sequences were prepared as described above and mixed in a 1:1 ratio of heavy and light chain expression vectors to Expi293 ( Antibodies were transiently expressed by transfection into ®F cells (Thermo Fisher) using the ExpiFectamine® 293 Transfection Kit. A purified antibody was prepared from the obtained culture supernatant by affinity purification using Protein A and used for subsequent evaluation.
Table 1 shows the sequence numbers of the variable regions and constant regions of the heavy and light chains of 30 clones of anti-human PD-L1 / human PD-1 complex protein antibodies (antibody sequence signal peptide and variable The boundary with the region followed the prediction results using the Signal Peptide prediction function of the GENETYX-SV/RC software (Ver.15.0.3).).

(Example 8) Specificity evaluation of anti-human PD-L1 / human PD-1 complex antibody Cell surface of 30 clones of anti-human PD-L1 / human PD-1 complex protein antibody produced in Example 7 Assessment of binding specificity to the human PD-L1/human PD-1 complex formed above was performed. Binding was assessed using a flow cytometer (iQue Screener, intellicyt). A solution obtained by adding BSA to D-PBS(-) to a final concentration of 0.1% (referred to as FACS buffer) was used for the dilution of cells and proteins to be added, and for the washing operation. For evaluation, DXB11s cells were used as a parent strain (parent/CHO), and human PD-L1 gene (nucleotide sequence: SEQ ID NO: 110, amino acid sequence: SEQ ID NO: 111) was introduced into the cell membrane surface. By introducing a stable expression strain (hPD-L1/CHO) forcibly expressing L1 and the full-length human PD-1 gene (nucleotide sequence: SEQ ID NO: 108, amino acid sequence: SEQ ID NO: 109), A stable expression strain (hPD-1/CHO) in which human PD-1 was forcibly expressed was prepared. hPD-L1/CHO was mixed with 10 μg/mL soluble human PD-1 extracellular domain (ECD) protein to preform the human PD-L1/human PD-1 complex protein on the cell surface. , hPD-1/CHO was mixed with 10 μg/mL soluble human PD-L1 extracellular domain (ECD) protein and left on ice for 30 minutes or longer. After adding 10 μL/well of the purified antibody adjusted to 20 μg/mL to a 384-well V-bottom plate (Greiner), 1 cell solution in which the above-mentioned human PD-L1/human PD-1 complex was previously formed was added. 10 μL/well was added so that 30000 cells per well. After incubating at 4°C for 30 minutes to bind the antibody, the supernatant was removed by aspiration after centrifugation. Subsequently, a washing operation of adding 50 μL/well of FACS buffer and then removing the supernatant by aspiration after centrifugation was repeated twice. Goat Anti-Human IgG Fc Cross-Adsorbed Secondary Antibody.DyLight 650 (Thermo Fisher Scientific, SA5-10137) was used as a secondary antibody for detecting binding.

The formation efficiency of the human PD-L1/human PD-1 complex protein on the cell surface was evaluated by the soluble human PD-1 ectodomain protein and biotin added to the soluble human PD-L1 ectodomain protein. was confirmed by adding and detecting Streptavidin-APC (Miltenyi Biotec, 130-106791).
Data analysis was performed using FlowJo software (Tomy Digital Biology).
The resulting binding data are shown in FIG. 5 and Table 2. Multiple antibodies were obtained with high binding selectivity for the PD-L1/PD-1 complex.

(Example 9) Preparation of anti-human PD-1 antibodies A plurality of anti-human PD-1 antibodies capable of binding to human PD-1 and inducing its immunosuppressive signal were prepared. PD1-17 described in Example 1, antibody949 (clone 949), clone 10, PDA0129 (heavy chain variable region SEQ ID NO: 99, light chain variable region SEQ ID NO: 100, heavy chain constant region SEQ ID NO: 18, light Four clones of anti-human PD-1 antibodies were prepared by a method known to those skilled in the art, with the addition of the chain constant region (SEQ ID NO: 38).

As a positive control, anti-human PD-1 monoclonal antibody 5C4 (heavy chain variable region SEQ ID NO: 101 and light chain variable region SEQ ID NO: 102 (see WO2006/121168), and heavy chain constant region SEQ ID NO: 105 and light chain constant region SEQ ID NO: 15) and Pembrolizumab (heavy chain variable region SEQ ID NO: :103 and light chain variable region SEQ ID NO:104 (see WO2016/137850), and heavy chain constant region SEQ ID NO:105 and light chain constant region SEQ ID NO:15) were also prepared by methods known to those skilled in the art.

(Example 10) Evaluation of PD-1 signal-inducing activity of anti-human PD-L1 / human PD-1 complex antibody (SHP-2 recruitment)
Anti-human PD-1 antibodies (clone949 and PDA0129) interact with the intracellular domain of PD-1 during PD-1 signal induction in the same manner as in Example 2 to determine whether they have PD-1 signal-inducing activity. The proximity of SHP2, a dephosphorylating enzyme that
The assay results are shown in FIG. It was suggested that the two types of anti-human PD-1 antibodies do not have clear SHP2 recruitment-inducing activity, that is, PD-1 signaling-inducing activity.

Whether the anti-human PD-L1 / human PD-1 complex antibody prepared in Example 7 has PD-1 signal-inducing activity was determined in the same manner as in Example 2, when PD-1 signal was induced. The proximity of SHP2, a phosphatase that interacts with the intracellular domain of -1, was evaluated as an index.
The assay results are shown in FIG. The anti-human PD-L1 / human PD-1 complex antibody, unlike the above-mentioned anti-human PD-1 antibodies (clone949 and PDA0129), has the activity of inducing SHP2 recruitment, that is, the activity of inducing PD-1 signaling. It has been shown.

(Example 11) Evaluation of ligand binding inhibitory activity of anti-human PD-1 antibody The inhibitory activity against the binding of 1 to human PD-L1 was evaluated. Evaluation of inhibitory activity indicates whether the binding strength of soluble human PD-L1 extracellular domain (ECD) protein binding to human PD-1-expressing cells is attenuated in the presence of anti-human PD-1 antibody. went to Binding was assessed using a flow cytometer (iQue Screener, intellicyt). A solution obtained by adding BSA to D-PBS(-) to a final concentration of 0.1% (referred to as FACS buffer) was used for the dilution of cells and proteins to be added, and for the washing operation. For the evaluation, a stable expression strain ( hPD-1/CHO) was used. A series of staining procedures were performed using a V-Bottom 96 Well Cell Culture Plate (costar, 3894). Antibody solution (100 μL/well) diluted to each concentration of 20, 4, and 0.8 μg/mL with FACS buffer was adjusted to 3 x 10 ⁶ cells/mL for hPD-1/CHO cells 50 μL/well (150000 cells/ wells) and placed on ice for 30 minutes to bind antibodies. Then, it was mixed with 10 μg/mL biotinylated soluble human PD-L1 extracellular domain (ECD) protein and left on ice for 15 minutes. After centrifugation, the supernatant was removed by aspiration, followed by the addition of 200 μL/well of FACS buffer, followed by centrifugation, followed by washing by aspiration and removal of the supernatant, which was repeated twice. Streptavidin-APC for detecting binding was diluted 1000 times and added at 100 μL/well and incubated on ice for 30 minutes. After centrifugation, remove the supernatant by aspiration, add 200 μL/well of FACS buffer, remove the supernatant after centrifugation, and finally suspend in 50 μL/well of FACS buffer. Combined data acquisition was performed. Analysis of the acquired data was performed using FlowJo software (Tomy Digital Biology).
As shown in Figure 8, PD1-17 and clone 10 were antibodies that inhibited the binding of human PD-L1 to human PD-1, while clone 949 and PDA0129 were antibodies that did not inhibit the binding.

(Example 12) Preparation of PD-1 agonist antibody using anti-human PD-L1 / human PD-1 complex antibody The present inventors prepared the anti-human PD-L1 / human PD-1 complex Based on the body antibodies, we investigated additional antibody molecular types with stronger agonistic activity against PD-1. Specifically, PD-L1-induced PD locally at immune synapses and TCR microclusters where endogenous PD-L1/PD-1 complexes are formed and exert their immunosuppressive regulatory function. We investigated a molecular type concept that could further enhance the immunosuppressive signal of -1. The present inventors considered that a bispecific antibody comprising an anti-PD-L1/PD-1 complex arm and an anti-PD-1 arm would satisfy the molecular type concept, and evaluated it.

Anti-human PD-1 antibodies from which designed bispecific antibodies were based were four clones of PD1-17, antibody949 (clone 949), clone 10 and PDA0129 described in Example 9. Table 3 summarizes whether these anti-human PD-1 antibodies shown in Example 11 have inhibitory activity on the binding of human PD-L1 to human PD-1.

The anti-PD-L1/PD-1 complex antibody, which is the source of the bispecific antibody, was selected from 30 clones prepared in Example 7 (Table 1) and used. Table 4 shows combinations of anti-PD-1 arms and anti-PD-L1/PD-1 complex arms in prepared bispecific antibodies, particularly those used in later examples.

Table 5 lists the variable regions and respective variable regions of the heavy and light chains of the antibodies from which the bispecific antibodies comprising the anti-PD-1 arm and the anti-PD-L1/PD-1 complex arm generated above are shown in Table 5. SEQ ID NO of the constant region is shown (The boundary between the signal peptide and the variable region of the antibody sequence was predicted using the Signal Peptide prediction function of the GENETYX-SV/RC software (Ver.15.0.3).) .

(Example 13) Evaluation of PD-1 signal-inducing activity of bispecific antibody containing anti-PD-L1/PD-1 complex arm and anti-PD-1 arm (SHP-2 recruitment)
Whether the bispecific antibody containing the anti-PD-L1/PD-1 complex arm and the anti-PD-1 arm has the PD-1 signal-inducing activity was examined in the same manner as in Example 2. The proximity of SHP2, a phosphatase that interacts with the intracellular domain of PD-1 upon signal induction, was evaluated.
The assay results are shown in FIG. It was shown that a bispecific antibody containing an anti-PD-L1/PD-1 complex arm and an anti-PD-1 arm has the activity of inducing SHP2 recruitment, that is, the activity of inducing PD-1 signaling. SHP2 recruitment was induced using bispecific antibodies, particularly antibody949 (clone 949) and PDA0129, with arms derived from anti-PD-1 antibodies that do not inhibit the binding of PD-1 and PD-L1. It was found that the activity was remarkably high.

(Example 14) Evaluation of PD-1 signal-inducing activity using suppression of NFAT activity as an indicator PD induced by a bispecific antibody containing an anti-PD-L1/PD-1 complex arm and an anti-PD-1 arm We evaluated whether -1 signaling has an immunosuppressive function using the activity of the transcription factor NFAT, which works downstream of T cell receptor signaling responsible for immune activation. Using the PD-1/PD-L1 Blockade Bioassay system (Promega), evaluation was carried out by taking advantage of the luminescence reaction of Luciferase occurring downstream of the NFAT response element. In this assay, the TCR stimulator on PD-L1 aAPC/CHO-K1 cells induces the activation signal of the TCR on PD-1 effector cells, resulting in NFAT activation and the luminescence of the reporter gene Luciferase. However, the induction of PD-1 signaling by PD-L1 almost completely suppresses NFAT activation. However, NFAT activation occurs when PD-1 signal induction is inhibited by the addition of PD-L1 inhibitory antibodies. The present inventors have previously found that in this assay, about 30% PD-L1 inhibition occurs when the final concentration of YW243.55S70, which is a PD-L1 inhibitory antibody, is added to 40 ng / mL. We assessed whether bispecific antibodies induced suppression of NFAT activation in .

Reactions were performed using Round well 384 Well White Flat Bottom Polystyrene TC-Treated Microplates (Corning, 3826). First, PD-L1 aAPC/CHO-K1 cells adjusted to 2.5 x 10 ⁵ cells/mL were added at 30 μL/well and incubated overnight at 37°C in 5% CO ₂ conditions to adhere, and then cultured. After removing the supernatant, 10 μL/well of an antibody solution mixed with bispecific antibodies at various concentrations was added so that the final concentration of YW243.55S70 was 40 ng/mL. After allowing to stand at room temperature for 15 minutes, 10 μL/well of PD-1 Effector Cells adjusted to 4.6875×10 ⁵ cells/mL was added and incubated at 37° C. and 5% CO ₂ for 6 hours. A Luciferase substrate solution of Bio-Glo Luciferase Assay System (Prmega, G7940) was added at 20 μL/well, allowed to stand at room temperature for 5 minutes, and then Luciferase luminescence was measured using a microplate reader EnVision Xcite (PerkinElmer).

The results of the evaluation are shown in FIG. The generated bispecific antibodies containing anti-PD-L1/PD-1 complex arms and anti-PD-1 arms were confirmed in the aforementioned NanoBRET®-based SHP-2 recruitment assay system. In addition to having SHP2 recruitment activity, it was also confirmed to have a function of suppressing NFAT activity as shown by this assay.

(Example 15) The design of each molecular type concept, the function of each arm, and their mechanism of action. FIG. 11 shows the presumed mechanism of action in FIG.
Both arms of the anti-human PD-L1/human PD-1 complex antibody (Fig. 11 top and Fig. 12 left) enhance the interaction of PD-L1 to PD-1 in addition to the targeting function to the immune synapse. By having the function, it was presumed to exert an agonistic action on PD-1 as shown in Example 10. This mechanism of action suggests not only anti-complex antibodies that can enhance the interaction of PD-L1 to PD-1, but also anti-complex antibodies that can enhance the interaction of other co-inhibitory ligands to other co-inhibitory molecules. It can also be applied to conjugated antibodies.
In bispecific antibodies comprising an anti-PD-L1/PD-1 complex arm and an anti-PD-1 arm (FIG. 11 bottom and FIG. 12 right), the anti-PD-L1/PD-1 complex arm is at least immune As long as it has a synapse-targeting function, it may or may not have a function of enhancing the interaction of PD-L1 with PD-1. In the bispecific antibody, the anti-PD-1 arm was presumed to exert an agonistic effect on PD-1. This mechanism of action suggests not only an anti-complex arm that can enhance the interaction of PD-L1 to PD-1, but also an anti-complex arm that can enhance the interaction of other co-inhibitory ligands to other co-inhibitory molecules. It can also be applied to composite arms.

Claims (15)

An antigen-binding molecule comprising a first antigen-binding domain,
An antigen-binding molecule, wherein said first antigen-binding domain is capable of specifically binding a first complex consisting of a first co-inhibitory molecule and a first co-inhibitory molecule ligand to said first co-inhibitory molecule. specific binding of said first antigen-binding domain to said first complex results in either or both of said first co-inhibitory molecule and said first co-inhibitory molecule ligand in said first complex 2. The antigen-binding molecule according to claim 1, which binds with intermolecular force. An antigen-binding molecule comprising a first antigen-binding domain,
said first antigen binding domain is capable of binding a first complex consisting of a first co-inhibitory molecule and a first co-inhibitory molecule ligand to said first co-inhibitory molecule, and said first antigen binding domain The binding activity to the first complex of the first co-inhibitory molecule that does not form the first complex and the first co-inhibitory molecule ligand that does not form the first complex An antigen-binding molecule that has higher binding activity to either or both. binding of the first antigen-binding domain to the first complex with either or both of the first co-inhibitory molecule and the first co-inhibitory molecule ligand in the first complex 4. The antigen-binding molecule according to claim 3, wherein the binding is due to intermolecular forces. 5. The antigen-binding molecule of claim 3 or 4, wherein said binding activity satisfies either one or both of the following conditions (a) and (b):
(a) cells of the first co-inhibitory molecule ligand in flow cytometry using a first cell that forcibly expresses the first co-inhibitory molecule but does not forcibly express the first co-inhibitory molecule ligand; The binding activity of the first antigen-binding domain to the first cell in the presence of the first soluble polypeptide containing the ectodomain is lower than in the absence of the first soluble polypeptide high;
(b) in flow cytometry using a second cell that forcibly expresses the first co-inhibitory molecule ligand but does not forcibly express the first co-inhibitory molecule, The binding activity of the first antigen-binding domain to the second cell in the presence of the second soluble polypeptide containing the domain is higher than that in the absence of the second soluble polypeptide expensive. 6. The antigen-binding molecule according to any one of claims 1 to 5, which is capable of activating a first intracellular signal downstream of said first co-inhibitory molecule at an immune synapse where said first complex is present. . 7. The antigen-binding molecule of claim 6, wherein activation of the first intracellular signal can suppress activation of T cells. 8. The immune synapse of claim 6 or 7, wherein said immune synapse is formed between a cell expressing MHC and said co-inhibitory molecule ligand and said T cell expressing said co-inhibitory molecule. antigen binding molecule. A third cell expressing the first co-inhibitory molecule and capable of measuring the intensity of the first intracellular signal, and expressing an antigen-independent T cell receptor activator and a ligand of the first co-inhibitory molecule and a fourth cell that is in contact with each other, and the first intracellular signal in the third cell is partially inhibited by an inhibitory antibody against the first co-inhibitory molecule ligand In the system for measuring the intensity of the first intracellular signal, the intensity of the first intracellular signal in the presence of the antigen-binding molecule is higher than that in the absence of the antigen-binding molecule 9. The antigen-binding molecule of any one of claims 6-8, wherein the antigen-binding molecule is relatively high. expressing the T cell receptor and the first co-inhibitory molecule, the intensity of a second intracellular signal that can be activated downstream of the T cell receptor can be measured, and the first intracellular signal and a sixth cell expressing an antigen-independent T cell receptor activator and the first co-inhibitory molecule ligand used under conditions that allow contact with each other, and
under conditions in which inhibition of the second intracellular signal in the fifth cell by the first co-inhibitory molecule ligand is partially inhibited by an inhibitory antibody to the first co-inhibitory molecule ligand; In the second intracellular signal intensity measurement system,
10. The antigen-binding molecule of any one of claims 6-9, wherein the intensity of the second intracellular signal in the presence of the antigen-binding molecule is lower than in the absence of the antigen-binding molecule. The combination of said first co-inhibitory molecule and said first co-inhibitory molecule ligand is a combination of PD-1 and PD-L1, a combination of PD-1 and PD-L2, a combination of BTLA and HVEM, TIGIT and CD155. combination, combination of TIGIT and CD112, combination of LAG-3 and MHC class II molecules, combination of CTLA4 and CD80, combination of CTLA4 and CD86, combination of TIM-3 and galectin-9, combination of TIM-3 and phosphatidylserine, TIM 11. The antigen-binding molecule according to any one of claims 1 to 10, which is any one selected from the group consisting of a combination of -3 and CEACAM-1, and a combination of TIM-3 and HMGB1. wherein the antigen-binding molecule is a multiple antigen-binding molecule further comprising a second antigen-binding domain;
12. Any one of claims 1-11, wherein said second antigen binding domain is capable of specifically binding a second co-inhibitory molecule capable of forming a second complex with a second co-inhibitory molecule ligand. The antigen-binding molecule according to . 13. The antigen of claim 12, wherein binding of said second antigen binding domain to said second co-inhibitory molecule does not compete with binding of said second co-inhibitory molecule ligand to said second co-inhibitory molecule. binding molecule. The combination of said second co-inhibitory molecule and said second co-inhibitory molecule ligand is a combination of PD-1 and PD-L1, a combination of PD-1 and PD-L2, a combination of BTLA and HVEM, TIGIT and CD155 combination, combination of TIGIT and CD112, combination of LAG-3 and MHC class II molecules, combination of CTLA4 and CD80, combination of CTLA4 and CD86, combination of TIM-3 and galectin-9, combination of TIM-3 and phosphatidylserine, TIM 14. The antigen-binding molecule according to claim 12 or 13, which is any one combination selected from the group consisting of a combination of -3 and CEACAM-1, and a combination of TIM-3 and HMGB1. 15. The antigen-binding molecule according to any one of claims 12 to 14, wherein said first antigen-binding domain and said second antigen-binding domain are antibody variable regions or target antigen-binding fragments thereof. .

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