EA046075B1 - ANTI-CD137 ANTIGEN-BINDING MOLECULES AND THEIR APPLICATIONS - Google Patents

Description

Field of technology

The present invention relates to aHTu-CD137 antigen binding molecules and methods of using them.

Prior Art

Cancer is a deadly disease that is difficult to completely cure, except in some cases. The results of the main method of treatment, which consists in the use of chemotherapeutic agents, cannot be considered satisfactory. It has been suggested that not only the heterogeneity of the cancer cells themselves, but also the tumor microenvironment plays an important role as a complicating factor in cancer treatment (NPL 1). Recently, it was shown that inoperable malignant melanoma and other similar diseases are potentially curable with an anti-CTLA-4 antibody that inhibits the immunosuppressive function of CTLA-4 and thereby promotes T cell activation (NPL 2). In 2011, a monoclonal antibody against human CTLA-4 (ipilimumab) was approved by the US Food and Drug Administration (FDA) as the world's first immune-activating antibody. In addition, inhibitory antibodies against PD-1 and PD-L1, other immune checkpoint molecules other than CTLA-4, have been reported to be therapeutic (NPL 3) and approved by the FDA.

It is known that T cells, which play an important role in tumor immunity, are activated by two signals: 1) binding of the T cell receptor (TCR) to an antigenic peptide represented by class I molecules of the major histocompatibility complex (MHC), and activation of the TCR; and 2) binding of a costimulatory molecule on the surface of T cells to its ligands on antigen presenting cells and activation of the costimulatory molecule. In addition, activation of costimulatory molecules belonging to the tumor necrosis factor receptor superfamily (TNFRSF), including CD137 (4-1BB), on the surface of T cells has been implicated as an important function for T cell activation (NPL 4 ).

The TNFRSF superfamily includes CD137, CD40, OX40, RANK, GITR and similar molecules. CD137 has been found to be expressed not only on the surface of T cells, but also on the surface of other immune cells such as dendritic cells (DCs), B cells, killer (NK) cells, macrophages and neutrophils (NPL 5).

Agonistic antibodies against CD137 have already demonstrated antitumor effects in a mouse model, and this has been shown to result from the activation of CD8-positive T cells and NK cells in experiments in a mouse model (NPL 6). However, side effects due to nonspecific hepatotoxicity of the agonistic anti-CD137 antibody have become clinical and non-clinical problems, hindering the required progress in drug development (NPL 7, NPL 8). The side effects are believed to be caused primarily by activation of immune cells in non-tumor, non-immune tissues such as the liver, which involves antibody binding to the Fcy receptor via the antibody constant region (NPL 9). On the other hand, it has been shown that in order for agonistic antibodies belonging to the anti-TNF receptor superfamily of antibodies to exhibit agonistic activity in vivo, the antibody must be cross-linked by cells expressing the Fcy receptor (FcyRII expressing cells) (NPL 10). That is, the binding of an agonistic anti-CD137 antibody to the Fcy receptor is involved in both the drug efficacy of the antitumor effect of the antibody and its side effects, such as hepatotoxicity. Thus, increasing binding between antibody and Fcy receptor would presumably increase drug efficacy but could also increase hepatotoxic side effects, and decreasing binding between antibody and Fcy receptor might reduce side effects but also decrease drug efficacy. To date, there have been no reports of agonistic antibodies against CD137 whose efficacy and side effects have been separated. Moreover, the antitumor effect of an agonistic anti-CD137 antibody is by no means a strong clinical benefit, and further enhancement of drug efficacy along with prevention of toxicity is desirable. Accordingly, it is desirable to develop a new drug that is capable of inducing antitumor immune responses while reducing side effects.

When a therapeutic antibody is administered to a living organism, it is desirable that its target antigen is expressed specifically only at the site of the lesion. However, in many cases the same antigen is also expressed in unaffected areas, that is, in normal tissues, and this may cause side effects that are undesirable from a treatment point of view. For example, although antibodies against tumor antigens can exhibit cytotoxic activity against tumor cells through antibody-dependent cellular cytotoxicity (ADCC), etc., they can also damage normal cells if the same antigen is expressed in normal cells. To solve the above-mentioned problems, attention has been focused on the phenomenon in which certain compounds are abundantly present in target tissues (such as tumor tissues), and technology has been developed to search for antigen-binding molecules with different antigen-binding activities depending on the

- 1 046075 sti on the concentration of such compounds (for example, PTL 1).

Sources cited.

Patent Literature:

WO 2013/180200.

Non-patent literature:

Hanahan, Cell, 144, 2011, 646-674,

Prieto, Clin Cancer Res. 18, 2012, 2039-2047,

Hamid, Expert Opin. Biol. Ther., 6, 2013, 847-861,

Summers, Nat Rev Immunol, 12, 2012, 339-351,

Vinay, Cellular & Molecular Immunology, 8, 2011, 281-284,

Houot, Blood, 114, 2009, 3431-3438,

Ascierto, Semin Oncol, 37, 2010, 508-516,

Dubrot, Cancer Immunol Immunother, 59, 2010, 1223-1233,

Schabowsky, Vaccine, 28, 2009, 512-522,

Li, Proc Natl Acad Sci USA, 110(48), 2013, 19501-19506.

Brief description of the invention

Technical problem

The present invention relates to anti-CD137 antigen-binding molecules and methods of using them.

Solution

To obtain anti-CD137 antigen-binding molecules that activate immunocytes, have cytotoxic or antitumor activity and at the same time exhibit reduced effect on non-tumor tissues, such as normal tissues, and have fewer side effects, and also to develop methods for their use, in the present invention prepare anti-CD137 antigen-binding molecules, characterized in that their binding activity to CD137 varies depending on various compounds (eg, small molecules) in target tissues (eg, tumor tissues), and develop methods of their use, pharmaceutical compositions and things like that. In one embodiment of the present invention, the anti-CD137 antigen binding molecules of the present invention have low side effects, and thus the dosage can be increased without concerns about side effects, and as a result, they can exhibit higher drug efficacy (cytotoxic activity or antitumor activity). activity).

Specifically, the present invention provides anti-CD137 antigen binding molecules, methods of using them, pharmaceutical compositions and the like, as described in the following examples.

[1]

Ahtu-CD137 antigen-binding molecule, which has CD 137-binding activity depending on the small molecule compound.

[2]

Ahtu-CD137 antigen-binding molecule according to claim [1], wherein the binding activity to CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM low molecular weight compound is two or more times higher compared to the binding activity with CD137 in the absence of a small molecule compound.

[2.1]

Ahtu-CD137 antigen-binding molecule according to claims [1] or [2], wherein the CD137 binding activity in the presence of 10 μM or more of the small molecule compound is two or more times greater than the CD137 binding activity in the absence of the small molecule compound.

[2.2]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.1], wherein the KD value for CD137 in the presence of 10 μM or more of the low molecular weight compound is 5x10 ^-7 M or less.

[2.3]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.2], wherein the KD value for CD137 in the absence of the small molecule compound is 5x10 ^-6 M or more.

[2.4]

Ahtu-CD137 antigen binding molecule according to claim [1], wherein the KD value for CD137 in a solution that is prepared such that the concentration of the low molecular weight compound is 10 μM or more is 5x10 ^-7 M or less, and the KD value for CD137 in solution to which the low molecular weight compound is not added is 1 x 10 ^-6 M or more.

[2.5]

Ahtu-CD137 antigen-binding molecule according to claim [1], wherein the KD value for CD137 is

- 2 046075 solution, which is prepared in such a way that the concentration of the small molecule compound is 10 μM or more, and the KD value of CD137 in a solution in which the small molecule compound is not added, each measured by the Biacore method within 24 hours after contact of CD137 and the antigen binding molecule anti-CD137 in solution.

[2.6]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.5], which forms a trimolecular complex with a low molecular weight compound and CD137.

[2.7]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.6], which binds to a CD137 molecule derived from a human or ape.

[2.8]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.7], in which the low molecular weight compound is adenosine-containing.

[2.9]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.8], in which the low molecular weight compound is ATP.

[3]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [2.9], which includes any combination of hypervariable regions HVR-H1, HVR-H2 and HVR-H3 selected from claims (a) to (k) below:

(a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 17;

(b) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 9, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 17;

(c) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 10, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 17;

(d) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 11, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 18;

(e) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 18;

(f) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 12, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 18;

(g) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 18;

(h) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 14, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 19;

(i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 20;

(j) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 16, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 20; and (k) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 14, and HVR-H3 containing the amino acid sequence of SEQ ID NO: 17.

[3.1]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [3], which includes any combination of hypervariable regions HVR-L1, HVR-L2 and HVR-L3 selected from the following claims (a) to (g):

(a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(b) HVR-L1 containing the amino acid sequence of SEQ ID NO: 22, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence

- 3 046075 sequence SEQ ID NO: 27;

(c) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 28;

(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 29;

(e) HVR-L1 containing the amino acid sequence of SEQ ID NO: 23, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(f) HVR-L1 containing the amino acid sequence of SEQ ID NO: 24, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; and (g) HVR-L1 containing the amino acid sequence of SEQ ID NO: 25, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27.

[4]

Ahtu-CD137 antigen binding molecule which includes any combination of hypervariable regions HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 selected from paragraphs (a) to (n) below:

(a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(b) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 9, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 22, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(c) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 10, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 22, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(d) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 11, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(e) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(f) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 12, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 28;

(g) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 29;

(h) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 14, HVR-H3 containing the amino acid sequence of SEQ ID NO: 19, HVR-L1 containing the amino acid sequence of SEQ ID NO: 23, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(i) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, HVR-H3 containing the amino acid sequence

- 4 046075 sequence SEQ ID NO: 20, HVR-L1 containing the amino acid sequence SEQ ID NO: 24, HVR-L2 containing the amino acid sequence SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence SEQ ID NO: 27;

(j) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, HVR-H3 containing the amino acid sequence of SEQ ID NO: 20, HVR-L1 containing the amino acid sequence of SEQ ID NO: 25, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(k) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 16, HVR-H3 containing the amino acid sequence of SEQ ID NO: 20, HVR-L1 containing the amino acid sequence of SEQ ID NO: 25, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27;

(m) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 14, HVR-H3 containing the amino acid sequence of SEQ ID NO: 19, HVR-L1 containing the amino acid sequence of SEQ ID NO: 24, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; and (n) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 14, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27.

[5]

Ahtu-CD137 antigen binding molecule including:

(a) a heavy chain variable region (VH) the sequence of which is at least 95% identical to any amino acid sequence of SEQ ID NO: 43-53; or (b) a light chain variable region (VL) the sequence of which is at least 95% identical to any amino acid sequence of SEQ ID NO: 54-60.

[5.1]

Ahtu-CD137 antigen binding molecule comprising any combination of VH and VL variable regions selected from (a) to (n) below:

(a) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 43, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 54;

(b) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 44, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 55;

(c) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 45, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 55;

(d) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 46, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 54;

(e) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 47, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 54;

(e) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 48, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 56;

(g) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 449, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 57;

(h) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 50, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 58;

(i) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 51, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 59;

(k) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 51, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 60;

- 5 046075 (l) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 52, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 60;

(l) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 50, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 59; and (m) VH having at least 95% identity with the amino acid sequence of SEQ ID NO: 53, and VL having at least 95% identity with the amino acid sequence of SEQ ID NO: 54.

[5.2]

Ahtu-CD137 antigen binding molecule comprising any combination of VH and VL variable regions selected from (a) to (n) below:

(a) VH containing the amino acid sequence of SEQ ID NO: 43, and VL containing the amino acid sequence of SEQ ID NO: 54;

(b) VH containing the amino acid sequence of SEQ ID NO: 44, and VL containing the amino acid sequence of SEQ ID NO: 55;

(c) VH containing the amino acid sequence of SEQ ID NO: 45, and VL containing the amino acid sequence of SEQ ID NO: 55;

(d) VH containing the amino acid sequence of SEQ ID NO: 46, and VL containing the amino acid sequence of SEQ ID NO: 54;

(e) VH containing the amino acid sequence of SEQ ID NO: 47, and VL containing the amino acid sequence of SEQ ID NO: 54;

(f) VH containing the amino acid sequence of SEQ ID NO: 48, and VL containing the amino acid sequence of SEQ ID NO: 56;

(g) VH containing the amino acid sequence of SEQ ID NO: 49, and VL containing the amino acid sequence of SEQ ID NO: 57;

(h) VH containing the amino acid sequence of SEQ ID NO: 50, and VL containing the amino acid sequence of SEQ ID NO: 58;

(i) VH containing the amino acid sequence of SEQ ID NO: 51, and VL containing the amino acid sequence of SEQ ID NO: 59;

(j) VH containing the amino acid sequence of SEQ ID NO: 51, and VL containing the amino acid sequence of SEQ ID NO: 60;

(k) VH containing the amino acid sequence of SEQ ID NO: 52, and VL containing the amino acid sequence of SEQ ID NO: 60;

(l) VH containing the amino acid sequence of SEQ ID NO: 50, and VL containing the amino acid sequence of SEQ ID NO: 59; and (n) VH containing the amino acid sequence of SEQ ID NO: 53, and VL containing the amino acid sequence of SEQ ID NO: 54.

[5.3]

Ahtu-CD137 antigen binding molecule whose value [binding activity (binding amount) to CD137 in the presence of 10 μM or more small molecule compound]/[binding activity (binding amount) to CD137 in the absence of small molecule compound] is equal to or greater than the value of the control antigen binding molecule wherein the control antigen binding molecule is an anti-CD137 antigen binding molecule comprising a combination of the hypervariable region HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27.

[5.4]

The Ahtu-CD137 antigen binding molecule of claim [5.3], wherein the control antigen binding molecule is an anti-CD137 antigen binding molecule comprising a combination of VH comprising the amino acid sequence of SEQ ID NO: 43 and VL containing the amino acid sequence of SEQ ID NO: 54.

[5.5]

Ahtu-CD137 antigen binding molecule, the value of which [binding activity (KD) with CD137 in the presence of 1 μM small molecule compound]/[binding activity (KD) with CD137 in the presence of 10 μM or more small molecule compound] is equal to or greater than the value of the control antigen binding molecule, where the control antigen binding molecule is an anti-CD137 antigen binding molecule comprising a combination of HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence

- 6 046075 identity of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVRL1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR- L3 containing the amino acid sequence SEQ ID NO: 27.

[5.6]

The Ahtu-CD137 antigen binding molecule of claim [5.5], wherein the control antigen binding molecule is an anti-CD137 antigen binding molecule comprising a combination of VH comprising the amino acid sequence of SEQ ID NO: 43 and VL containing the amino acid sequence of SEQ ID NO: 54.

[5.7]

Ahtu-CD137 antigen binding molecule which has CD137 antigen binding activity dependent on a small molecule compound, wherein the anti-CD137 antigen binding molecule competes with the antigen binding molecule of any one of claims [3] to [5.2] for binding to CD137 in the presence of 10 μM or more , 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more of a low molecular weight compound.

[5.8]

Ahtu-CD137 antigen binding molecule which has CD137 antigen binding activity dependent on a small molecule compound, wherein the anti-CD137 antigen binding molecule binds to the same CD137 epitope associated with the antigen binding molecule of any one of claims [3] to [5.2] in the presence of 10 µM or more, 50 µM or more, 100 µM or more, 150 µM or more, 200 µM or more, or 250 µM or more of a low molecular weight compound.

[5.8A]

Ahtu-CD137 antigen binding molecule according to any one of claims [5.3] to [5.8], wherein the low molecular weight compound is an adenosine-containing compound.

[5.8B]

Ahtu-CD137 antigen binding molecule according to any one of paragraphs [5.3] to [5.8A], wherein the low molecular weight compound is ATP.

[5.9]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [5.8B], which is a monoclonal antibody or an antigen binding fragment thereof.

[5.10]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [5.9], which is a human antibody, a humanized antibody or a chimeric antibody, or an antigen binding fragment thereof.

[5.11]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [5.10], which is a full length IgG1 antibody.

[5.12]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [5.11], which contains an altered Fc region in which at least one amino acid is altered, wherein the altered Fc region has increased binding activity to FcYRIIb compared to the original Fc region, which does not contain amino acid changes.

[5.13]

Ahtu-CD137 antigen-binding molecule according to claim [5.12], in which the binding activity of the altered Fc with FcYRIIb is equal to or higher than that of the control Fc region, and the control Fc region is a human IgG1 Fc region containing a combination of amino acid substitutions G236N/H268D/A330K according to EU numbering.

[5.14]

Ahtu-CD137 antigen binding molecule according to claim [5.12] or [5.13], wherein the Fc control region contains the amino acid sequence SEQ ID NO: 153.

[5.15]

Ahtu-CD137 antigen binding molecule according to claim [5.12], wherein the at least one amino acid change is at least one amino acid substitution selected from the group consisting of G236N, H268D and A330K according to EU numbering.

[5.16]

Ahtu-CD137 antigen binding molecule according to claims [5.12] or [5.15], wherein at least one amino acid change is a combination of amino acid substitutions G236N/H268D/A330K in accordance with EU numbering.

[5.17]

The Ahtu-CD137 antigen binding molecule according to any one of claims [5.12] to [5.16], wherein the original Fc region is derived from the Fc region of human IgG1.

- 7 046075

[5.18]

The Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [5.17], which comprises an altered Fc region in which at least one amino acid is altered, wherein the anti-CD137 antigen binding molecule has an increased isoelectric point (pI) compared to the isoelectric point of the original anti-CD137 antigen binding molecule containing the original Fc region, which does not contain amino acid changes.

[5.19]

The Ahtu-CD137 antigen binding molecule according to claim [5.18], wherein the at least one amino acid change is a change in an amino acid residue that can be exposed on the surface of the original Fc region.

[5.20]

Ahtu-CD137 antigen binding molecule according to claims [5.18] or [5.19], wherein at least one amino acid change is:

(i) replacing at least one amino acid residue having a negative side chain charge in the original Fc region with an amino acid residue having no side chain charge, (ii) replacing at least one amino acid residue having no side chain charge in the original Fc region, with an amino acid residue having a positive side chain charge, and/or (iii) replacing at least one amino acid residue having a negative side chain charge in the original Fc region with an amino acid residue having a positive side chain charge chains.

[5.21]

The Ahtu-CD137 antigen binding molecule according to any one of claims [5.18] to [5.20], wherein the at least one amino acid change is a combination of amino acid substitutions, and wherein the amino acid substitutions are located at positions that are conformationally close to each other.

[5.22]

The Ahtu-CD137 antigen binding molecule according to any one of claims [5.18] to [5.21], wherein the binding activity of the altered Fc region to the Fcy receptor (FcyR) is not significantly reduced compared to the binding activity of the original Fc region.

[5.23]

Ahtu-CD137 antigen binding molecule according to claim [5.22], wherein the Fcy receptor (FcyR) is FcYRIIb.

[5.24]

Ahtu-CD137 antigen binding molecule according to any one of claims [5.18] to [5.23], wherein the at least one amino acid change is at least one amino acid substitution selected from the group consisting of Q311R, P343R and D413K according to numbering EU.

[5.25]

Ahtu-CD137 antigen binding molecule according to any one of claims [5.18] to [5.24], wherein at least one amino acid change is (i) a P343R amino acid substitution, (ii) a combination of Q311R/P343R amino acid substitutions, or (iii) a combination of amino acid substitutions replacements Q311R/D413K, according to EU numbering.

[6]

The Ahtu-CD137 antigen binding molecule of any one of claims [1] to [5.25], which comprises an altered Fc region, wherein the altered Fc region comprises any combination of amino acid changes selected from the following:

L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K;

K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K;

L234Y/P238D/T250V/V264I/T307P/A330K/P343R/D413K;

L234Y/P238D/V264I/A330K/P343R/D413K;

L234Y/G237D/P238D/T250V/T307P/A330K/P343R/D413K;

L234Y/G237D/P238D/A330K/P343R/D413K;

L235W/G236N/H268D/Q295L/K326T/A330K/Q311R/P343R;

L234Y/P238D/T250V/V264I/T307P/A330K/Q311R/P343R;

L234Y/P238D/V264I/A330K/Q311R/P343R;

L234Y/G237D/P238D/T250V/T307P/A330K/Q311R/P343R;

L234Y/G237D/P238D/A330K/Q311R/P343R;

L235W/G236N/H268D/Q295L/K326T/A330K/P343R;

K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R;

L235W/G236N/H268D/Q295L/K326T/A330K/D413K;

K214R/G236N/H268D/A330K/P343R;

- 8 046075

K214R/L235W/G236N/H268D/A330K/P343R;

K214R/G236N/H268D/A330K/D413K;

K214R/G236N/H268D/A330K/P343R/D413K;

K214R/L235W/G236N/H268D/A330K/P343R/D413K;

K214R/G236N/H268D/A330K/Q311R;

K214R/L235W/G236N/H268D/A330K/Q311R;

K214R/G236N/H268D/A330K/Q311R/P343R;

K214R/L235W/G236N/H268D/A330K/Q311R/P343R;

K214R/G236N/H268D/A330K/Q311R/D413K;

K214R/L235W/G236N/H268D/A330K/Q311R/D413K; And

K214R/L235W/G236N/H268D/Q295L/K326T/A33OK/Q311R.

EU numbering.

[6.1]

The Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [6], wherein the altered Fc region is derived from the Fc region of human IgG1.

[6.2]

The Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [6.1], wherein the altered Fc region further includes deletions at positions 446 and 447 in accordance with EU numbering.

[7]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [6.2], which includes a heavy chain constant region containing any of the amino acid sequences of SEQ ID NO: 64-85.

[7.1]

Ahtu-CD137 antigen binding molecule containing any combination of VH, VL, CH and CL regions selected from the following (1)-(xxxHn):

(i) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 64, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(ii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 66, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(iii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 67, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(iv) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 68, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(v) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 69, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(vi) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 70, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(vii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 71, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(viii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 73, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(ix) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(x) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xi) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xii) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xiii) VH containing the amino acid sequence SEQ ID NO: 43, CH containing ami

- 9 046075 amino acid sequence SEQ ID NO: 84, VL containing the amino acid sequence SEQ ID NO: 54, and CL containing the amino acid sequence SEQ ID NO: 63;

(xiv) VH containing the amino acid sequence of SEQ ID NO: 43, CH containing the amino acid sequence of SEQ ID NO: 85, VL containing the amino acid sequence of SEQ ID NO: 54, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 65, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 72, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 74, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xix) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 77, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xx) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 79, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxiii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 81, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxiv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 83, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 59, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 72, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 74, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxix) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 75, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxx) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 77, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxxi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 78, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxxii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 79, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxxiii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 80, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

- 10 046075 (xxxiv) VH containing the amino acid sequence SEQ ID NO: 51, CH containing the amino acid sequence SEQ ID NO: 81, VL containing the amino acid sequence SEQ ID NO: 60, and CL containing the amino acid sequence SEQ ID NO: 63 ;

(xxxv) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 82, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxxvi) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 83, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63;

(xxxvii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 84, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63; and (xxxviii) VH containing the amino acid sequence of SEQ ID NO: 51, CH containing the amino acid sequence of SEQ ID NO: 85, VL containing the amino acid sequence of SEQ ID NO: 60, and CL containing the amino acid sequence of SEQ ID NO: 63.

[8]

An isolated nucleic acid encoding an anti-CD137 antigen-binding molecule according to any one of claims [1] to [7.1].

[9]

A vector containing a nucleic acid according to claim [8].

[10]

A host cell containing a nucleic acid according to paragraph [8] or a vector according to paragraph [9].

[eleven]

A method for producing an anti-CD137 antigen-binding molecule, which includes culturing a host cell according to claim [10] so as to obtain an anti-CD137 antigen-binding molecule.

[12]

An immunoconjugate comprising an anti-CD137 antigen-binding molecule according to any one of claims [1][7.1] and a cytotoxic agent.

[13]

A pharmaceutical composition comprising an anti-CD137 antigen-binding molecule according to any one of paragraphs [1] to [7.1] or an immunoconjugate according to paragraph [12]; and a pharmaceutically acceptable carrier.

[14]

Ahtu-CD137 antigen binding molecule according to any one of paragraphs [1] to [7.1] or an immunoconjugate according to paragraph [12], which are intended for use as a pharmaceutical agent.

[14.1]

Ahtu-CD137 antigen-binding molecule according to any one of paragraphs [1]-[7.1], immunoconjugate according to paragraph [12] or a pharmaceutical composition according to claim [13], which are intended for use in the treatment of cancer.

[14.2]

Ahtu-CD137 antigen binding molecule, immunoconjugate or pharmaceutical composition according to claim [14.1], wherein the tumor is a solid tumor infiltrated by B cells, dendritic cells, natural killer cells, macrophages and/or CD8 positive T cells.

[14.3]

Ahtu-CD137 antigen binding molecule, immunoconjugate or pharmaceutical composition according to claim [14.1], wherein the tumor is a solid tumor infiltrated by regulatory T cells (Treg).

[15]

Ahtu-CD137 antigen-binding molecule according to any one of paragraphs [1]-[7.1], immunoconjugate according to paragraph [12] or a pharmaceutical composition according to claim [13], which are intended for use in activating immune cells.

[15.1]

Ahtu-CD137 antigen binding molecule, immunoconjugate or pharmaceutical composition according to claim [15], wherein the immune cells are B cells, dendritic cells, natural killer cells, macrophages and/or T cells.

[15.2]

Ahtu-CD137 antigen-binding molecule according to any one of paragraphs [1] to [7.1] or a pharmaceutical composition according to paragraph [13], which are intended to activate immune cells in tumor tissue.

[15.3]

Ahtu-CD137 antigen binding molecule or pharmaceutical composition according to claim [15.2], wherein the immune cells are B cells, dendritic cells, natural killer cells, macrophages and/or T cells.

[15.4]

Ahtu-CD137 antigen-binding molecule according to any one of paragraphs [1]-[7.1], immunoconjugate according to paragraph [12]

- 11 046075 or a pharmaceutical composition according to claim [13], which are intended for use in cell destruction.

[16]

Ahtu-CD137 antigen-binding molecule according to any one of paragraphs [1]-[7.1], immunoconjugate according to paragraph [12] or a pharmaceutical composition according to claim [13], whose level of immune activation in non-tumor tissue is lower than the level of immune activation of an anti-CD137 antigen-binding molecule that does not have CD137-binding activity dependent on a small molecule compound.

[16.1]

Ahtu-CD137 antigen-binding molecule, immunoconjugate or pharmaceutical composition according to claim [16], in which the non-tumor tissue is lymph nodes, spleen and/or liver.

[16.2]

The Ahtu-CD137 antigen binding molecule of any one of claims [1] to [7.1] or the immunoconjugate of claim [12], which does not significantly bind to CD137 expressed in non-tumor tissue.

[16.3]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [7.1] or immunoconjugate according to claim [12], which have a longer half-life in the blood compared to an anti-CD137 antigen binding molecule that does not have CD137 binding activity , depending on the low molecular weight compound.

[17]

Ahtu-CD137 antigen-binding molecule according to any one of paragraphs [1]-[7.1], immunoconjugate according to paragraph [12] or a pharmaceutical composition according to claim [13], which have a reduced level of side effects compared to an anti-CD137 antigen binding molecule that does not have small molecule dependent CD137 binding activity.

[17.1]

Ahtu-CD137 antigen-binding molecule, immunoconjugate or pharmaceutical composition according to claim [17], in which the side effects are increased AST, increased ALT, fever, nausea, acute hepatitis, hepatopathy, splenomegaly, enteritis, purulent inflammation of the skin, decreased neutrophils, decreased lymphocytes , decreased platelets, transaminase expression and/or hyperbilirubinemia.

[18]

Ahtu-CD137 is an antigen-binding molecule that has small molecule dependent agonist activity.

[18.1]

Ahtu-CD137 antigen binding molecule according to claim [18], wherein the agonist activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM small molecule compound is two or more times greater than the agonist activity against CD137 in the absence of a small molecule compound.

[18.2]

Ahtu-CD137 antigen-binding molecule according to claims [18] or [18.1], wherein the anti-CD137 agonist activity in the presence of 10 μM or more of the small molecule compound is twofold or more greater than the anti-CD137 agonist activity in the absence of the small molecule compound.

[18.3]

Ahtu-CD137 antigen-binding molecule according to claims [18] or [18.1], wherein the anti-CD137 agonist activity in the presence of 50 μM or more of the small molecule compound is twofold or more greater than the anti-CD137 agonist activity in the absence of the small molecule compound.

[18.4]

Ahtu-CD137 antigen-binding molecule according to claims [18] or [18.1], wherein the anti-CD137 agonist activity in the presence of 250 μM or more of the small molecule compound is twofold or more greater than the anti-CD137 agonist activity in the absence of the small molecule compound.

[18.5]

The Ahtu-CD137 antigen binding molecule according to any one of claims [18] to [18.4], wherein agonist activity against CD137 is assessed by the amount of IL-2 and/or IFN-γ produced by CD137 expressing cells.

[18.6]

Ahtu-CD137 antigen binding molecule according to claim [18.5], wherein the CD137-expressing cells are isolated human peripheral blood mononuclear cells (PBMCs) or human PBMC-derived T cells.

[18.7]

The Ahtu-CD137 antigen binding molecule according to any one of claims [18] to [18.4], wherein agonistic activity against CD137 is assessed by a reporter gene assay.

- 12 046075

[18.8]

Ahtu-CD137 antigen binding molecule according to claim [18], which exhibits CD137 agonist activity in a solution that is prepared such that the final concentration of the small molecule compound is 50 μM or more, and substantially does not exhibit CD137 agonist activity in solution , to which no low molecular weight compound is added.

[18.9]

Ahtu-CD137 antigen binding molecule according to claim [18.8], wherein the anti-CD137 agonist activity in a solution that is prepared such that the final concentration of the small molecule compound is 50 μM or more, and the anti-CD137 agonist activity in a solution to which the small molecule compound is not added compound, each assessed for the amount of IL-2, IFN-γ and/or IL-6 as measured within 72 hours after contact of a cell expressing CD137 and an anti-CD137 antigen-binding molecule in solution.

[18.10]

Ahtu-CD137 antigen binding molecule according to claim [18.8], wherein the anti-CD137 agonist activity in a solution that is prepared such that the final concentration of the small molecule compound is 50 μM or more, and the anti-CD137 agonist activity in a solution to which the small molecule compound is not added compound, each is assessed by the luciferase luminescence signal, which is measured within 6 hours after contact of T cells expressing the NFkappaB-luciferase reporter construct and CD137 with an anti-CD137 antigen-binding molecule.

[18.11]

The Ahtu-CD137 antigen binding molecule according to any one of claims [18] to [18.10], wherein the low molecular weight compound is an adenosine-containing compound.

[18.12]

Ahtu-CD137 antigen binding molecule according to any one of paragraphs. [18]-[18.11], in which the low molecular weight compound is ATP.

[19]

Ahtu-CD137 antigen binding molecule according to any one of claims [1] to [7.1], which has agonistic activity against CD137, dependent on a small molecule compound.

[19.1]

Ahtu-CD137 antigen binding molecule according to claim [19], wherein the agonistic activity against CD137 in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM small molecule compound is two or more times higher than the agonistic activity against CD137 in the absence of a small molecule compound.

[19.2]

Ahtu-CD137 antigen-binding molecule according to claims [19] or [19.1], wherein the CD137 agonist activity in the presence of 10 μM or more of the small molecule compound is two or more times greater than the CD137 agonist activity in the absence of the small molecule compound.

[19.3]

Ahtu-CD137 antigen-binding molecule according to claim [19] or [19.1], wherein the CD137 agonist activity in the presence of 50 μM or more of the small molecule compound is two or more times greater than the CD137 agonist activity in the absence of the small molecule compound.

[19.4]

Ahtu-CD137 antigen-binding molecule according to claim [19] or [19.1], wherein the CD137 agonist activity in the presence of 250 μM or more of the small molecule compound is two or more times greater than the CD137 agonist activity in the absence of the small molecule compound.

[19.5]

Ahtu-CD137 antigen-binding molecule according to claim [19] or [19.1], wherein CD137 agonist activity is assessed by the amount of IL-2 and/or IFN-γ produced by the CD137-expressing cell.

[19.6]

The Ahtu-CD137 antigen binding molecule of claim [19.5], wherein the CD137-expressing cell is an isolated human peripheral blood mononuclear cell (PBMC) or a T cell derived from a human PBMC.

[19.7]

Ahtu-CD137 antigen binding molecule according to any one of claims[19] or [19.4], in which the agonistic activity of CD137 is assessed using a reporter gene assay.

[19.8]

Ahtu-CD137 antigen binding molecule according to claim [19], which exhibits CD137 agonist activity in a solution that is prepared such that the final concentration of the small molecule compound is 50 μM or more, and substantially does not exhibit CD137 agonist activity in solution , to which no low molecular weight compound is added.

- 13 046075

[19.9]

Ahtu-CD137 antigen binding molecule according to claim [19.8], wherein the anti-CD137 agonist activity in a solution that is prepared such that the final concentration of the small molecule compound is 50 μM or more, and the anti-CD137 agonist activity in a solution to which the small molecule compound is not added compound, each assessed by the amount of IL-2, IFNγ and/or IL-6 measured within 72 hours after contact of a cell expressing CD137 and an anti-CD137 antigen-binding molecule in solution.

[19.10]

Ahtu-CD137 antigen binding molecule according to claim [19.8], wherein the anti-CD137 agonist activity in a solution that is prepared such that the final concentration of the small molecule compound is 50 μM or more, and the anti-CD137 agonist activity in a solution to which the small molecule compound is not added compound, each is assessed by a luciferase luminescence signal, which is measured within 6 hours after contact of T cells expressing the NFkappaB-luciferase reporter construct and CD137 with an anti-CD137 antigen-binding molecule.

[19.11]

The Ahtu-CD137 antigen binding molecule according to any one of claims [19] to [19.10], wherein the low molecular weight compound is an adenosine-containing compound.

[19.12]

Ahtu-CD137 antigen binding molecule according to any one of paragraphs. [19]-[19.11], in which the low molecular weight compound is ATP.

[20]

An agonistic antigen binding molecule comprising an altered Fc region, wherein the altered Fc region comprises at least one amino acid change that results in an increase in isoelectric point (pI) compared to the isoelectric point (pI) of the original agonistic antigen binding molecule containing the original Fc region, and wherein the agonistic antigen binding molecule has increased agonistic activity compared to the agonistic activity of the parent agonistic antigen binding molecule.

[20.1]

The agonistic antigen-binding molecule of claim [20], wherein the at least one amino acid change is a change in an amino acid residue that can be exposed on the surface of the parent Fc region.

[20.2]

Agonistic antigen-binding antibody according to claims[20] or [20.1], wherein the change in at least one amino acid is:

(i) replacing at least one amino acid residue having a negative side chain charge in the original Fc region with an amino acid residue having no side chain charge, (ii) replacing at least one amino acid residue having no side chain charge in the original Fc region, with an amino acid residue having a positive side chain charge, and/or (iii) replacing at least one amino acid residue having a negative side chain charge in the original Fc region with an amino acid residue having a positive side chain charge chains.

[20.3]

An agonistic antigen-binding molecule according to any one of claims [20] to [20.2], wherein the at least one amino acid change is a combination of amino acid substitutions, and wherein the amino acid substitutions are located at positions conformationally close to each other.

[20.4]

An agonistic antigen-binding molecule according to any one of claims[20] -[20.3], in which the binding activity of the altered Fc region to the Fcy receptor is not significantly reduced compared to the binding activity of the original Fc region.

[20.5]

An agonistic antigen-binding molecule according to claim [20.4], wherein the Fcy receptor is FcYRIIb.

[20.6]

The antigen-binding agonist molecule according to any one of claims [20] to [20.4], wherein the at least one amino acid change is at least one amino acid substitution selected from the group consisting of Q311R, P343R and D413K, in accordance with EU numbering .

[20.7]

An agonistic antigen-binding molecule according to any one of claims[20] -[20.6], wherein at least one amino acid change is a change in amino acid (i) P343R/D413K, (ii) Q311R/P343R, (iii) P343R, (iv) D413K, (v) Q311R, or (vi) Q311R/ D413K or combination thereof according to

- 14 046075 EU numbering.

[20.8]

An agonistic antigen binding molecule according to any one of claims [20] to [20.7], which is an anti-CD137 antigen binding molecule.

[20.9]

An agonistic antigen-binding molecule according to any one of claims [20] to [20.8], which is an anti-CD137 antibody.

[21]

A method for producing an agonistic antigen-binding molecule containing an altered Fc region, comprising:

introducing into the original Fc at least one amino acid change that results in an increase in the isoelectric point (pI) compared to that of the original agonistic antigen binding molecule containing the original Fc region, wherein the agonistic activity of the agonistic antigen binding molecule containing the altered Fc region is increased compared to with that of the original agonistic antigen-binding molecule.

[21.1]

The method according to claim [21], in which the agonistic activity of the agonistic antigen-binding molecule for the antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM of the low molecular weight compound is two times or more higher than the agonistic activity for the antigen in absence of low molecular weight compound.

[21.2]

Method according to claim [21] or [21.1], wherein the agonistic activity of the agonistic antigen-binding molecule for an antigen in the presence of 10 μM or more of the small molecule compound is twice or greater than the agonistic activity for the antigen in the absence of the small molecule compound.

[21.3]

Method according to claim [21] or [21.1], wherein the agonistic activity of the agonistic antigen-binding molecule for an antigen in the presence of 50 μM or more of the small molecule compound is twice or greater than that of the agonistic antigen in the absence of the small molecule compound.

[21.4]

Method according to claim [21] or [21.1], wherein the agonistic activity of the agonistic antigen binding molecule for an antigen in the presence of 250 μM or more of the small molecule compound is two times or more greater than that of the agonistic antigen binding molecule in the absence of the small molecule compound.

[21.5]

The method according to any one of claims [21] to [21.4], wherein the agonistic activity for the antigen is assessed by the amount of IL-2 and/or IFN-γ produced by cells expressing the antigen.

[21.6]

The method according to claim [21.5], wherein the antigen-expressing cell is an isolated human peripheral blood mononuclear cell (PBMC) or a T cell derived from a human PBMC.

[21.7]

The method according to any one of claims [21] to [21.4], wherein the agonistic activity against the antigen is assessed by reporter gene analysis.

[21.8]

The method according to any one of claims [21] to [21.7], further comprising:

(i) obtaining an expression vector that includes an appropriate promoter operatively linked to a gene encoding an agonistic antigen-binding molecule obtained by the method of any one of claims [21] to [21.7], (ii) introducing the vector into a host cell and culturing the cell host to obtain the agonistic antigen-binding molecule, and (iii) isolating the agonistic antigen-binding molecule from the host cell culture.

[21.9]

The method according to any one of claims [21] to [21.8], wherein an anti-CD137 antigen-binding molecule is provided.

[21.10]

The method according to any one of claims [21] to [21.9], wherein an anti-CD137 antibody is provided.

[21.11]

The method according to any one of claims [21.1] to [21.10], wherein the low molecular weight compound is an adenosine-containing compound.

[21.12]

The method according to any one of paragraphs. [21.1]-[21.11], in which the low molecular weight compound is ATP.

- 15 046075

[22]

A method of increasing the agonistic activity of an agonistic antigen binding molecule containing an Fc region, which includes introducing into the Fc region at least one amino acid change that results in an increase in the isoelectric point (pI) compared to the agonistic activity of the parent antigen binding molecule containing the parent Fc region.

[22.1]

The method according to claim [22], wherein the agonistic activity of the agonistic antigen-binding molecule towards the antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM low molecular weight compound is two or more times higher than the agonistic activity in relation to the antigen in the absence of a low molecular weight compound.

[22.2]

Method according to claim [22] or [22.1], wherein the agonistic activity of the agonistic antigen-binding molecule toward an antigen in the presence of 10 μM or more of the small molecule compound is two or more times greater than the agonistic activity toward the antigen in the absence of the small molecule compound.

[22.3]

Method according to claim [22] or [22.1], wherein the agonistic activity of the agonistic antigen-binding molecule toward an antigen in the presence of 50 μM or more of the small molecule compound is two or more times greater than the agonistic activity toward the antigen in the absence of the small molecule compound.

[22.4]

Method according to claim [22] or [22.1], wherein the agonistic activity of the agonistic antigen-binding molecule toward an antigen in the presence of 250 μM or more of the small molecule compound is two or more times greater than the agonistic activity toward the antigen in the absence of the small molecule compound.

[22.5]

The method according to any one of claims [22] to [22.4], wherein the agonistic activity towards the antigen is assessed by the amount of IL-2 and/or IFN-γ produced by the antigen-expressing cell.

[22.6]

The method according to claim [22.5], wherein the cell expressing the antigen is an isolated human peripheral blood mononuclear cell (PBMC) or a T cell derived from a human PBMC.

[22.7]

The method according to any one of claims [22] to [22.4], wherein the agonistic activity towards the antigen is assessed by reporter gene analysis.

[22.8]

The method according to any one of claims [22] to [22.7], which includes an anti-CD137 antigen-binding molecule.

[22.9]

The method according to any one of claims [22] to [22.8], which includes an anti-CD137 antibody.

[22.10]

The method according to any one of claims [22.1] to [22.9], wherein the low molecular weight compound is an adenosine-containing compound.

[21.11]

The method according to any one of paragraphs [22.1] - [22.10], in which the low molecular weight compound is ATP.

[23]

A method of using at least one amino acid change to increase the agonistic activity of an antigen binding molecule containing an Fc region, wherein the amino acid change results in an increase in the isoelectric point (pI) compared to the isoelectric point of the original agonistic antigen binding molecule containing the original Fc region.

[23.1]

The method according to claim [23], wherein the agonistic activity of the agonistic antigen-binding molecule towards the antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM low molecular weight compound is two or more times higher than the agonistic activity in relation to the antigen in the absence of a low molecular weight compound.

[23.2]

Method according to claim [23] or [23.1], wherein the agonistic activity of the agonistic antigen-binding molecule toward an antigen in the presence of 10 μM or more of the small molecule compound is two or more times greater than the agonistic activity toward the antigen in the absence of the small molecule compound.

[23.3]

Method according to claim [23] or [23.1], wherein the agonistic activity of the agonistic antigen-binding molecule toward an antigen in the presence of 50 μM or more low molecular weight co

- 16 046075 unity is two or more times higher than the agonist activity towards the antigen towards the antigen in the absence of the small molecule compound.

[23.4]

Method according to claim [23] or [23.1], wherein the agonistic activity of the agonistic antigen-binding molecule toward an antigen in the presence of 250 μM or more of the small molecule compound is two or more times greater than the agonistic activity toward the antigen relative to the antigen in the absence of the small molecule compound.

[23.5]

The method according to any one of claims [23] to [23.4], wherein the agonistic activity towards the antigen is assessed by the amount of IL-2 and/or IFN-γ produced by the antigen-expressing cell.

[23.6]

The method of claim [23.5], wherein the antigen-expressing cell is an isolated human peripheral blood mononuclear cell (PBMC) or a T cell derived from a human PBMC.

[23.7]

The method according to any one of claims [23] to [23.4], wherein the agonistic activity towards the antigen is assessed using a reporter gene assay.

[23.8]

The method according to any one of claims [23] to [23.7], which includes an anti-CD137 antigen-binding molecule.

[23.9]

The method according to any one of claims [23] to [23.8], which includes an anti-CD137 antibody.

[23.10]

The method according to any one of claims [23.1] to [23.9], wherein the low molecular weight compound is a compound containing adenosine.

[23.11]

The method according to any one of paragraphs [23.1] - [23.10], in which the low molecular weight compound is ATP.

[24]

A method of screening for an antigen binding domain or an antigen binding molecule that has antigen binding activity dependent on a small molecule compound, the method comprising:

(a) contacting an antigen binding domain or antigen binding molecule or a library of antigen binding domains or antigen binding molecules with a fusion molecule in the presence of a small molecule compound where in the fusion molecule two or more antigen units are fused to one unit of a fusion partner, (b) placing the antigen binding domain, or antigen binding molecule associated with the antigen within the molecule fused in step (a), in the absence or presence of a low concentration of the small molecule compound, and (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b).

[24.1]

The method according to [24], in which the fusion partner molecule is a dimeric Fc region.

[24.2]

The method of [24.1], wherein the Fc region comprises a first Fc subunit and a second Fc subunit, wherein one antigen unit is fused to each of the first and second Fc subunits.

[24.3]

The method of claim [24.1] or [24.2], wherein one antigen unit is fused to the N-terminus of each of the first and second Fc subunits.

[24.4]

The method according to any one of claims [24] to [24.3], wherein the library of antigen binding domains or antigen binding molecules is a library of phages.

[24.5]

The method according to any one of claims [24] to [24.4], wherein the phages included in the phage library are phages having two or more antigen binding domains or antigen binding molecules on their surface.

[24.6]

The method according to any one of claims [24] to [24.5], wherein the phages included in the phage library are phages having a defect in the helper phage-derived pIII gene.

[25]

A method of screening for an antigen binding domain or an antigen binding molecule that has antigen binding activity dependent on two or more different small molecules, the method comprising the following steps:

(a) contacting the antigen binding domain, or antigen binding molecule, or bib

- 17 046075 libraries of antigen binding domains or antigen binding molecules with an antigen in the presence of a first small molecule compound, (b) placing the antigen binding domain or antigen binding molecule associated with the antigen from step (a), in the absence or presence of a low concentration of the first small molecule compound, and (c ) isolating the antigen binding domain or antigen binding molecule dissociated in step (b), (d) contacting the antigen binding domain or antigen binding molecule isolated in step (c) with the antigen in the presence of a second low molecular weight compound, (e) placing the antigen binding domain or antigen binding molecule, associated with the antigen in step (d) in the absence or presence of a low concentration of a second small molecule compound, and (e) isolating the antigen binding domain or antigen binding molecule dissociated in step (e), wherein the method does not include between steps (c) and (d) amplification of a gene encoding the antigen binding domain or antigen binding molecule isolated in step (c).

[25.1]

The method according to claim [25], wherein the library of antigen binding domains or antigen binding molecules is a library of phages.

[26]

A method for screening an antigen binding domain or an antigen binding molecule that has antigen binding activity dependent on a small molecule compound, the method comprising the following steps:

(a) contacting a naïve library of antigen binding domains or antigen binding molecules with an antigen in the presence of a small molecule compound, (b) placing an antigen binding domain or antigen binding molecule coupled to the antigen in step (a) in the absence of the small molecule compound or in its presence but at a low concentration, and (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b), wherein the naïve library is a library of phages presenting on their surface two or more antigen binding domains or two or more antigen binding molecules.

[27]

A method for screening an antigen binding domain or an antigen binding molecule that has antigen binding activity dependent on a small molecule compound, the method comprising the following steps:

(a) contacting a library of antigen binding domains or antigen binding molecules with an antigen in the presence of a small molecule compound, (b) placing an antigen binding domain or antigen binding molecule coupled to the antigen in step (a) in the absence of the small molecule compound or in its presence but at a low concentration, and (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b), wherein the library is a library of phages having a defect in the pIII gene derived from the helper gene.

[28]

A method for screening an antigen binding domain or an antigen binding molecule that has antigen binding activity dependent on a small molecule compound, the method comprising the following steps:

(a) contacting a library of antigen binding domains or antigen binding molecules with an antigen in the presence of a small molecule compound, (b) placing an antigen binding domain or antigen binding molecule coupled to the antigen in step (a) in the absence of the small molecule compound or in its presence but at a low concentration, and (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b), wherein the library is a library comprising phages obtained by increasing the expression of the antigen binding domain or antigen binding molecule using a small molecule additive that increases the level of expression from a promoter that regulates the expression of the antigen binding domain or antigen-binding molecule.

[28.1]

The screening method according to claim [28], wherein the low molecular weight additive is isopropyl β-thiogalactopyranoside or arabinose.

- 18 046075

[28.2]

The method according to any one of claims [24] to [28.1], wherein the low molecular weight compound is an adenosine-containing compound.

[28.3]

The method according to any one of claims [24] to [28.2], wherein the low molecular weight compound is ATP.

[29] An antigen-binding molecule that has antigen-binding activity that is dependent on the concentration of a tumor tissue-specific compound, with antigen-binding activity in the presence of 100 μM of the compound being two times or more higher than antigen-binding activity in the absence of the compound.

[29.1] The antigen-binding molecule according to claim [29], the KD value of which in the presence of 100 μM of the compound is 5x 10 ^-7 M or less.

[29.2] Antigen-binding molecule according to claim [29] or [29.1], the KD value of which in the absence of a compound is 1x 10 ^-6 M or more.

[29.3] An antigen-binding molecule according to any one of claims [29] to [29.2], which has neutralizing activity against an antigen.

[29.4] An antigen-binding molecule according to any one of claims [29] to [29.3], which has cytotoxic activity against cells expressing the antigen.

[29.5] The antigen-binding molecule of any one of claims [29] to [29.4], wherein the antigen is an antigen expressed or secreted by any of a tumor cell, an immune cell, or a stromal cell in a tumor tissue.

[29.6] The antigen-binding molecule according to any one of claims [29] to [29.5], wherein the compound is an adenosine-containing compound.

[29.7] An antigen-binding molecule according to any one of claims [29] to [29.6], which contains an Fc region.

[29.8] The antigen binding molecule of claim [29.7], wherein the Fc region is a mutated Fc region comprising an amino acid change, wherein the mutated Fc region has increased binding activity to at least one Fcy receptor selected from the group consisting of FcYRIa, FcYRIIa, FcYRIIb, and FcYRIIIa, compared with the wild-type Fc region.

[29.9] The antigen binding molecule according to any one of claims [29] to [29.8], wherein the antigen binding molecule is an antibody or antibody fragment.

[30] A pharmaceutical composition comprising the antigen binding molecule of any one of claims [29][29.9] and a pharmaceutically acceptable carrier.

[30.1] The pharmaceutical composition according to claim [30], which is intended for the treatment of a tumor.

[30.2] The pharmaceutical composition of claim [30.1], which has reduced cytotoxic activity in non-tumor tissue compared to a pharmaceutical composition containing a control antigen-binding molecule.

[30.3] A pharmaceutical composition according to claims [30.1] or [30.2], which has a reduced level of side effects compared to a pharmaceutical composition containing a control antigen-binding molecule.

[30.4] The pharmaceutical composition according to claims [30.2] or [30.3], wherein the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of the tumor tissue-specific compound.

[31] A method for producing an antigen-binding molecule for use in the treatment of a tumor, which includes the step of selecting an antigen-binding molecule whose antigen-binding activity in the presence of 100 μM of a tissue-specific tumor compound is two times or more higher than the antigen-binding activity in the absence of the compound.

[32] A method for preparing a pharmaceutical composition for use in the treatment of a tumor, which includes the step of mixing an antigen-binding molecule according to any one of claims [29] to [29.9] with a pharmaceutically acceptable carrier.

[33] An antigen-binding molecule having antigen-binding activity that is dependent on the concentration of a target tissue-specific compound, wherein antigen-binding activity in the presence of 1 μM of such compound is reduced by two times or more compared to antigen-binding activity in the presence of a sufficient amount of the compound.

[33.1] The antigen-binding molecule according to claim [33], wherein the KD value in the presence of 1 μM of such compound is 2x 10 ^-7 M or more.

[33.2] Antigen-binding molecule according to claim [33] or [33.1], and the KD value in the presence of 1 μM of such a compound is 1x 10 ^-7 M or less.

[33.3] The antigen-binding molecule according to any one of claims [33] to [33.2], wherein the compound is a tissue-specific tumor compound.

[33.4] The antigen-binding molecule of claim [33.3], wherein the compound is an adenosine-containing compound.

[33.5] An antigen-binding molecule according to any one of claims [33] to [33.4], which has an increased

- 19 046075 plasma retention capacity and/or has a reduced ability to accumulate antigen in plasma compared to the control antigen binding molecule.

[33.6] The antigen-binding molecule of claim [33.5], which is an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of the target tissue-specific compound.

[33.7] An antigen-binding molecule according to any one of claims [33] to [33.6], which is an antibody or antibody fragment.

[34] A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims [33][33.7] and a pharmaceutically acceptable carrier.

[35] A method for producing an antigen binding molecule that has an increased plasma retention capacity and/or a reduced plasma antigen accumulation capacity compared to a control antigen binding molecule, the method comprising the steps of (a) producing an antigen binding molecule whose antigen binding activity increases as increasing the concentration of the target tissue-specific compound, and (b) measuring the plasma retention ability and/or plasma antigen accumulation ability of the antigen-binding molecule obtained according to item (a).

[35.1] The method of claim [35], which includes the step of selecting an antigen-binding molecule whose antigen-binding activity in the presence of 1 μM of the target tissue-specific compound is two times or more lower than the antigen-binding activity in the presence of a sufficient amount of the compound.

[35 .2] Method according to claims [35] or [35.1], wherein the control antigen-binding molecule is an antigen-binding molecule that does not have antigen-binding activity dependent on the concentration of the target tissue-specific compound.

[36] A method for preparing a pharmaceutical composition, which includes the step of mixing an antigen-binding molecule according to any one of claims [33] to [33.7] with a pharmaceutically acceptable carrier.

[37] A method for measuring the concentration of ATP in a solution, which includes the steps of (i) contacting a split Luc/HEK293 cell expressing P2Y11 with the solution, and (ii) measuring luciferase activity in the cell.

[37.1] The method of claim [37], which further includes the step of contacting a solution containing a luciferase substrate with a cell.

[37.2] Method according to paragraphs [37] or [37.1], in which the solution is an intercellular fluid in tissue in vivo.

[37.3] The method of claim [37.2], wherein the tissue is tumor tissue.

[37.4] The method of claim [37.2] or [37.3], wherein step (i) is the step of transplanting a split Luc/HEK293 cell expressing P2Y11 into tissue in vivo.

Brief description of the figures

Fig. 1. Diagram showing the agonist activity of various anti-CD137 antibodies tested using Jurkat cells in the presence or absence of ATP.

The X axis shows the antibody concentration (µg/ml), the Y axis shows the relative light units.

Fig. 2. Diagram showing the agonistic activity of various anti-CD137 antibodies tested using Jurkat cells in the presence or absence of ADP.

The X axis shows the antibody concentration (µg/ml), the Y axis shows the relative light units.

Fig. 3. Diagram showing the agonistic activity of various anti-CD137 antibodies tested using human T cells in the presence or absence of ADP-beta-S (ADP-beta-S).

Fig. 4. Diagram showing the agonist activity of dBBAT119-P253/dBBAT119LLamLib (anti-CD137 small molecule switchable antibody) or NS1-P253 (non-switchable anti-CD137 antibody) tested using human T cells in the presence or absence of ADP-beta-S .

The X-axis shows the antibody concentration (µg/ml), the Y-axis shows the amount of IFN-γ produced (ng/ml).

Fig. 5. Diagram showing the ATP-dependent antigen binding activity of various anti-CD137 antibodies (switchable anti-CD137 antibody with improved binding activity) tested by phage enzyme-linked immunosorbent assay (ELISA).

The Y axis shows the S/N (signal to noise) absorption ratio in the presence/absence of ATP, and the X axis shows the S/N ratio in the presence/absence of antigen.

Fig. 6. Diagram showing the binding activity of various anti-CD137 antibody variants (dBAT119H-P253/dBAT119L-LamLib) to human CD137 in the presence or absence of ATP.

The top row shows binding activity to human CD137 in the absence of ATP, and the bottom row shows binding activity to human CD137 in the presence of ATP.

Fig. 7. Diagram showing the agonist activity of dBBAT119H-P253/dBBAT119LLamLib, dBBATk119H024-P253/dBBATk119L020-LamLib, IC17HdK-hIgG1/IC17L-k0 (control) or NS1

- 20 046075

P253 (non-switchable aHTu-CD137 antibody) using human T cells in the presence or absence of ADP-beta-S.

Fig. 7A shows the results of testing in the absence of ADP-beta-S, FIG. 7B shows the results of testing in the presence of ADP-beta-S.

The X axis shows the antibody concentration (μg/ml), the Y axis shows the amount of IFN-γ produced (ng/ml).

Fig. 8. Diagram showing the agonist activity of various switchable anti-CD137 antibodies tested using the Jurkat 4-1BB reporter gene assay in the presence or absence of ATP.

Fig. 8A shows the test results in the absence of ATP, FIG. 8B shows the results of testing in the presence of ATP.

Fig. 9. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence of ATP due to increased binding activity of heavy chain constant regions to Fcy receptors tested using peripheral blood mononuclear cells.

Fig. 9A shows agonist activity determined using test results in the absence of ATP, FIG. 9B shows the results of testing in the presence of ATP.

Fig. 9A shows agonist activity determined using the amount of IL-2 produced as an indicator, FIG. 9B shows agonist activity determined using the amount of IFN-β produced as an indicator.

Fig. 10. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence of ATP due to increased binding activity of heavy chain constant regions to Fcy receptors or increased pI of heavy chain constant regions, as analyzed using human peripheral blood mononuclear cells.

Fig. 10A shows agonist activity determined using the amount of IL-2 produced as an indicator, Figure 10B shows agonist activity determined using the amount of IFN-γ produced as an indicator.

Fig. 11. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased heavy chain constant region binding activity to Fcy receptors, determined using human peripheral blood mononuclear cells.

Fig. 11A shows agonist activity determined using the amount of IL-2 produced as an indicator, FIG. 11B shows agonist activity determined using the amount of IFN-γ produced as an indicator.

Fig. 12. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased heavy chain constant region binding activity to Fcy receptors, determined using human peripheral blood mononuclear cells.

Fig. 12A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 12B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 13. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased binding activity of heavy chain constant regions to Fcy receptors, determined using human peripheral blood mononuclear cells.

Fig. 13A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 13B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 14. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased binding activity of heavy chain constant regions to Fcy receptors, as determined by a study using human peripheral blood mononuclear cells.

Fig. 14A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 14B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 15. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased binding activity of heavy chain constant regions to Fcy receptors, as determined by a study using human peripheral blood mononuclear cells.

Fig. 15A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 15B shows agonist activity determined using

- 21 046075 amount of IFN-γ produced as an indicator.

Fig. 16. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 16A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 16B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 17. Diagram showing the enhancement of agonistic activity of various switchable anti-CD137 antibodies in the presence or absence of ATP due to increased pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 17A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 17B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 18. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 18A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 18B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 19. Diagram showing the enhancement of agonistic activity of various switchable anti-CD137 antibodies in the presence or absence of ATP due to increased pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 19A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 19B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 20. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 20A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 20B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 21. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 21A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 21B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 22. Diagram showing the enhancement of agonistic activity of various switchable anti-CD137 antibodies in the presence or absence of ATP by increasing the pI value of heavy chain constant regions, determined using human peripheral blood mononuclear cells.

Fig. 22A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 22B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 23. Diagram showing the enhancement of agonistic activity of various anti-CD137 switch antibodies in the presence or absence of ATP due to increased binding activity of heavy chain constant regions to Fcy receptors, as determined by a study using human peripheral blood mononuclear cells.

Fig. 23A shows agonist activity determined by the amount of IL-2 produced as an indicator; fig. 23B shows agonist activity determined by the amount of IFN-γ produced as an indicator.

Fig. 24. Graph showing plasma concentrations of various switched and non-switched anti-CD137 antibodies tested using human CD137 knockin mice.

All Fc from mIgG1.

Fig. 25. Diagram showing plasma concentrations of various switchable and non-switchable

- 22 046075 aHTu-CD137-keyed antibodies studied using human CD137 knockin mice.

All Fc from MB110.

Fig. 26. Graph showing plasma concentrations of various switched and non-switched anti-CD137 antibodies assayed using human CD137 knockin mice.

All Fc from MV492.

Fig. 27. Diagram showing the antitumor effect of A375-mIgG1/B167-mYr on a mouse model transplanted with MC38 cells.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 28. Diagram showing organ weights of a mouse model transplanted with MC38 cells after administration of antibodies (NO1-mIgG1 or A375-mIgG1/B167-ml0r).

Fig. 28A shows the lymph node mass, and FIG. 28B shows the mass of the spleen.

Fig. 29. Diagram showing the degree of T cell activation in the lymph nodes of a mouse model transplanted with MC38 cells after administration of NO1-mIgG1 or A375-mIgG1/B167-ml0r antibodies.

Fig. 29A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 29B shows the percentage of ICOS positive T cells among CD8 positive T cells, FIG. 29B shows the percentage of granzyme B positive T cells among CD8 positive T cells.

Fig. 30. Diagram showing the degree of T cell activation in the spleen of a mouse model transplanted with the MC38 cell line after administration of NO1-mIgG1 or A375-mIgG1/B167-ml0r.

Fig. 30A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 30B shows the percentage of ICOS positive T cells among CD8 positive T cells, FIG. 30B shows the percentage of granzyme B positive T cells among CD8 positive T cells.

Fig. 31. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NO1-mIgG1 or A375-mIgG1/B167-ml0r.

Fig. 31A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 31B shows the percentage of granzyme B positive T cells among CD8 positive T cells.

Fig. 32. Diagram showing the antitumor effect of A356-MB110/B040-mUR in a mouse model transplanted with the MC38 cell line.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 33. Diagram showing organ mass in a mouse model transplanted with the MC38 cell line after administration of NS2-MB110 or Α356-ΜΒ110/Β040^0γ

Fig. 33A shows the lymph node mass, and FIG. 33B shows the mass of the spleen.

Fig. 34. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS2-MB110 or A3 56-MB110/B040-ml0r.

Fig. 34A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 34B shows the percentage of ICOS positive T cells among CD8 positive T cells.

Fig. 35. Diagram showing the antitumor effect of A372-mIgG1/B040-ml0r in a mouse model transplanted with the MC38 cell line.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 36. Lymph node cell count (FIG. 36A) and spleen weight (FIG. B) in a mouse model transplanted with the MC38 cell line after administration of A372-mIgG1/B040-ml0r.

Fig. 37. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of A372-mIgG1/B040-ml0r (percentage of granzyme B positive T cells among CD8 positive T cells).

Fig. 38. Diagram showing the antitumor effect of A372-MB110/B040-mUR in a mouse model transplanted with the MC38 cell line.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 39. Diagram showing organ weights of a mouse model transplanted with MC38 cells after administration of NS2-MB110 or A372-MB110/B040-ml0r.

Fig. 39A shows the lymph node mass, FIG. 39B shows the mass of the spleen.

Fig. 40. Graph showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS2-MB110 or A372-MB110/B040-ml0r (percentage of PD-1 positive T cells among CD8 positive T cells ).

Fig. 41. Diagram showing the antitumor effect of A372-MB110/B040-mUR in a mouse model transplanted with the MC38 cell line.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 42. Diagram showing lymph node cell number and spleen organ weight in a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A372-MB492/B040-ml0r.

Fig. 42A shows the number of lymph node cells, FIG. 42B shows the mass of the spleen.

Fig. 43. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A372-MB492/B040-ml0r (percentage of granzyme B-positive T cells among CD8-positive T cells).

Fig. 44. Diagram showing the antitumor effect of A486-MB492/B167-ml0r or A488MB492/B226-ml0r in a mouse model transplanted with the MC38 cell line.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 45. Diagram showing lymph node cell number and spleen weight in a mouse model transplanted with the MC38 cell line after administration of NS1-MB492, A486-MB492/B167ml0r, or A488-MB492/B226-ml0r.

Fig. 45A shows the number of cells in a lymph node, FIG. 45B shows the mass of the spleen.

Fig. 46. Diagram showing the level of effector cell infiltration in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-MB492, A486-MB492/B167ml0r or A488-MB492/B226-ml0r (percentage of CD3-positive and CD8-positive T cells among CD45-positive T cells).

Fig. 47. Diagram showing the antitumor effect of A489-MB492/B223-ml0r in a mouse model transplanted with the MC38 cell line.

Each dot represents the mean tumor volume for a group (n=5).

Fig. 48. Diagram showing the number of lymph node cells and the number of cells in the spleen lymphocyte fraction in a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A489-MB492/B223-ml0r.

Fig. Figure 48A shows the number of lymph node cells; Figure 48B shows the number of cells in the spleen lymphocyte fraction.

Fig. 49. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-MB492 or A489-MB492/B223-ml0r (percentage of CD8 positive T cells among CD45 positive T cells).

Fig. 50. Diagram showing the antitumor effect of A548-mIgG1/B256-ml0r and A551mIgG1/B256-ml0r in a mouse model transplanted with the MC38 cell line.

Fig. 50A shows the antitumor effect of A548-mIgG1/B256-ml0r, FIG. 50B shows the antitumor effect of A551-mIgG1/B256-ml0r.

Fig. 51. Diagram showing organ weights in a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1, A548-mIgG1/B256-ml0r, or A551-mIgG1/B256-ml0r.

Fig. 51A shows the lymph node mass, FIG. 51B shows the mass of the spleen.

Fig. 52. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1, A548-mIgG1/B256-ml0r or A551mIgG1/B256-ml0r.

Fig. 52A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 52B shows the percentage of granzyme B positive cells among CD8 positive T cells.

Fig. 53. Diagram showing the antitumor effect of A551-MB110/B379-ml0r in a mouse model transplanted with the MC38 cell line.

Fig. 54. Diagram showing organ weights in a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1 or A551-MB110/B379-ml0r.

Fig. 54A shows a lymph node mass, FIG. 54B shows the mass of the spleen.

Fig. 55. Diagram showing the degree of T cell activation in the spleen of a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1 or Α551-ΜΒ110/Β379^0γ

Fig. 55A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 55B shows the percentage of ICOS positive T cells among CD8 positive T cells, FIG. 55B shows the percentage of granzyme B positive T cells among CD8 positive T cells.

Fig. 56. Diagram showing the degree of T cell activation in the liver of a mouse model transplanted with the MC38 cell line after administration of NS1-mIgG1 or A551-MB 110/B379-ml0r.

Fig. 56A shows the percentage of PD-1 positive T cells among CD8 positive T cells, FIG. 56B shows the percentage of ICOS positive T cells among CD8 positive T cells, FIG. 56B shows the percentage of granzyme B positive T cells among CD8 positive T cells.

Fig. 57. Diagram showing the agonistic activity of various anti-CD137 antibodies using Jurkat cells in the presence or absence of L-kynurenine.

The X-axis shows the antibody concentration (µg/ml) and the Y-axis shows the relative light units.

Fig. 58. Diagram showing the agonist activity of various anti-CD137 antibodies using 4-1BB Jurkat cells in the presence or absence of a small molecule compound (ATP or FDP).

The X-axis shows the antibody concentration (µg/ml) and the Y-axis shows the relative light units.

Fig. 59. Diagram showing the ATP response (ATP concentration dependent luciferin luminescence) in P2Y11 split Luc/HEK293 cells obtained to determine extracellular ATP levels.

- 24 046075

Fig. 60. Diagram showing the in vivo ATP response (ATP concentration-dependent luciferin luminescence) in P2Y11 split Luc/HEK29 cells that were transplanted subcutaneously into mice.

Fig. 61. Diagram showing fluorescence imaging results of mice subcutaneously transplanted with P2Y11 split Luc/HEK293 cells and predetermined concentrations of ATP, and FM3A tumor-bearing mice subcutaneously transplanted with P2Y11 split Luc/HEK293 cells. Labels in the ventral region of mice indicate detected luminescence.

Fig. 62. Diagram showing the ATP concentration-dependent binding activity (KD value) of anti-hIL6R antibodies MRAH-G4T1/MRAL-k0 (control antibody) and H0002G4T1/L1058-lam1, H0041-G4T1/L1088-lam1, H0052-G4T1/L1083 -lam1 (all are switchable antibodies) against hIL6R.

Fig. 63. Diagram showing the ADP concentration-dependent binding activity (KD value) of anti-hIL6R antibodies MRAH-G4T1/MRAL-k0 (control antibody) and H0002G4T1/L1058-lam1, H0041-G4T1/L1088-lam1, H0052-G4T1/L1083 -lam1 (all are switchable antibodies) against hIL6R.

Fig. 64. Diagram showing AMP concentration-dependent binding activity (KD value) of anti-hIL6R antibodies MRAH-G4T1/MRAL-k0 (control antibody) and H0002G4T1/L1058-lam1, H0041-G4T1/L1088-lam1, H0052-G4T1/L1083 -lam1 (all are switchable antibodies) against hIL6R.

Fig. 65. Diagram showing ATP concentration-dependent ADCC activity of anti-hIL6R antibodies MRAH-mFa55/MRAL-mk0 (control antibody) and H0002-mFa55/L1058-ml0, H0041mFa55/L1088-ml0, H0052-mFa55/L1083-ml0 (all switchable) antibodies).

Fig. 66. Diagram showing the in vivo antitumor activity of anti-hIL6R antibodies MRAH-mFa55/MRAL-mk0 (control antibody) and H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0, H0052-mFa55/L1083-ml0 (all are switchable antibodies). IC17Hdk-mFa55/IC17L-mk1 is a negative control antibody.

Fig. 67. Diagram showing comparison of plasma kinetics of anti-hIL6R antibody MRAHmFa55/MRAL-mk0 (control antibody) in normal mice and in hIL6R transgenic mice. The vertical axis of the graph shows the concentration of antibody in plasma.

Fig. 68. Diagram showing a comparison of the plasma kinetics of the anti-hIL6R antibody H0002mFa55/L1058-ml0 (switchable antibody) in normal mice and in hIL6R transgenic mice. The vertical axis of the graph shows the concentration of antibody in plasma.

Fig. 69. Diagram showing a comparison of the plasma kinetics of the anti-hIL6R antibody H0041mFa55/L1088-ml0 (switchable antibody) in normal mice and in hIL6R transgenic mice. The vertical axis of the graph shows the concentration of antibody in plasma.

Fig. 70. Diagram showing a comparison of the plasma kinetics of the anti-hIL6R antibody H0052mFa55/L1083-ml0 (switchable antibody) in normal mice and in hIL6R transgenic mice. The vertical axis of the graph shows the concentration of antibody in plasma.

Fig. 71. Diagram showing the accumulation of antigens in hIL6R transgenic mice after administration of each of the anti-hIL6R antibodies: non-switchable antibody MRAH-mFa55/MRAL-mk0 (control antibody) and anti-hIL6R switchable antibodies H0002-mFa55/L1058-ml0, H0041- mFa55/L1088ml0, H0052-mFa55/L1083-ml0 (all are switchable antibodies). The vertical axis of the graph shows the plasma concentration of soluble hIL6R. IC17Hdk-mFa55/IC17L-mk1 (indicated as KLH-mFa55 in the figure) was used as a negative control antibody.

Fig. 72. Diagram showing in vivo antitumor activity of anti-hIL6R non-switchable antibodies MRAH-mFa55/MRAL-mk0 (control antibody) and anti-hIL6R switchable antibodies H0002-mFa55/L1058-ml0 and H0041-mFa55/L1088-ml0 (both switchable antibodies). IC17Hdk-mFa55/IC17L-mk1 is a negative control antibody.

Fig. 73. Diagram showing comparison of the plasma kinetics of anti-hIL6R non-switchable antibody MRAH-mFa55/MRAL-mk0 (control antibody) and anti-hIL6R switchable antibodies H0002-mFa55/L1058-ml0 and H0041-mFa55/L1088-ml0 (both switchable antibodies). The vertical axis of the graph shows the concentration of antibody in plasma.

Fig. 74. Diagram showing the accumulation of antigens after administration of each of the anti-hIL6R antibodies: non-switchable antibody MRAH-mFa55/MRAL-mk0 (control antibody) and anti-hIL6R switchable antibodies H0002-mFa55/L1058-ml0 and H0041-mFa55/L1088- ml0 (both are switchable antibodies). The vertical axis of the graph shows the plasma concentration of soluble hIL6R. IC17Hdk-mFa55/IC17L-mk1 (labeled KLH-mFa55 in the figure) was used as a negative control antibody.

Fig. 75. Diagram showing in vivo antitumor activity of anti-hIL6R non-switchable antibody MRAH-mFa55/MRAL-mk0 (control antibody) and anti-HIL6R switchable antibodies H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 (both switchable antibodies). IC17Hdk-mFa55/IC17L-mk1 is a negative control antibody.

- 25 046075

Fig. 76. Diagram showing a comparison of the plasma kinetics of the aHTu-hIL6R non-switchable antibody MRAH-mFa55/MRAL-mk0 (control antibody) and the anti-AL6R switchable antibody H0052-mFa55/L1083-ml0 (switchable antibody). The vertical axis of the graph shows the concentration of antibody in plasma.

Fig. 77. Diagram showing the accumulation of antigens after administration of each of the anti-hIL6R antibodies: non-switchable antibody MRAH-mFa55/MRAL-mk0 (control antibody) and anti-L6R switchable antibody H0052-mFa55/L1083-ml0 (switchable antibody). The vertical axis of the graph shows the plasma concentration of soluble hIL6R. IC17Hdk-mFa55/IC17L-mk1 (labeled KLH-mFa55 in the figure) was used as a negative control antibody.

Fig. 78. Diagram showing the ATP concentration-dependent activity of the anti-PD1 antibodies mPD1F2VH-mF18/mPD1F2VL-mk1 (control antibody) and H5029-mFa31/L3021-ml0 (switch antibody) to inhibit PD-1/PDL-1 binding.

Fig. 79. Diagram showing the ATP concentration-dependent activity of the anti-PD1 antibodies mPD1F2VH-mF18/mPD1F2VL-mk1 (control antibody) and H5041-mFa31/L3021-ml0 (switch antibody) to inhibit PD-1/PDL-1 binding.

Fig. 80. Diagram showing the AMP concentration-dependent in vitro neutralizing activity of the anti-PD1 antibodies mPD1F2VH-mF18/mPD1F2VL-mk1 (control antibody), H5029mFa31/L3021-ml0 and H5041-mFa31/L3021-ml0 (both switch antibodies).

Fig. 81. Diagram showing the in vitro ATP concentration-dependent neutralizing activity of the anti-PD1 antibodies mPD1F2VH-mF18/mPD1F2VL-mk1 (control antibody) and H5029mFa31/L3021-ml0 and H5041-mFa31/L3021-ml0 (both switch antibodies).

Fig. 82. Diagram showing in vivo anti-tumor activity of anti-PD1 antibodies mPD1F2VH-mFa55/mPD1F2VL-mk1 (control antibody) and H5041-mFa55/L3023-ml0 (switch antibody). IC17Hdk-mFa55/IC17L-mk1 is a negative control antibody.

Fig. 83. Diagram showing the activity of anti-PD1 antibodies mPD1F2VH-mFa55/mPD1F2VLmk1 (control antibody) and H5041-mFa55/L3023-ml0 (switch antibody) in eliminating PD-1-expressing cells from (A) tumor and (B) spleen. In the figure, the isotype represents the negative control antibody (IC17Hdk-mFa55/IC17L-mk1).

Fig. 84. Diagram showing the binding mechanism of ATP and the Fab fragment of the anti-hIL6R switch antibody H0041L1088. This figure shows ATP as a ball-and-stick model and the amino acid residues that interact with ATP are shown as a stick-and-stick model. The dotted lines indicate hydrogen bonds between the antibody and ATP.

Fig. 85. Diagram showing the amino acid sequence of the extracellular domain of hIL6R (shIL6R) mapped to the anti-hIL6R epitope of the switch antibody H0041L1088. In the figure, the amino acid residues shaded in gray are residues (epitope residues) of shIL6R containing one or more non-hydrogen atoms located 4.2 angstroms or less from ATP or Fab H0041L1088 in the crystal structure.

Fig. 86. Diagram showing details of binding between shIL6R and Fab fragment ATP bound H0041L1088. In the figure, the antibody's heavy chain is shown in black, the light chain in gray, and shIL6R in white. In the figure, ATP is shown as a hinge model, and the shIL6R epitope residue within 4.2 angstroms of the antibody or ATP and the antibody paratope residue within 4.2 angstroms of the epitope residue are shown using the rod model. Dashed lines indicate hydrogen bonds between the antibody and shIL6R. To clarify the interaction with ATP, only F298 shIL6R is shown using a spherical model.

Fig. 87. Diagram showing a structure different from the structure in FIG. 86 rotated 180° (rear view).

Fig. 88. Diagram showing the agonistic activity of various switchable anti-CD137 antibodies tested using the Jurkat 4-1BB reporter gene assay in the presence of ATP.

Fig. 89. Diagram showing comparison of plasma kinetics for each of the anti-CD137 switch antibodies A375-SCF041aPh/B167-Lamlib and A375-MY201aPh/B167-Lamlib. The vertical axis of the graph shows the plasma concentration of each antibody.

Fig. 90. Diagram showing the antitumor effect of each of A375/B167-SCF041aPh and A375/B167-MY201aPh in a mouse model generated by transplantation of the LLC1/OVA/GPC3 cell line into hCD137KI/mFcYR2bKO/hFcYR2bTg#90 mice.

Each dot shows the average tumor volume in the group (n=5).

Fig. 91. Diagram showing the agonistic activity of various switchable anti-CD3 antibodies examined by reporter gene assay using T cell activation Bioassay (NFAT) in the presence of ATP.

- 26 046075

Embodiments of the present invention

I. Definitions.

The term binding activity refers to the strength of the total amount of non-covalent interactions between one or more binding sites of a molecule (eg, antibody) and its binding partner (eg, antigen). In the present invention, binding activity is not strictly limited to a 1:1 interaction between members of a binding pair (eg, antibody and antigen). For example, if members of a binding pair reflect a 1:1 monovalent interaction, the binding activity is specifically referred to as intrinsic binding affinity. If a member of a binding pair is capable of both monovalent binding and multivalent binding, the binding activity is the sum of each binding strength. The binding activity of molecule X towards its partner Y can typically be represented by the dissociation constant (KD) or the binding amount of test compound per unit amount of ligand (hereinafter may be referred to as binding amount). Those skilled in the art will appreciate that, in general, a lower dissociation constant (KD) value indicates higher binding activity, and a higher amount of analyte binding per unit amount of ligand or binding amount means higher binding activity. Binding activity can be measured by conventional methods known in the art, including those described in the present invention. Specific illustrations and examples of embodiments of the present invention for measuring binding activity are described below.

The concept of an antigen binding molecule or antibody with matured binding activity or an antigen binding molecule or antibody with increased binding activity refers to an antibody with one or more changes in one or more hypervariable regions (HVRs) compared to the original antigen binding molecule or parent antibody that is not bear such changes; such compounds acquire improved binding activity of the antigen-binding molecule or antibody to the antigen.

The terms '^4^^:0137 antigen-binding molecule against or anti-CD137 antibody and antigen-binding molecule that binds to CD137 or antibody that binds to CD137 refer to an antigen-binding molecule or antibody that is capable of binding to CD137 with sufficient binding activity, in whereby an antigen binding molecule or antibody can be used as a diagnostic and/or therapeutic agent for targeting CD137. In some embodiments, the anti-CO137 antibody binds to an epitope of CD137 that is conserved among CD137 species.

The term anti-CD137 antigen binding activity or an anti-CD137 antibody having small molecule dependent CD137 binding activity means an antigen binding molecule or antibody exhibiting increased binding activity to CD137 in the presence of the small molecule compound compared to binding activity to CD137 in the absence of the small molecule compound. In one embodiment of the present invention, the presence of a small molecule compound refers to a condition in which the small molecule compound is present in a concentration of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more . In one embodiment of the present invention, the degree of binding activity of the anti-CD137 antigen binding molecule or antibody to an unrelated non-CD137 protein in the presence of a small molecule compound is less than about 10% of the binding of the antigen binding molecule or antibody to CD137 as measured by, e.g. radioimmunoassay (RIA, radioimmunoassay) or surface plasmon resonance (SPR, surface plasmon resonance). In some embodiments of the present invention, in the presence of a small molecule compound, the anti-CD137 antigen binding molecule or antibody has a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (for example, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, for example, from 10 ^-6 M to 10 ^-10 M, from 10 ^-7 M to 10 ^-9 M, for example, from 10 ^-7 M to 10 ^-8 M).

In the present invention, the term antigen-binding molecule is used in its broadest sense and refers to a molecule that specifically binds to an antigenic determinant. In one embodiment of the present invention, the antigen binding molecule is an antibody, an antibody fragment, or an antibody derivative.

In the context of the present invention, the term agonistic antigen binding molecule or agonistic antibody in the context of the present invention is an antigen binding molecule or antibody that significantly induces or enhances the biological activity of the antigen to which it binds (eg, CD137 and CD3).

Therefore, if the antigen is, for example, CD137, such an antigen binding molecule or antibody having an agonistic effect is called a CD137 agonistic antigen binding molecule or a CD137 agonistic antibody, respectively.

- 27 046075

In the context of the present invention, the term antibody is used in the broadest sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) and antibody fragments, as long as they exhibit the necessary antigen-binding activity.

In the context of the present invention, the term antibody fragment refers to a molecule, other than an intact antibody, that includes a portion of the intact antibody that binds 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 (eg scFv); and multispecific antibodies formed from antibody fragments.

An antigen-binding molecule that binds to the same epitope or an antibody that binds to the same epitope as a control antigen-binding molecule or control antibody refers to an antibody or antigen-binding molecule that blocks the binding of a control antibody or control antigen-binding molecule to its antigen in a competition assay by 50% or more, and conversely, the control antibody blocks binding of the antibody to its antigen in the competition assay by 50% or more. An example of competitive analysis is given in the present invention. In one embodiment of the present invention, if a control antigen-binding molecule or control antibody exhibits antigen-binding activity depending on the small molecule compound, the competition assay is performed in the presence of the small molecule compound.

The term chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The concept of an antibody class is determined by the type of constant domain or constant region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, _IgG4 , IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively.

The term effector functions refers to those biological activities attributable to the Fc region of an antibody that vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity); phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor); and activation of B cells.

The term cytotoxicity refers to an activity that inhibits or interferes with cellular function and/or causes cell death or destruction. Cytotoxicity may be, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and T cell-mediated cytotoxicity; There may also be cytotoxicity caused by cytotoxic agents (eg, radioisotopes and chemotherapeutic agents) such as immunoconjugates.

In the context of the present invention, the term Fc region is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least part of a constant region. The concept includes native sequence Fc regions and Fc region variants. In one embodiment of the present invention, the Fc region of the human IgG heavy chain is located from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) Fc region may or may not be present. Unless otherwise specified in the present invention, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., ed. National Institutes of Health, Bethesda, MD, 1991.

The term Fc region variant as used herein includes an amino acid sequence that differs from the native Fc region sequence by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution compared to the native Fc region sequence or the Fc region of the parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the native sequence of the region Fc or in the Fc region of the parent polypeptide. In the present invention, the variant Fc region may preferably have at least about 80% homology to the native Fc region sequence and/or the Fc region of the parent polypeptide, and most preferably at least about 90% homology, more preferably at least about 95% homology .

In the present invention, amino acid changes or substitutions in the Fc region or constant region may be represented by a combination of amino acids expressed in the EU numbering system.

- 28 046075

For example, S424N indicates a substitution at position 424 of the EC numbering from serine (Ser) to asparagine (Asn). EU424N means a change at position 424 in EU numbering from an amino acid (any type) to asparagine (Asn).

In the present invention, the term Fc region-containing antibody means an antibody that contains an Fc region. The C-terminal lysine (residue 447 in the EU numbering system) or the C-terminal glycine lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or recombinant engineering of the nucleic acid encoding the antibody. Accordingly, a composition containing an antibody having an Fc region in accordance with the present invention may contain an antibody with G446-K447, with G446 and without K447, with all G446-K447 removed, or a mixture of the three types of antibodies described above.

In the present invention, the terms full-length antibody, intact antibody, and whole antibody are used interchangeably to refer to an antibody having a structure substantially similar to that of a native antibody or having heavy chains that contain an Fc region or a variant Fc region as described herein.

A human antibody is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human body or a human cell, or obtained from a non-human source that uses human antibody repertoires or other human antibody-encoding sequences. This definition of human antibody specifically excludes a humanized antibody containing non-human antigen-binding moieties.

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

In the present invention, a human acceptor framework is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as described below. A human acceptor framework derived from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence, or it may contain changes in the amino acid sequence. In some embodiments of the present invention, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments of the present invention, the human VL acceptor framework sequence is identical to a human immunoglobulin VL framework sequence or a human consensus framework sequence.

In the present invention, the term human consensus framework region means the most frequently occurring amino acid residues when selecting human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable domain sequences. Typically, the subset of sequences is the subset described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, 1991, volumes 1-3, Bethesda, MD. In one embodiment of the present invention, the VL subgroup is kappa I, as described in Kabat et al., supra. In one embodiment of the present invention, the VH subgroup is subgroup III, as described in Kabat et al., supra.

The term humanized antibody refers to a chimeric antibody containing amino acid residues from non-human HVR and amino acid residues from human FR. In some embodiments of the present invention, a humanized antibody may include substantially all of at least one, typically two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of the non-human antibody, and all or substantially all from FR correspond to those of human antibodies. The humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. A humanized form of an antibody, for example a non-human antibody, refers to an antibody that has been humanized.

The term variable region or variable domain refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody typically have similar structures, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, ^6th ed., WH Freeman and Co., 2001, p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind a particular antigen can be isolated using the VH or VL domain from the antibody that binds the antigen for library screening

- 29 046075 complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150, 1993, 880-887; Clarkson et al., Nature 352, 1991, 624-628.

The term hypervariable region or HVR in the context of the present invention refers to each of the regions of the variable domain of an antibody that are hypervariable in sequence (complementarity determining regions or CDRs) and/or form structurally defined loops (hypervariable loops). ) and/or contain residues in contact with the antigen (antigen contacts). Antibodies typically contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Examples of HVRs in this document include:

(a) hypervariable loops found at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3 ) (Chothia, Lesk, J. Mol. Biol. 196, 1987, 901-917);

(b) CDRs carrying amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Bethesda, MD, 1991);

(c) antigen contacts at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) ( MacCallum et al., J. Mol. Biol., 262, 1996, 732-745);

(d) combinations (a), (b) and/or (c), including amino acid residues HVR 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered in the present invention according to Kabat et al., supra. In the present invention, HVR residues or other residues in a variable domain (eg, FR residues) and amino acid changes or substitutions of such residues may be represented in combination using the Kabat numbering system. For example, N99 means asparagine (Asn) at position 99 in the Kabat numbering, and N99A means a substitution at position 99 in Kabat numbering from asparagine (Asn) to alanine (Ala).

As used herein, an immunoconjugate is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

In the present invention, the term cytotoxic agent refers to a substance that inhibits or interferes with the expression of cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (eg, ²¹¹ At, ¹³¹ 1, ¹²⁵ 1, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and Lu radioactive isotopes); chemotherapeutic agents or drugs (eg, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents described below.

The concept of an isolated antibody is an antibody that has been separated from components of its natural environment. In some embodiments of the present invention, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse chromatography). phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. In 848, 2007, 79-87.

The term isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present outside of a chromosome or has a chromosomal location that differs from its natural chromosomal location.

In the present invention, the term vector refers to a nucleic acid molecule capable of propagating with another nucleic acid to which it is linked. The concept includes a vector as a self-replicating nucleic acid structure, as well as a vector included in the genome of the host cell into which it was introduced. Some vectors are capable of directing the expression of the nucleic acids to which they are operably linked. Such vectors are referred to in the present invention as expression vectors.

The term anti-CD137 antigen binding molecule encoded nucleic acid refers to one or more nucleic acid molecules that encode the polypeptide(s) constituting the antigen binding molecule. The term isolated nucleic acid encoding an anti-CD137 antibody refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecule(s) in one vector or in separate vectors, and such molecule Nucleic acid (molecules) present at one or more sites in a host cell.

The terms host cell, host cell line, and host cell culture are used interchangeably.

- 30 046075 is replaceable and refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and the progeny derived from it, regardless of the number of passages. The offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. In the present invention, these concepts also include mutant progenies that have the same function or biological activity as those tested or selected from the originally transformed cells.

In the context of the present invention, the term monoclonal antibody refers to an antibody obtained from a population of essentially homogeneous antibodies, i.e. The individual antibodies composing the population are identical and/or bind to the same epitope, with the exception of possible variant antibodies, for example those containing naturally occurring mutations or occurring during production of the monoclonal antibody preparation, such variants are usually present in minute quantities. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the designation monoclonal refers to the nature of the antibody as being derived from an essentially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present description can be produced by various methods, including, but not limited to, hybridoma method, recombinant DNA methods, phage display methods and methods using transgenic animals containing all or part of the immunoglobulin loci human, as well as other methods for producing monoclonal antibodies, examples of the use of which are described in the present invention.

The term naked antibody refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or to a radiolabel. The naked antibody may be present in the pharmaceutical composition.

The concept of native antibodies refers to natural immunoglobulin molecules of various structures. For example, native IgG antibodies are approximately 150,000 dalton heterotetrameric glycoproteins consisting of two identical light chains and two identical heavy chains linked by a disulfide bond. From the N- 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). Likewise, from N- 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 light chain constant domain (CL). The light chain of an antibody can be classified into one of two types, called kappa (k) and lambda (λ), based on the amino acid sequence of the corresponding constant domain.

The concept of percentage (%) of amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be achieved by various methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

The ALIGN-2 sequence alignment computer program was developed by Genentech, Inc., and the source code has been filed with user documentation at the United States Copyright Office, Washington, DC 20559, where it is registered under Copyright Number US TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. ALIGN-2 can be compiled for use on UNIX operating systems, including UNIX Digital Operating System V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used to compare amino acid sequences, the percentage identity of a given amino acid sequence to amino acid sequence A with/against amino acid sequence B (which can alternatively be stated as a given amino acid sequence A that has or contains a certain percentage of amino acid sequence identity

- 31 046075 lot sequence with, with or against a given amino acid sequence B) is calculated as follows:

100-fold X/Y fraction, where X is the number of amino acid residues scored as identical matches by the ALIGN-2 sequence alignment program when aligning A and B by that program, and where Y is the total number of amino acid residues in B. Note that if the length amino acid sequence A is not equal in length to amino acid sequence B, the percentage identity of the amino acid sequence A to B will not equal the percentage identity of the amino acid sequence B to A. Unless specifically stated otherwise, all percent amino acid sequence identity values used in the present invention are obtained directly from the description in the previous paragraph, using the ALIGN-2 computer program.

The term pharmaceutical composition refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to whom the composition is to be administered.

The term pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical composition, other than the active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.

The term effective amount of an agent, such as a pharmaceutical composition, refers to an amount effective in dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

The term individual or subject means a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and other primates such as monkeys), rabbits, and rodents (e.g., mice and rats) . In some embodiments of the present invention, the individual or subject is a human.

As used herein, the term CD137 refers to any naturally occurring CD137 from any vertebrate, including mammals, such as primates (humans) and rodents (eg, mice and rats), unless otherwise noted. The term covers unprocessed full-length CD137 as well as any form of CD137 that results from cellular processing. The term also includes naturally occurring variants of CD137, such as splice variants or allelic variants. The amino acid sequence of exemplary human full-length CD137 is shown in SEQ ID NO: 1 (NCBI reference sequence: NP_001552.2), and the exemplary amino acid sequence of exemplary human CD137 extracellular region is shown in SEQ ID NO: 2. The amino acid sequence of exemplary full-length mouse CD137 is shown in SEQ ID NO: 3 (NCBI reference sequence: NP_035742.1), the amino acid sequence of an exemplary extracellular region of mouse CD137 is shown in SEQ ID NO: 4. The amino acid sequence of an example full-length monkey CD137 is shown in SEQ ID NO: 5 (NCBI reference sequence: ABY47575 .1), the amino acid sequence of an example extracellular region of monkey CD137 is shown in SEQ ID NO: 6.

CD137 is a member of the tumor necrosis factor (TNF) receptor family. It is also designated as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB and ILA. In addition to expression on activated CD4' and CD8+ T cells, CD137 is expressed on B cells, dendritic cells, natural killer (NK) cells and NK T cells, macrophages, monocytes, neutrophils, CD4 ⁺ CD25+ regulatory T cells, and endothelial cells vessels. Expression in tumor cells has also been described (Labiano et al. Oncoimmunology, 24, 2015, e1062967). The natural ligand of CD137, CD137L, is presented by antigen presenting cells such as B cells, monocytes/macrophages and dendritic cells (Watts et al., Annu. Rev. Immunol., 23, 2005, 23-68). Through ligand interaction, CD137 causes an increase in TCR-induced T cell proliferation, cytokine production, functional maturation, suppression of apoptosis, and long-term survival of CD8+ T cells (Nam et al., Curr. Cancer Drug Targets, 5, 2005, 357-363 ; Watts, et al., Annu. Rev. Immunol., 23, 2005, 23-68).

The terms carcinoma, cancer, and malignant refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.

The term tumor refers to all neoplastic cells of growth and proliferation, malignant or benign, and to all precancerous and malignant cells and tissues. The terms carcinoma, cancer, malignant, cell proliferation disorder, proliferative disorder and tumor are not mutually exclusive in the context of the present invention.

The terms cell proliferation disorder and proliferative disorder refer to disorders that involve some degree of abnormal cell proliferation. In one embodiment of the present invention, the cell proliferation disorder is cancer. In one of the

- 32 046075 ants of the present invention, the proliferative disorder is cancer.

In the context of the present invention, the concept of treatment (and its grammatical variations such as treat) refers to a clinical intervention in an attempt to change the condition of an individual, and can be carried out either for prevention or during the course of ongoing clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of onset or recurrence of disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of a disease or slow the progression of a disease.

II. Compositions and methods (anti-CD137 agonistic antigen-binding molecules).

In one embodiment, the present invention is based in part on anti-CD137 agonistic antigen-binding molecules and their uses. In some embodiments, the present invention provides antibodies that bind to CD137. The antibodies of the present invention may exhibit immune cell activating effects, cytotoxicity, or antitumor activity and are therefore useful, for example, in the diagnosis or treatment of cancer.

A. Typical anti-CD137 antigen-binding molecules or antibodies.

In one embodiment, the present invention provides isolated antigen-binding molecules or antibodies that bind to CD137. In some embodiments of the present invention, anti-CD137 antigen-binding molecules or antibodies have CD137-binding activity dependent on a small molecule compound;

bind to the extracellular region of CD137;

form a ternary complex together with a low molecular weight compound and CD137;

bind to human-derived CD137 and monkey-derived CD137;

are agonists in relation to the activity of CD137;

exhibit agonistic activity towards CD137 in the presence of a low molecular weight compound;

have low agonistic activity towards CD137 in the absence of a small molecule compound; and/or exhibit virtually no CD137 agonist activity in the absence of the small molecule compound.

Binding Activity of Antigen-Binding Molecules or Antibodies In some embodiments of the present invention, the binding activity of antigen-binding molecules or antibodies of the present invention in the presence of a small molecule compound has a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less , 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (for example, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, for example from 10 ^-6 M to 10 ^-10 M, from 10 ^-7 M to 10 ^-9 M, for example from 10 ^-7 M to 10 ^-8 M).

In one embodiment of the present invention, the binding activity of antigen-binding molecules or antibodies is measured using a radioimmunoassay (RIA) using a radiolabeled antigen and is expressed as KD. In one embodiment of the present invention, an RIA is performed with a Fab version of the test antibody and its antigen. For example, the affinity of a Fab solution for an antigen is measured by equilibrating the Fab with a minimal concentration of ( ^|25 [)-labeled antigen in the presence of a series of titrations of unlabeled antigen, followed by capture of the bound antigen using a plate coated with anti-Fab antibodies (see, for example, Chen et al. et al., J Mol Biol 293, 1999, 865-881). To establish conditions for the MICROTITER (registered trademark) assay, multiwell plates (Thermo Scientific) are coated overnight with 5 μg/ml anti-Fab capture antibody (Capel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked 2% (w/v) bovine serum albumin solution in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc, catalog number 269620), 100 pM or 26 pM [ ¹² 5[|-antigen is mixed with serial dilutions of the Fab fragment of interest (for example, corresponds to the evaluation of the anti-VEGF antibody, Fab-12, in the publication Presta et al., Cancer Res. 57, 1997, 4593-4599). The Fab to be tested is then incubated overnight; however, incubation may be longer (eg, approximately 65 hours) to ensure that equilibrium reaction is achieved. The mixtures are then transferred to a gripper plate for incubation at room temperature (eg, 1 hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (trade name)) in SFB. After the plates are dry, 150 μl/well of scintillator (MICROSCINT-20™; Packard) is added and the emission from the plates is counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that exhibit rates less than or equal to 20% of maximum binding are selected for use in competitive binding assays.

- 33 046075

In one embodiment of the present invention, ligand capture methods are used to measure the binding activity of an antibody, for example, using BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare, Uppsala, Sweden), which are based on assay methods surface plasmon resonance as the measurement principle. Control software BIACORE (registered trademark) is used to operate the devices. In one embodiment of the present invention, an amine coupling kit (GE Healthcare, Upisal, Sweden) is used, following the manufacturer's instructions, to ensure that the ligand capture molecule, e.g. anti-tag antibody, anti-IgG antibody, protein A, etc., is used. was recorded on a sensor chip (GE Healthcare, Uppsala, Sweden) coated with carboxymethyldextran. The ligand-capturing molecule is diluted with 10 mM sodium acetate at the appropriate pH and injected at the appropriate flow rate for the appropriate injection time. Binding activity measurements are carried out using a buffer containing 0.05% polysorbate 20 (also known as Tween-20 (registered trademark)) as the measurement buffer at a flow rate of 10-30 microliters per minute, with the measurement temperature preferably maintained at level 25°C or 37°C. For a measurement made with an antigen and/or Fc receptor captured by a ligand-binding molecule as a ligand, the antigen and/or Fc receptor is injected to allow the target amount to be captured, and then a serial dilution of the antibody (test compound) prepared is injected using a measurement buffer.

In one embodiment of the present invention, the measurement results are analyzed using BIACORE (registered trademark) Evaluation Software. Calculation of kinetic parameters is performed by fitting association and dissociation sensorgrams simultaneously using a 1:1 binding model and the association rate (kon or ka), dissociation rate (koff or kd) and equilibrium dissociation constant (KD) can be calculated. For weak binding activity, particularly for cases where dissociation is rapid and kinetic parameters are difficult to calculate, a steady state model can be used to calculate the equilibrium dissociation constant (KD). As additional parameters regarding binding activity, the amount of binding test compound per unit amount of ligand can be calculated by dividing the binding amount of test compound (resonance unit: RU) at a particular concentration by the amount of ligand captured.

Small molecule dependent binding activity.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody has small molecule dependent CD137 binding activity. In one non-limiting embodiment of the present invention, the antigen binding molecule or anti-CD137 antibody has higher binding activity for CD137 in the presence of the small molecule compound compared to binding activity for CD137 in the absence of the small molecule compound. In another embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody has increased binding activity to CD137 in the presence of a high concentration of a small molecule compound compared to CD137 binding activity in the presence of a small molecule compound at a low concentration. In one preferred embodiment of the present invention, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of a small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more more, multiple of 1x 10 ³ or more, multiple of 2x 10 ³ or more, multiple of 3x 10 ³ or more, multiple of 5x10 ³ or more, multiple of 1x10 ⁴ or more, multiple of 2x10 ⁴ or more, multiple of 3x10 ⁴ or more, multiple of 5x10 ⁴ or more or a multiple of 1x 10 ⁵ or more compared to the binding activity in the absence of the low molecular weight compound. In another preferred embodiment of the present invention, the binding activity of the anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of a small molecule compound is greater than 2-fold, greater than 3-fold, greater than 5-fold, greater than 10-fold, greater than 15 times, more than 20 times, more than 25 times, more than 30 times, more than 50 times, more than 100 times, more than 200 times, more than 300 times, more than 500 times , more than 1000 times, more than 2000 times, more than 3000 times, more than 5000 times, more than 10000 times, more than 20000 times, more than 30000 times, more than 50000 times or more than 100,000 times the binding activity in the absence of the low molecular weight compound.

The concentration of the small molecule compound can be any arbitrary concentration as long as a difference in the binding activity of the anti-CD137 antigen binding molecule or antibody is detected. In one embodiment of the present invention, the concentration of the low molecular weight compound in the presence of the low molecular weight compound and/or in the presence of

- 34 046075 high concentration of a low molecular weight compound is, for example, 100 nM or more, 500 nM or more, 1 μM or more, 3 μM or more, 5 μM or more, 10 μM or more, 50 μM or more, 100 μM or more , 150 μM or more, 200 μM or more, 250 μM or more, 300 μM or more, 400 μM or more, 500 μM or more, or 1 mM or more. Alternatively, the concentration may be defined as an amount sufficient for the anti-CD137 antigen binding molecule or antibody to exhibit maximum binding activity. Additionally, in one embodiment of the present invention, the concentration of the small molecule compound in the presence of the low concentration of the small molecule compound may be, for example, 500 μM or less, 250 μM or less, 200 μM or less, 150 μM or less, 100 μM or less, 50 µM or less, 10 µM or less, 1 µM or less, 500 nM or less, 100 nM or less, 50 nM or less, or 10 nM or 1 nM or less. The case where the concentration of the low molecular weight compound is zero, or the actual concentration is zero, can also be selected as a low concentration embodiment of the present invention.

In the present invention, the term real concentration equal to zero means, for example, a concentration that is so small that it cannot be detected using current technology, although the low molecular weight compound is present.

In one embodiment of the present invention, the CD137 binding activity in the presence of a small molecule compound at a concentration of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM is 2-fold or greater, 5-fold or greater, 10-fold. times or more, 15 times or more, 16 times or more, 17 times or more, 18 times or more, 19 times or more, 20 times or more, compared to CD137 binding activity in the absence of low molecular weight compound.

In one embodiment of the present invention, the binding activity of the anti-CD137 antigen-binding molecule or anti-CD137 antibody in the presence of a small molecule compound at a concentration of 10 μM or more is 2-fold or more, 5-fold or more, 10-fold or more, 15-fold or more , 16 times or more, 17 times or more, 18 times or more, 19 times or more, 20 times or more compared to CD137 binding activity in the absence of the small molecule compound. In one embodiment of the present invention, the binding activity of the anti-CD137 antigen-binding molecule or anti-CD137 antibody in the presence of a small molecule compound at a concentration of 100 μM or more is 2-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 16 times or more, 17 times or more, 18 times or more, 19 times or more, 20 times or more compared to CD137 binding activity in the absence of the small molecule compound.

In one embodiment of the present invention, the binding activity (KD) of an anti-CD137 antigen-binding molecule or antibody against CD137 in the presence of a small molecule compound in an amount of 10 μM or more has a dissociation constant (KD) of 9 x 10 ⁷ M or less, 8 x 10 ^-7 M or less, 7x10 ^-7 M or less, 6x10 ^-7 M or less, 5x10 ^-7 M or less, or 4x10 ^-7 M or less, or preferably a dissociation constant (KD) of 5x10 ^-7 M or less. In another embodiment of the present invention, the binding activity (KD) of an anti-CD137 antigen-binding molecule or anti-CD137 antibody in the absence of a small molecule compound is too large to be calculated using Biacore (weak binding activity), or it has a dissociation constant (KD) of 1 x 10 ^-7 M or more, 5x10 ^-7 M or more, 7x10 ^-7 M or more, 8x10 ^-7 M or more, 9x10 ^-7 M or more, 1 x 10 ^-6 M or more, 2x10 ^-6 M or more than 3x10 ^-6 M or more, or 4x10 ^-6 M or more, or preferably a dissociation constant (KD) of 1x 10 ^-6 M or more. In another embodiment of the present invention, the binding activity (KD) of an anti-CD137 antigen-binding molecule or antibody to CD137 in the presence of a small molecule compound in an amount of 100 μM or more is 9x10 ^-7 M or less, 8x10 ^-7 M or less, 7x10 ^-7 M or less, 6x 10 ^-7 M or less, 5x 10 ^-7 M or less, 4x 10 ^-7 M or less, 3x 10 ^-7 M or less, 2x 10 ^-7 M or less, or 1x10 ^-7 M or less , or preferably, the dissociation constant (KD) is 2x10 ^-7 M or less. In another embodiment of the present invention, the binding activity (KD) of an anti-CD137 antigen-binding molecule or anti-CD137 antibody in the absence of a small molecule compound is too high to be calculated using Biacore (weak binding activity), or is a dissociation constant (KD) of 1x10 ^-7 M or more, 5x10 ^-7 M or more, 7x10 ^-7 M or more, 8x10 ^-7 M or more, 9x10 ^-7 M or more, 1x 10 ^-6 M or more, 2x10 ^-6 M or more, 3x 10 ^-6 M or more, or 4x 10 ^-6 M or more, or preferably, dissociation constant (KD) 1x 10 ^-6 M or more.

In one embodiment of the present invention, the binding activity (KD) of an anti-CD137 antigen-binding molecule or anti-CD137 antibody in the presence of a small molecule compound in an amount of 10 μM or more is a dissociation constant (KD) of 8x10 ^-8 M or less, and a binding activity (KD) relative to CD137 in the absence of a small molecule compound is too large to be calculated using Biacore (weak binding

- 35 046075 calling activity). In another embodiment of the present invention, the binding activity (KD) of an anti-CD137 antigen-binding molecule or antibody against CD137 in the presence of a small molecule compound in an amount of 100 μM is a dissociation constant (KD) of 2x10 ^-8 M or less, and binding activity against CD137 in the absence of the small molecule compound is too large to be calculated using Biacore (weak binding activity).

In one embodiment, the present invention provides an anti-CD137 antigen-binding molecule or antibody having a value of [binding activity (binding amount) to CD137 in the presence of a small molecule compound at a concentration of 10 μM or more]/[binding activity (binding amount) by relative to CD137 in the absence of the small molecule compound)] is the same as or greater than the value for the control anti-CD137 antigen-binding molecule. In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody having a value of [binding activity (amount of binding) for CD137 in the presence of a small molecule compound at 100 μM or more]/[binding activity (amount of binding) for CD137 in absence of small molecule compound)] is the same as or greater than the value of the control anti-CD137 antigen-binding molecule. In any of the above embodiments of the present invention, the control anti-CD137 antigen binding molecule can be selected from hhtu-CD137 antibodies containing HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167, A372/B040, A356/B040, A486/B167, A487 /B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 or A549/B167, described in table. 17.

In one embodiment of the present invention, the control anti-CD137 antigen binding molecule is an antibody comprising the amino acid sequences A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/ B376, A551/B256, A551/B379, A555/B379, A548/B256 or A549/B167, described in table. 17 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the control antigen binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acid sequences as HVR -H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167. In another embodiment of the present invention, the control anti-CD137 antigen binding molecule is an anti-CD137 antibody comprising A375/B167 as a heavy chain variable region/light chain variable region combination. In another preferred embodiment of the present invention, the control antigen binding molecule is an anti-CD137 antibody comprising HVRH1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acid sequences as HVR-L3. H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A551/B379. In another embodiment of the present invention, the control anti-CD137 antigen binding molecule is an anti-CD137 antibody comprising A551/B379 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the control antigen binding molecule contains heavy and light chain constant regions of human origin (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region, human Lamlib λ chain (SEQ ID NO: 63) as light chain constant region).

In one embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody that has a binding activity (bound amount) for CD137 in the absence of a small molecule compound that is the same as or lower than the binding activity of a control anti-CD137 antigen binding molecule, as well as binding activity ( bound amount) to CD137 in the presence of a small molecule compound at a concentration of 10 μM or more is equal to or greater than the binding activity of a control anti-CD137 antigen-binding molecule to CD137 under the same conditions. In another embodiment, the present invention provides an anti-CD137 antigen-binding molecule or antibody that has binding activity for CD137 in the absence of a small molecule compound that is the same as or less than the binding activity of a control anti-CD137 antigen-binding molecule, and also has binding activity (bound amount) for CD137 in the presence of a small molecule compound at a concentration of 10 μM or more is equal to or more than the binding activity (bound amount) of control anti-CD137 binding activity to CD137 under the same conditions. In any of the above embodiments of the present invention, the control anti-CD137 antigen binding molecule can be selected from anti-CD137 antibodies comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acids

- 36 046075 sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167, A372/B040, A356/B040, A486/B167, A487 /B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 or A549/B167, described in table. 17.

In one embodiment of the present invention, the control anti-CD137 antigen-binding molecule is an anti-CD137 antibody comprising the amino acid sequence A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 or A549/B167, described in table. 17 as a heavy chain variable region/light chain variable region combination. In another preferred embodiment of the present invention, the control anti-CD137 antigen binding molecule is an anti-CD137 antibody comprising the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVRL2 and HVR-L3 regions having the same amino acid sequences as and HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167. In another embodiment of the present invention, the control anti-CD137 antigen binding molecule is an anti-CD137 antibody comprising A375/B167 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the control antigen binding molecule is an anti-CD137 antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acid sequences as HVR-L3. H1, HVRH2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A551/B379. In another embodiment of the present invention, the control anti-CD137 antigen binding molecule is an anti-CD137 antibody comprising A551/B379 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the control antigen binding molecule contains human-derived heavy and light chain constant regions (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region, human Lamlib λ chain (SEQ ID NO: 63) as the constant region light chain region.

In one embodiment, the present invention provides an anti-CD137 antigen-binding molecule or antibody having a value of [binding activity (KD) for CD137 in the presence of a small molecule compound at a concentration of 1 μM]/[binding activity (KD) for CD137 at presence of a small molecule compound at a concentration of 10 μM or more] is the same or greater than the value for the control anti-CD137 antigen-binding molecule. In another embodiment, the present invention provides an anti-CD137 antigen-binding molecule or antibody having a value of [binding activity (KD) for CD137 in the presence of a small molecule compound at a concentration of 1 μM]/[binding activity (KD) for CD137 in the presence of a small molecule compound at a concentration of 100 μM or more] is the same or greater than the value for the control antigen-binding molecule. In any of the above embodiments of the present invention, the control antigen binding molecule can be selected from anti-CD137 antibodies containing HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acid sequences as HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488 /B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 or A549/B167, described in table. 17.

In one embodiment of the present invention, the control antigen-binding molecule is an antibody comprising the amino acid sequence A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/ B256, A551/B379, A555/B379, A548/B256 or A549/B167, described in table. 17, as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the control antigen binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acid sequences as HVR-H1, HVR- H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167. In another embodiment of the present invention, the control antigen binding molecule is an antibody comprising A375/B167 as a heavy chain variable region/light chain variable region combination. In another embodiment of the present invention, the control antigen binding molecule is an antibody comprising HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 having the same amino acid sequence as HVR-H1, HVR -H2, HVR-H3, HVR-Ll, HVR-L2 and HVR-L3 contained in A551/B379. In another embodiment of the present invention, the control antigen binding molecule is an antibody comprising A551/B379 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the control antigen binding molecule comprises human-derived heavy and light chain constant regions (e.g., G1T3 (SEQ ID NO: 138) as the heavy chain constant region, human Lamlib λ chain (SEQ ID NO: 63) in as the light chain constant region.

In one embodiment of the present invention, the binding activity of anti

- 37 046075

CD137 antibodies against CD137, in the presence, absence, high concentration and/or low concentration of the small molecule compound are measured, for example, by a ligand capture method using BIACORE (registered trademark) T200 surface plasmon resonance as the measurement principle.

Details of an example method for measuring the binding activity of an anti-CD137 antibody against CD137 are described below. In one embodiment of the present invention, the binding activity of an anti-CD137 antibody against CD137 against CD137 is assessed by the BIACORE (registered trademark) T200 method. In a preferred embodiment of the present invention, this method uses 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ and 0.05% Tween 20 as running buffer and is carried out at 37°C. In one embodiment of the present invention, such measurement is performed after the antibody has been captured as a ligand on the ligand-capturing molecule. Specifically, the required amount (e.g., approximately 100 RU, 200 RU, 300 RU, 400 RU, or 500 RU) of antibody is captured by reacting with an antibody solution prepared using a running buffer with a chip prepared in advance by immobilizing Sure Protein A (GE Healthcare ) on the Series S Sensor Chip CM3 (GE Healthcare).

In a preferred embodiment of the present invention, about 100 to 500 RU, preferably about 250 to 400 RU, of the antibody is captured. CD137 binding activity in the presence or absence of the small molecule compound is then assessed by reacting a CD137 solution prepared using running buffer added with the small molecule compound to a target concentration (e.g., 1 μM, 10 μM, 50 μM, or 100 μM) or solution CD137 is prepared using a running buffer that does not contain the small molecule compound. Although the concentration of CD137 in a CD137 solution can be determined accordingly, for example, when hCD137-HisBAP (see Example 1-1) is used as an antigen, the measurement is carried out using antigen concentrations of 0 nM, 15.625 nM, 62.5 nM, 250 nM and 1000 nM, respectively. In one embodiment of the present invention, the dissociation constant (KD) of the uhtu-CD137 antibody from human CD137 is calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (L/M/sec) and the dissociation rate constant kd (1/sec) are calculated by globally fitting the sensogram obtained by measurement using the 1:1 Langmuir binding model. The dissociation constant KD (M/L) is calculated from these values.

Additional exemplary assay methods for measuring the binding activity of an anti-CD137 antibody to CD137 will be described in detail below. Binding of anti-CD137 antibodies to human CD137 was assessed using Biacore T200. Binding to human CD137 was measured using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 0.05% Tween 20 running buffer and measured at 37°C. First, an antibody of approximately 250 to 400 RU is captured by contacting an antibody solution prepared using running buffer with a chip on which Sure Protein A (GE Healthcare) is immobilized on a Series S CM3 sensor chip (GE Healthcare). Then, a solution of human CD137 prepared using a running buffer supplemented with ATP at a target concentration (e.g., 1 μM, 10 μM, 50 μM, or 100 μM), or a solution of human CD137 prepared using a running buffer that does not contain ATP, enters interaction to assess CD137 binding activity in the presence or absence of ATP. hCD137-HisBAP was prepared by the method described in Example (1-1) and used as human CD137 antigen, measured at antigen concentrations of 0 nM, 15.625 nM, 62.5 nM, 250 nM and 1000 nM, respectively. The chip is regenerated using 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) and measured by repeated antibody capture. The dissociation constant of each antibody against human CD137 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (L/M/sec) and the dissociation rate constant kd (1/sec) are calculated by globally fitting the sensogram obtained by measurement using the 1:1 Langmuir binding model. The dissociation constant KD (M/L) is calculated from these values.

In one embodiment of the present invention, the binding activity of an anti-CD137 antibody against CD137 (preferably human CD137) may also be referred to by another name, namely the amount of CD137 bound per unit of antibody. Specifically, using the sensorgrams obtained by the above BIACORE (registered trademark) T200 assay method, the amount of CD137 binding to the antibody (RU) is divided by the amount of captured antibody to calculate the amount of CD137 binding per unit amount of antibody. In one embodiment of the present invention, the binding activity of an anti-CD137 antibody against CD137 (preferably human CD137) can also be measured by the method described in Examples 5-3 or 6-2.

The terms small molecule and small molecule compound refer to a naturally occurring chemical substance other than the biopolymers present in a living organism, or to

- 38 046075 to a chemical substance of non-natural origin. Preferably, it is a target tissue-specific compound or a non-naturally occurring compound, but is not limited to these. In one embodiment of the present invention, the small molecule compound of the present invention is a cancer tissue-specific compound or a cancer tissue-specific metabolite. In the present invention, the term cancer tissue-specific compound refers to a compound that exists in tumor tissue but not in non-tumor tissue. In the present invention, the term cancer is used to describe malignant neoplasms, which may be with or without metastases. The term metabolism refers to the chemical changes that occur in body tissue, including assimilation and catabolism. Assimilation refers to the biosynthesis or accumulation of molecules, while catabolism refers to the breakdown of molecules. A metabolite is an intermediate or product resulting from the metabolism of a substance.

In the present invention, the term target tissue means any tissue in a living body to which the antigen binding molecule of the present invention is to be delivered. The target tissue may be histologically different, such as tissue from different organs, or pathologically different tissue, such as normal tissue and diseased tissue. In some embodiments of the present invention, the target tissue is tumor tissue. In contrast, non-target tissue(s) are tissues in a living organism other than the target tissue.

The term tumor tissue means tissue that includes at least one tumor cell. Typically, tumor tissue produces a population of tumor cells that make up the main body of the tumor (parenchyma) and the connective tissues and blood vessels that exist between the tumor cells and the tumor-supporting tissue (stroma). In some cases they are clearly distinguishable, but there are cases where they are mixed up. In some cases, there are cells such as immune cells that infiltrate into the tumor tissue. In contrast, the term non-tumor tissue means tissue of a living organism that is different from the tumor tissue(s). Healthy tissue/normal tissue represents such non-tumor tissue.

An example, which does not limit the scope of the present invention, is a cancer tissue-specific compound or cancer tissue-specific metabolite used in the present invention, at least one compound selected from the compounds detailed below, which can be suitably illustrated. The term at least one compound includes, in addition to the case where the binding activity against an antigen by the same antigen-binding domains described below depends on one type of cancer tissue-specific compound or cancer tissue-specific metabolite, the case where the binding activity depends on several types specific to the cancer tissue compounds or cancer tissue-specific metabolites.

In the present invention, the term tissue-specific target compound refers to a compound that is specifically present in a target tissue compared to a non-target tissue. In some embodiments of the present invention, the tissue-specific target compound may be a compound defined by the qualitative tissue specificity of the target, such as being present in the target tissue but not in non-target tissues, or present in non-target tissue but not in tissue-target. targets. In another embodiment of the present invention, the tissue-specific target compound may be a compound quantified by the tissue specificity of the target, such as being present in the target tissue at a concentration that is different (eg, higher concentration or lower concentration) compared to tissue not being a target. In certain embodiments of the present invention, the tissue-specific target compound is present in the target tissue at a concentration that is greater than, for example, 1.05 times or more, 1.1 times or more, 1.15 times or more, 1. 2 times or more, 1.25 times or more, 1.3 times or more, 1.35 times or more, 1.4 times or more, 1.45 times or more, 1.5 times or more, 1.55 times or more, 1.6 times or more, 1.65 times or more, 1.7 times or more, 1.75 times or more, 1.8 times or more , 1.85 times or more, 1.9 times or more, 1.95 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2, 3 times or more, 2.4 times or more, 2.5 times or more, 3 times or more, 5 times or more, 10 times or more, 50 times or more, 100 times or more , 10 ³ times or more, 10 ⁴ times or more, 10 ⁵ times or more, 10 ⁶ times or more compared to non-target tissue. In a certain embodiment of the present invention, the target tissue-specific compound, relative to the non-target tissue, is present in the target tissue at a concentration that is statistically significantly higher or lower (i.e., as analyzed using Welch's t test or rank sum test Wilcoxon, p value less than 0.05 and/or q value less than 0.10). In a certain embodiment of the present invention, the target tissue-specific compound is a tumor tissue-specific compound.

In a certain embodiment of the present invention, the tissue-specific target compound is a metabolite specifically produced by a tumor cell. Meta

- 39 046075 pain can be a product that is formed in the process of metabolism necessary for life (primary metabolite), or a product formed as a result of metabolism that is not necessary for life (secondary metabolite). Examples of primary metabolites may include sugars, proteins, lipids, nucleic acids, and the like. Examples of secondary metabolites include antibiotics and dyes. The metabolite may be a biopolymer or a small molecule. In a certain embodiment of the present invention, the biopolymer is a molecule with a molecular weight of about 5000 or more that is composed of one or more types of repeating units, including, for example, polysaccharides, polypeptides and polynucleotides. In a certain embodiment of the present invention, small molecules are molecules having a molecular weight of about 500 or less, and are chemical substances present in a living organism. In another embodiment of the present invention, the tissue-specific tumor compound is a small molecule metabolite specifically produced in tumor cells (Eva Gottfried, Katrin Peter, Marina P. Kreutz, From Molecular and Modular Tumor Therapy, 3(2), 2010, 111-132). In another embodiment of the present invention, the tumor tissue-specific compound is a metabolite that is specifically produced by a cell infiltrating the tumor tissue (e.g., an immune cell) or a stromal cell (e.g., a cancer associated fibroblast (CAF)) present in tumor tissue. Examples of immune cells infiltrating tumor tissue include dendritic cells, suppressive dendritic cells, regulatory T cells, exhausted T cells, myeloma-derived suppressor cells (MDSCs), and the like. In another embodiment of the present invention, a metabolite that is produced by cells present in tumor tissue (eg, tumor cells, immune cells, stromal cells, etc.) that is released from the cell after they die by apoptosis or necrosis or the like process , may also be classified as tumor tissue-specific compounds of the present invention.

To identify tissue-specific tumor junctions, analysis at the transcriptomic level (see, for example, Dhanasekaran et al., Nature, 41 2, 2001, 822-826; Lapointe et al., Proc. Natl. Acad. Sci. USA, 101, 2004 , 811-816; Perou et al., Nature, 406, 2000, 747-752), analysis at the proteomic level (see, for example, Ahram et al., Mol. Carcinog., 33, 2002, 9-15; Hood et al., Mol. Cell. Proteomics, 4, 2005, 1741-1753) and metabolism analysis based on metabolomic profiling (metabolomics) can be used appropriately. That is, to identify the metabolite in the test sample, high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) (Brindle et al., J. Mol. Recognit., 10, 1997, 182-187)), mass spectrometry (GC /MS and LC/MS) (Gates, Sweeley, Clin. Chem., 24, 1978, 1663-1673) and metabolic profiling using the ELISA method, and similar suitable methods, which can be used alone and/or in combination.

In a certain embodiment of the present invention, the tumor tissue-specific compound is at least one compound selected from the group consisting of: nucleosides having a purine ring structure, amino acids and their metabolites, lipids and their metabolites, primary metabolites of carbohydrate metabolism, and nicotinamide and its metabolites. In another embodiment of the present invention, the tumor tissue-specific compound is at least one compound selected from paragraphs (1) to (6), including:

(1) nucleosides having a purine structure, such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and inosine;

(2) amino acids such as alanine, glutamic acid and aspartic acid;

(3) amino acid metabolites such as kynurenine, anthranilic acid, 3-hydroxykynurenine and kynurenic acid;

(4) arachidonic acid metabolites such as prostaglandin E2;

(5) primary metabolites of the glycolytic pathway or Krebs cycle, such as lactic acid, succinic acid and citric acid; and, (6) nicotinamide metabolites such as 1-methylnicotinamide.

(1) Nucleosides having a purine structure, such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and inosine.

It is known that when tumor cells die, a large amount of intracellular ATP leaks. Consequently, the concentration of ATP in tumor tissue is significantly higher than in normal tissue (PLoS One. (2008) 3, e2599). AMP is metabolized by cell surface enzymes such as extracellular 5'-nucleotidase (eco-5'-nucleotidase) (CD73) (Resta, Thompson, Immunol. Rev. 161, 1998, 95-109; Sadej et al., Melanoma Res. 16, 2006, 213-222). Adenosine is a purine nucleoside that is constantly present in the extracellular environment at low concentrations, but a marked increase in extracellular adenosine concentration has been reported in hypoxic tissues found in solid tumors (Blay, Hoskin, Cancer Res., 57, 1997, 2602-2605). CD73 is expressed on the surface of tumors and immune cells (Kobie et al., J. Immunol. 177, 2006, 6780-6786) and is increasingly

- 40 046075 This activity is found in breast cancer (Canbolat et al., Breast Cancer Res. Treat. 37, 1996, 189-193), stomach cancer (Durak et al., Cancer Lett. 84, 1994, 199-202), pancreatic cancer (Flocke, Mannherz, Biochim. Biophys. Acta 1076, 1991, 273-281) and glioblastoma (Bardot et al., Br. J. Cancer, 70, 1994, 212-218). It has been suggested that the accumulation of adenosine in tumor tissue results from increased dephosphorylation of AMP by cytoplasmic 5'-nucleotidase (Headrick, Willis, Biochem. J., 261, 1989, 541-550). In addition, regulatory T cells infiltrating tumor tissue also express ATPase and produce adenosine (Proc. Natl. Acad. Sci. USA, 103 (35), 2006, 13132-13137; Curr. Med. Chem., 18, 2011, 5217-5223). The adenosine produced is believed to maintain tumor tissues in an immunosuppressive environment through adenosine receptors such as the A2A receptor (Curr. Med. Chem., 18, 2011, 5217-5223). Thus, ATP, ADP, AMP, adenosine and the like, which are believed to accumulate in high concentration in tumor tissue due to purine nucleotide metabolism, are examples of tumor tissue-specific compounds used in the present invention. In addition, because adenosine is broken down into inosine by adenosine deaminase, inosine accumulates in high concentrations.

In a certain embodiment of the present invention, nucleosides having a purine ring structure include adenosine-containing compounds. In certain embodiments of the present invention, adenosine-containing compounds include, for example, adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), adenosine[/-thio]triphosphate (ATPγS) and the like. In another embodiment of the present invention, nucleosides having a purine ring structure include inosine, which is a metabolite of adenosine.

Moreover, in a certain embodiment of the present invention, nucleosides having a purine ring structure include commercially available nucleosides having a purine ring structure, such as ADP-beta-S (Sigma Inc.) and the like.

(2) Amino acids such as alanine, glutamic acid and aspartic acid.

The rate of uptake of glutamine, which acts as a nitrogen carrier in vivo, is increased in tumor cells, and this incorporation of glutamine and the associated conversion to glutamic acid and lactic acid (destruction of glutamine (glutaminolysis)) is thought to be a feature of tumor cells (Mazurek, Eigenbrodt, Anticancer Res. 23, 2003, 1149-1154; Mazurek et al., J. cell Physiol. 181, 1999, 136-146). In cancer patients, plasma glutamine levels decrease and glutamic acid concentrations increase (Droge et al., Immunobiology, 174, 1987, 473-479), and studies of the metabolism of ^13C -labeled glucose in lung cancer tissue have shown a correlation between concentrations of ^13C -labeled glucose. C succinic acid, ¹³ C labeled alanine, ¹³ C labeled glutamic acid and C labeled citrate. For these reasons, alanine, glutamic acid, aspartic acid and other similar compounds that are believed to accumulate in high concentrations in tumor tissue, for example due to glutamine degradation, are examples of tumor tissue-specific compounds used in the present invention.

(3) Amino acid metabolites such as kynurenine, anthranilic acid, 3-hydroxykynurenine and kynurenic acid.

Indoleamine 2,3-dioxygenase (IDO, Indoleamine 2,3-dioxygenase) is a tryptophan-metabolizing enzyme that is highly expressed in many cancers such as melanoma, colon cancer, kidney cancer and other similar diseases (Uyttenhove et al., Nat. Med., 9, 2003, 1269-1274)). IDO catalyzes the conversion of tryptophan to kynurenine. In gliomas that do not express IDO, kynurenine is produced from tryptophan by liver tryptophan 2,3-dioxygenase (TDO) (Opitz et al., Nature, 478 (7368), 2011, 197-203). IDO is also expressed on dendritic cells infiltrating tumor tissue, and dendritic cells also produce kynurenine (J. Immunol. 181, 2008, 5396-5404). In addition, IDO is also expressed in myeloid-derived suppressor cells (MDSCs) of tumor tissues, and MDSCs also produce kynurenine (Yu et al., J. Immunol., 190, 2013, 3783-3797). Kynurenine is converted to anthranilic acid by kynureninase and to 3-hydroxykynurenine by kynurenine 3hydroxylase. Both anthranilic acid and 3-hydroxykynurenine are converted to 3hydroxyanthranilic acid, a precursor to NAD. Kynurenine is converted to kynurenic acid by kynurenine aminotransferase. For these reasons, kynurenine and its metabolite, that is, anthranilic acid, 3-hydroxykynurenine, kynurenic acid, etc., are examples of tumor tissue-specific compounds used in the present invention, in particular tumor cell-specific metabolites.

(4) Arachidonic acid metabolites such as prostaglandin E2.

Prostaglandin E2 (PGE2) promotes the growth of colon cancer cells and inhibits their apoptosis (Sheng et al., Cancer Res., 58, 1998, 362-366). Regarding PGE2 synthetases, COX-1 has generally been found to be constitutively expressed in almost all tissues, while COX-2 is induced by several inflammatory cytokines and oncogenes in tumors (Warner, Mitchell, FASEB J., 18,

- 41 046075

2004, 790-804). Overexpression of COX-2 has been reported to be associated with poor prognosis in breast cancer (Denkert et al., Clin. Breast Cancer, 4, 2004, 428-433) and rapid progression of ovarian cancer (Denkert et al., Mod. Pathol., 19, 2006, 1261-1269). In addition, regulatory T cells infiltrating tumor tissue also produce PGE2 (Curr. Med. Chem., 18, 2011, 5217-5223). For these reasons, arachidonic acid metabolites such as PGE2 are examples of tumor tissue-specific compounds, particularly tumor cell-specific metabolites or metabolites specific to immune cells infiltrating tumor tissue. In addition to PGE2, the production of thromboxane A2 (TXA2) is increased in tumor tissues, such as colon cancer (J. Lab. Clin. Med., 122, 1993, 518-523).

(5) Primary metabolites of the glycolytic pathway or Krebs cycle, such as lactic acid, succinic acid and citric acid.

The glycolytic phenotype, characterized by the activation of glycolytic (Embden-Meyerhoff pathway) enzymes such as pyruvate kinase, hexokinase and lactate dehydrogenase (LDH), is traditionally known as the Warburg effect, characteristic of solid tumors. Lactic acid, which is the end product of glycolysis, as well as succinic and citric acids formed during the Krebs cycle, accumulate in tumor tissues (Teresa et al., Mol. Cancer, 8, 2009, 41-59). For these reasons, lactic acid, succinic acid, citric acid and other similar compounds, which are primary metabolites formed during glycolysis, are examples of tumor tissue-specific compounds, in particular tumor cell-specific metabolites mentioned in the present invention. In addition, it is known that due to cell death, succinate, which is present in high concentrations in cells, is released from the cells (Nature Immunology, 9, 2008, 1261-1269). It is believed that the cause is an increased concentration of succinic acid in tumor tissues, where cell death often occurs.

(6) Nicotinamide metabolites such as 1-methylnicotinamide.

Nicotinamide N-methyltransferase is known to be expressed at high levels in a variety of human tumor tissues. It is also known that 1-methylnicotinamide, which is a stable metabolite of nicotinamide produced by this enzyme, is secreted outside tumor cells (Yamada et al., J. Nutr. Sci. Vitaminol., 56, 2010, 83-86). For this reason, 1methylnicotinamide and other similar compounds, which are believed to accumulate in high concentrations in tumor tissue as a result of nicotinamide metabolism, are examples of tumor tissue-specific compounds mentioned in the present invention.

The antigen binding molecule of the present invention contains an antigen binding domain. A domain of any structure can be used as an antigen-binding domain as long as it binds to the target antigen. In one embodiment of the present invention, the antigen binding domains of the present invention include, for example, heavy chain and/or light chain variable regions of antibodies, avimers containing a module (domain A) of about 35 amino acids contained in various cell membrane proteins in a living organism ( WO 2004/044011 and WO 2005/040229), adnectins containing the 10Fn3 domain of fibronectin, which is a glycoprotein expressed on the cell membrane (WO 2002/032925), affibodies using the 58 amino acid IgG binding domain of protein A as a scaffold (WO 1995/001937), designed ankyrin repeat proteins (DARPin, Designed Ankyrin Repeat protein), using the ankyrin repeat (AR, ankyrin repeat), which is a repeating sequence of 33 amino acids, as a base (WO 2002/020565), ankylins containing lipocalin, such as neutrophil gelatinase-associated lipocalin (NGAL) as a base (WO 2003/029462), variable lymphocyte receptors (VLR, variable lymphocyte receptor), which are proteins that function in the adaptive immune systems of jawless vertebrates such as Lampetra japonica and Eptatretus, and contain a leucine-rich-repeat (LRR) module (WO 2008/016854) and other similar compounds. In a certain embodiment of the present invention, the antigen binding domain of the present invention comprises the variable regions of the heavy and light chains of an antibody. In another embodiment of the present invention, the antigen binding domain of the present invention comprises, for example, a single chain Fv domain, single chain antibodies, Fv, scFv2 (single chain Fv2), Fab or F(ab') ₂ .

HVR and variable region.

In one embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody that comprises at least one, at least two, or all three HVR VH sequences selected from: (a) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising any amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (c) HVR-H3, comprising any amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20. In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody includes: (a) HVR-H1, containing an amino acid sequence

- 42 046075 SEQ ID NO: 7; (b) HVR-H2 containing any amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (c) HVR-H3 containing any amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule is an antibody comprising an amino acid sequence of the sequences A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 or A549/B167, presented in table. 17 as a heavy chain variable region/light chain variable region combination. In a preferred embodiment of the present invention, the antigen binding molecule is an anti-CD137 antibody comprising the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 regions having the same amino acid sequences as HVR-H1 , HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 contained in A375/B167. In another embodiment of the present invention, the anti-CD137 antigen binding molecule is an anti-CD137 antibody comprising A375/B167 as a heavy chain variable region/light chain variable region combination. In another preferred embodiment of the present invention, the antigen binding molecule is an anti-CD137 antibody comprising regions HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVRL2 and HVR-L3 containing the same amino acid sequences as HVR-H1, HVR-H2, HVRH3, HVR-L1, HVR-L2 and HVR-L3 contained in A551/B379.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 17.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 9; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 17.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 17.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 18.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 18.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 18.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 18.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 19.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 20.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 20.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the following regions (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3, comprising any amino acid sequence of SEQ ID NO: 17.

- 43 046075

In another embodiment, the present invention provides an aHTu-CD137 antigen binding molecule or antibody comprising at least one, at least two or all three HVR VL sequences selected from: (a) HVR-L1 including any amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 or 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising any amino acid sequence selected from SEQ ID NO: 27, 28 or 29.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three HVR VH sequences selected from (i) HVR-H1, comprising amino acid sequence SEQ ID NO: 7; (ii) HVR-H2 comprising an amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) HVR-H3 comprising any amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20; (b) a VL domain comprising at least one, at least two or all three HVR VL sequences selected from (i) HVR-L1 comprising any amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 and 25; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (iii) HVR-L3, comprising any amino acid sequence selected from SEQ ID NO: 27, 28 or 29.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 9; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID

- 44 046075

NO: 10; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 11; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 8; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 16; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid following

- 45 046075 sequence SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 7; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (e) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In a certain embodiment of the present invention, any one or more amino acids of the above-described anti-CD137 antibody are replaced at the following HVR positions:

In HVR-H2 (SEQ ID NO: 30): positions 5, 6, 7, 10, 13, 14 and/or 17

In HVR-H3 (SEQ ID NO: 31): position 3 and/or 6

In HVR-L1 (SEQ ID NO: 32): positions 4, 5, 9 and/or 11

In HVR-L3 (SEQ ID NO: 33): positions 6, 7 and/or 8.

In a certain embodiment of the present invention, the substitutions provided by the present invention are conservative substitutions. In a particular embodiment of the present invention, any one or more of the following substitutions may be made in any combination:

In HVR-H2 (SEQ ID NO: 8): K5H or S; S6G; T7S; E10Y; D13E; S14Q; V17G or L.

In HVR-H3 (SEQ ID NO: 17): A3P, K or I; F6E.

In HVR-L1 (SEQ ID NO: 21): R4S; Y5T; Y9F; E11N.

In HVR-L3 (SEQ ID NO: 27): E6P; H7A; Q8I.

All possible combinations of the above substitutions are included in the consensus sequences SEQ ID NO: 30, 31, 32 and 33 for HVR-H2, HVR-H3, HVR-L1 and HVR-L3, respectively.

In any of the above embodiments of the present invention, the anti-CD 137 antigen binding molecule or antibody is humanized. In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody includes HVR regions according to any of the above embodiments of the present invention, and further comprises a human acceptor framework region, for example, a human immunoglobulin framework region or a human consensus framework region. In another embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody includes HVR regions as in any of the above embodiments of the present invention and further comprises a heavy chain variable region (VH) or a light chain variable region (VL) containing a framework sequence ( FR, framework). In one embodiment of the present invention, the framework region FR1 in the variable heavy chain region contains the amino acid sequence of SEQ ID NO: 35, FR2 in the variable region of the heavy chain contains the amino acid sequence of SEQ ID NO: 36, FR3 in the variable heavy chain region contains the amino acid sequence of SEQ ID NO: 37 and FR4 in the heavy chain variable region contains the amino acid sequence of SEQ ID No: 38. In one embodiment of the present invention, FR1 in the light chain variable region contains the amino acid sequence of SEQ ID NO: 39, FR2 in the light chain variable region contains the amino acid sequence SEQ ID NO: 40, FR3 in the light chain variable region contains the amino acid sequence of SEQ ID NO: 41 and FR4 in the light chain variable region contains the amino acid sequence of SEQ ID NO: 42.

In another embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53. In some embodiments, the VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, contains substitutions (e.g., contains conservative substitutions), insertions, or deletions relative to the reference sequence , but an anti-CD137 antigen-binding molecule or antibody containing such a sequence retains the ability to bind CD137. In some embodiments, a total of 1 to 10 amino acids have been replaced, inserted, and/or deleted in SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53. In some In embodiments of the present invention, substitutions, insertions or deletions occur in regions outside the HVR (ie, frame regions (FR)). Optionally, the anti-CD137 antibody contains the VH sequence of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53, including post-translational modifications of this sequence. In one of the

- 46 046075 embodiments of the present invention VH contains one, two or three HVRs selected from: (a) HVR-H1 containing the amino acid sequence SEQ ID NO: 7; (b) HVR-H2 comprising any one amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 and 16; and (c) HVR-H3 comprising any one amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy chain or light chain into pyroglutamic acid by pyroglutamylation.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody, wherein the antibody comprises a light chain variable domain (VL) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60. In some embodiments, a VL sequence that is at least 90% identical , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, contains substitutions (eg, contains conservative substitutions), insertions or deletions relative to the reference sequence, but anti-CD137 an antigen-binding molecule or antibody containing such a sequence retains the ability to bind CD137. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 54, 55, 56, 57, 58, 59, or 60. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the HVR (i.e., frame regions (FR)). Optionally, the anti-CD137 antigen binding molecule or antibody contains the VL sequence of SEQ ID NO: 54, 55, 56, 57, 58, 59 or 60, including post-translational modifications of this sequence. In one embodiment of the present invention, VL contains one, two or three HVRs selected from: (a) HVR-L1 containing an amino acid sequence selected from SEQ ID NO: 21, 22, 23, 24 and 25; (b) HVR-L2 containing the amino acid sequence SEQ ID NO: 26; (c) HVR-L3 comprising any one amino acid sequence selected from SEQ ID NOs: 27, 28 and 29. Post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

In another embodiment, the present invention provides an anti-CD137 antigen binding molecule or antibody, wherein the antigen binding molecule or antibody comprises VH as in any of the embodiments of the present invention presented above, and VL as in any of the embodiments of the present invention presented above.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody includes the VH and VL sequences of SEQ ID NO: 43 and SEQ ID NO: 54, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 44 and SEQ ID NO: 55, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 45 and SEQ ID NO: 55, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 46 and SEQ ID NO: 54, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 47 and SEQ ID NO: 54, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 48 and SEQ ID NO: 56, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 49 and SEQ ID NO: 57, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 50 and SEQ ID NO: 58, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 51 and SEQ ID NO: 59, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 51 and SEQ ID NO: 60, respectively, including post-translational modifications of these sequences.

- 47 046075

In one embodiment of the present invention, the aHTu-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 52 and SEQ ID NO: 60, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody includes the VH and VL sequences of SEQ ID NO: 50 and SEQ ID NO: 59, respectively, including post-translational modifications of these sequences.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody comprises the VH and VL sequences of SEQ ID NO: 53 and SEQ ID NO: 54, respectively, including post-translational modifications of these sequences.

The above post-translational modifications include, but are not limited to, modification of glutamine or glutamate at the N-terminus of the heavy chain or light chain to pyroglutamic acid by pyroglutamylation.

SEQ ID NOs corresponding to the amino acid sequences of the preferred heavy chain variable region and light chain variable region and their HVR1, 2 and 3 for each anti-CD137 antigen binding molecule or antibody of the present invention are shown in the table below.

Table 1

Variable SEQ ID NO SEQ ID NO of hypervariable region e heavy chain/light chain regions variable region (HVR) Heavy chain Easy chains Sh H2 nz L1 L2 L3 А375/В167 43 54 7 8 17 21 26 27 А372/В040 44 55 7 9 17 22 26 27 А356/В040 45 55 7 10 17 22 26 27 А486/В167 46 54 7 AND 18 21 26 27 А487/В167 47 54 7 8 18 21 26 27 А488/В226 48 56 7 12 18 21 26 28 А489/В223 49 57 7 13 18 21 26 29 А548/В376 50 58 7 14 19 23 26 27 A551/B256 51 59 7 15 20 24 26 27 А551/В379 51 60 7 15 20 25 26 27 А555/В379 52 60 7 16 20 25 26 27 А548/В256 50 59 7 14 19 24 26 27 А549/В167 53 54 7 14 17 21 26 27

If the anti-CD137 antigen binding molecule or antibody of the present invention contains glutamine as the N-terminal amino acid of the heavy chain or light chain, this amino acid may be replaced by glutamic acid. If the anti-CD137 antibody provided in the present invention contains glutamic acid as an amino acid at the N-terminus of the heavy chain or light chain, this amino acid can be replaced with glutamine.

In a preferred embodiment of the present invention, anti-CD137 antigen binding molecules or antibodies comprising the above-described HVRs, heavy chain variable regions and/or light chain variable regions all have small molecule dependent binding activity for CD137 as described above.

Constant regions.

In another embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies comprise a constant region. The constant region may be a heavy chain constant region (including an Fc region), a light chain constant region, or both. In another embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies comprise an Fc region. In some embodiments of the present invention, the constant region is a native sequence region. Examples of heavy chain constant regions derived from native antibodies include, for example, human IgG1 heavy chain constant region (SEQ ID NO: 61, 62), human IgG2, human IgG3, human IgG4, and the like. Examples of light chain constant regions derived from native antibodies include, for example, the kappa chain

- 48 046075 human, human lambda circuit (for example, SEQ ID NO: 63) and the like.

In the present invention, the term "original constant region" or "original Fc region" refers to the constant region or Fc region before the introduction of the amino acid changes described in the present invention. The term parent antigen binding molecule refers to an antigen binding molecule that contains a parent constant region or a parent Fc region. In some embodiments of the present invention, the original Fc region is a native sequence Fc region (or a native antibody Fc region). Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc. Antibodies can be of human or monkey origin (e.g., cynomolgus, rhesus, marmoset , chimpanzee or baboon). Native antibodies can also have natural mutations. The many allotypes of IgG sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, each of which can be used in the present invention. In one embodiment of the present invention, the parent Fc region is an Fc region derived from the human IgG1 constant region represented by SEQ ID NO: 61, 62, or 182.

In one embodiment of the present invention, anti-CD137 antigen binding molecules or antibodies have an increased isoelectric point (pI, isoelectric point) compared to anti-CD137 antigen binding molecules or antibodies that contain a native Fc region sequence or parent Fc region. In some embodiments of the present invention, Fc region variants include at least one amino acid change. In other embodiments of the present invention, the amino acid change results in an increase in the isoelectric point (pI) of the variant Fc region compared to the original Fc region. Without relying on any theory, it is assumed that the pH of biological fluids (for example, plasma) is in the neutral pH range. In biological fluids, the overall positive charge of an antigen-binding molecule or antibody with an increased pI value increases due to the increase in pI, and as a result, the antigen-binding molecule or antibody is more strongly attracted by the physicochemical Coulomb interaction to the surface of the endothelial cell, which has an overall negative charge compared to the antigen-binding molecule or an antibody that does not have an increased pI value. Thus, agonistic antigen-binding molecules (or antibodies) or antigen-binding agonistic antigen-binding molecules (or antibodies) can approach the surface of cells that express the Fc-gamma receptor, resulting in increased binding of the antigen-binding molecules or antibodies to the cells expressing the Fc-gamma receptor . For those anti-CD137 agonistic antigen-binding molecules or antibodies that exhibit anti-CD137 agonistic activity based on a contribution through Fcgamma receptor binding activity, anti-CD137 agonistic antigen-binding molecules or antibodies having increased binding to Fcgamma receptor expressing cells due to pI-increasing amino acid changes may exhibit stronger anti-CD137 agonist activity compared to anti-CD137 agonistic antigen-binding molecules or antibodies that do not have amino acid changes that increase the isoelectric point (pI).

In the present invention, pI may be a theoretical or experimentally determined value. The pI value can be determined, for example, using isoelectric focusing, known to those skilled in the art. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx, etc.). When calculating, the properties of the antibody can be reflected in the calculation formula. For example, (i) typically the Cys conserved in the antibody forms a disulfide bond and carries no electrical charge in the side chain; therefore, such Cys can be excluded from the calculation, and only Cys in the free form that does not form a disulfide bond can be included in the calculation. Alternatively, (ii) the charge state or isoelectric point of the antibodies may change due to post-translational modifications; therefore, the calculation formula can be modified as follows to account for such post-translational modifications: (a) when the N-terminus of the heavy chain is Q (glutamine), the N-terminal amino group is excluded from the calculation, assuming that pyroglutamylation occurs, (b) when the C-terminus the heavy chain is K (lysine), K (one residue only) is excluded from the calculation, assuming truncation occurs; and (c) the side chains of all Cs (cysteines) present in normally conserved positions are excluded from the calculation, assuming that all of these Cs form disulfide bonds within the molecule. In one preferred embodiment of the present invention, both of the above-described provisions (i) and (ii) can be reflected in the calculation formula.

In one embodiment of the present invention, the pI value may be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 , or more by at least 0.6, 0.7, 0.8, 0.9, or more by at least 1.0, 1.1, 1.2, 1.3, 1.4 , 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2 .5, 3.0 or more, compared to the pI value before modification.

In one embodiment of the present invention, amino acid changes related to increasing and methods for increasing the pI of an antigen-binding molecule or antibody, in detail

- 49 046075 are described in the present invention in section III. Compositions and methods (agonistic antigen-binding molecules containing a variant Fc region with an increased isoelectric point (pI)). Those skilled in the art will understand that any amino acid changes and methods for increasing pI described in section III. The compositions and methods (agonistic antigen-binding molecules containing a variant Fc region with an increased isoelectric point (pI)) can be applied to anti-CD137 antigen-binding molecules or antibodies.

In one embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies have a variant Fc region with an increased pI value, and the variant Fc region contains at least one amino acid change at at least one position selected from the group consisting of positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 according to EU numbering. In other embodiments of the present invention, variants of the Fc region with increased pI value include Arg or Lys at each selected position.

In other embodiments of the present invention, the anti-CD137 antigen binding molecules or antibodies have a variant Fc region with an increased pI value, and the variant Fc region contains at least one amino acid change at at least one position selected from the group consisting of positions 311, 343 and 413 according to EU numbering. In other embodiments of the present invention, variants of the Fc region with an increased pI value include an amino acid change at position 311, 343, or 413 of the EU numbering. In another embodiment of the present invention, variants of the Fc region with increased pI value include Arg or Lys at each selected position.

In another embodiment, the present invention provides anti-CD137 antigen-binding molecules or antibodies comprising a variant of the Fc region with an increased pI value that contains amino acid changes in any of the following (1)-(3): (1) at positions 311 and 343; (2) in provisions 311 and 413; and (3) in provisions 343 and 413 according to EU numbering. In other embodiments of the present invention, variants of the Fc region with an increased pI value contain an Arg or Lys at each selected position.

In one embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention include a variant Fc region that contains the amino acid change(s) presented in table. 2 below.

table 2

Amino acid changes to increase the pI of the Fc region

Number Amino acid substitutions (EU numbering) 1 P343R/D413K 2 Q311R/P343R 3 P343R 4 D413K 5 Q311R 6 Q311R/D413K

In one embodiment, the present invention provides anti-CD137 antigen binding molecules or antibodies containing a variant Fc region obtained by introducing amino acid changes into the Fc region having a native sequence. In one embodiment of the present invention, the variant Fc regions have increased binding activity to at least one Fc-gamma receptor selected from the group consisting of Fcgamma RIa, Fc-gamma RIIa, Fc-gamma RIIb, Fc-gamma RIIIa and Fc- gamma RIIIb, compared to an Fc region having a native sequence or original Fc region. Preferably, the variant Fc regions have increased binding activity for Fc-gamma RIIb compared to an Fc region having a native sequence or original Fc region. An anti-CD137 antibody containing a variant Fc region with increased binding activity to Fc-gamma RIIb is reported to have increased agonist activity compared to an anti-CD137 antibody containing a native sequence Fc region. In one embodiment of the present invention, the amino acid changes to increase the binding activity towards Fc-gamma RIIb, for example, can use the amino acid changes described in WO 2012/115241, WO 2014/030728, WO 2014/163101 and/or WO 2017/104783. In one preferred embodiment of the present invention, the changes to increase Fc-gamma RIIb binding activity are amino acid changes at at least one position selected from the group consisting of positions 234, 235, 236, 237, 238, 264, 268 , 295, 326 and 330 according to EU numbering.

- 50 046075

Fc-gamma receptors (referred to herein as Fc-gamma, Fc-gamma R, or FcgR receptors) refer to receptors that can bind to the Fc region of monoclonal antibodies IgG1, IgG2, IgG3, and IgG4, and essentially mean any member of the family of proteins encoded by Fc-gamma receptor genes. In humans, this family includes Fc-gamma RI (CD64), including the Fc-gamma RIa, Fc-gamma RIb and Fc-gamma RIc isoforms; Fc-gamma RII (CD32), including isoforms Fc-gamma RIIa (including allotypes H131 (type H) and R131 (type R)), Fc-gamma RIIb (including Fc-gamma RIIb-1 and Fcgamma RIIb-2) and Fc -gamma RIIc; and Fc-gamma RIII (CD16), including isoforms Fc-gamma RIIIa (including allotypes V158 and F158), and Fc-gamma RIIIb (including allotypes Fc-gamma RIIIb-NA1 and Fc-gamma RIIIb-NA2) and any Fc-gamma Human Rs, Fc gamma isoforms or allotypes yet to be discovered. Fc-gamma RIIb1 and Fc-gamma RIIb2 were found to be splice variants of human Fc-gamma RIIb. In addition, a splice variant called Fc-gamma RIIb3 has been reported (J Exp Med, 170, 1989, 1369-1385). In addition to these splice variants, human Fc-gamma RIIb includes AAI46679.1, registered in NCBI, and all splice variants registered in NCBI, namely NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1 and NP_003992. 3. In addition, human Fc-gamma RIIb includes all previously described genetic polymorphisms, as well as Fc-gamma RIIb (Arthritis Rheum. 48, 2003, 3242-3252; Kono et al., Hum. Mol. Genet. 14, 2005, 2881- 2892; Kyogoju et al., Arthritis Rheum. 46, 2002, 1242-1254) and every genetic polymorphism that will be known in the future.

There are two allotypes in Fc-gamma RIIa, one in which the amino acid at position 131 of Fc-gamma RIIa is a histidine (type H), and the other in which the amino acid at position 131 is replaced by arginine (type R) (Warrmerdam, J. Exp. Med. 172 , 1990, 19-25).

Fc-gamma R includes, but is not limited to, human, mouse, rat, rabbit and monkey Fc-gamma R and can be derived from any organism. Mouse Fc-gamma R includes Fc-gamma RI (CD64), Fc-gamma RII (CD32), Fc-gamma RIII (CD16), and Fc-gamma RIII-2 (CD16-2), as well as any mouse Fc-gamma R isoforms Fc-gamma R, but the list is not limited to them.

In another embodiment, the present invention provides anti-CD137 antigen-binding molecules or antibodies comprising a variant Fc region with increased binding activity for Fc-gamma RIIb, which includes amino acid changes in any of the following (1)-(8): (1) in provisions 234, 238, 264 and 330; (2) in provisions 234, 238 and 330; (3) in provisions 234, 237, 238 and 330; (4) in provisions 236, 268 and 330; (5) in provisions 235, 236, 268, 295, 326 and 330; according to EU numbering.

In one embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention comprise a variant Fc region comprising the amino acid changes set forth in Table 1 below. 3. In another embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention contain a variant Fc region that further includes, in addition to the amino acid change(s) described in table. 2 (amino acid changes including an increase in the pI region of the Fc), any combination of amino acid changes indicated in table. 3 below.

Table 3

Amino acid changes to increase Fc-gamma RIIb binding activity in the Fc region

Number Amino acid substitutions (EU numbering) 1 L23 4Y/P23 8D/V264I/A3 3 0K 2 L234Y/P238D/A330K 3 L23 4Y/G23 7D/P23 8D/A3 3 0K 4 G236N/H268D/A330K 5 L235W/G236N/H268D/Q295L/K326T/A330K

In one embodiment, the present invention provides variants of the Fc region that have at least one amino acid change and have binding activity for Fc-gamma RIIb that is equivalent to or greater than that of the control Fc region. In one embodiment of the present invention, an Fc control region is an Fc region containing any combination of amino acid changes listed in Table 1 above. 3. In one preferred embodiment of the present invention, the Fc control region is an Fc region contained in the heavy chain constant region of TT14 (SEQ ID NO: 149), TT16 (SEQ ID NO: 150), MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154). In one preferred embodiment of the present invention, the Fc control region is an Fc region contained in the heavy chain constant region of MY201 (SEQ ID NO: 153) or MY518 (SEQ ID NO: 154).

In another embodiment of the present invention, the present invention provides isolated agonistic antigen-binding molecules or antibodies that contain var.

- 51 046075 Fc region ant with increased binding activity to the Fc gamma receptor (preferably Fc gamma RIIb) and increased pI. In one embodiment of the present invention, the variant Fc regions contain at least two amino acid changes compared to the original Fc regions. As described above, an antigen binding molecule or antibody with an elevated pI is more strongly attracted by physicochemical Coulomb interaction to the surface of the endothelial cell, which has an overall negative charge, compared to an antigen binding molecule or antibody that does not have an increased pI. Therefore, for those agonistic antigen-binding molecules or antibodies that exhibit agonistic activity based on the contribution of binding activity to the Fc-gamma receptor (preferably Fc-gamma RIIb), the agonistic activity of the antigen-binding molecules or antibodies can be increased by combining the amino acid change(s) to increase Fc-gamma receptor (preferably Fc-gamma RIIb) and amino acid change(s) to increase pI.

In one embodiment of the present invention, anti-CD137 antigen binding molecules or antibodies comprise a variant Fc region that includes both amino acid changes to increase Fc gamma receptor binding activity (e.g., Fc gamma RIIb) and amino acid changes to increase isoelectric point (pI) as described above. As described above, an antigen-binding molecule or antibody with an increased pI is more strongly attracted by physicochemical Coulomb interaction to the surface of the endothelial cell, which has an overall negative charge, compared to an antigen-binding molecule or antibody that does not have an increased isoelectric point (pI). Therefore, for those anti-CD137 agonistic antigen-binding molecules or antibodies that exhibit agonistic activity against CD137 based on the contribution of binding activity to the Fc-gamma receptor (preferably Fcgamma RIIb), the agonistic activity of the anti-CD137 antigen-binding molecules against or antibodies can be increased by combining amino acid changes to increase Fcgamma receptor (preferably Fcgamma RIIb) and amino acid changes to increase pI.

In one embodiment, the present invention provides polypeptides comprising a variant Fc region with increased binding activity for Fc-gamma RIIb and an increased pI value, which includes at least three amino acid changes, including (a) at least one an amino acid change at at least one position selected from the group consisting of positions 234,235, 236, 237, 238, 264, 268, 295, 326 and 330 according to EU numbering, and (b) at least two amino acid changes according to in at least two positions selected from the group consisting of provisions 311, 343 and 413, according to EU numbering. In another embodiment, the present invention provides polypeptides containing a variant Fc region with increased binding activity for Fc-gamma RIIb and an increased pI value, which include amino acid changes according to any of the following paragraphs (1)-(26):

(1) provisions 235, 236, 268, 295, 326, 330, 343 and 413;

(2) provisions 214, 235, 236, 268, 295, 326, 330, 343 and 413;

(3) provisions 234, 238, 250, 264, 307, 330, 343 and 413;

(4) provisions 234, 238, 264, 330, 343 and 413;

(5) provisions 234, 237, 238, 250, 307, 330, 343 and 413;

(6) provisions 234, 237, 238, 330, 343 and 413;

(7) provisions 235, 236, 268, 295, 326, 330, 311 and 343;

(8) provisions 234, 238, 250, 264, 307, 330, 311 and 343;

(9) provisions 234, 238, 264, 330, 311 and 343;

(10) provisions 234, 237, 238, 250, 307, 330, 311 and 343;

(11) provisions 234, 237, 238, 330, 311 and 343;

(12) provisions 235, 236, 268, 295, 326, 330 and 343;

(13) provisions 214, 235, 236, 268, 295, 326, 330 and 343;

(14) provisions 235, 236, 268, 295, 326, 330 and 413;

(15) provisions 214, 236, 268, 330 and 343;

(16) provisions 214, 235, 236, 268, 330 and 343;

(17) provisions 214, 236, 268, 330 and 413;

(18) provisions 214, 236, 268, 330, 343 and 413;

(19) provisions 214, 235, 236, 268, 330, 343 and 413;

(20) provisions 214, 236, 268, 330 and 311;

(21) provisions 214, 235, 236, 268, 330 and 311;

(22) provisions 214, 236, 268, 330, 311 and 343;

(23) provisions 214, 235, 236, 268, 330, 311 and 343;

(24) provisions 214, 236, 268, 330, 311 and 413;

(25) provisions 214, 235, 236, 268, 330, 311 and 413;

(26) provisions 214, 235, 236, 268, 295, 326, 330 and 311, according to EU numbering.

- 52 046075

In one embodiment of the present invention, the variant Fc regions of the present invention include any combination of amino acid changes presented in Table below. 4.

Table 4

Number Amino acid substitutions (EU numbering) 1 L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K 2 K214R/L23 5 W/G23 6N/H268D/Q295L/K3 26T/AZ 3 OK/RZ 43R/D41ЗК 3 L23 4Y/P23 8D/T25 0V/V264I/T3 07Р/AZ 3 OK/РЗ 43R/D41 ЗК 4 L234Y/P238D/V264I/A330K/P343R/D413K 5 L234Y/G237D/P238D/T250V/T307P/A330K/P343R/D413K 6 L234Y/G237D/P238D/A330K/P343R/D413K 7 L235W/G236N/H268D/Q295L/K326T/A330K/Q311R/P343R 8 L234Y/P238D/T250V/V264I/T307P/A330K/Q3 11R/P343R 9 L234Y/P238D/V264I/A330K/Q311R/P343R 10 L234Y/G237D/P238D/T250V/T307P/A330K/Q311R/P343R AND L234Y/G237D/P238D/A330K/Q311R/P343R 12 L235W/G236N/H268D/Q295L/K326T/A330K/P343R 13 K214R/L23 5 W/G23 6N/H268D/Q295L/K3 26T/AZ 3 OK/RZ 43R 14 L23 5 W/G23 6N/H268D/Q295L/K326T/A3 30K/D41 ZK 15 K214R/G23 6N/H268D/A3 3 OK/RZ 43R 16 K214R/L23 5 W/G23 6N/H268D/A3 3 OK/RZ 43R 17 К214R/G23 6N/H268D/A3 3 0K/D41 ЗК 18 K214R/G23 6N/H268D/A3 3 OK/RZ 43R/D41 ZK 19 K214R/L23 5 W/G23 6N/H268D/A3 3 OK/RZ 43R/D41 ZK 20 K214R/G236N/H268D/A330K/Q311R 21 K214R/L23 5 W/G23 6N/H268D/A3 3 0K/Q311R 22 K214R/G236N/H268D/A330K/Q311R/P343R 23 K214R/L23 5 W/G23 6N/H268D/A3 3 0K/Q311R/P3 43R 24 К214R/G23 6N/H268D/A3 3 0K/Q311R/D41 ЗК 25 K214R/L23 5 W/G23 6N/H268D/A3 3 0K/Q311R/D41 ZK 26 K214R/L23 5 W/G23 6N/H268D/Q295L/K3 26T/AZ 3 0K/Q311R

In one embodiment of the present invention, Fc region variants containing any combination of amino acid changes described in table above. 4 do not contain the amino acid at position 447 according to the EU numbering.

Those skilled in the art will recognize that at least one amino acid change to increase binding activity for Fc-gamma R (including Fc-gamma RIIb) compared to the original Fc region, as described or proposed, for example, in WO 2013/047752, WO 2013/125667, WO 2014/030728, WO 2014/163101 or WO 2017104783, and at least one amino acid change to increase pI compared to the original Fc region, as described or proposed, for example, in WO 2017/104783, WO 2017 /046994 and any combination of these amino acid changes can be used in addition to the changes presented as illustrations above.

In addition, amino acid changes made for other purposes can be combined in the variant Fc region described in the present invention. For example, amino acid substitutions that increase FcRn-binding activity (Hinton et al., J. Immunol. 176(1), 2006, 346-356; Dall'Acqua et al., J. Biol. Chem. 281(33), 2006 , 23514-23524; Petkova et al., Intl. Immunol. 18(12), 2006, 1759-1769; Zalevsky et al., Nat. Biotechnol. 28(2), 2010, 157-159; WO 2006/019447; WO 2006/053301; WO 2009/086320), and amino acid substitutions can be added to improve heterogeneity or stability of antibodies (WO 2009/041613). In another embodiment, polypeptides with the property of promoting antigen clearance, which are described in WO 2011/122011, WO 2012/132067, WO 2013/046704 or WO 2013/180201, polypeptides with the property of specifically binding to a target tissue, which are described in WO 2013/180200 , polypeptides with the property of multiple binding to multiple antigen molecules, which are described in WO 2009/125825, WO 2012/073992 or WO 2013/047752, can be combined with the Fc region variant described in the present invention. In another embodiment, for the purpose of conferring binding activity to other antigens, the amino acid changes described in EP 1752471 and EP 1772465 can be combined into the CH3 variant Fc region described in the present invention.

In one embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention comprise a heavy chain constant region comprising any one amino acid sequence selected from SEQ ID NOs: 64-85. Preferably, the anti-CD137 antigen binding molecules or antibodies of the present invention comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 75 or 82.

In one preferred embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention containing the above-described variant

- 53 046075 Fc region ant, have the CD137-binding activity described above, depending on the small molecule compound.

In one embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention comprise the following variable region and constant region: a variable region comprising the above-described HVR region, a heavy chain variable region and/or a light chain variable region; and the Fc region variant described above. In one preferred embodiment of the present invention, the anti-CD137 antigen-binding molecules or antibodies of the present invention can be any anti-CD137 antibody selected from the antibodies described in table. 52.

In another embodiment, the present invention provides antigen-binding molecules or antibodies that bind to the same epitope on CD137 as the anti-CD137 antigen-binding molecules or antibodies of the present invention in the presence of a low molecular weight compound (for example, in the presence of 10 μM or more, 50 µM or more, 100 µM or more, 150 µM or more, 200 µM or more, or 250 µM or more of a low molecular weight compound). For example, in one embodiment, the present invention provides those antigen binding molecules or antibodies that bind to the same epitope with anti-CD137 antigen binding molecules or antibodies containing A375/B167, A372/B040, A356/B040, A486/B167, A487 /B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 and/or A549/B167, described in table. 17, as a heavy chain variable region/light chain variable region combination. In one embodiment of the present invention, the anti-CD137 antigen binding molecules or antibodies of the present invention having CD137 binding activity that is dependent on the small molecule dependent antigen binding activity recognize an epitope formed by a complex formed from an antigen (eg, CD137) and low molecular weight compound (for example, ATP).

In another embodiment, the present invention provides antigen-binding molecules or antibodies that compete for binding to CD137 with anti-CD137 antigen-binding molecules or antibodies of the present invention in the presence of a low molecular weight compound (e.g., in the presence of 10 μM or more, 50 μM or more, 100 μM or more, 150 µM or more, 200 µM or more, or 250 µM or more of a low molecular weight compound). For example, in one embodiment of the present invention, they compete for the CD137 binding site with anti-CD137 antigen-binding molecules or antibodies containing A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 and/or A549/B167, described in table. 17, as a heavy chain variable region/light chain variable region combination.

In another embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody according to any of the above embodiments of the present invention is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment of the present invention, the anti-CD137 antibody is an antibody fragment, for example, an Fv, Fab, Fab', scFv, diabody or F(ab') ₂ fragment. In another embodiment of the present invention, the antibody is a full length antibody, for example, an intact IgG1 antibody or an antibody of another class or isotype as described herein.

In another embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody according to any of the above embodiments of the present invention may include any of the functions, individually or in combination, as described in sections 1-7 below:

1. Agonistic activity of an anti-CD137 antigen-binding molecule or antibody.

In a certain embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody of the present invention has CD137 agonist activity. CD137 signaling not only stimulates IFN-γ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998), but also increases their survival and dendritic cell (DC) activation. as indicated by activation of co-stimulatory molecules and cytokine secretion (Choi et al., 2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137 is best characterized as a co-stimulatory molecule that regulates TCR-induced activation as in CD4'. and in CD8' T cell subsets. When combined with TCR activation, anti-CD137 agonist antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the sensitivity of T cells to activation-induced cell death (reviewed in Snelc et al., 2011). Of these phenomena, the physiological phenomena observed after CD137 signaling on T cells are mediated by downstream signals activated by CD137 signaling such as TRAF2, TRAF1, particularly NF-kappaB, JNK, Erk, Akt, survivin, Bcl- XL and/or Bcl-2 (Ward-Kavanagh et al., Immunity, 44, 2016, 1005).

In one embodiment of the present invention, the terms anti-CD137 agonistic antigen binding molecule or anti-CD137 agonistic antibody mean an antigen binding molecule or antibody that, upon binding to CD137, transduces the CD137 signal and thus

- 54 046075 specifically induces or enhances the secretion of IFN-gamma, proliferation and increased survival of NK cells; DC activation indicates upregulation of cytokine and co-stimulatory molecule secretion; TCR induction; T cell proliferation; and/or secretion of lymphokines. In another embodiment of the present invention, the terms anti-CD137 agonistic antigen binding molecule or anti-CD137 agonistic antibody mean an antigen binding molecule or antibody that transduces the CD137 signal by binding to CD137 on T cells and significantly induces NF-kappaB T cell activation. In addition, the antigen binding molecule or antibody exhibits agonistic activity against CD137 means that any of the above physiological phenomena is observed when the antigen binding molecule or antibody binds to CD137. The method for measuring CD137 agonistic activity is described in detail below in section B. Assays.

In a certain embodiment of the present invention, the anti-CD137 antigen-binding molecule or antibody of the present invention has small molecule dependent agonist activity against CD137. In one non-limiting embodiment of the present invention, the CD137 agonist activity of an anti-CD137 antigen binding molecule or anti-CD137 antibody in the presence of a small molecule compound is greater than the CD137 agonist activity in the absence of a small molecule compound. In another embodiment of the present invention, the CD137 agonist activity of an anti-CD137 antigen binding molecule or antibody in the presence of a high concentration of a small molecule compound is greater compared to the CD137 agonist activity in the presence of a low concentration of a small molecule compound. In another embodiment of the present invention, the CD137 agonistic activity of the anti-CD137 antigen binding molecule or antibody in the presence of the small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold greater or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1x 10 ³ times or more, 2x10 ³ times or more, 3x10 ³ times or more, 5x10 ³ times or more, 1x10 ⁴ times or more, 2x10 4 times ^or more, 3x10 ⁴ times or more, 5x10 ⁴ times or more, or 1x10 ⁵ times or more compared to CD137 agonist activity in the absence of the small molecule compound.

Any suitable concentration may be selected as the small molecule concentration as long as a difference in the binding activity of the anti-CD137 antigen binding molecule or antibody is detected. In one embodiment of the present invention, the anti-CD137 antigen-binding molecule or antibody transduces the CD137 signal by binding to CD137 on the surface of a cell. Therefore, one skilled in the art will appreciate that an anti-CD137 antigen-binding molecule or antibody that has small molecule-dependent binding activity to CD137 has small-molecule-dependent CD137 agonist activity. However, on the other hand, since the methods for measuring binding activity and agonist activity are different, one of ordinary skill in the art will appreciate that the concentration of the small molecule compound at which differences in binding activity are detected may be different from the concentration of the small molecule compound at which differences in agonist activity are detected ( for example, for an anti-CD137 antigen-binding molecule or antibody whose CD137 binding activity in the presence of a small molecule compound at a concentration of 10 μM is 2-fold or more greater than the CD137 binding activity in the absence of the small molecule compound, the CD137 agonist activity (test value) in the presence small molecule compound at a concentration of 10 μM may be less than 2-fold compared to CD137 agonist activity (test value) in the absence of small molecule compound). In addition, those skilled in the art will appreciate that the determination of agonist activity may vary depending on the assay for CD137 agonist activity (see section B. Assays).

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody (i) exhibits agonistic activity against CD137 in the presence of a small molecule compound in an amount of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM, and (ii ) exhibits essentially no CD137 agonistic activity in the absence of the small molecule compound or has low CD137 agonistic activity in the absence of the small molecule compound (compared to the presence of the small molecule compound).

In one embodiment of the present invention, if the agonistic activity of an anti-CD137 antigen binding molecule or antibody is assessed using a) Agonistic Activity Assay (AAAs), described in detail in section B. Assays, the anti-CD137 agonistic antigen binding molecule or antibody (i) exhibits agonistic activity activity against CD137 in the presence of the small molecule compound at a concentration of 250 μM, and (ii) has low agonistic activity against CD137 in the absence of the small molecule compound (compared to the presence of the small molecule compound). In another embodiment of the present invention, the aHTu-CD137 agonist antigen-binding molecule or antibody (i) exhibits CD137 agonist activity in the presence of the small molecule compound at a concentration of 250 μM, and (ii) exhibits substantially no CD137 agonist activity. in the absence of a low molecular weight compound.

In one embodiment of the present invention, if the agonistic activity of an anti-CD137 antigen binding molecule or antibody is assessed using item b) Agonistic activity assay (reporter gene assay) discussed in detail in section B. Assays, anti-CD137 antigen binding molecule or antibody (i) exhibit CD137 agonist activity in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM small molecule compound, and (ii) have essentially no CD137 agonist activity or have reduced agonist activity in the absence of the small molecule compounds (compared to that in the presence of a low molecular weight compound). The antibody concentrations in the reporter gene assay can be selected arbitrarily, for example, the final antibody concentration is 0, 0.001, 0.01, 0.1.1 or 10 μg/ml. In a preferred embodiment of the present invention, the final concentration of the antibody is 0.1 μg/ml or 1 μg/ml.

In one embodiment of the present invention, when the final antibody concentration is 0.1 μg/ml in b) Agonist activity assay (reporter gene assay), discussed in detail in section B. Assays, (i) agonist activity (relative light unit ( RLU) of an anti-CD137 antigen-binding molecule or anti-CD137 antibody in the presence of 10 μM small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more compared to (ii) CD137 agonist activity (relative light unit) in the absence low molecular weight compound. In one embodiment of the present invention, if the final antibody concentration is 0.1 μg/ml in b) Agonist activity assay (reporter gene assay), discussed in detail in section B. Assays, (i) CD137 agonist activity (relative light unit) of antigen binding molecule or antibody against CD137 in the presence of 100 μM small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or greater than, 50-fold or greater, 60-fold or greater, 70-fold or greater, 80-fold or greater, or 90-fold or greater than (ii) CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In one embodiment of the present invention, when the final antibody concentration is 0.1 μg/ml in b) Agonist activity assay (reporter gene assay), discussed in detail in section B. Assays, (i) CD137 agonist activity (c relative light units (RLU)) of an anti-CD137 agonist antigen-binding molecule or antibody in the presence of 250 μM small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold, or more than, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more compared to (ii) CD137 agonist activity ( in relative light units) in the absence of a low molecular weight compound. In any of the above embodiments of the present invention, additionally, 0.1 μg/ml of anti-CD137 antigen-binding molecule or antibody exhibits substantially no CD137 agonistic activity in the absence of the small molecule compound.

In one embodiment of the present invention, when the final antibody concentration is 1 μg/ml in b) Agonist activity assay (reporter gene assay), discussed in detail in section B. Assays, (i) CD137 agonist activity (in relative light units (RLU)) anti-CD137 agonistic antigen-binding molecule or antibody in the presence of 10 μM small molecule compound is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more compared to (ii) CD137 agonist activity (in relative light units) in the absence of a low molecular weight compound. In one embodiment of the present invention, if the final antibody concentration is 0.1 μg/ml in b) Agonist activity assay (reporter gene assay), discussed in detail in section B. Assays, (i) CD137 agonist activity (in relative terms) light units (RLU)) of anti-CD137 antigen-binding molecule or antibody in the presence of 100 μM small molecule compound is 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, or 90-fold or more compared to (ii) CD137 agonist activity (in relative terms) light units) in the absence of a low molecular weight compound. In one embodiment of the present invention, if the final antibody concentration is 0.1 μg/ml in b) Agonistic activity assay (reporter gene assay), discussed in detail in section B. Assays, (i) agonistic

- 56 046075 ical activity against CD137 (in relative light units (RLU)) of aHmu-CD137 antigen-binding molecule or antibody in the presence of 250 μM low molecular weight compound is greater than 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more (ii) CD137 agonist activity (in relative light units) in the absence of the small molecule compound. In any of the above embodiments of the present invention, additionally, 1 μg/ml of anti-CD137 antigen-binding molecule or antibody exhibits substantially no CD137 agonist activity in the absence of the small molecule compound.

2. Antibody fragments.

In some embodiments of the present invention, the antibody provided is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv and scFv fragments, as well as other fragments described below. A review of antibody fragments was published by Hudson et al. Nat. Med. 9, 2003, 129-134. For a review of scFv fragments, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, ed. Rosenburg and Moore, (Springer-Verlag, New York), 113, 1994, 269-315; see also WO 93/16185; US 5,571,894 and US 5,587,458. For a discussion of Fab and F(ab') ₂ fragments containing salvage receptor binding epitope residues and having an increased half-life in vivo, see US 5,869,046.

Diabodies and antibody fragments with two antigen binding sites can be bivalent or bispecific. See, for example, EP 404097; WO 1993/01161; Hudson et al., Nat. Med. 9, 2003, 129134; Hollinger et al., Proc. Natl. Acad. Sci. USA 90, 1993, 6444-6448. Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9, 2003, 129-134.

Single domain antibodies are antibody fragments containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, for example, US Pat. No. 6,248,516).

Antibody fragments can be produced by various methods, including, but not limited to, proteolytic cleavage of the intact antibody, as well as production by recombinant host cells (eg, E. coli or phage) as described herein.

3. Chimeric and humanized antibodies.

In some embodiments of the present invention, the antibody provided is a chimeric antibody. Some chimeric antibodies are described, for example, in US 4816567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81, 1984, 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region derived from mouse, rat, hamster, rabbit, or a non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a class switched antibody in which the class or subclass has been changed from the class of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

In some embodiments of the present invention, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the original non-human antibody. Typically, a humanized antibody includes one or more variable domains in which the HVRs, eg, CDRs (or portions thereof) are derived from a non-human antibody, and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may also optionally contain at least a portion of a human constant region. In some embodiments of the present invention, certain FR residues in a humanized antibody are replaced with corresponding residues of a non-human antibody (eg, an antibody from which the HVR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

Humanized antibodies and methods for their production are discussed, for example, Almagro and Fransson, Front. Biosci. 13, 2008, 1619-1633, and also described, for example, in the publications of Riechmann et al., Nature 332, 1988, 323-329; Queen et al., Proc. Nat'l Acad. Sci. USA 86, 1989, 10029-10033; US 5821337, US 7527791, US 6982321, US 7087409; Kashmiri et al., Methods 36, 2005, 25-34 (describing specificity determining region (SDR) transplantation); Padlan, Mol. Immunol. 28, 1991, 489-498 (description of translation); Dall'Acqua et al., Methods 36, 2005, 43-60 (describing FR shuffling); Osbourn et al., Methods 36, 2005, 61-68; Klimka et al., Br. J. Cancer, 83, 2000, 252-260 (describing a directed selection approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the best fit method (see, for example, Sims et al. J. Immunol. 151, 1993, 2296); framework region derived from the consensus sequence of human antibodies of a certain subset of the light or heavy chain variable regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89,

- 57 046075

1992, 4285; and Presta et al. J. Immunol., 151:2623 (1993)); mature (somatically mutated) human or germline human frameworks (see, for example, Almagro and Fransson, Front. Biosci. 13, 2008, 1619-1633); and framework regions obtained from FR screening libraries (See, for example, Vasa et al., J. Biol. Chem. 272, 1997, 10678-10684; Rosok et al., J. Biol. Chem. 271, 1996, 1996 22611-22618).

4. Human antibodies.

In some embodiments, the antibody provided herein is a human antibody. Human antibodies can be obtained using various techniques known in the art. Human antibodies are generally described in the publications of van Dijk, van de Winkel, Curr. Opin. Pharmacol. 5, 2001, 368-374, and Lonberg, Curr. Opin. Immunol. 20, 2008, 450-459.

Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact human variable region antibodies in response to an antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci, or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for producing human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23, 2005, 1117-1125. See also, for example, US 6,075,181 and US 6,150,584, which describe XENOMOUSE™ technology; US patent 5,770,429 describing HuMab technology (registered trademark); patent US 7041870 describing K-M MOUSE technology (registered trademark), and patent application publication US 2007/0061900 describing VelociMouse technology (registered trademark). Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with another human constant region.

Human antibodies can also be obtained using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, for example, Kozbor J. Immunol., 133, 1984, 3001); Brodeur et al. in: Monoclonal Antibody Production Techniques and Applications, 1987, 51-63, Marcel Dekker, Inc., New York); Boerner et al., J. Immunol., 147, 1991, 86.) Human antibodies produced using human B cell hybridoma technology are also described by Li et al., Proc. Natl. Acad. Sci. USA, 103, 2006, 3557-3562. Additional methods are described, for example, in US 7189826 (describing the production of human IgM monoclonal antibodies by hybridoma cell lines); Ni, Xiandai Mianyixue, 26(4), 2006, 265-268 (description of human cell-human cell hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers, Brandlein, Histology and Histopathology, 20(3), 2005, 927-937, and Vollmers, Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3), 2005 , 185-191.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

5. Antibodies derived from libraries.

Antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for constructing phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are discussed, for example, in the work of Hoogenboom et al., Methods in Molecular Biology 178, 2001, 1-37, ed. O'Brien et al., Human Press, Totowa, NJ, and also described, for example, by McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 1991, 624-628; Marks et al., J. Mol. Biol. 222, 1992, 581-597; Marks, Bradbury, Methods in Molecular Biology 248, 2003, 161-175 (ed. Lo, Human Press, Totowa, NJ); Sidhu et al., J. Mol. Biol. 338(2), 2004, 299-310; Lee et al., J. Mol. Biol. 340(5), 2004, 10731093; Fellowes, Proc. Natl. Acad. Sci. USA 101(34), 2004, 12467-12472; Lee et al., J. Immunol. Methods 284(1-2), 2004, 119-132.

In some phage display methods, the VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage, as described by Winter et al., Ann. Rev. Immunol., 12, 1994, 433-455. Phage typically displays antibody fragments as either single chain Fv fragments (scFv) or Fab fragments. Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, the naïve repertoire can be cloned (eg from humans) to provide a single source of antibodies to a wide range of foreign as well as self-antigens without any immunization, as described by Griffiths et al., EMBO J, 12, 1993, 725-734. Finally, naïve libraries can also be prepared synthetically by cloning disordered V gene segments from

- 58 046075 human cells and using random sequence PCR primers to encode highly variable CDR3 regions and to perform in vitro rearrangements as described by Hoogenboom and Winter, J. Mol. Biol., 227, 1992, 381-388. Patent publications describing human antibody phage libraries include, for example, US 5750373 and patent application publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/02 92936 and 2009/0002360 .

Antibodies or antibody fragments isolated from human antibody libraries are regarded as human antibodies or human antibody fragments in the present invention.

Antigen binding molecules or antibodies having antigen binding activity dependent on the small molecule compound of the present invention can be selected by screening a library of antigen binding molecules. The combinatorial libraries described above can be used as such a library.

A library of antigen binding molecules may have an unbiased repertoire of antigen binding molecules (naive library) or may have a biased repertoire of antigen binding molecules. Examples of the latter type of library include a library of antigen-binding molecules that are precontained with binding activity for a particular compound. In one embodiment of the present invention, the library of antigen binding molecules is a library of antigen binding molecules in which amino acid changes are previously made to impart binding activity for a particular compound. Examples of libraries of this type include those described, for example, in international patent application publication WO 2015/083764.

6. Multispecific antibodies.

In some embodiments, the present invention provides an antibody that is multispecific, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites. In some embodiments of the present invention, one of the binding specificities is for CD137 and the other is for any other antigen. In some embodiments of the present invention, bispecific antibodies can bind to two different epitopes of CD137. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD137. Bispecific antibodies can be obtained as full-length antibodies or antibody fragments.

In one embodiment of the present invention, the anti-CD137 antigen-binding molecules or antibodies of the present invention are bispecific antibodies, one moiety that binds to CD137, dependent on a small molecule compound, and the other moiety that binds to a non-CD137 antigen. Antigen other than CD137 is not particularly limited in structure. In other words, the antigen may be an inorganic or organic compound. Representative antigens are described in the present invention (for example, in section IV.

Compositions and methods (antigen-binding molecules, the binding activity of which to antigens varies depending on the concentration of the low molecular weight compound), B. Antigen). In one embodiment of the present invention, the antigens are preferably antigens expressed in cancer cells, immune cells, stromal cells or the like in cancerous tissues or inflammatory tissues.

Methods for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, Cuello, Nature 305, 1983, 537; WO 93/08829; Traunecker et al., EMBO J. 10, 1991, 3655), projection-to-recess design (see, for example, US 5731168). Multispecific antibodies can also be produced by creating electrostatic driving effects to create Fc heterodimeric antibody molecules (WO 2009/089004 A1); cross-linking two or more antibodies or fragments (see, for example, US 4676980; Brennan et al., Science, 229, 1985, 81); the use of leucine zippers to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5), 1992, 1547-1553); using diabody technology to produce bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90, 1993, 6444-6448); using single chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., 152, 1994, 5368); and the production of trispecific antibodies, as described, for example, by Tutt et al. J. Immunol. 147, 1991, 60.

Engineered antibodies with three or more functional antigen binding sites, including multivalent octopus antibodies, are also included in the present invention (see, for example, US 2006/0025576 A1).

The present invention also provides a Dual Acting Fab or DAF (Dual Acting Fab) containing an antigen binding site that binds to CD137 as well as another different antigen (see, for example, US 2008/0069820).

7. Antibody variants.

- 59 046075

In some embodiments, the present invention provides variations in the amino acid sequence of the provided antibodies. For example, it may be desirable to improve the affinity and/or other biological properties of the antibody. Variants of the amino acid sequence of an antibody can be obtained by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution can be used to produce the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

a) Options for substitutions, insertions and deletions.

In some embodiments, the present invention provides antibody variants containing one or more amino acid substitutions. Sites of interest for replacement mutagenesis include the HVR and FR regions. Conservative substitutions are shown in table. 1 under the heading Preferred Substitutions. More significant changes are presented in table. 1 under the heading Examples of Substitutions and are further described below with reference to classes of amino acid side chains. Amino acid substitutions can be introduced into the antibody of interest, and the products can be tested for the desired activity, such as maintaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

Table 5

Original balance Examples of substitutions Preferred Substitutions Ala (A) Val; Leu; lie Val Arg(R) Lys; Gin; Asn Lys Asn(N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gin (Q) Asn; Glu Asn Glu(E) Asp; Gin Asp Gly (G) Ala Ala His(H) Asn; Gin; Lys; Arg Arg He(I) Leu; Val; Met; Ala; Ph; Norleucine Leu Leu (L) Norleucine; lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met(M) Leu; Ph; lie Leu Phe(F) Trp; Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Ph; Thr; Ser Phe Val(V) lie; Leu; Met; Ph; Ala; Norleucine Leu

Amino acids can be grouped according to side chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues affecting side chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions result in the replacement of a member of one of these classes with another class.

One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, the resulting variant(s) selected for further study may have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and/or will substantially retain certain biological properties of the parent antibodies. A typical replacement option is an affinity matured antibody that can be readily produced, for example, using phage display-based affinity generation methods such as those described in the present invention. Briefly, one or more HVR residues are mutated and antibody variants are displayed on phage and screened for specific biological activities (e.g.

- 60 046075 measures, affinity).

Changes (eg, substitutions) can be made in the HVR, for example, to improve the affinity of the antibody. Such changes can be made at HVR hot spots, that is, at residues encoded by codons that undergo mutations at high rates during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207, 2008, 179-196), and/or in residues that contact the antigen, wherein the resulting VH or VL variant is tested for affinity. Affinity maturation by design and iterative selection from secondary libraries has been described, for example in Hoogenboom et al. in the book: Methods in Molecular Biology 178, 2001, 1-37, ed. O'Brien et al., Human Press, Totowa, NJ. In some affinity maturation embodiments of the present invention, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then a secondary library is created. This library is screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR-targeting approaches in which multiple HVR residues (e.g., 4–6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using mutagenesis or alanine scanning modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In some embodiments of the present invention, substitutions, insertions or deletions may occur in one or more HVRs as long as such changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes (eg, conservative substitutions as provided herein) that do not significantly reduce affinity can be made to the HVR. Such changes may, for example, occur outside the antigen-contacting residues in the HVR. In some embodiments of the present invention, of the VH and VL sequence variants presented above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A useful method for identifying residues or regions of an antibody that may be targeted for mutagenesis is called alanine scanning mutagenesis, which is described by Cunningham and Wells, Science, 244, 1989, 1081-1085. In this method, a residue or group of target residues (eg, charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (eg, alanine or polyalanine) to determine whether there is an effect on the antibody interaction with antigen. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be analyzed to determine points of contact between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or excluded as replacement candidates. The options can be checked to determine whether they contain the required properties.

Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal inserts include an antibody with an N-terminal methionyl residue. Other insertional variants of an antibody molecule include the fusion of an enzyme (eg, for ADEPT) or a polypeptide that increases the half-life of the antibody in plasma, at the N- or C-terminus of the antibody.

b) Glycosylation options.

In some embodiments of the present invention, the provided antibody is modified to increase or decrease the degree of glycosylation of the antibody. The addition or removal of glycosylation sites to an antibody can be accomplished by a proven method of altering the amino acid sequence in such a way as to create or remove one or more glycosylation sites.

If the antibody includes an Fc region, the carbohydrate attached to it may be altered. Native antibodies produced by mammalian cells typically contain a branched biantennary oligosaccharide that is typically N-linked to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15, 1997, 26-32. The composition may contain various oligosaccharides, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the stem of the biantennary oligosaccharide structure. In some embodiments of the present invention, modifications to the oligosaccharide in the antibody of the present invention can be made to create antibody variants with certain improved properties.

In one embodiment, the present invention provides antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by the method

- 61 046075 MALDI-TOF mass spectrometry as described in WO 2008/077546, for example at Asn297, refers to the asparagine residue located near position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately +/- 3 amino acids upstream or downstream of position 297, that is, between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylated variants may have improved ADCC function. See, for example, US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to defucosylated or fucose-deficient antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al. J. Mol. Biol. 336, 2004, 1239-1249; Yamane-Ohnuki et al., Biotech. Bioeng. 87, 2004, 614.

Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249, 1986, 533-545; US 2003/0157108 A1; WO 2004/056312 A1, Adams et al., especially in example 11), and knockout cell lines such as the alpha-1,6-fucosyltransferase gene, FUT8, CHO knockout cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87 , 2004, 614; Kanda Y. et al., Biotechnol. Bioeng., 94(4), 2006, 680-688; WO 2003/085107).

The antibody variants further comprise bisected oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US 6602684 (Umana et al.); US 2005/0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.).

c) Fc region variants.

In some embodiments of the present invention, one or more amino acid modifications may be introduced into the Fc region of an antibody of the present invention, thereby creating a variant Fc region (which may also be referred to as an altered Fc region). A variant Fc region may comprise a human Fc region sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc region) including an amino acid modification (eg, substitution) at one or more amino acid positions.

In some embodiments, the present invention contemplates a variant antibody that possesses some, but not all, effector functions, making it a desirable candidate for applications in which the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody has lost the ability to bind Fc gamma R (thus likely lacking ADCC activity) but retains FcRn binding activity. The primary cells for mediating ADCC, NK cells, express only Fc gamma RIII, whereas monocytes express Fc gamma RI, Fc gamma RII, and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table. 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9, 1991, 457-492. Examples of methods for analyzing ADCC activity in vitro, the list of which is not limited to these examples, are given in US 5500362 (see, for example, Hellstrom I. et al. Proc. Nat'l Acad. Sci. USA 83, 1986, 70597063; Hellstrom I et al. al., Proc. Nat'l Acad. Sci. USA 82, 1985, 1499-1502; US 5821337 (see Bruggemann M. et al., J. Exp. Med. 166, 1987, 1351-1361). Alternatively, non-radioactive assay methods may be used (see, for example, the ACT1™ Liquid Cytometry Non-Radioactive Cytotoxicity Assay (CellTechnology, Inc., Mountain View, CA; and the CytoTox 96 (Registered Trademark) Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the test molecule can be assessed in vivo, for example, in an animal model such as described in the publication of Clynes et al. Proc. Nat'l Acad. Sci. USA 95, 1998, 652-656. C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore does not have CDC activity. See, for example, C1q and C3c binding by ELISA in WO 2006/029879 and WO 2005/100402. A CDC assay can be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202, 1996, 163; Cragg M.S. et al., Blood 101, 2003, 1045-1052; Cragg M.S. , Glennie M.J., Blood 103, 2004, 2738-2743). FcRn binding and in vivo clearance/half-life determination can also be performed using methods known in the art (see, e.g., Petkova S.B. et al., Int'l. Immunol. 18(12), 2006, 1759-1769) .

- 62 046075

Antibodies with reduced effector function include antibodies with a substitution of one or more of residues 238, 265, 269, 270, 297, 327 and 329 in the Fc region (US 6737056). These Fc mutants include Fc mutants with substitutions at two or more positions of amino acids 265, 269, 270, 297 and 327, including the so-called Fc DANA mutant with residues 265 and 297 replaced by alanine (US 7332581).

Several antibody variants with increased or decreased binding to FcR have been described. (See, for example, US 6737056; WO 2004/056312; Shields et al., J. Biol. Chem. 9(2), 2001, 6591-6604).

In some embodiments, the antibody variant includes an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering).

In some embodiments of the present invention, changes are made to the Fc region that result in altered (i.e., increased or decreased) C1q binding and/or complement dependent cytotoxicity (CDC), for example, as described in US 6194551, WO 99/51642 and Idusogie et al. J. Immunol. 164, 2000, 4178-4184.

Antibodies with an extended half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117, 1976, 587; Kim et al., J. Immunol. 24, 1994 , 249), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include variants with substitutions at one or more residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, substitution of residue 434 in the Fc region (US 7371826).

See also Duncan, Winter, Nature 322, 1988, 738-740; US 5648260; US 5624821; WO 94/29351 regarding examples of other Fc region variants.

In one embodiment of the present invention, the binding activity for each human Fc-gamma receptor (Fc-gamma R) of the Fc region of an antibody (including a variant Fc region (the same applies hereinafter)) can be measured by a ligand capture method using, such as BIACORE (registered trademark) T200, which use surface plasmon resonance analysis as the measurement principle.

Details of an example method for measuring the binding activity of the Fc region of an antibody to various human Fc-gamma receptors (Fc-gamma Rs) are described below. In one embodiment of the present invention, the binding activity of the Fc region towards Fc-gamma R is assessed using BIACORE (registered trademark) T200. In a preferred embodiment of the present invention, this measurement is carried out at 25°C using measurement buffer (50 mM phosphate, 150 mM NaCl, P20 0.05 w/v%, pH 7.4. In particular, about 1000 RU antibody containing the variant Fc region is first captured on the sensor chip using CaptureSelect (trademark) Fab-human lambda-biotin conjugate (ThermoFisher Scientific) immobilized as the ligand capture molecule. Human Fc-gamma R is diluted with measurement buffer to 8 nM for Fc-gamma RIa and up to 1000 nM for other Fc-gamma R, and allow binding to the captured antibody.The binding activity of each antibody to each Fc-gamma R is assessed by calculating the amount of bound Fc-gamma R per unit amount of antibody ( RU) using Biacore T200 Evaluation Software 2.0 In one embodiment of the present invention, the binding activity of the Fc region of an antibody to various human Fc-gamma receptors (Fc-gamma R) can be measured by the method described in Example 7-4.

In one preferred embodiment of the present invention, the Fc-gamma R used for the measurement method described above may be an extracellular domain of Fc-gamma R obtained by the method described below. First, the synthesis of the Fc-gamma R extracellular domain gene is carried out by a method known to those skilled in the art. For this synthesis, the sequences of each Fcgamma R are prepared based on information registered with NCBI. More specifically, the Fc-gamma RI sequence is derived from the NCBI sequence (database number NM000566.3), the Fc-gamma RIIa sequence is derived from the NCBI sequence (database number NM_001136219.1), the sequences for Fc-gamma RIIb are derived based on the NCBI sequence (database number NM_004001.3), the sequence for Fc-gamma RIIIa is derived from the NCBI sequence (database number NM_001127593.1); A His tag is added to the C-terminus. Polymorphic sites for Fc-gamma RIIa are obtained with reference to J. Exp. Med., 172, 1990, 19-25, and polymorphic sites for Fc-gamma RIIIa are obtained with reference to J. Clin. Invest., 100, 1997, 1059-1070. The resulting gene fragments are inserted into an expression vector for animal cells to obtain expression vectors. Such expression vectors are transiently introduced into FreeStyle293 cells (Invitrogen), derived from human embryonic kidney cancer cells, and allow the protein of interest to be expressed. The culture supernatant is collected and filtered through a 0.22 μm filter, then purified essentially using the four steps described below. At the first stage, cation exchange column chromatography (SP Sepharose FF) is carried out; at the second stage - column affinity chromatography for His tags (HisTrap HP); at the third stage - gel filtration column chromatography

- 63 046075 (Superdex200); and, the fourth stage - aseptic filtration. It should be noted that for Fc-gamma RI, the first step is anion exchange column chromatography using QSepharose FF. The concentration of the purified protein is calculated based on the absorbance coefficient calculated by measuring the absorbance at 280 nm using a spectrophotometer and using PACE or a similar method to obtain the values (Protein Science, 4, 1995, 2411-2423).

In one embodiment of the present invention, the binding activity of the Fc region of an antibody to human FcRn can be measured by a ligand capture method using, for example, BIACORE (registered trademark) T200, the measurement principle of which is based on surface plasmon resonance analysis methods.

Details of an example method for measuring the binding activity of the Fc region of an antibody to human FcRn are described below. In one embodiment of the present invention, the binding activity of the Fc region of an antibody to FcRn is assessed using BIACORE (registered trademark) T200. In a preferred embodiment of the present invention, this measurement is carried out at 25°C using measurement buffer (50 mM phosphate, 150 mM NaCl, P20 - 0.05 w/v%; pH 6.0). Specifically, about 400 RU of antibody containing the Fc region is first captured on a sensor chip on which CaptureSelect (trade mark) Human Fab-lambda Kinetics Biotin Conjugate (ThermoFisher Scientific) is immobilized as a ligand capture molecule, and then human FcRn is diluted buffer for measurements, it is allowed to communicate with it. The FcRn binding activity of each antibody is assessed by calculating the KD (M) using the Biacore T200 Evaluation Software 2.0 steady state model. In one preferred embodiment of the present invention, the human FcRn protein used in this measurement is obtained in accordance with the method described in example 2 of WO 2010107110, used as a sample. In one embodiment of the present invention, the binding activity of the Fc region of an antibody to various human FcRns can be measured by the method described in Example 7-5.

d) Cysteine Engineered Antibody Options In some embodiments of the present invention, it may be desirable to develop cysteine engineered antibodies, eg, Thio Mab, in which one or more antibody residues are replaced with cysteine residues. In one embodiment of the present invention, the substituted residues are located at accessible sites in the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described below in the present invention. In some embodiments of the present invention, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) light chain; A118 (EU numbering) heavy chain; and S400 (EU numbering) heavy chain Fc region. Engineered antibodies using cysteine can be prepared as described, for example, in US 7521541.

e) Antibody derivatives.

In some embodiments, the present invention provides an antibody that can be further modified to include additional non-protein fragments that are known in the art and readily available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene copolymer and maleic anhydride, polyamino acids (homopolymers or random copolymers) and dextran or poly(vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide-ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization may be determined based on a number of circumstances, such as the specific properties or functions of the antibody to be improved, whether the antibody derivative is intended to be used in therapy under certain conditions, etc.

In another embodiment, the present invention provides conjugates of an antibody and a non-protein moiety that can be selectively heated by radiation. In one embodiment of the present invention, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102, 2005, 11600-11605). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-protein moiety to a temperature that kills cells proximal to the non-protein moiety that does not contain the antibody.

B. Recombination and composition methods.

- 64 046075

Antibodies can be produced using recombination methods and compositions, for example, described in US 4816567. In one embodiment, the present invention provides an isolated nucleic acid encoding an anti-CD137 antigen binding molecule or antibody described in the present invention. Such a nucleic acid may encode an amino acid sequence comprising a VL and/or an amino acid sequence containing a VH of an antibody (eg, the light and/or heavy chains of an antibody). In another embodiment, the present invention provides one or more vectors (eg, expression vectors) comprising such a nucleic acid. In another embodiment, the present invention provides a host cell comprising such a nucleic acid. In one such embodiment of the present invention, the host cell contains (eg, has been transformed): (1) a vector containing a nucleic acid encoding an amino acid sequence containing a VL antibody and an amino acid sequence containing a VH antibody, or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing a VL antibody, and a second vector containing a nucleic acid that encodes an amino acid sequence containing a VH antibody. In one embodiment of the present invention, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (eg, Y0, NS0, Sp2/0 cells). In one embodiment, the present invention provides a method for producing an anti-CD137 antigen-binding molecule or antibody, which comprises culturing a host cell containing a nucleic acid encoding an antibody as defined above under conditions suitable for expression of the anti-CD137 antigen-binding molecule or antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

To recombinantly produce an anti-CD137 antigen binding molecule or antibody, the nucleic acid encoding the antibody, for example as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells described in the present invention. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5648237, US 5789199, US 5840523 (see also Charlton, Methods in Molecular Biology, 2003, volume 248, 245-254 (ed. B.K.C. Lo, ed. Humana Press, Totowa, NJ), which describe the expression of antibody fragments in E. coli). Once expressed, the antibody can be isolated from the bacterial cell biomass into a soluble fraction and can be further purified.

In addition to prokaryotes, suitable hosts for the cloning or expression of antibody-encoding vectors include eukaryotic microorganisms such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been humanized, resulting in the production of an antibody with a partially or fully glycosylated human pattern. See publications Nat. Biotech., 22, 2004, 1409-1414; Li et al., Nat. Biotech. 24, 2006, 210-215.

Host cells useful for expression of the glycosylated antibody also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include insect cells, and plant cells are also applicable. Numerous strains of baculoviruses have been identified that can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells.

Plant cells can also be used as host cells. See, for example, US 5959177, US 6040498, US 6420548, US 7125978 and US 6417429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plant cells).

Animal cells can also be used as host cells. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples include mammalian host cell lines, namely the monkey kidney cell line CV1 transformed with SV40 virus (COS-7); human embryonic kidney line (293 or 293 as described, for example, in Graham et al., J. Gen Virol. 36, 1977, 59); baby hamster kidney cells (BHK, baby hamster kidney); mouse Sertoli cells (TM4 cells as described, for example, in Mather, Biol. Reprod. 23, 1980, 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383, 1982, 44-68; MRC 5 cells; FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77,

- 65 046075

1980, 4216); myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of some mammalian host cell lines useful for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, vol. 248, 2003, 255-268, ed. B.K.C. Lo, Humana Press, Totowa, New Jersey.

B. Research methods.

Ahtu-CD137 antigen binding molecules or antibodies provided in the present invention can be identified, screened or characterized for their physical/chemical properties and/or biological activity using various assay methods known in the art.

1. Binding analysis and other methods.

In one embodiment of the present invention, an antigen binding molecule or antibody as described herein is tested for antigen binding activity, for example, by known methods such as ELISA, Western blotting, etc.

In another embodiment of the present invention, a competition assay in the presence of a small molecule compound can be used to identify an antigen binding molecule or antibody that competes for binding to CD137 with anti-CD137 antigen binding molecules or antibodies, including antibodies A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 and/or A549/B167, described in table. 17, as a heavy chain variable region/light chain variable region combination in the presence of a small molecular compound (for example, in the presence of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or lower molecular weight compound). In some embodiments of the present invention, such competing antigen binding molecules or antibodies bind to the same epitope (e.g., a linear epitope or conformational epitope) that is bound by anti-CD137 antigen binding molecules, including A375/B167, A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376, A551/B256, A551/B379, A555/B379, A548/B256 and/or A549/B167, described in table. 17 as a heavy chain variable region/light chain variable region combination. For detailed descriptions of examples of methods for mapping the epitope to which an antigen-binding molecule or antibody binds, see Morris Epitope Mapping Protocols, in Methods in Molecular Biology volume 66, 1966, Humana Press, Totowa, NJ). In one embodiment of the present invention, anti-CD137 antigen-binding molecules or antibodies as used herein, having CD137-binding activity that is dependent on antigen-binding activity that in turn is dependent on the small molecule compound, recognize an epitope formed by a complex formed from the antigen (eg , CD137) and a low molecular weight compound (for example, ATP).

In a typical antibody competition assay, immobilized CD137 is incubated in the presence of a small molecule compound (e.g., in the presence of 10 μM or more, 50 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, or 250 μM or more small molecule compound) in a solution containing a first labeled antibody that binds to CD137 (e.g., anti-CD137 antibodies including A372/B040, A356/B040, A486/B167, A487/B167, A488/B226, A489/B223, A548/B376 , A551/B256, A551/B379, A555/B379, A548/B256 and/or A549/B167 described in Table 17 as a heavy chain variable region/light chain variable region combination), and a second unlabeled antibody that is tested on the ability to compete for binding to CD137 with the first antibody. The second antibody may be present in the supernatant of the hybridoma. As a control, immobilized CD137 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing binding of the first antibody to CD137, excess unbound antibody is removed and the amount of label bound to immobilized CD137 is measured. If the amount of label bound to immobilized CD137 is significantly reduced in the test sample compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to CD137. See Harlow and Lane, Antibodies: A Laboratory Manual, chapter 14, 1988, Cold Spring Harbor Laboratory, New York). Those skilled in the art will appreciate that the assay can be performed in a similar manner to the assay of antigen-binding molecules other than antibodies.

2. Activity analyses.

In one embodiment, the present invention provides assays to identify the biological activity of anti-CD137 antigen-binding molecules or antibodies having biological activity. Biological activity may include, for example, CD137 agonist activity; plasma half-life; antitumor activity; and weak or suppressed systemic response in tissues other than tumors. Antigen-binding molecules or antibodies having such biological activity in vivo and/or in vitro are also provided. In some embodiments, the antigen binding molecule (eg, anti-CD137 antigen binding molecule) or antibody of the present invention is tested for such biological activity.

- 66 046075

a) Agonist Activity Assay (AAAs) In one embodiment of the present invention, CD137 agonist activity is measured by contacting CD137-expressing cells with an α-anti-CD137 antigen-binding molecule or antibody in a solution to which a small molecule compound is added or not. In one embodiment of the present invention, CD137 agonist activity in a solution to which the small molecule compound is added and CD137 agonist activity in a solution to which the small molecule compound is not added are assessed by the amount of cytokine produced (e.g., the amount of cytokine produced IL-2, IFN-γ and/or IL-6), which is measured for 18 hours, 24 hours, 36 hours, 48 hours or 72 hours after solution contact of CD137-expressing cells and anti-CD137 antigen-binding molecule or antibody. In one embodiment of the present invention, the solution to which the small molecule compound is added is adjusted such that the concentration of the small molecule compound after adjustment is 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM. In another embodiment of the present invention, the CD137 expressing cells used are isolated human peripheral blood mononuclear cells (PBMCs) or T cells expanded from isolated human PBMCs.

In one embodiment, the present invention uses human PBMCs isolated from the blood of a healthy donor by centrifugation at 400xg for 30 minutes at room temperature. Preferably, human PBMCs isolated in the next two steps are used. In the first step, Leucosep (Greiner Bio-One) fortified with Ficoll-Paque PLUS (GE Healthcare) is centrifuged at 1000xg for 1 min at room temperature, then the blood is diluted with PBS and centrifuged at 400xg for 30 min at room temperature. . At the second stage, the buffy coat from the tube after centrifugation is collected and washed with 60 ml of PBS (Wako).

A detailed description of an example method for measuring CD137 agonist activity using human PBMCs is described below. It should be noted that even though the following example uses ATP as the small molecule compound for illustration, this does not exclude other small molecule compounds. In one embodiment of the present invention, isolated human PBMCs are diluted to a density of 5x10 ⁶ /ml with culture medium (5% human serum (SIGMA), 95% AIM-V (Thermo Fischer Scientific)). The isolated human PBMCs are then contacted with an anti-human CD3ε antibody and/or an anti-human CD28 antibody, whereby CD137 expression is induced in the human PBMCs. Preferably, 50 μl of 0.04 μg/ml anti-human CD3ε antibody (BD Co., clone SP34) and 20 μg/ml anti-human CD28 antibody (BD, clone: CD28.2), diluted in culture medium, are added to the isolated PBMCs human (100 µl with a cell density of 5x 10 ⁶ / ml).

To human PBMCs to which anti-human CD3ε antibodies and/or anti-human CD28 antibodies have been added, (i) culture medium with or without ATP is then further added; and (ii) an anti-CD137 antigen-binding molecule or antibody. Preferably, 25 μl of culture medium with or without ATP is added. Preferably, 25 μl of anti-CD137 antigen binding molecule or antibody is added at a concentration of 40 μg/ml. More preferably, (i) and (ii) above are added approximately 6 hours after contact of human PBMCs with the anti-human CD3ε antibody and/or the anti-human CD28 antibody. In one embodiment of the present invention, preferably the amount of IL-2 produced is measured before the amount of IFN-γ is produced. In one embodiment of the present invention, the amount of IL-2 production is measured for about 24 hours after contact of human PBMCs with an anti-human CD3ε antibody and/or an anti-human CD28 antibody. Preferably, the amount of IL-2 production is measured approximately 24 hours after contact of human PBMCs with an anti-human CD3ε antibody and/or an anti-human CD28 antibody and approximately 18 hours after addition of an anti-CD137 antigen-binding molecule or antibody.

In another embodiment of the present invention, the amount of IFN-γ produced is measured approximately 48 hours after exposure of human PBMCs to an anti-human CD3ε antibody and/or an anti-human CD28 antibody. Preferably, the amount of IFN-γ produced is measured within about 48 hours after contact of human PBMCs with an anti-human CD3ε antibody and/or with an anti-human CD28 antibody and about 42 hours after the addition of an anti-CD137 antigen-binding molecule or antibody. In one embodiment of the present invention, the amount of IL-2 produced and/or the amount of IFN-γ produced is determined by measuring the amount of IL-2 produced and/or the amount of IFN-γ produced in the collected culture supernatant. In one embodiment of the present invention, human PBMCs spiked with anti-human CD3ε antibody and/or anti-human CD28 antibody are left in an incubator under 5% CO2 at 37°C until all measurements are completed.

Details of another example of a method for measuring CD137 agonistic activity using human PBMCs are described below. Isolated human PBMC are diluted to cell density

- 67 046075

5x10 ⁶ /ml culture medium (5% human serum (SIGMA), 95% AIM-V (Thermo Fischer Scientific)). Then the density of human PBMCs is adjusted to 5x10 ⁶ /ml and 100 μl is seeded into the wells of a 96-well plate (flat-bottomed with a lid) (Corning). Human PBMCs are then exposed to induce CD137 expression. For example, CD137 expression in human PBMCs is induced by adding 50 μl of an anti-human CD3ε antibody at a concentration of 0.04 μg/ml (BD Co., clone SP34) and an anti-human CD28 antibody at a concentration of 20 μg/ml (BD Co., clone CD28. 2), diluted with culture medium.

CD137 expression is then induced in human PBMCs, the plate is shaken and left for 6 hours at 37°C in an incubator under 5% CO ₂ atmosphere. Then 25 μl of 2 mM ATP (SIGMA) diluted in medium, or medium without ATP alone, and 25 μl of each antibody at a concentration of 40 μg/ml are added to each well, and the plate is shaken and left for 18 hours at 37°C in an incubator in an atmosphere of 5% CO2. A portion of the culture supernatant is then collected and the amount of IL-2 contained in the culture supernatant is quantified using the Human IL-2 DuoSet ELISA kit (R&D systems) or the Human IL-2 ELISA Set (BD Biosciences). After collecting the culture supernatant, the plate is again left for 24 hours at 37°C in an incubator under an atmosphere of 5% CO2. A portion of the culture supernatant is then collected and the IFN-γ contained in the culture supernatant is quantified using the IFN-γ DuoSet ELISA kit (R&D systems) or the Human IFN-γ ELISA Development kit (PeproTech). The ELISA method is generally used according to the protocol supplied with the kit. When working with the Human IL-2 DuoSet ELISA kit (R&D systems) and the Human IFN-γ ELISA Development kit (R&D systems), the appearance and cessation of staining is performed in accordance with the protocol using a substrate solution (R&D systems) containing H ₂ O ₂ , tetramethylbenzidine and 1 N H ₂ SO ₄ (Wako). When working with the Human IL-2 ELISA Set (BD Biosciences), stop staining using 1 N H2SO4 (Wako).

When working with the IFN-γ ELISA Development kit (PeproTech), the onset and cessation of staining is carried out using a solution of TMB Chromogen Solution (Thermo Fischer Scientific) and 1 N H ₂ SO ₄ (Wako). Absorbance measurement is then performed using EnVision (PerkinElmer), and the amounts (pg/ml) of IL-2 and IFN-γ in the culture supernatant are respectively calculated using a calibration curve constructed according to the protocol. In this PBMC assay, CD137 agonist activity can be expressed as the fold change in the amount of IL-2 and IFN-γ in the culture supernatant compared to a negative control antibody (an antibody that does not bind to CD137). In one embodiment of the present invention, CD137 agonist activity is measured in accordance with the methods described in Examples 5-5-1 and 5-5-2.

In one embodiment of the present invention, if CD137 agonist activity is assessed by the amount of cytokine produced (e.g., the amount of IL-2, IFN-γ, and/or IL-6 produced) in human PBMCs, the anti-CD137 antigen-binding molecule or antibody may be regarded as a manifestation of agonistic activity against CD137 in the presence of a low molecular weight compound if the amount of cytokine produced in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM low molecular weight compound upon addition of an anti-CD137 antigen-binding molecule or antibody is greater than 1 .01 times or more, 1.02 times or more, 1.03 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, 1.1 times or more, 1.11 times or more, 1.12 times or more, 1.13 times or more, 1.14 times or more than or 1.15 times or more, 1.5 times or more, 2 times or more, or 3 times or more the amount of cytokine produced when the negative control antibody was added.

In one embodiment of the present invention, CD137 agonist activity is assessed by the amount of IL-2 produced in human PBMCs, where an anti-CD137 antigen-binding molecule or antibody can be assessed as having CD137 agonist activity in the presence of a small molecule compound if the amount of IL-2 produced 2 in the presence of 10 µM, 50 µM, 100 µM, 150 µM, 200 µM or 250 µM small molecule compound, if the anti-CD137 antigen-binding molecule or antibody added is 1.01 times or more, 1.02 times or more, in 1.03 times or more, or 1.05 times or more, 1.05 times or more in a preferred embodiment of the present invention compared to the amount of IL-2 produced when a negative control antibody is added.

In one embodiment of the present invention, when CD137 agonist activity is assessed by the amount of IFN-γ produced in a human PBMC assay, the anti-CD137 antigen binding molecule or antibody can be assessed as having CD137 agonist activity in the presence of the small molecule compound when the amount of IFN produced -γ in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM small molecule compound with the addition of an anti-CD137 antigen-binding molecule or antibody

- 68 046075 1.1 times or more, 1.11 times or more, 1.12 times or more, 1.13 times or more, 1.14 times or more, 1.15 times or more more, 1.15 times or more in a preferred embodiment of the present invention compared to the amount of IFN-γ produced when adding a negative control antibody.

In another embodiment of the present invention, among the anti-CD137 antigen-binding molecules or antibodies found to exhibit agonistic activity against CD137 in the presence of a small molecule compound in the above-mentioned negative control comparison, antibodies that are assessed as not exhibiting agonistic activity in against CD137 or having reduced CD137 agonist activity in the absence of the small molecule compound (compared to that in the presence of the small molecule compound). In particular, when (ii) below is greater than (i), the hhtu-CD137 antigen binding molecule or antibody is assessed as having no CD137 agonist activity or having more reduced CD137 agonist activity in the absence of the small molecule compound:

(i) [cytokine produced in the absence of small molecule when anti-CD137 antigen binding molecule or antibody is added]/[cytokine produced in the absence of small molecule when antibody is added - negative control] (ii) [cytokine produced in the presence of small molecule when added anti-CD137 antigen-binding molecule or antibody]/[the amount of cytokine produced in the presence of a small molecule compound with the addition of a negative control antibody].

In another embodiment of the present invention, when the CD137 agonistic activity of the first antigen binding molecule or anti-CD137 antibody and the second antigen binding molecule or anti-CD137 antibody is compared using the amounts of cytokines produced (e.g., amounts of IL-2, IFN-γ, and/or IL-6 production ) in a human PBMC assay, the first antigen-binding molecule or anti-CD137 antibody can be assessed as having higher agonistic activity than the second antigen-binding molecule or anti-CD137 antibody if (i) the amount of cytokines produced when the first antigen-binding molecule or anti-CD137 antibody is added in the presence 10 µM, 50 µM, 100 µM, 150 µM, 200 µM or 250 µM small molecule compound 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.04 times or more more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, 1.1 times or more, 1.11 times or more, 1.12 times or more, 1.13 times or more, 1.14 times or more or 1.15 times or more, 1.5 times or more, 2 times or more or 3 times or more compared to (ii) the amount of cytokines produced when a second antigen-binding molecule or anti-CD137 antibody was added under the same conditions.

In one embodiment of the present invention, if CD137 agonistic activity is assessed by the amount of IL-2 produced in a human PBMC assay, the first anti-CD137 antigen-binding molecule or antibody may be assessed as having higher agonistic activity than the second anti-CD137 antigen-binding molecule or antibody. antibody if (i) the amount of cytokine production upon addition of the first antigen binding molecule or anti-CD137 antibody in the presence of 10 µM, 50 µM, 100 µM, 150 µM, 200 µM or 250 µM small molecule compound is greater than 1.01 times or more .02 times or more, 1.03 times or more, or 1.04 times or more, or 1.04 times or more, in a preferred embodiment of the present invention compared to (ii) the amount of IL-2 produced when a second antigen binding agent is added molecules or antibodies against CD137 under the same conditions.

In one embodiment of the present invention, if CD137 agonistic activity is assessed by the amount of IFN-γ production in a human PBMC assay, the first anti-CD137 antigen-binding molecule or antibody may be assessed as having higher agonistic activity than the second anti-CD137 antigen-binding molecule or antibody. antibody if (i) the amount of cytokines produced upon addition of the first antigen binding molecule or anti-CD137 antibody in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM small molecule compound is greater than 1.05 times or more, 1 .06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, or 1.1 times or more, or 1.1 times or more in a preferred embodiment of the present invention compared to (ii) adding a second antigen-binding molecule or anti-CD137 antibody under the same conditions. In one embodiment of the present invention, the first anti-CD137 antigen-binding molecule or antibody is an antigen-binding molecule or antibody containing the original Fc region, and the second anti-CD137 antigen-binding molecule is an antigen-binding molecule or antibody containing a variant Fc region.

In one embodiment of the present invention, the method described in Examples 5-5-1 and 5-5-2 can be used to measure CD137 agonist activity using human PBMCs, and the method described in Example 2-6 can be used to measure CD137 agonist activity using T cells expanded from isolated human PBMCs.

- 69 046075

In one embodiment of the present invention, CD137 agonist activity can be measured by contacting CD8-positive or CD4-positive T cells isolated from human PBMCs with an antigen-binding molecule or anti-CD137 antibody in a solution to which the small molecule compound is added or not. At this time, additional cells expressing FcYRIIb can be added to the solution. In another embodiment, CD137 agonist activity can be measured by contacting B cells (which can be isolated from human PBMCs or a known B cell line can be used) with an anti-CD137 antigen binding molecule or antibody. In one embodiment of the present invention, CD137 agonist activity can be assessed by the amount of cytokine production (eg, the amount of IL-2, IFN-γ, and/or the amount of IL-6 produced), which is measured after contact in a CD8- positive T cells, CD4 positive T cells, or B cells with antigen binding molecule or anti-CD137 antibody.

In one embodiment of the present invention, agonist activity assays using human peripheral blood mononuclear cells (PBMCs) are isolated from blood collected from healthy individuals. Thus, in any of the above embodiments of the present invention, it will be understood by those skilled in the art that the results of assays using assays may vary for each blood sample donor. Taking this into account, antibodies that do not exhibit agonistic activity on some or the majority of human PBMCs isolated from multiple donors cannot be considered to exhibit CD137 agonistic activity, even if the criteria for agonistic activity are met in another portion of human PBMCs. In addition, when the assay according to the method described above is performed on human PBMCs isolated from multiple donors and the criteria for CD137 agonist activity are met in most donors, it can be concluded that CD137 agonist activity is exhibited. In another embodiment of the present invention, when an assay in accordance with the method described above is performed on human PBMCs isolated from multiple donors, the mean or median assay value (e.g., amounts of IL-2, IFN-γ, and/or IL-6) may be used to determine the presence or absence of CD137 agonist activity. In one embodiment of the present invention, when the assay is performed according to the above method on human PBMCs isolated from multiple donors, the number of donors is, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 or more.

b) Agonist Activity Assay (Reporter Gene Assay) In one embodiment of the present invention, CD137 agonist activity is assessed using a reporter gene assay in a solution to which a small molecule compound is added or not. In one embodiment of the present invention, CD137 agonist activity in a solution to which the small molecule compound is added and CD137 agonist activity in a solution to which the small molecule compound is not added are respectively assessed by the luciferase luminescence signal measured after contact T -cells expressing an NF-kappaB-luciferase and CD137 reporter construct with an antigen-binding molecule against CD137, and left for a certain time in the appropriate solution. In one embodiment of the present invention, the solution to which the small molecule compound is added is adjusted such that the concentration of the small molecule compound after adjustment is 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM. T cells expressing the NF-kappaB-luciferase and CD137 reporter construct are preferably GloResponse™ NF-kappaB-Luc2/4-1BB Jurkat cells (Promega, CS196004).

Details of a typical method for measuring CD137 agonist activity using a reporter gene assay are described below. In the examples below, ATP is used as an example of a small molecule compound, but other small molecule compounds are not excluded. In one embodiment of the present invention, the concentration of FcyRIIB-expressing cells is first adjusted to 5x10 ⁴ /ml with medium (CHO culture medium (90% Ham's F12, 10% PBS)), and left overnight at 37°C in an incubator under an atmosphere of 5% CO ₂ . In one embodiment of the present invention, not only cells that potently express FcgRIIB, but also cell lines that endogenously express FcgRIIB, such as B cell lines, B cells isolated from a living organism, can be used as FcgRIIB-expressing cells. and so on. In a preferred embodiment of the present invention, the FcYRIIB-expressing cells are FcyRIIB CHO-K1 cells (Promega). Then, after removing the medium by aspiration, the concentration of GloResponseTM NF-kappaB-Luc2/4-1BB Jurkat cells (hereinafter 4-1BB Jurkat) is adjusted to 2ε10 ⁶ /ml with another medium (99% RPMI, 1% FSB). In a preferred embodiment of the present invention, 25 μl of GloResponse™ NF-kappaB-Luc2/4-1BB media-corrected Jurka cells are added to 200 μl of media-corrected FcYRIIB-expressing cells. Then, 25 μl of each anti-CD137 antigen binding molecule is diluted with the above medium (99% RPMI, 1% PBS) to achieve the desired concentration (e.g., final concentration 0, 0.001, 0.01, 0.1,

- 70 046075

1, 10 μg/ml), and then add 25 μl of ATP solution diluted with the above medium (99% RPMI, 1% PBS) to obtain the desired concentration (for example, the final concentration is 0, 10, 50, 100, 150, 200, 250 µM). In one embodiment of the present invention, the luciferase luminescence signal is measured after 2 hours or less, 4 hours or less, 6 hours or less, 24 hours or less after addition of the anti-CD137 antigen binding molecule to 4-1BB Jurkat cells. In a preferred embodiment of the present invention, the 4-1BB Jurkat is maintained at 37°C for 6 hours in an incubator under an atmosphere of 5% CO ₂ . After this, 75 μl of BioGlo reagent is added and luminescence is measured using a plate reader. In one preferred embodiment of the present invention, the Bio-Glo reagent is the Bio-Glo Luciferase Assay System (buffer and substrate). In one embodiment of the present invention, to maintain a constant temperature during the reaction, the 4-1BB Jurkat cells can be left at room temperature for 5 minutes, 10 minutes, 15 minutes, or 20 minutes after removal from the incubator. In a preferred embodiment of the present invention, the 4-1BB Jurkat is left at room temperature for 15 minutes after removal from the incubator. In one embodiment of the present invention, the luminescence value of 4-1BB Jurkat cells to which anti-CD137 antigen-binding molecule has been added is divided by the luminescence value of 4-1BB Jurkat cells to which no anti-CD137 antigen-binding molecule has been added, and this value is taken as the fold index induction (relative light unit) and is used as an indicator to evaluate the CD137 agonist activity of each antigen-binding molecule.

Another example of a method for measuring CD137 agonist activity using a reporter gene assay is described below. To each well of a 96-well plate, add 200 μl of FcyRIIB CHO-K1 cells (Promega), adjusted by adding medium to a concentration of 5x10 ⁴ / ml, and leave overnight at 37°C in an incubator in an atmosphere of 5% CO2. CHO medium (90% Ham's F12, 10% PBS) is used as a culture medium. The medium is then completely removed by suction, and 25 μl of GloResponse™ NF-kB-Luc2/4-1BB Jurkat cells at an adjusted concentration of 2x10 ⁶ /ml in assay medium (99% RPMI, 1% PBS) is added to each well. Then add 25 µl of each antigen solution diluted with assay medium to a final concentration of 0, 0.001, 0.01, 0.1, 1 or 10 µg/ml, and finally add 25 µl of ATP solution diluted with assay medium to 0 or 250 µM. After the plate is left for 6 hours at 37°C in a 5% CO ₂ incubator, it is left at room temperature for 15 minutes and then 75 μl of Bio-Glo reagent is added to each well. For example, the Bio-Glo Luciferase Assay System (buffer and substrate) can be used as a Bio-Glo reagent. Luminescence in each well is then measured using a plate reader. The value obtained by dividing the luminescence value in each well by the luminescence value in the well to which no antibody was added is determined as the induction factor. In a reporter gene assay, CD137 agonistic activity can be assessed by the fold change (as a relative light unit) in luminescence in wells with each antibody added, compared with the amount of luminescence in wells to which no antibody was added.

Another example of a method for measuring CD137 agonist activity using a reporter gene assay is described below. To each well of a 384-well plate, add 10 μl of FcyRIIB CHO-K1 cells (Promega), adjusted to a concentration of 2x10 ⁶ / ml using assay medium (99% RPMI, 1% PBS). Then, 10 μl of an antibody solution containing ADP, or an antibody solution containing ATP, or an antibody solution not containing ATP or ADP, is added to each well. After this, 10 μl of the GloResponse™ NF-kB-Luc2/4-1BB Jurkat cell line, adjusted to 2x10 ⁶ /ml with assay medium (99% RPMI, 1% PBS), is added to each well. The final concentration of ADP is 50 µM and the final concentration of ATP is 50 µM. After the plate is left at 37°C for 6 hours in a 5% CO ₂ incubator, it is left at room temperature for 15 minutes and 30 μl of Bio-Glo reagent is added to each well. The Bio-Glo reagent uses the Bio-Glo Luciferase Assay System (buffer and substrate). Luminescence in each well is then measured using a plate reader. CD137 agonist activity can be assessed as the relative light unit (also called luminescence fold or fold change) of luminescence in wells with each antibody added, compared to the amount of luminescence in wells to which no antibody was added.

Another example of a method for measuring CD137 agonist activity using a reporter gene assay is provided below. Add 10 μl of FcyRIIB CHO-K1 cells (Promega) to each well of a 384-well plate, adjusted to a concentration of 4x10 ⁵ /ml using assay medium (99% RPMI, 1% PBS). Then, 10 μl of an antibody solution containing ADP, or an antibody solution containing ATP, or an antibody solution not containing ATP or ADP, is added to each well. After this, 10 μl of the GloResponse™ NF-kBLuc2/4-1BB Jurkat cell line, adjusted to 2x10 ⁶ /ml with assay medium (99% RPMI, 1% PBS), is added to each well. The final concentration of ADP is 10 µM and the final concentration of ATP is 10 µM. After

- 71 046075 After the plate is left at 37°C for 6 hours in an incubator with 5% CO ₂ atmosphere, it is left at room temperature for 15 minutes and 30 μl of Bio-Glo reagent is added to each well. The Bio-Glo Luciferase Assay System (buffer and substrate) is used as a Bio-Glo reagent. Luminescence in each well is then measured using a plate reader. CD137 agonist activity can be assessed as the relative light unit (also called luminescence fold or fold change) of luminescence in wells with each antibody added, compared to the amount of luminescence in wells to which no antibody was added.

In one embodiment of the present invention, when CD137 agonist activity is measured using a reporter gene assay, an anti-CD137 antigen binding molecule or antibody may be assessed to exhibit CD137 agonist activity in the presence of a small molecule compound if (i) CD137 agonist activity (relative light unit) in the presence of 10 µM, 50 µM, 100 µM, 150 µM, 200 µM or 250 µM low molecular weight compound 1.1 times or more, 1.2 times or more, 1.3 times or more , 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more , 60 times or more, 70 times or more, 80 times or more, or 90 times or more compared to (ii) CD137 agonist activity (relative light unit) in the absence of the small molecule compound. In one embodiment of the present invention, the resulting concentration of antibody in (i) and (ii) above is 0, 0.001, 0.01, 0.1, 1 or 10 μg/ml, in a preferred embodiment of the present invention it is 0.1 µg/ml or 1 µg/ml.

In another embodiment of the present invention, if an antigen binding molecule or anti-CD137 antibody is added at a specific concentration (for example, at a final concentration of 0.001, 0.01, 0.1, 1 or 10 μg/ml, or 0.1 μg/ml or 1 μg /ml), an anti-CD137 antigen-binding molecule or antibody is assessed as having substantially no anti-CD137 agonistic activity in the absence of the small molecule compound if the fold induction (relative light unit) in the absence of the small molecule compound is 10 or less, 9 or less, 8 or less, or 5 or less in a preferred embodiment of the present invention, and substantially 1 in a more preferred embodiment of the present invention. In one embodiment of the present invention, the induction fold (relative light unit) in the absence of the small molecule compound is substantially equal to 1, meaning those cases where the induction fold (relative light unit) in the absence of the small molecule compound is less than 2 times, less than 1, 9 times, less 1.8 times, less than 1.7 times, less than 1.6 times, less than 1.5 times, less than 1.4 times, less than 1.3 times, less than 1.2 times or less than 1.1 times.

c) Concentration in plasma.

In one embodiment of the present invention, the blood kinetics of an anti-CD137 antigen-binding molecule or antibody of the present disclosure is measured and/or compared using human CD137 knockin mice. A human CD137 knockin mouse is generated, for example, by replacing the mouse CD137 gene with a human CD137 gene by introducing a replacement vector with the human CD137 gene into mouse embryonic stem cells (ES cells). In one embodiment of the present invention, the CD137 antigen binding molecule or antibody of the present invention is administered by a single intravenous injection to human CD137 knockin mice, and blood is collected several times over time, starting immediately after administration until about 5 days, 10 days, 15 days, 20 days, 25 days or 30 days after administration. In a preferred embodiment of the present invention, the anti-CD137 antigen-binding molecule or antibody of the present invention is administered by a single intravenous injection to human CD137 knockin mice, and blood is collected several times over a period of time from 5 minutes to 28 days after administration. The plasma is quickly separated from the collected blood, and the plasma antibody concentration is measured by electrochemiluminescence (ECL). In one embodiment of the present invention, plasma antibody concentration can be measured by the method described in Example 6-3-2.

When measuring plasma half-life using a human CD137 knockin mouse, if the anti-CD137 antigen-binding molecule or antibody disappears from the plasma more slowly than the control molecule, the anti-CD137 antigen-binding molecule or antibody is judged to have improved kinetics in the blood than the control molecule . In addition, if an anti-CD137 antigen-binding molecule or antibody having small molecule-dependent binding activity (switchable molecule or switchable antibody) disappears from the plasma more slowly than an anti-CD137 antigen-binding molecule or antibody that is independent of the small molecule (non-switchable molecule or non-switchable antibody), it can be concluded that the switchable molecule (or switchable antibody) does not bind to CD137 expressed in non-tumor tissue compared to the non-switchable molecule (non-switchable antibody).

d) Antitumor activity.

In one embodiment of the present invention, the anti-CD137 antigen-binding

- 72 046075 a molecule or antibody is tested for its ability to inhibit cell growth or proliferation in vivo. In some embodiments of the present invention, the anti-CD137 antigen binding molecule or antibody is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as allograft models or xenograft models, can be used for such testing. In an example xenograft system, human tumor cells are administered to an appropriately immunocompromised non-human animal, such as an athymic nude mouse. The antibody of the present invention is administered to an animal. The ability of an antibody to inhibit or reduce tumor growth is measured. In some embodiments of the present invention, in the above xenograft system, the human tumor cells are tumor cells from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Freiburg, Germany). In some embodiments of the present invention, human tumor cells are administered to an animal (not human) with a corresponding immunodeficiency by subcutaneous injection or transplantation into a suitable site, such as mammary fat pad.

In one embodiment of the present invention, the antitumor activity of the anti-CD137 antigen-binding molecules or antibodies of the present invention can be measured and/or compared using a syngeneic tumor cell transplant model based on the human CD137 knockin mice described above. A cancer cell line may be suitably selected for use in the test, preferably the mouse colon cancer cell line MC38. In one embodiment of the present invention, the MC38 cell line is transplanted subcutaneously into the abdominal region of the mouse, and the model is considered established when the tumor volume reaches approximately 50-300 mm ³ . Once the model is established, mice transplanted with MC38 cells are assigned to groups and then treated with an anti-CD137 antigen-binding molecule or antibody. In one embodiment of the present invention, tumor volume measurements are performed at a frequency of one to two times per week to assess antitumor activity. Tumor volume is calculated using the following equation:

tumor volume = (major axis x minor axis x minor axis)/2

In one embodiment of the present invention, the antitumor activity of an anti-CD137 antigen-binding molecule or antibody can be tested and evaluated in accordance with the method described in Example 6-4.

In one embodiment of the present invention, anti-CD137 antigen-binding molecules or antibodies are recognized as having antitumor activity when the tumor volume in the anti-CD137 antigen-binding molecule or antibody treatment group is less than that in the vehicle group, or the increase in tumor volume is less in the group. administration of anti-CD137 antigen-binding molecules or antibodies to animals is less than in the group of animals receiving a solvent.

e) Systemic reaction.

In one embodiment of the present invention, the systemic response to anti-CD137 antigen-binding molecules or antibodies of the present invention is measured and/or compared using a syngeneic tumor cell transplant model based on the human CD137 knockin mice described above. The organs to measure the systemic response can be selected accordingly and may include the liver, spleen and/or lymph nodes. In one embodiment of the present invention, the systemic response is assessed by removing the liver, spleen and/or lymph nodes from human CD137 knockin mice at an appropriate time after administration of anti-CD137 antigen-binding molecules or antibodies. If the organ to be removed is the spleen and/or lymph nodes, this organ is weighed and/or the cells in the lymphocyte fraction are counted. When counting, preferably the lymphocyte fraction after hemolysis is used for the spleen, and the lymphocyte fraction obtained by homogenization is used for the lymph nodes. If the organ to be removed is the liver, I count the cells in the lymphocyte fraction obtained using the Liver dissociation kit, mouse (Milteny Biotec). In addition, T cell analysis using liquid cytometry (FCM, flowcytometry) can be performed on lymphocyte fractions from each organ (liver, spleen and/or lymph nodes). The FCM assay uses granzyme B expression or PD-1 expression or ICOS expression in CD8 alpha-positive T cells or the ratio of CD8 alpha-positive T cells to CD45 positive cells. In one embodiment of the present invention, the systemic response to anti-CD137 antigen-binding molecules or antibodies can be tested and assessed in accordance with the method described in Example 6-4.

In one embodiment of the present invention, anti-CD137 antigen-binding molecules or antibodies are recognized as causing a suppressed systemic response and/or suppressed immune cell activation in non-tumor tissues (eg, liver, spleen and/or lymph nodes) compared to a control molecule when group in which mice were injected with anti-CD137 antigen-binding molecules or antibodies noted a lower value when assessed above

- 73 046075 described different indicators compared to a group in which mice were injected with the same amount of a control molecule. In addition, if a group receiving administration of an anti-CD137 antigen-binding molecule or an antibody having binding activity dependent on a small molecule compound (switch molecule or switch antibody) shows a lower value when evaluating the various indicators described above than that of the group in which mice were administered anti-CD137 antigen-binding molecules or antibodies that do not have small molecule-dependent binding activity (nonswitchable molecule or nonswitchable antibody), the switchable molecule (or switchable antibody) are recognized as causing a suppressed systemic response and/or suppressed immune cell activation in non-tumor tissues ( for example, in the liver, spleen and/or lymph nodes) compared to a non-switchable molecule (or a non-switchable antibody).

It will be appreciated that any of the measurement methods described above can be performed using the immunoconjugates of the present invention instead of or in addition to anti-CD137 antigen-binding molecules or antibodies.

D. Immunoconjugates.

The present invention also provides immunoconjugates containing anti-CD137 antigen-binding molecules or antibodies conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (for example, protein toxins, enzymatically active bacterial toxins, and also of fungal, plant or animal origin, or fragments thereof) or radioactive isotopes.

In one embodiment of the present invention, the immunoconjugate is an antibody-drug conjugate (ADC), where the antibody is conjugated to one or more drugs, including, but not limited to, a maytansinoid (see US 5208020, US 5416064 , EP 0 425 235 B1); auristatin, such as the drug moieties of monomethyl auristatin DE and DF (MMAE and MMAF) (see US 5635483, US 5780588, US 7498298); dolastatin; calicheamicin or a derivative thereof (see US 5712374, US 5714586, US 5739116, US 5767285, US 5770701, US 5770710, US 5773001, US 5877296; Hinman et al., Cancer Res. 53, 1993, 3336-3342 ; Lode et al. ., Cancer Res. 58, 1998, 2925-2928); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13, 2006, 477-523; Jeffrey et al., Bioorganic & Med. Chem. Letters 16, 2006, 358362; Torgov et al. , Bioconj. Chem. 16, 2005, 717-721; Nagy et al., Proc. Natl. Acad. Sci. USA 97, 2000, 829-834; Dubowchik et al., Bioorg. & Med. Chem. Letters 12, 2002, 1529-1532; King et al., J. Med. Chem. 45, 2002, 4336-4343; US 6630579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecin; CC1065.

In another embodiment of the present invention, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and tricothecins.

In another embodiment of the present invention, the immunoconjugate includes an antibody as described in the present invention, conjugated to a radioactive atom to form a radioactive immunoconjugate. Various radioactive isotopes are available to produce radioconjugates. Examples are ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and radioactive epitopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as Tc-99sh or ¹²³ I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), e.g. iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Antibody-cytotoxic agent conjugates can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane ( IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the immunotoxin ricin can be prepared as described in Vitetta et al., Science 238, 1987, 1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MXDTPA) is a typical chelating agent for conjugating a radionuclide to an antibody. See WO 94/11026. The linker may be a cleavable linker that promotes the release of cytotoxins

- 74 046075 chemical drug in a cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52, 1992, 127-131; US 5,208,020).

Immunoconjugates or ADCs in the present invention clearly include, but are not limited to, conjugates such as those obtained using cross-linker reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP , SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone) benzoate), which are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL, USA).

E. Methods and compositions for diagnosis and detection.

In some embodiments, any of the anti-CD137 antigen binding molecules or antibodies provided herein are useful for detecting the presence of an anti-CD137 antigen binding molecule in a biological sample. In the present invention, the term detection means quantitative or qualitative detection. In some embodiments of the present invention, the biological sample includes a cell or tissue.

In one embodiment of the present invention, the anti-CD137 antigen binding molecule or antibody is provided for use in a diagnostic and detection method. In another embodiment, the present invention provides a method for detecting CD137 in a biological sample. In some embodiments of the present invention, the method includes contacting a biological sample with an anti-CD137 antigen-binding molecule or antibody, as described in the present invention, under conditions allowing the anti-CD137 antigen-binding molecule or antibody to bind to CD137, and determining whether a complex is formed between an anti-CD137 antigen-binding molecule or an antibody with CD137. Such a method may be an in vitro or in vivo method. In one embodiment of the present invention, the anti-CD137 antigen-binding molecule or antibody is used to select subjects suitable for therapy with the anti-CD137 antigen-binding molecule or antibody, for example, if CD137 is a biomarker for patient selection.

Typical disorders that can be diagnosed using the antibody of the present invention include cancer.

In some embodiments, the present invention provides anti-CD137-labeled antigen-binding molecules or antibodies. Labels include, but are not limited to, tags or moieties that are detected directly (such as fluorescent, chromophore, electron dense, chemiluminescent, and radioactive tags), as well as moieties such as enzymes or ligands that are detected indirectly, e.g. through an enzymatic reaction or molecular interaction. Examples of labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores, for example, such as chelates of rare earth elements or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone , luceriferases, e.g. firefly luciferase and bacterial luciferase (US 4737456), luciferin, 2,3dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g. glucose oxidase, galactose oxidase and glu goat-6phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin tags, bacteriophage tags, stable free radicals, etc.

E. Pharmaceutical compounds.

Pharmaceutical formulations of the anti-CD137 antigen binding molecule or antibody of the present invention are prepared by mixing such antibody, having the desired purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980) in in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as

- 75 046075 polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers in the present invention also include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g. soluble human hyaluronidase PH-20 glycoproteins such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Some examples of sHASEGP and methods of their use, including rHuPH20, are described in US patent publications No. 2005/0260186 and US No. 2006/0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Examples of lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, the latter formulations including histidine acetate buffer.

The active ingredients can be encapsulated in microcapsules prepared, for example, by coacervation or interfacial polymerization methods, e.g. hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are described in Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles, such as films or microcapsules.

Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

G. Methods of therapy and medicinal compositions.

Any of the antigen-binding molecules or antibodies against CD137 provided in the present invention can be used in therapeutic methods.

In one aspect, the present invention provides antigen-binding molecules or antibodies against CD137 for use as a drug. In the present invention, examples of the drug specifically include drugs for inducing antitumor effects, such as inhibiting neovascularization in tumors, inhibiting tumor cell proliferation, depleting tumor B cells, etc., by activating cells resulting from the binding of anti -CD137 antigen-binding molecules or antibodies with CD137 expressed on immune cells such as T cells. In other embodiments, the present invention provides anti-CD137 antigen-binding molecules or antibodies for use in the treatment of tumors. In some embodiments, the present invention provides certain anti-CD137 antigen-binding molecules or antibodies for use in a treatment method. In some embodiments, the present invention provides certain anti-CD137 antigen-binding molecules or antibodies for use in a method of treating an individual with cancer, comprising administering to the individual an effective amount of an anti-CD137 antigen-binding molecule or antibody. In other embodiments of the present invention, examples of a tumor include a solid tumor that has been infiltrated by B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells).

In other embodiments, the present invention provides anti-CD137 antigen binding molecules or antibodies for use in a method of activating immune cells in an individual, comprising administering to the individual an effective amount of an anti-CD137 antigen binding molecule or antibody to activate immune cells, such as B cells, dendritic cells, natural killer cells, macrophages and/or CD8-positive T cells (more specifically, these immune cells that have infiltrated tumor tissue).

In other embodiments, the present invention provides anti-CD137 antigen-binding molecules or antibodies for use in a method of damaging cells (eg, tumor cells) in an individual, comprising administering to the individual an effective amount of an anti-CD137 antigen-binding molecule or antibody. In some embodiments of the present invention, examples of tumors include a solid tumor that has been infiltrated by B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells). In any of the above embodiments of the present invention, the individual is preferably a human.

In other embodiments, the present invention provides for the use of anti-CD137 antigen-binding molecules or antibodies to produce a drug. In one embodiment of the present invention, the drug is intended to treat a tumor (depending on the context, it may be more appropriate to call this cancer (the same applies hereinafter)). In other embodiments of the present invention, the drug is for use in a method of treating a tumor (or cancer, depending on the context),

- 76 046075 wherein the method includes administering to an individual with a tumor (or cancer, depending on the context), an effective amount of a drug. In other embodiments of the present invention, the drug is intended to induce an antitumor effect, such as inhibiting neovascularization in tumors, inhibiting tumor cell proliferation, depleting tumor B cells, etc., by activating cells resulting from the binding of an anti-CD137 antigen-binding molecule or antibody with CD137 expressed on immune cells such as T cells. In other embodiments of the present invention, the drug is for use in a method for, for example, inhibiting neovascularization in tumors, inhibiting tumor cell proliferation, depleting tumor B cells, etc. by activating cells by binding an anti-CD137 antigen-binding molecule or antibody to CD137 expressed on immune cells, such as T cells, in an individual, the method comprising administering to the individual an effective amount of a drug. In any of the above embodiments of the present invention, the individual is preferably a human.

In other embodiments, the present invention provides a method for treating cancer. In one embodiment, the methods include administering to an individual with a tumor an effective amount of an anti-CD137 antigen-binding molecule or antibody. In other embodiments of the present invention, examples of a tumor include a solid tumor that is infiltrated by B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells).

In other embodiments, the present invention provides methods for activating immune cells in an individual. In one embodiment of the present invention, the method includes administering to a subject an effective amount of an anti-CD137 antigen-binding molecule or antibody. In other embodiments of the present invention, immune cells include immune cells such as B cells, dendritic cells, natural killer cells, macrophages, and/or CD8 positive T cells (more specifically, these immune cells that have infiltrated tumor tissue) .

In other embodiments, the present invention provides methods for damaging cells (particularly tumor cells) in an individual. In one embodiment, the present invention provides methods that include administering to a subject an effective amount of an anti-CD137 antigen-binding molecule or antibody. In some embodiments of the present invention, examples of a tumor include a solid tumor that is infiltrated by B cells, dendritic cells, natural killer cells, macrophages, CD8 positive T cells, and/or regulatory T cells (Treg cells). In any of the above embodiments of the present invention, the individual may be a human.

In other embodiments of the present invention, pharmaceutical preparations containing anti-CD137 antigen binding molecules or antibodies that are intended for use in the methods of treatment described above are used in the treatment, and the drugs may contain an effective amount of an anti-CD137 antigen binding molecule or antibody that has a reduced level of immunological activation in non-tumor tissue compared to an anti-CD137 antigen-binding molecule, which does not have CD137 small-molecule dependent antigen-binding activity.

In one embodiment of the present invention, non-tumor tissues include lymph nodes, spleen and/or liver.

In other embodiments of the present invention, pharmaceutical preparations containing an anti-CD137 antigen-binding molecule or antibody for use in the methods of treatment, treatment, and medicaments described above may contain an effective amount of an anti-CD137 antigen-binding molecule or antibody that does not substantially bind to CD137 expressed in non-tumor tissue.

In other embodiments of the present invention, pharmaceutical preparations containing an anti-CD137 antigen binding molecule or antibody for use in the above-described methods of treatment, use in treatment, wherein the pharmaceutical preparations may contain an effective amount of an anti-CD137 antigen binding molecule or antibody that has an increased half-life in blood compared to an anti-CD137 antigen-binding molecule or antibody that does not have small molecule dependent CD137 binding activity.

In other embodiments of the present invention, pharmaceutical preparations containing an anti-CD137 antigen binding molecule or antibody for use in the above-described methods of treatment, use in treatment, wherein the pharmaceutical preparations may contain an effective amount of an anti-CD137 antigen binding molecule or antibody that has a low level of side effects compared to an anti-CD137 antigen-binding molecule or antibody that does not have small molecule dependent CD137 binding activity.

In other embodiments of the present invention, side effects include increased AST, increased ALT, fever, nausea, acute hepatitis, liver damage, splenomegaly, enteritis, purulent skin inflammation, neutropenia, lymphopenia, thrombopenia, trans expression

- 77 046075 saminase and/or hyperbilirubinemia.

In one embodiment of the present invention, the anti-CD137 antigen-binding molecule of the present invention has low side effects, and thus the dosage can be increased without concern for side effects, so as a result they can exhibit higher drug efficacy (cytotoxic activity or antitumor activity).

In other embodiments, the present invention provides pharmaceutical compositions containing any of the anti-CD137 antigen-binding molecules or antibodies provided in the present invention, for example, for use in any of the above therapeutic methods. In one embodiment of the present invention, the pharmaceutical composition contains any of the anti-CD137 antigen-binding molecules or antibodies provided in the present invention and a pharmaceutically acceptable carrier.

The antigen-binding molecule or antibody of the present invention can be administered by any suitable route, including parenteral, intrapulmonary and intranasal, and, if local treatment is necessary, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be accomplished by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. The present invention contemplates various dosing regimens, including, but not limited to, single or multiple administration at different times, bolus administration and pulse infusion.

The antibodies of the present invention can be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration and other factors known to the practitioner. The antibody is optional, but may be formulated with one or more agents that are currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. They are generally used in the same dosages and administered by the methods described in the present invention, or from about 1 to 99% of the dosages described in the present invention, or in any dosage and in any manner that is empirically/clinically determined to be applicable.

For the prevention or treatment of a disease, the appropriate dose of an antibody of the present invention may depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylaxis or therapy, previous therapy, the patient's medical history and response. for the antibody, as well as at the discretion of the attending physician. The antibody is usually administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of antibody may be the starting dose of the candidate antibody to be administered to the patient, e.g., as one or several separate administrations, or by continuous infusion. One typical daily dose can range from about 1 microgram/kg to 100 mg/kg or more, depending on the factors mentioned above. With repeated administrations over several days or longer, depending on the condition, treatment will usually continue until the desired suppression of disease symptoms occurs. One exemplary antibody dosage may range from about 0.05 mg/kg to about 10 mg/kg. Thus, the patient may be administered one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof). Such doses may be administered intermittently, for example, every week or every three weeks (eg, when the patient receives from about two to about twenty, or for example, about six doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of this therapy is easily monitored using conventional methods and assays.

It will be appreciated that any of the above formulations or therapeutic methods can be carried out using an immunoconjugate as described herein instead of or in addition to an anti-CD137 antigen-binding molecule or antibody.

3. Received products.

Another aspect of the present disclosure provides for the provision of an article containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above. The article includes a container and an associated label or package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. Containers can be made from various materials such as glass or plastic. The container contains the composition itself and may have a sterile access port (for example, the container may be an intravenous solution bag or vial

- 78 046075 no. with a stopper that can be pierced with a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. The product in this embodiment of the present invention may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively or additionally, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials commercially and user-desirable, including other buffers, diluents, filters, needles and syringes.

The term package insert is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information regarding the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

It is understood that any of the above industrial products may include an immunoconjugate of the present invention instead of or in addition to an anti-CD137 antigen-binding molecule or antibody.

III. Compositions and methods (agonistic antigen-binding molecules and cytotoxic antigen-binding molecules containing a variant Fc region with an increased isoelectric point (pI)).

In one embodiment, the present invention provides agonistic antigen-binding molecules or antibodies containing an increased isoelectric point (pI) variant Fc region and methods of using them. In some embodiments of the present invention, polypeptides comprising a variant Fc region with an increased isoelectric point (pI) contain at least one amino acid change in the original Fc region. In other embodiments of the present invention, each such amino acid change causes an increase in the isoelectric point (pI) of the variant Fc region compared to the original Fc region. Without being bound by any theory, the inventors propose that the pH of biological fluids (eg, plasma) is in the neutral pH range. In biological fluids, the net positive charge of an antigen-binding molecule or antibody with increased pI increases due to the increase in pI, and as a result, the antigen-binding molecule or antibody is more strongly attracted by the physicochemical Coulomb interaction to the surface of the endothelial cell, which has a net negative charge compared to the antigen-binding molecule or an antibody that does not have an increased pI. Thus, agonistic antigen-binding molecules (or antibodies) or antigen-binding agonistic antigen-binding molecules (or antibodies) can approach the surface of cells that express the Fc-gamma receptor, resulting in increased binding of the antigen-binding molecules or antibodies to the cells expressing the Fc-gamma receptor. For those agonistic antigen-binding molecules or antibodies that exhibit agonistic activity due to binding activity to the Fc-gamma receptor, agonistic antigen-binding molecules or antibodies having increased binding to cells expressing the Fcgamma receptor due to pI-increasing amino acid changes may exhibit more potent agonistic activity. activity compared to agonistic antigen-binding molecules or antibodies that do not have amino acid changes that increase pI. In one embodiment of the present invention, the agonistic antigen binding molecules are anti-CD137 antigen binding molecules or anti-CD3 antigen binding molecules. In another embodiment of the present invention, the agonistic antigen binding molecules are anti-CD137 antibodies or anti-CD3 antibodies.

In another embodiment, the present invention provides cytotoxic antigen-binding molecules or antibodies containing an increased isoelectric point (pI) variant Fc region and methods for using them. In biological fluids, the net positive charge of an antigen-binding molecule or antibody with an increased pI increases due to the increase in pI, and as a result, the antigen-binding molecule or antibody is more strongly attracted by the physicochemical Coulomb interaction to the surface of the endothelial cell, which has a net negative charge compared to the antigen-binding molecule or antibody, without increased pI. Thus, cytotoxic antigen-binding molecules (or antibodies) or antigen-bound cytotoxic antigen-binding molecules (or antibodies) can approach the surface of cells that express the Fc-gamma receptor, resulting in increased binding of the antigen-binding molecules or antibodies to cells expressing the Fc-gamma receptor. gamma. For those cytotoxic antigen-binding molecules or antibodies that exhibit cytotoxic activity due to binding activity to the Fc-gamma receptor, cytotoxic antigen-binding molecules or antibodies having increased binding due to increased pI to cells expressing the Fc-gamma receptor, amino acid changes may exhibit stronger ago

- 79 046075 nistic activity compared to cytotoxic antigen-binding molecules or antibodies that do not have amino acid changes that increase pI.

In one embodiment of the present invention, the cytotoxicity exhibited by the cytotoxic antigen-binding molecules includes, for example, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), which are caused by effector cells.

In the present invention, pI can be determined theoretically or experimentally. The pI value can be determined, for example, using isoelectric focusing, known to those skilled in the art. Theoretically, the pI value can be calculated, for example, using software for gene and amino acid sequence analysis (Genetyx, etc.). In the present invention, pI can be either theoretical or experimentally determined. The pI value can be determined, for example, using isoelectric focusing, known to those skilled in the art. The theoretical pI value can be calculated, for example, using gene and amino acid sequence analysis software (Genetyx, etc.). When calculating, the properties of the antibody can be reflected in the calculation formula. For example, (i) typically, Cys conserved in an antibody forms a disulfide bond and carries no electrical charge in the side chain; therefore, such Cys can be excluded from the calculation, and only Cys in the free form that does not form a disulfide bond can be included in the calculation. Alternatively, (ii) the charge state or isoelectric point of the antibodies may be changed due to post-translational modifications; therefore, the calculation formula can be modified to take into account the following post-translational modifications: (a) when the N-terminus of the heavy chain is Q (glutamine), the N-terminal amino group is excluded from the calculation, assuming that pyroglutamylation occurs, (b) when the C-terminus of the heavy chain is K (lysine), K (one residue only) is excluded from the calculation, assuming truncation occurs; (c) the side chains of all C (cysteines) present in normally conserved positions are excluded from the calculation, assuming that all of these Cs form disulfide bonds within the molecule. In one preferred embodiment of the present invention, both points (i) and (ii) described above can be reflected in the calculation formula.

In one embodiment of the present invention, the pI value may be increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more by at least 0.6, 0.7, 0.8, 0.9, or more by at least 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2, 5, 3.0 or more compared to what it was before the modification.

In some embodiments of the present invention, the pI-increasing amino acid may be exposed on the surface of the variant Fc region. In the present invention, an amino acid that can be exposed on a surface generally refers to an amino acid residue located on the surface of a polypeptide constituting a variant Fc region. An amino acid residue located on the surface of a polypeptide refers to an amino acid residue whose side chain can be in contact with solvent molecules (which are typically water molecules). However, the side chain need not be completely in contact with the solvent molecules, and when even part of the side chain is in contact with the solvent molecules, the amino acid is defined as the amino acid residue located on the surface. Amino acid residues located on the surface of a polypeptide also include amino acid residues located close to the surface, and thus may be influenced by the electrical charge from another amino acid residue whose side chain, even partially, is in contact with the solvent molecules. Those skilled in the art can generate a homology model of the polypeptide, for example, using commercially available software. Alternatively, methods known to those skilled in the art, such as X-ray crystallography, can be used. Amino acid residues that can be detected on the surface are determined, for example, using the coordinates of a three-dimensional model and a computer program such as InsightII (Accelrys). Open regions can be determined using algorithms known in the art (eg, Lee and Richards (J. Mol. Biol. 55, 1971, 379-400); Connolly (J. Appl. Cryst. 16, 1983, 548-558 )). The affected areas on the surface can be identified using protein modeling software to obtain 3D structural information. Software available for such purposes includes, for example, SYBYL Biopolymer Module software (Tripos Associates). When the algorithm requires a user input size parameter, the probe size used in the calculation can be set to approximately 1.4 Angstroms (A) or less in radius. In addition, Pacios has described methods for determining exposed surface areas using personal computer software (Comput. Chem. 18(4), 1994, 377-386; J. Mol. Model. 1, 1995, 46-53). Based on such information, as described above, appropriate amino acid residues located on the surface of the polypeptide that constitutes the variant Fc region can be selected.

Methods for increasing the pI by introducing a single amino acid substitution in the constant region of an antibody are not particularly limited and, for example, can be implemented based on the methods described in WO 2014/145159. As amino acid substitutions for introduction into the constant region, we prefer

- 80 046075 it is advisable to introduce amino acid substitutions (substitution) to reduce the number of amino acids having a negative charge (for example, aspartic acid or glutamic acid), while increasing the number of amino acids having a positive charge (for example, arginine or lysine), as in the case of the variable region .

Although there is no particular limitation, sites at which amino acid side chains can be exposed on the surface of the antibody molecule are preferred sites for introducing amino acid substitutions into the constant region. Preferred examples include methods for introducing multiple amino acid substitutions in combination at sites that may be exposed on the surface of an antibody molecule. In another embodiment, it is preferred that the plurality of amino acid substitutions made are at sites proximal to each other in the three-dimensional structure. The multiple amino acid substitutions that are introduced are preferably positively charged amino acids, which may in some cases result in a condition where multiple positive charges are transferred to the proximal portions of the three-dimensional structure, although they are not limited to this. The definition of proximal sites in a three-dimensional structure in the present invention is not strictly limited, but, for example, it may mean a state in which a single amino acid substitution or multiple amino acid substitutions are introduced at a distance from each other of 45 angstroms or less, 40 angstroms or less, 30 angstroms or less, 20 Angstroms or less, preferably 15 Angstroms or less, or more preferably 10 Angstroms or less. Determining whether amino acid substitutions of interest are located at sites exposed on the surface of the antibody molecule or whether multiple amino acid substitution sites are located proximally can be determined by known methods such as X-ray crystallography.

In one embodiment of the present invention, variant Fc regions with increased pI contain at least one position selected from the group consisting of positions 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, EU numbering. In other embodiments of the present invention, variants of the Fc regions with increased pI contain Arg or Lys at each selected position.

In other embodiments of the present invention, the increased pI Fc region variants contain at least one amino acid change at at least one position selected from the group consisting of EU numbering positions 311, 343, and 413. In other embodiments of the present invention, variants of the Fc regions with increased pI contain an amino acid change at positions 311, 343, or 413 according to the EU numbering. In other embodiments of the present invention, variants of the Fc regions with increased pI contain Arg or Lys at each selected position.

In another embodiment, the present invention provides polypeptides containing an increased pI variant Fc region that includes amino acid changes at any of the following (1)-(3): (1) at positions 311 and 343; (2) in positions 311 and 413; (3) in positions 343 and 413 according to EU numbering. In other embodiments of the present invention, variants of the Fc regions with increased pI contain Arg or Lys at each selected position.

A method for increasing the pI of a protein is, for example, reducing the amount of amino acids with a negatively charged side chain (eg, aspartic acid and glutamic acid) and/or increasing the amount of amino acids with a positively charged side chain (eg, arginine, lysine and histidine) at neutral pH. Amino acids with a negatively charged side chain have a negative charge, represented by -1, at pH conditions that are significantly higher than the pKa of their side chain, which is a theory well known to those skilled in the art. For example, the theoretical pKa value for the side chain of aspartic acid is 3.9, and the side chain has a negative charge, represented as -1 under neutral pH conditions (e.g., in a pH 7.0 solution). Conversely, amino acids with a positively charged side chain have a positive charge, represented as +1, provided the pH is substantially lower than the pKa of their side chain. For example, the theoretical pKa value for the arginine side chain is 12.5, and the side chain has a positive charge, represented as +1 under neutral pH conditions (e.g., in a pH 7.0 solution). Meanwhile, it is known that amino acids whose side chain has no charge at neutral pH (for example, in a solution with pH 7.0) include 15 types of natural amino acids, that is, alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan and tyrosine. The authors note that amino acids to increase pI may be unnatural amino acids.

Based on the above, a method of increasing the pI of a protein under neutral pH conditions (e.g., in a pH 7.0 solution) can impart a +1 charge change to the protein of interest, for example, by replacing amino acids with uncharged side chains of aspartic acid or glutamic acid (whose side chain has a negative charge -1) in the amino acid sequence of a protein. Additionally, a change in +1 charge can be imparted to a protein, for example by replacing arginine or lysine (whose side chain has a positive +1 charge) with amino acids whose side chain has no charge. Moreover, a +2 charge change can be imparted to a protein at a time by replacing arginine or lysine

- 81 046075 (whose side chain has a positive charge of +1) to aspartic acid or glutamic acid (whose side chain has a negative charge of -1). In another embodiment, to increase the pI of a protein, amino acids with a side chain that does not have a charge and/or amino acids with a positively charged side chain can be added to the amino acid sequence of the protein, or amino acids with a side chain that does not have a positive charge, and/or preferably amino acids with a negatively charged side chain present in the amino acid sequence of a protein can be deleted. It is understood that, for example, the N-terminal and C-terminal amino acid residues of a protein have a charge derived from the main chain (NH3+ amino group at the N-terminus and a COO carbonyl group at the C ^- terminus) in addition to the charges from their side chains. Thus, the pI of a protein can also be increased by performing some addition, deletion, substitution or insertion on the backbone functional groups.

Substituting an amino acid to increase pI includes, for example, replacing an amino acid whose side chain has no charge with an amino acid with a negatively charged side chain, replacing an amino acid with a positively charged side chain with an amino acid whose side chain has no charge, and replacing an amino acid with an uncharged side chain. having a positively charged side chain, to an amino acid with a negatively charged side chain in the amino acid sequence of the parent Fc region, which are performed alone or in appropriate combinations.

The insertion or addition of an amino acid to increase the pI includes, for example, the insertion or addition of an amino acid whose side chain is uncharged and/or the insertion or addition of an amino acid having a positively charged side chain in the amino acid sequence of the parent Fc region, which are performed alone or in appropriate combinations .

Deletion of an amino acid to increase pI includes, for example, deletion of an amino acid having a side chain without a charge and/or deletion of an amino acid having a negatively charged side chain in the amino acid sequence of the parent Fc region, which are performed individually or in appropriate combinations.

In one embodiment of the present invention, natural amino acids used to increase pI can be classified as follows: (a) an amino acid with a negatively charged side chain can be Glu (E) or Asp (D); (b) an amino acid whose side chain has no charge can be Ala (A), Asn (N), Cys (C), Gln (Q), Gly (G), His (H), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) or Val (V); (c) The amino acid with a positively charged side chain can be His (H), Lys (K) or Arg (R). In one embodiment of the present invention, the amino acid insertion or substitution after modification is Lys (K) or Arg (R).

In another embodiment, the present invention provides isolated agonistic antigen binding molecules or antibodies comprising variant Fc region sequences with increased Fc gamma receptor binding activity (preferably Fc gamma RIIb binding activity) and increased isoelectric point pI. In some embodiments of the present invention, a variant Fc region of the present invention contains at least two amino acid changes in the original Fc region. As described above, an antigen binding molecule or antibody having an elevated pI is more strongly attracted by the physicochemical Coulomb interaction to the surface of the endothelial cell, which has an overall negative charge, compared to an antigen binding molecule or antibody that does not have an elevated pI. Therefore, for agonistic antigen-binding molecules or antibodies that exhibit agonistic activity based on the influence of binding activity at the Fc-gamma receptor (preferably Fc-gamma RIIb), it is possible to increase the agonistic activity of the antigen-binding molecules or antibodies by combining amino acid changes to increase the Fc-gamma receptor (preferably Fc-gamma RIIb) and amino acid changes (changes) to increase pI. The term Fc-gamma receptor is defined in Section II. Compositions and methods (agonist antigen-binding molecules against CD137) are used in a similar manner.

In one embodiment, the present invention provides polypeptides comprising Fc region variants with increased FcYRIIb binding activity and increased pI, comprising at least three amino acid changes, including: (a) at least one amino acid change at at least one position, selected from the group consisting of: 231, 232, 233, 234, 235, 236, 237, 238, 239, 264, 266, 267, 268, 271, 295, 298, 325, 326, 327, 328, 330, 331 , 332, 334 and 396, EU numbering, and (b) at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431, EU numbering.

In one embodiment, the present invention provides a polypeptide comprising a variant Fc region that results in increased FcYRIIb binding activity and increased pI, wherein the polypeptide contains at least 3 amino acid changes comprising (a) at least one amino acid change of at least in one position

- 82 046075 selected from the group consisting of positions 234, 235, 236, 237, 238, 264, 268, 295, 326 and 330 according to EU numbering, and (b) at least 2 amino acid mutations in at least 2 positions, selected from the group consisting of provisions 311, 343 and 413 by EU numbering.

In another embodiment, the present invention provides a polypeptide comprising a variant Fc region that results in increased FcγRIIb binding activity and increased pI, comprising at least one amino acid change at any one of positions (1) to (26) (EU numbering) :

(1) provisions 235, 236, 268, 295, 326, 330, 343 and 413;

(2) provisions 214, 235, 236, 268, 295, 326, 330, 343 and 413;

(3) provisions 234, 238, 250, 264, 307, 330, 343 and 413;

(4) provisions 234, 238, 264, 330, 343 and 413;

(5) provisions 234, 237, 238, 250, 307, 330, 343 and 413;

(6) provisions 234, 237, 238, 330, 343 and 413;

(7) provisions 235, 236,268, 295, 326, 330, 311 and 343;

(8) provisions 234, 238, 250, 264, 307, 330, 311 and 343;

(9) provisions 234, 238, 264, 330, 311 and 343;

(10) provisions 234, 237, 238, 250, 307, 330, 311 and 343;

(11) provisions 234, 237, 238, 330, 311 and 343;

(12) provisions 235, 236, 268, 295, 326, 330 and 343;

(13) provisions 214, 235, 236, 268, 295, 326, 330 and 343;

(14) provisions 235, 236, 268, 295, 326, 330 and 413;

(15) provisions 214, 236, 268, 330 and 343;

(16) provisions 214, 235, 236, 268, 330 and 343;

(17) provisions 214, 236, 268, 330 and 413;

(18) provisions 214, 236, 268, 330, 343 and 413;

(19) provisions 214, 235, 236, 268, 330, 343 and 413;

(20) provisions 214, 236, 268, 330 and 311;

(21) provisions 214, 235, 236, 268, 330 and 311;

(22) provisions 214, 236, 268, 330, 311 and 343;

(23) provisions 214, 235, 236, 268, 330, 311 and 343;

(24) provisions 214, 236, 268, 330, 311 and 413;

(25) provisions 214, 235, 236, 268, 330, 311 and 413;

(26) provisions 214, 235, 236, 268, 295, 326, 330 and 311.

In one embodiment of the present invention, a variant Fc region of the present invention includes the amino acid changes presented in Table below. 6.

Table 6

Amino acid changes to increase the pI of the Fc region

Numbers Amino acid changes (by EU numbering) 1 P343R/D413K 2 Q311R/P343R 3 P343R 4 D413K 5 Q311R 6 Q311R/D413K

In one embodiment of the present invention, in addition to the amino acid changes described in table. 6 (amino acid changes that increase Fc pI), the variant Fc region of the present invention includes the amino acid changes described in table. 7 below.

Table 7 Amino acid changes that increase FcγRIIb-binding activity of the Fc region

Numbers Amino acid changes (by EU numbering) 1 L23 4Y/P23 8D/V264I/AZ 3 0K 2 L234Y/P238D/A330K 3 L23 4Y/G23 7D/P23 8D/A3 3 0K 4 G236N/H268D/A330K 5 L235W/G236N/H268D/Q295L/K326T/A330K

- 83 046075

In one embodiment of the present invention, the variant Fc region of the present invention includes the amino acid changes described in table. 8 below. In a preferred embodiment of the present invention, the variant Fc region of the present invention includes an amino acid deletion at EU numbering position 447 in addition to the amino acid changes described in Table below. 8. In another preferred embodiment of the present invention, the variant Fc region of the present invention includes amino acid deletions at EU numbering positions 446 and 447 in addition to the amino acid changes described in Table below. 8.

Table 8

No Amino acid substitution (EU numbering) 1 L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D413K 2 K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R/D41ЗК 3 L234Y/P238D/T250V/V264I/T307P/A330K/P343R/D413K 4 L234Y/P238D/V264I/A330K/P343R/D413K 5 L234Y/G237D/P238D/T250V/T307P/A330K/P343R/D413K 6 L234Y/G237D/P238D/A330K/P343R/D413K 7 L235W/G236N/H268D/Q295L/K326T/A330K/Q311R/P343R 8 L234Y/P238D/T250V/V264I/T307P/A330K/Q311R/P343R 9 L234Y/P238D/V264I/A330K/Q311R/P343R 10 L234Y/G237D/P238D/T250V/T307P/A330K/Q311R/P343R eleven L234Y/G237D/P238D/A330K/Q311R/P343R 12 L235W/G236N/H268D/Q295L/K326T/A330K/P343R 13 K214R/L235W/G236N/H268D/Q295L/K326T/A330K/P343R 14 L235W/G236N/H268D/Q295L/K326T/A330K/D413K 15 K214R/G236N/H268D/A330K/P343R 16 K214R/L235W/G236N/H268D/A330K/P343R 17 К214R/G236N/H268D/A330K/D41 ЗК 18 K214R/G236N/H268D/A330K/P343R/D413K 19 K214R/L235W/G236N/H268D/A330K/P343R/D41 ZK 20 K214R/G236N/H268D/A330K/Q311R 21 K214R/L235W/G236N/H268D/A330K/Q311R 22 K214R/G236N/H268D/A330K/Q311R/P343R 23 K214R/L235W/G236N/H268D/A330K/Q311R/P343R 24 К214R/G236N/H268D/A330K/Q311R/D41 ЗК 25 K214R/L235W/G236N/H268D/A330K/Q311R/D41 ZK 26 K214R/L235W/G236N/H268D/Q295L/K326T/A330K/Q311R

In addition to the changes exemplified above, those skilled in the art will appreciate that at least one amino acid change that increases FcyR binding activity, including FcyRIIb, compared to the original Fc region, for example, as described or proposed in WO 2013/ 047752, WO 2013/125667, WO 2014/030728, WO 2014/163101 or WO 2017104783, at least one amino acid change that increases the pI compared to the original Fc region can be used, for example as described or proposed in WO 2017/104783 or WO 2017/046994, and combinations of these amino acid changes.

In one embodiment of the present invention, the agonistic antigen binding molecule or antibody comprises both a variant Fc region and an antigen binding domain. In another embodiment of the present invention, the antigen is a membrane antigen. In another embodiment of the present invention, the antigen is a receptor expressed on the surface of a cell. In some embodiments of the present invention, the parent Fc region is derived from human IgG1. In another embodiment of the present invention, the polypeptide is an antibody. In another embodiment of the present invention, the polypeptide is an Fc fusion protein.

- 84 046075

In one embodiment, the present invention provides a method for producing an agonistic antigen-binding molecule that includes a variant Fc region (an increased isoelectric point (pI) variant Fc region) as described in detail above.

In one embodiment of the present invention, the method of the present invention has the following features: introducing into the parent Fc region at least one amino acid change (as described above) that results in an increase in the isoelectric point (pI) compared to the isoelectric point (pI) of the parent an agonistic antigen-binding molecule containing a parent Fc region; identifying and isolating an agonistic antigen binding molecule in which the agonistic activity of the agonistic antigen binding molecule including the Fc region variant resulting from the above introduction is increased compared to that of the original agonistic antigen binding molecule.

In one embodiment of the present invention, the method of the present invention has the features of obtaining an expression vector containing a suitable promoter operably linked to a gene encoding the above-mentioned agonistic antigen-binding molecule identified and isolated as above, introducing the vector into a host cell, culturing the host cells for producing an agonistic antigen-binding molecule and isolating said agonistic antigen-binding molecule from the host cell culture.

In one embodiment of the present invention, the method of the present invention produces an agonistic antigen-binding molecule whose agonistic activity against an antigen in the presence of 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, or 250 μM low molecular weight compound is 2-fold or more greater. , 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more, the agonistic activity against the antigen in the absence of the small molecule compound.

In one embodiment of the present invention, the method of the present invention can produce an agonistic antigen-binding molecule whose agonistic activity against an antigen in the presence of 10 μM or more of a low molecular weight compound is 2-fold or more, 3-fold or more, 5-fold or more higher. , 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more compared with agonistic activity towards the antigen in the absence of a low molecular weight compound.

In one embodiment of the present invention, the method of the present invention can produce an agonistic antigen-binding molecule whose agonistic activity against an antigen in the presence of 50 μM or more of a low molecular weight compound is 2-fold or more, 3-fold or more, 5-fold or more higher. , 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more compared with agonistic activity towards the antigen in the absence of a low molecular weight compound.

In one embodiment of the present invention, the method of the present invention can produce an agonistic antigen-binding molecule whose agonistic activity towards an antigen in the presence of 250 μM or more of a low molecular weight compound is 2-fold or more, 3-fold or more, 5-fold or more higher , 10 times or more, 20 times or more, 30 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, or 90 times or more compared with agonistic activity towards the antigen in the absence of a low molecular weight compound.

In one embodiment of the present invention, in such a method, the agonistic activity of an agonistic antigen-binding molecule towards an antigen can be assessed by the amount of IL-2 and/or IFN-γ produced by the antigen-expressing cells.

In one embodiment of the present invention, in such a method, the agonistic activity of an agonistic antigen-binding molecule towards an antigen can be assessed by the amount of IL-2 and/or IFN-γ produced by isolated human peripheral blood mononuclear cells (PBMCs) or T cells derived from from human peripheral blood mononuclear cells (PBMCs).

In one embodiment of the present invention, in such a method, the agonistic activity of an agonistic antigen-binding molecule towards an antigen can be assessed by reporter gene assay.

IV. Compositions and methods (antigen-binding molecules, the binding activity of which to antigens varies depending on the concentration of the low molecular weight compound).

A. Antigen-binding molecules, the binding activity of which to antigens varies depending on the concentration of the low molecular weight compound.

In one embodiment, the present invention provides antigen binding

- 85 046075 molecules whose antigen-binding activity varies depending on the concentration of the low-molecular compound. The molecules may also be referred to as small molecule dependent antigen binding molecules (having antigen binding activity dependent on the concentration of the small molecule compound). In some embodiments of the present invention, the antigen binding molecules are antibodies. In some embodiments, the antigen binding molecules of the present invention have antigen binding activity that increases as the concentration of the small molecule compound becomes higher. In some embodiments, the antigen binding molecules of the present invention have antigen binding activity that decreases as the concentration of the small molecule compound becomes higher. In one embodiment of the present invention, when the binding activity of an antigen binding molecule in the presence of a small molecule compound is compared with the binding activity of an antigen binding molecule in the absence of a small molecule compound, one of the two values is higher than the other value. In another embodiment of the present invention, when the binding activity of an antigen binding molecule in the presence of a high concentration of a small molecule compound is compared with the binding activity of an antigen binding molecule in the presence of a low concentration of a small molecule compound, one of the two values is higher than the other value. In some embodiments of the present invention, the small molecule compound of the present invention is a target tissue-specific compound. In other embodiments of the present invention, the small molecule compound of the present invention is a tumor tissue-specific compound.

In another embodiment, the present invention provides antigen-binding molecules having high plasma retention properties. In another embodiment, the present invention provides antigen binding molecules having antigen binding activity that increases as the concentration of the small molecule compound increases. In some embodiments of the present invention, the small molecule compound is a tissue-specific target compound. In other embodiments of the present invention, the antigen binding molecules have increased antigen binding activity in the target tissue compared to antigen binding activity in the non-target tissue. In one embodiment of the present invention, the antigen binding molecules are antibodies. Without being limited to any particular theory, the change in plasma kinetics described above can be explained as follows. When the antigen-binding activity of an antigen-binding molecule is high depending on the concentration of the target tissue-specific compound, the antigen-binding activity of the antigen-binding molecule in tissues other than the target tissue will be lower. As a result, the antigen-dependent elimination (clearance) of this antigen-binding molecule in tissues other than the target tissue will also become lower. Lower antigen-dependent elimination (clearance) in most tissues of the living body (that is, in tissues other than the target tissue) generally results in an increased ability to retain the antigen-binding molecule in plasma. Determination of whether the antigen binding molecule of the present invention has a high plasma retention capacity can be made by comparison with a control antigen binding molecule. In some embodiments of the present invention, antigen-binding molecules that have increased antigen-binding activity when the concentration of the target tissue-specific compound becomes higher have a higher plasma retention capacity compared to a control antigen-binding molecule. In one embodiment of the present invention, the control antigen-binding molecule does not have antigen-binding activity that is dependent on the concentration of the small molecule compound. In some embodiments of the present invention, an antigen binding molecule that does not have small molecule concentration dependent antigen binding activity is an antigen binding molecule for which the difference in antigen binding activity in the presence of the small molecule compound and in the absence of the small molecule compound is a factor of 2 or less than 1. 8 times or less, 1.5 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. For comparison, it is preferred that the antigen binding activity of the antigen binding molecules of the present invention and the control antigen binding molecules in the presence of a sufficient amount of the small molecule compound is substantially the same.

The present inventors believe that the amount of antigen-dependent elimination of antigen-binding molecule detected in vivo varies according to the quantitative balance of antigens and antigen-binding molecules present in the plasma. In general, it is believed that the more antigens are present in the plasma/the fewer antigen-binding molecules are present in the plasma, the more detectable the antigen-dependent elimination of the antigen-binding molecule becomes. On the contrary, it is believed that the fewer antigens are present in the plasma/the more antigen-binding molecules are present in the plasma, the less detectable antigen-dependent elimination becomes.

- 86 046075 tion of antigen-binding molecule. The antigen-binding molecules of the present invention do not necessarily have to exhibit high plasma retention under all conditions, but it is sufficient that the antigen-binding molecules exhibit high plasma retention under suitable conditions when sufficient antigen-dependent elimination is detected. If the amount of antigen in the plasma is too small, plasma retention capacity can be assessed after increasing the amount of antigen by any artificial means.

In another embodiment, the present invention provides antigen-binding molecules having a low ability to accumulate antigen in plasma. In another embodiment of the present invention, antigen binding molecules have antigen binding activity that increases as the concentration of the small molecule compound becomes higher. In some embodiments of the present invention, the small molecule compound is a target tissue-specific compound. In other embodiments of the present invention, the antigen binding molecules have increased antigen binding activity in the target tissue compared to antigen binding activity in the non-target tissue. In one embodiment of the present invention, the antigen binding molecules are antibodies. Without being limited to any particular theory, the change in plasma kinetics described above can be understood as follows. When the antigen-binding activity of an antigen-binding molecule is high depending on the concentration of the target tissue-specific compound, the antigen-binding activity of the antigen-binding molecule in tissues other than the target tissue will be lower. As a result, this antigen-binding molecule will have a reduced ability to form an antigen-antibody complex in tissues other than the target tissue. In general, it is known that when an antigen is bound by an antigen-binding molecule, such as an antibody, the clearance of the antigen becomes lower and the concentration of the antigen in the plasma increases (the antigen accumulates). The reduced ability to form an antigen-antibody complex in most tissues of a living organism (that is, in tissues other than the target tissue) generally results in reduced accumulation of antigen (in other words, a reduced ability of the antigen-binding molecule to accumulate antigen). Determination of whether the antigen binding molecule of the present invention has a reduced ability to accumulate antigen in plasma can be made by comparison with a control antigen binding molecule. In some embodiments of the present invention, antigen-binding molecules that exhibit increased antigen-binding activity when the concentration of the target tissue-specific compound becomes higher have a reduced ability to accumulate antigen in plasma compared to a control antigen-binding molecule. In one embodiment of the present invention, the control antigen-binding molecule does not have antigen-binding activity that is dependent on the concentration of the small molecule compound. In some embodiments of the present invention, an antigen binding molecule that does not have small molecule concentration dependent antigen binding activity is an antigen binding molecule for which the difference in antigen binding activity in the presence of the small molecule compound and in the absence of the small molecule compound is different, for example, by a factor of 2 or less, 1.8 times or less, 1.5 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. When compared, it is preferred that the antigen binding activity of the antigen binding molecules of the present invention and the control antigen binding molecules in the presence of a sufficient amount of the compound are substantially the same.

In the present invention, we note that the amount of antigen-antibody complex formed in vivo depends on the amount of antigen and antigen-binding molecule present in the plasma. It is generally believed that the more antigens/antibodies present in the plasma, the more antigen-antibody complexes are formed. Conversely, it is believed that the fewer antigens/antibodies present in the plasma, the fewer antigen-antibody complexes are formed. The antigen-binding molecules of the present invention do not necessarily need to exhibit a low ability to accumulate antigen in plasma under all conditions, but it is sufficient that the antigen-binding molecules exhibit a low ability to accumulate antigen in plasma under suitable conditions when a sufficient amount of antigen-antibody complex is formed. If the amount of antigen in the plasma is too small, the ability to accumulate antigen in the plasma can be assessed after increasing the amount of antigen by any artificial methods.

In some embodiments of the present invention, the difference in antigen binding activity depending on the concentration of the small molecule compound of the antigen binding molecules of the present invention is greater, such as two times or more, three times or more, five times or more, ten times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1x 10 ³ times or more, 2x10 ³ times or more, 3x10 ³ times or more, 5x10 ³ times or more, 1 x 10 ⁴ times or more, 2x10 ⁴ times or more, 3x 10 ⁴ times or more, 5x 10 ⁴ times or more than or 1x 10 ⁵ times or more.

- 87 046075

In some embodiments of the present invention, the binding activity of antigen binding molecules can be expressed by a KD (dissociation constant) value. In other embodiments of the present invention, when the KD value for an antigen binding molecule in the presence of a small molecule compound is compared with the KD value for an antigen binding molecule in the absence of a small molecule compound, one value is less than the other value. In another embodiment of the present invention, if the KD value of an antigen binding molecule in the presence of a high concentration of a small molecule compound is compared with the KD value of an antigen binding molecule in the presence of a low concentration of a small molecule compound, one value is less than the other value. In other embodiments of the present invention, the difference in KD values of the antigen-binding molecules is greater, for example, two times or more, three times or more, five times or more, 10 times or more, 20 times or more, 30 times or more more, 50 times or more, 100 times or more, times or more, 300 times or more, 500 times or more, 1x 10 ³ times or more, 2x 103 times or more, 3x 10 ³ times or more, 5x103 times or more, ^1x104 times or more, 2x104 times or more, ^3x104 times or more, ^{5x104 times} or more, or ^1x105 times or more. The KD value may be less, for example, 9x10 ^-7 M or less, 8x10 ^-7 M or less, 7x10 ^-7 M or less, 6x10 -7 M or less, ^{5x10 -7 M} or less, 4x10 ^-7 M or less, 3x10 ^-7 M or less, 2x10 ^-7 M or less, 1x 10 ^-7 M or less, 9x10 ^-8 M or less, 8x10 -8 M or less, ^{7x10 -8 M} or less, 6x10 ^-8 M or less, 5x10 ^-8 M or less, 4x10 ^-8 M or less, 3x10 ^-8 M or less, 2x10 ^-8 M or less, 1x 10 ^-8 M or less, 9x10 ^-9 M or less, 8x10 ^-9 M or less, 7x10 ^-9 M or less, 6x10 ^-9 M or less, 5x10 ^-9 M or less, 4x10 ^-9 M or less, 3x10 -9 M or less, ^{2x10 -9 M} or less, 1x 10 ^-9 M or less, 9x10 ^-10 M or less, 8x10 ^-10 M or less, 7x10 ^-10 M or less, 6x10 -10 M or less, 5x10 ^-10 M or less, ^{4x10 -10 M} or less, 3x10 ^-10 M or less, 2x 10 ^-10 M or less, 1 x 10 ^-10 M or less. The KD value may be greater, for example, 1 x 10 ^-8 M or more, 2 x 10 ^-8 M or more, 3 x 10 ^-8 M or more, 4 x 10 ^-8 M or more, 5 x 10 ^-8 M or more, 6 x 10 ^-8 M or more, 7x 10 ^-8 M or more, 8x 10 ^-8 M or more, 9x 10 ^-8 M or more, 1 x 10 ^-7 M or more, 2x 10 ^-7 M or more, 3x 10 ^-7 M or more , 4x 10 ^-7 M or more, 5x 10 ^-7 M or more, 6x 10 ^-7 M or more, 7x 10 ^-7 M or more, 8x 10 ^-7 M or more, 9x 10 ^-7 M or more, 1 x 10 ^-6 M or more, 2x 10 ^-6 M or more, 3x 10 ^-6 M or more, 4x10 ^-6 M or more, 5x10 ^-6 M or more, 6x10 ^-6 M or more, 7x10 ^-6 M or more, 8x10 ^-6 M or more, 9x 10 ^-6 M or more.

In another embodiment of the present invention, the binding activity of antigen binding molecules can be expressed by a kd value (dissociation rate constant) instead of a KD value.

In another embodiment of the present invention, the binding activity of antigen binding molecules can be expressed by the amount of antigen that has bound to the antigen binding molecule. For example, by surface plasmon resonance techniques, the binding amount of an antigen-binding molecule immobilized on a sensor chip and the binding amount of antigen that binds thereto are measured as a resonance unit (RU). Antigen-binding activity can be expressed using the bound amount of antigen derived from it as an indicator, or alternatively can be expressed using a value obtained by dividing the amount of antigen bound by the amount of antigen-binding molecule bound (i.e., the amount of antigen bound by unit amount of antigen-binding molecule) as an indicator. Specific methods for measuring and calculating the amount of binding are described in the examples below. In some embodiments of the present invention, if the amount of bound antigen in the presence of a small molecule compound is compared with the amount of bound antigen in the absence of a small molecule compound, one of the values is greater than the other. In another embodiment of the present invention, if the amount of bound antigen in the presence of a high concentration of a small molecule compound is compared with the amount of bound antigen in the presence of a low concentration of a small molecule compound, or one value is greater than the other value. In other embodiments of the present invention, the difference in the amount of antigen bound is greater, for example, two times or more, three times or more, five times or more, 10 times or more, 20 times or more, 30 times or more , 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1x 10 ³ times or more, 2x 10 ³ times or more, x 10 ³ times or more, 5x 10 ³ times or more, 1x 10 ⁴ times or more, 2x 10 ⁴ times or more, 3x 10 4 times or more, 5x 10 ^{4 times} or more, or 1x 10 ⁵ times or more. The amount of higher antigen bound may be, for example, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1 or more. The amount of lower antigen bound may be, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less than, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, 0.001 or less.

- 88 046075

In some embodiments of the present invention, KD values, kd values, or associated quantity values provided in the present invention are measured or calculated by performing surface plasmon resonance analysis at 25°C or 37°C (for example, see examples in the present invention).

The concentration of the small molecule compound may be arbitrarily selected as long as a difference in binding activity to the antigen binding molecule can be detected. In some embodiments of the present invention, a high concentration includes, for example, 1 nM or higher concentration, 3 nM or higher concentration, 10 nM or higher concentration, 30 nM or higher concentration, 100 nM or higher concentration, 300 nM or higher concentration, 1 µM or higher concentration, 3 µM or higher concentration, 10 µM or higher concentration, 30 µM or higher concentration, 100 µM or higher concentration, 300 µM or higher concentration, 1 mM or more high concentration, 3 mM or higher concentration, 10 mM or higher concentration, 30 mM or higher concentration, 100 mM or higher concentration, 300 mM or higher concentration, 1 M or higher concentration. In another embodiment of the present invention, the high concentration may be a concentration that provides a sufficient amount to allow each antigen binding molecule to exhibit maximum binding activity. In one embodiment of the present invention, 1 micromolar, 10 µM, 100 µM, 1 mM, or an amount sufficient for each antigen binding molecule to exhibit maximum binding activity may be selected as the high concentration in the present invention. In some embodiments of the present invention, the low concentration includes, for example, 1 mM or lower concentration, 300 μM or lower concentration, 100 μM or lower concentration, 30 μM or lower concentration, 10 μM or lower concentration, 3 μM or lower concentration, 1 µM or lower concentration, 300 nM or lower concentration, 100 nM or lower concentration, 30 nM or lower concentration, 10 nM or lower concentration, 3 nM or lower concentration, 1 nM or more low concentration, 300 pM or lower concentration, 100 pM or lower concentration, 30 pM or lower concentration, 10 pM or lower concentration, 3 pM or lower concentration, 1 pM or lower concentration, etc. Alternatively, the low concentration in the present invention may be the concentration at which each antigen binding molecule exhibits minimal binding activity. The case of virtually zero concentration (i.e., no low molecular weight compound) can be selected as one of the low concentration options. In one embodiment of the present invention, a concentration of 1 mM, 100 μM, 10 μM, 1 μM is a concentration at which each antigen binding molecule of the present invention exhibits minimal binding activity, or the absence of a small molecule compound may be selected as the low concentration in the present invention . In another embodiment of the present invention, the ratio of high concentration to low concentration may be selected, for example, three times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1x10 ³ times the value or more, 1x10 ⁴ times the value or more, 3x10 ⁴ times the value or more, 1x10 ⁵ times the value or more, 3x10 ⁵ times the value or more, 1x10 ⁶ times the value or more, 3x10 ⁶ -fold the value or more, 1x10 ⁷ times the value or more, 3x10 ⁷ times the value or more, 1x 108 times the value or more, 3x10 ⁸ times the value or more, 1x10 ⁹ times the value or more, 3x10 ⁹ times the value or more, 1x10 ¹⁰ times or more, 3x10 ¹⁰ times or more, 1x10 ¹¹ times or more, 3x 10 times or more, 1x10 ¹² times or more.

In another embodiment of the present invention, the antigen-binding molecules of the present invention exhibit cytotoxicity against cells expressing their antigens. When a target cell expresses an antigen on its surface and the antigen-binding molecule of the present invention binds to it, the cell may be damaged. The cellular damage may be that caused by effector cells, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and there may be complement-induced damage such as, for example, complement-dependent cytotoxicity (CDC, complementdependent cytotoxicity). In another embodiment, the damage may be such as damage caused by a cytotoxic agent (eg, radioisotopes or chemotherapeutic agents) as immunoconjugates. Cytotoxicity in the present invention may include actions to induce cell death, actions to inhibit cell proliferation, actions to damage cell functions, etc. The cytotoxicity of the present invention may include death inducing actions

- 89 046075 cells, actions to suppress cell proliferation, actions to damage cell functions, etc. In the presence of a sufficient amount of the antigen binding molecule of the present invention, damage may be caused by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more , 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90%, or more than 95% or more of the cells expressing the antigen. The measurement of such cytotoxic activity can be performed as a comparison with a measurement in the absence of an antigen binding molecule of the present invention or in the presence of a negative control antigen binding molecule. The present invention provides examples of cytotoxicity assays.

In another embodiment of the present invention, the antigen-binding molecules of the present invention exhibit neutralizing activity against their antigens. Neutralizing activity means the activity of neutralizing (or inhibiting, blocking) biological activity associated with antigens. In one embodiment of the present invention, the biological activity is provided by binding between the ligand and the receptor. In some embodiments of the present invention, the antigen binding molecules of the present invention inhibit such binding between a ligand and a receptor. In the presence of a sufficient amount of the antigen binding molecule of the present invention, the biological activity can be inhibited, for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35%. or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The measurement of such inhibitory activity can be performed as a comparison with a measurement in the absence of an antigen binding molecule of the present invention or in the presence of a negative control antigen binding molecule. The present invention provides specific methods for measuring neutralizing activity.

In another embodiment of the present invention, the antigen binding molecules of the present invention include an Fc region. In other embodiments of the present invention, the antigen binding molecules of the present invention include a constant region. The constant region may be a heavy chain constant region (including the Fc region), a light chain constant region, or both. In some embodiments of the present invention, the Fc region is a native sequence region. Examples of a heavy chain constant region derived from native sequence antibodies include, for example, human IgG1 heavy chain constant region (SEQ ID NO: 219), human IgG2 (SEQ ID NO: 220), human IgG3 (SEQ ID NO: 221) , human IgG4 (SEQ ID NO: 222), etc. Other examples of the heavy chain constant region include heavy chain constant regions such as SEQ ID NO: 215, SEQ ID NO: 217. Examples of the light chain constant region derived from native sequence antibodies include, for example, kappa chain light chain constant regions person. (SEQ ID NO: 186, SEQ ID NO: 199, SEQ ID NO: 218), human lambda circuit (SEQ ID NO: 189, SEQ ID NO: 216), etc.

In another embodiment of the present invention, the Fc region may be a variant Fc region obtained by introducing amino acid change(s) into the Fc region with a native sequence. In some embodiments of the present invention, the Fc region variant has increased binding activity to at least one Fc gamma receptor selected from the group consisting of Fc gamma RIa, Fc gamma RIIa, Fc gamma RIIb, and Fc gamma RIIIa, compared to the Fc region with the native sequence.

In other embodiments of the present invention, the antigen binding molecules of the present invention are antibodies. In some embodiments of the present invention, the antibodies are monoclonal antibodies, including chimeric antibodies, humanized antibodies, or human antibodies. In one embodiment of the present invention, the antibodies are antibody fragments, for example, Fv, Fab, Fab', scFv, diabodies or F(ab') ₂ fragments, etc. In another embodiment of the present invention, the antibodies are full-length antibodies, for example, intact IgG1 antibodies, intact IgG4 antibodies, or another class or isotype of antibodies as described in the present invention.

In another embodiment of the present invention, the antigen-binding molecules of the present invention form a ternary complex together with the small molecule compound and the antigen. In some embodiments of the present invention, the antigen binding molecules are antibodies. In one embodiment of the present invention, the antigen binding molecules of the present invention bind to the small molecule compound via the heavy chain CDR1, CDR2, CDR3. In one embodiment of the present invention, the antigen binding molecules of the present invention have a binding motif for a small molecule compound. The binding motif for a small molecule compound is derived, for example, from at least one amino acid located at position 33, at position 52, at position 52a, at position 53, at position 55, at position 56, at position 58, at position 95, in position 96, in position 98, in position 100a, in position 100b, in position 100c according to numbering

- 90 046075

Kabat. In other embodiments of the present invention, the antigen binding molecules of the present invention bind to a small molecule compound through at least one amino acid through a group located at position 33, at position 52, at position 52a, at position 53, at position 55, at position 56 , at position 58, at position 95, at position 96, at position 98, at position 100a, at position 100b, at position 100c according to Kabat numbering The antigen can further bind to a complex formed by the binding of an antigen-binding molecule of the present invention and a small molecule compound . In addition, the small molecule compound may be present at the interface between the antigen binding molecule and the antigen and may bind to both. Formation of a ternary complex by the antigen-binding molecule of the present invention together with the small molecule compound and the antigen can be confirmed, for example, by crystallography, as described below, or such methods. In some embodiments of the present invention, the small molecule compound is an adenosine-containing compound.

In one embodiment of the present invention, the small molecule compound of the present invention is a tissue-specific target compound. In other embodiments of the present invention, the target tissue-specific compound is a tumor tissue-specific compound. Exemplary target tissue-specific compounds and tumor tissue-specific compounds are described in the present invention (eg, II. Compositions and Methods (Agonistic Antigen-Binding Molecules Against CD137), A. Exemplary Antigen-Binding Molecules or Antibodies against CD137, [Small Molecular Compound-Dependent Binding Activity]).

B. Antigen.

In the present invention, the antigens are not significantly limited in their structure, provided that they contain epitopes to which the antigen-binding molecules of the present invention bind. Antigens can be inorganic or organic substances. In some embodiments, examples of antigens include: 17-IA, 41BB, 4Dc, 6-keto-PGF1a, 8-roo-PGF2a, 8-oxo-dG, adenosine A1 receptor, A33, ACE, ACE-2, activin , activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1antitrypsin, alpha-U/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL , AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic peptide, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulating factor (BlyS), BACE , BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BSA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM , BLK, BMP, BMP2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associated antigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/ 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152 , CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF , CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14 , CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin, tumor associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (decay accelerating factor), des (1-3 )-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB- 1), EMA, EMMPRIN, ENA endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX , fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 ( myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFRalpha3, GITR, glucagon, Glut4, glycoprotein Ilb/IIIa (GPIIb/IIIa ), GM-CSF, gp130, gp72, GrO, hormone

- 91 046075 growth hormone release, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA) ), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I- 309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL- 1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin chain A, insulin chain B, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5 (alpha V), integrin alpha5/beta1, integrin alpha5/ beta3, integrin alpha6, integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2 , kallikrein L3, kallikrein, kallikrein L3, kallikrein keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, left-handed, Lewis-Y antigen, Lewis-Y antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin receptor beta, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA -DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAS1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, mullerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4 or -6, neurotrophin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG -3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, pGe, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76 , RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g. T cell receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, PLAP-like testicular alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan specific, TGF-beta-RI (ALK-5), TGF-betaRII, TGF-beta-RIIb, TGF-beta-RIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2 , TNF, TNF-alpha, TNF-alphabeta, I^-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK -B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L ( RELT), TNFRSF1A (TNF RI CD120a, p5560), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 ( DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE/RANK ODF ligand , OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI) , TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, ART1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand ), TR-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR1 (Toll-like receptor 1), TLR2, TLR3 , TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis-Y associated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM -1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A , WNT10B, WNT11, WNT16, XCL1,

- 92 046075

XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, A beta, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1 .5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a , C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA and S1P. In some embodiments of the present invention, examples of antigens include hormone and growth factor receptors. In some embodiments of the present invention, antigens are those that are expressed or secreted by cells present in tumor tissues (eg, tumor cells, immune cells, stromal cells, or the like).

B. Activities, compositions and methods.

In one embodiment of the present invention, the cytotoxic activity of the present invention includes, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and T-cell cytotoxicity activity. CDC activity refers to the cytotoxic activity of the complement system. On the other hand, ADCC activity refers to the activity of damaging target cells through effector cells, which occurs when antigen binding molecules bind to antigens present on the cell surface of target cells, and effector cells further bind to antigen binding molecules. Whether an antigen binding molecule of interest has ADCC activity or whether it has CDC activity can be determined using known methods (eg, Current Protocols in Immunology, Chapter 7, 1993. Immunologic studies in humans, ed. Coligan et al.).

In one embodiment of the present invention, neutralizing activity in the present invention refers to the activity of an antigen binding molecule to inhibit a biological activity by binding to a molecule involved in that biological activity. In some embodiments of the present invention, biological activity is provided by binding of a ligand to a receptor. In some embodiments of the present invention, the antigen-binding molecule binds to a ligand or receptor, thereby inhibiting binding between the ligand and the receptor. When the antigen binding molecule is an antibody, the antigen binding molecule having such neutralizing activity is called a neutralizing antibody. The neutralizing activity of a test substance can be measured by comparing the biological activity in the presence of a ligand under conditions where the test substance is present or absent.

In one embodiment, the present invention provides methods for producing an antigen-binding molecule whose antigen-binding activity varies depending on the concentration of the compound. When the compound is a tissue-specific target compound, the present invention provides methods for producing an antigen-binding molecule that specifically exerts its effect in the target tissue. In some embodiments of the present invention, the target tissue is tumor tissue. When the compound is a tumor tissue specific compound, the present invention provides methods for producing an antigen binding molecule for use in the treatment of tumors. In some embodiments of the present invention, these production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a compound is different from its antigen binding activity in the absence of the compound. In some embodiments of the present invention, these production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a high concentration of the compound is different from its antigen binding activity in the presence of a low concentration of the compound.

In some embodiments of the present invention, these production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a high concentration of the compound is different from its antigen binding activity in the presence of a low concentration of the compound. In some embodiments of the present invention, these production methods include the steps of (a) obtaining antigen binding activity of an antigen binding molecule in the presence of a compound, (b) obtaining antigen binding activity of an antigen binding molecule in the absence of a compound, and (c) selecting an antigen binding molecule whose antigen binding activity in the presence of a compound is different. on its antigen-binding activity in the absence of the compound. In some embodiments of the present invention, these production methods include the steps of (a) obtaining antigen binding activity of an antigen binding molecule in the presence of a high concentration of the compound, (b) obtaining antigen binding activity

- 93 046075 antigen binding molecule in the presence of a low concentration of the compound, and (c) selecting an antigen binding molecule whose antigen binding activity in the presence of a high concentration of the compound is different from its antigen binding activity in the presence of a low concentration of the compound.

In another embodiment, the present invention provides methods for producing an antigen binding molecule whose antigen binding activity increases as the concentration of the compound becomes higher. In another embodiment, the present invention provides methods for producing an antigen-binding molecule whose antigen-binding activity decreases as the concentration of the compound becomes higher. The production methods include the step of selecting an antigen-binding molecule whose antigen-binding activity in the presence of the compound is higher than its antigen-binding activity in the absence of the compound. In another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the absence of the compound is lower than its antigen binding activity in the presence of the compound. In another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the absence of the compound is higher than its antigen binding activity in the presence of the compound. In another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a compound is lower than its antigen binding activity in the absence of the compound. In yet another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a high concentration of the compound is higher than its antigen binding activity in the presence of a low concentration of the compound. In another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a low concentration of the compound is lower than its antigen binding activity in the presence of a high concentration of the compound. In yet another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a low concentration of the compound is greater than its antigen binding activity in the presence of a high concentration of the compound. In another embodiment of the present invention, the production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a high concentration of the compound is lower than its antigen binding activity in the presence of a low concentration of the compound.

In one embodiment, the present invention provides methods for producing an antigen-binding molecule having high plasma retention. In some embodiments of the present invention, the production methods include the step of producing an antigen binding molecule whose antigen binding activity increases as the concentration of the compound becomes higher. In other embodiments of the present invention, the production methods include the steps of (a) producing an antigen-binding molecule whose antigen-binding activity increases as the concentration of the compound becomes higher; and (b) measuring the plasma retention capacity of the antigen-binding molecule obtained in step (a). In some embodiments of the present invention, the compound is a target tissue-specific compound. In other embodiments of the present invention, the compound is a compound specific for tumor tissue. Determination of whether the antigen binding molecule of the present invention has a high plasma retention capacity can be made by comparison with a control antigen binding molecule. In some embodiments of the present invention, those antigen-binding molecules whose antigen-binding activity increases as the concentration of the target tissue-specific compound becomes higher have a higher plasma retention capacity compared to the control antigen-binding molecule. In one embodiment of the present invention, the control antigen binding molecule does not have compound concentration dependent antigen binding activity. In some embodiments of the present invention, an antigen binding molecule that does not have compound concentration dependent antigen binding activity is an antigen binding molecule for which the difference in antigen binding activity in the presence of the compound and in the absence of the compound is less, for example, 2 times or less than 1. 8 times or less, 1.5 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. From a comparative point of view, it is preferable that the antigen binding activity of the antigen binding molecules of the present invention and the control antigen binding molecules in the presence of a sufficient amount of the compound is substantially the same.

In another embodiment, the present invention provides methods for producing an antigen-binding molecule having a low ability to accumulate antigen in plasma. In some embodiments of the present invention, the production methods include the step of producing

- 94 046075 antigen-binding molecule, the antigen-binding activity of which increases as the concentration of the compound becomes higher. In other embodiments of the present invention, the production methods include the steps of (a) producing an antigen-binding molecule whose antigen-binding activity increases as the concentration of the compound becomes higher; and (b) measuring the plasma antigen storage capacity of the antigen binding molecule obtained in (a). In some embodiments of the present invention, the compound is a target tissue-specific compound. In other embodiments of the present invention, the compound is a compound specific for tumor tissue. Determination of whether the antigen binding molecule of the present invention has a low ability to accumulate antigen in plasma can be made by comparison with a control antigen binding molecule. In some embodiments of the present invention, those antigen-binding molecules whose antigen-binding activity increases as the concentration of the target tissue-specific compound increases have a lower ability to accumulate antigen in plasma compared to the control antigen-binding molecule. In one embodiment of the present invention, the control antigen binding molecule does not have compound concentration dependent antigen binding activity. In some embodiments of the present invention, an antigen binding molecule that does not have compound concentration dependent antigen binding activity is an antigen binding molecule for which the difference in antigen binding activity in the presence of the compound and in the absence of the compound is less, for example, 2 times or less than 1. 8 times or less, 1.5 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. From a comparative point of view, it is preferable that the antigen binding activity of the antigen binding molecules of the present invention and the control antigen binding molecules in the presence of a sufficient amount of the compound is substantially the same.

In some embodiments of the present invention, for antigen binding molecules produced by the methods of the present invention, the difference in antigen binding activity as a function of the concentration of the compound is greater, for example, two times or more, three times or more, five times or more, 10 times times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, 1000 times or more more, 2000 times or more, 3000 times or more, 5000 times or more, 10000 times or more, 20000 times or more, 30000 times or more, 50000 times or more, or 100000 times or more.

In some embodiments of the present invention, antigen binding activity can be expressed using a dissociation constant (KD) value or a dissociation rate constant (kd) value. Alternatively, it may be expressed using the amount of antigen bound to the antigen binding molecule, as described above. In another embodiment of the present invention, the cytotoxic activity or neutralizing activity of an antigen binding molecule can be used as an alternative indicator of antigen binding activity.

In some embodiments of the present invention, the increased antigen binding activity is, for example, a KD value of 9x10 ^-7 M or less, 8x10 ^-7 M or less, 7x10 ^-7 M or less, 6x10 ^-7 M or less, 5x10 ^-7 M or less, 4x10 ^-7 M or less, 3x10 ^-7 M or less, 2x10 ^-7 M or less, 1x 10 ^-7 M or less, 9x10 -8 M or less, ^{8x10 -8 M} or less, 7x10 ^-8 M or less, 6x10 ^-8 M or less, 5x10 ^-8 M or less, 4x10 ^-8 M or less, 3x10 ^-8 M or less, 2x10 -8 M or less, 1 x ^{10 -8 M} or less, 9x10 ^-9 M or less , 8x10 ^-9 M or less, 7x10 ^-9 M or less, 6x10 ^-9 M or less, 5x10 ^-9 M or less, 4x10 -9 M or less, ^{3x10 -9 M} or less, 2x10 ^-9 M or less, 1 x 10 ^-9 M or less, or, for example, the amount of bound antigen is 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more , 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 1 or more.

In some embodiments of the present invention, the reduced antigen binding activity is, for example, a KD value of 1x10 ^-8 M or more, 2x10 ^-8 M or more, 3x10 ^-8 M or more, 4x10 ^-8 M or more, 5x10 ^-8 M or more , 6x10 ^-8 M or more, 7x10 ^-8 M or more, 8x10 ^-8 M or more, 9x 10 ^-8 M or more, 1x 10 -7 M or more, 2x ^{10 -7 M} or more, 3x 10 ^-7 M or more, 4x 10 ^-7 M or more, 5x10 ^-7 M or more, 6x10 ^-7 M or more, 7x10 ^-7 M or more, 8x10 ^-7 M or more, 9x10 ^-7 M or more, 1x 10 ^{- 6} M or more, 2x10 ^-6 M or more, 3x10 ^-6 M or more, 4x10 ^-6 M or more, 5x10 ^-6 M or more, 6x10 ^-6 M or more, 7x10 ^-6 M or more, 8x10 ^-6 M or more, 9x10 ^-6 M or more, or, for example, the amount of bound antigen is 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0 .09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, 0.001 or less.

The concentration of the compound can be selected over a wide range, provided that the difference in

- 95 046075 binding activity for an antigen binding molecule can be detected. Examples of concentration (high and low) are presented in the present invention. In some embodiments of the present invention, examples of high concentration include, for example, a concentration of 1 μM or higher, a concentration of 3 μM or higher, a concentration of 10 μM or higher, a concentration of 30 μM or higher, a concentration of 100 μM or higher, a concentration of 300 μM or higher, concentration 1 mM or higher. In another embodiment of the present invention, the high concentration may be a concentration that provides a sufficient amount to allow each antigen binding molecule to exhibit maximum binding activity. In some embodiments of the present invention, examples of low concentrations include, for example, a concentration of 1 mM or lower, a concentration of 300 μM or lower, a concentration of 100 μM or lower, a concentration of 30 μM or lower, a concentration of 10 μM or lower, a concentration of 3 μM or lower, a concentration 1 µM or lower. In another embodiment of the present invention, the low concentration may be the concentration at which each antigen binding molecule exhibits minimal binding activity. The case of essentially zero concentration (ie no compound) may be one of the low concentration cases.

In another embodiment of the present invention, these production methods include the step of selecting an antigen binding molecule whose antigen binding activity in the presence of a compound is different from its antigen binding activity in the absence of the compound from a library of antigen binding molecules. A library of antigen-binding molecules can be a free repertoire of antigen-binding molecules (an unexposed selection of molecules) or can be a repertoire of specifically selected antigen-binding molecules. Examples of libraries of this type include a library of antigen-binding molecules that are pre-conditioned to bind to a particular compound. In some embodiments of the present invention, the library of antigen binding molecules is a library of antigen binding molecules into which amino acid changes are introduced to impart binding activity to a particular compound in advance. Examples of libraries of this type include those described, for example, in WO 2015/083764.

In some embodiments of the present invention, the production methods further comprise step (d) of producing a nucleic acid that encodes the antigen binding molecule selected in step (c), step (e) of introducing the nucleic acid from step (d) into a host cell , and (e) culturing the host cell as in (e) such that the antigen binding molecule is expressed. The nucleic acid of claim (d) may be one or more nucleic acids and may be included in one or more vectors (eg, an expression vector). In some embodiments of the present invention, the production methods further comprise the step of claim (g) of isolating the antigen binding molecule from the cell culture of claim (e).

Antigen binding molecules produced by the production methods of the present invention are also included in the present invention.

In another embodiment, the present invention provides pharmaceutical preparations comprising an antigen binding molecule provided in the present invention. In one embodiment of the present invention, the aforementioned pharmaceutical preparations further comprise a pharmaceutically acceptable carrier. In other embodiments, the present invention provides pharmaceutical preparations for use in the treatment of tumors.

In another embodiment, the present invention provides methods for preparing a pharmaceutical preparation comprising admixing an antigen binding molecule of the present invention with a pharmaceutically acceptable carrier. In other embodiments, the present invention provides methods for producing a pharmaceutical preparation for the treatment of tumors.

In one embodiment, the present invention provides an antigen-binding molecule that, when administered to a living organism, exhibits stronger antigen-binding activity in tumor tissue than in non-tumor tissue. Such a difference in response need not be observed for every dose of the antigen-binding molecule, but rather that the difference be observed over a certain dose range. In another embodiment of the present invention, target cells (cells expressing the antigen) are more severely damaged in tumor tissue than in non-tumor tissue. In another embodiment of the present invention, target cells are damaged at a lower dose in tumor tissue than in non-tumor tissue. In another embodiment of the present invention, the therapeutic effect is observed at a lower dose than the dose at which the side effect is observed. In the present invention, the therapeutic effect is the development of an antitumor effect (eg, tumor regression, induction of tumor cell death or inhibition of tumor cell proliferation, etc.), and the side effect is the occurrence of an undesirable phenomenon in normal tissues (eg, damage to normal tissues).

In one embodiment of the present invention, the degree of effectiveness of the drug

- 96 046075 The effective agent provided by the antigen binding molecule of the present invention is different depending on whether the antigen binding molecule has compound concentration dependent (ie, varying with compound concentration) antigen binding activity. In some embodiments of the present invention, the antigen binding molecules of the present invention are an antigen binding molecule whose antigen binding activity increases as the concentration of the compound becomes higher. In some embodiments of the present invention, the control antigen binding molecule is an antigen binding molecule that does not have compound concentration dependent antigen binding activity. In some embodiments of the present invention, the compound is a tumor tissue-specific compound. In some embodiments of the present invention, an antigen binding molecule that does not have compound concentration dependent antigen binding activity is an antigen binding molecule for which the difference in antigen binding activity in the presence of the compound and in the absence of the compound is, for example, a factor of 2 or less than 1. 8 times or less, 1.5 times or less, 1.3 times or less, 1.2 times or less, or 1.1 times or less. Preferably, the antigen binding activity of the antigen binding molecules of the present invention and the control antigen binding molecules in the presence of a sufficient amount of the compound is substantially the same.

In one embodiment of the present invention, the antigen binding molecule of the present invention and the control antigen binding molecule have different drug effects. In yet another embodiment of the present invention, the drug effects are different in tissues where the concentration of the compound is low. Examples of tissue with low concentrations of the compound include non-tumor tissues such as normal tissues. The antigen binding molecule may be presented in the form of a pharmaceutical preparation containing the antigen binding molecule. In some embodiments of the present invention, in tissue with a low concentration of the compound, the antigen binding molecule of the present invention has reduced activity to damage a target cell (cell expressing the antigen) compared to a control antigen binding molecule. In some embodiments of the present invention, in tissue with a low concentration of the compound, the antigen binding molecule of the present invention requires a higher dose to damage the cell compared to the control antigen binding molecule. In some embodiments of the present invention, in tissue with a low concentration of the compound, the antigen binding molecule of the present invention exhibits a reduced level of side effect compared to a control antigen binding molecule. In some embodiments of the present invention, in tissue with a low concentration of the compound, the dose at which a side effect is observed is higher for the antigen binding molecule of the present invention compared to the control antigen binding molecule. These differences in response are not necessarily observed for every tissue (eg, for all tissues exhibiting a low concentration of the compound), but rather for differences to be observed for some tissues. In some embodiments of the present invention, the side effect is an undesirable event in normal tissue (eg, damage to normal tissue).

In one embodiment of the present invention, the drug effects of the antigen binding molecule of the present invention and the control antigen binding molecule are substantially the same. In some embodiments of the present invention, the effects as a drug are substantially the same in tissue where the concentration of the compound is high. Examples of tissue with high concentrations of the compound include tumor tissue. The antigen binding molecule may be presented in the form of a pharmaceutical preparation containing the antigen binding molecule. In some embodiments of the present invention, in tissue with a high concentration of the compound, the activity of damaging the target cell (the cell expressing the antigen) is essentially the same for the antigen binding molecule of the present invention and the control antigen binding molecule. In some embodiments of the present invention, in tissue with a high concentration of the compound, the dose required to damage the cell is substantially the same for the antigen binding molecule of the present invention and the control antigen binding molecule. In some embodiments of the present invention, in tissue with a high concentration of the compound, the level of therapeutic effect is substantially the same for the antigen binding molecule of the present invention and the control antigen binding molecule. In some embodiments of the present invention, in tissue with a high concentration of the compound, the dose at which a therapeutic effect is observed is substantially the same for the antigen binding molecule of the present invention and the control antigen binding molecule. In some embodiments of the present invention, the therapeutic effect is an antitumor effect (eg, tumor regression, induction of tumor cell death, or inhibition of tumor cell proliferation, etc.).

In another embodiment, the present invention provides an antigen-binding

- 97 046075 molecule for use as a medicine. In yet another embodiment, the present invention provides an antigen binding molecule for use in the treatment of a tumor. In another embodiment, the present invention provides for the use of an antigen binding molecule in the preparation of a medicament. In yet another embodiment, the present invention provides for the use of an antigen binding molecule to treat a tumor. In a further embodiment, the present invention provides a method of treating a tumor, comprising administering an effective amount of an antigen binding molecule to an individual with a tumor. The individual according to any of the above embodiments of the present invention is preferably a human.

Methods for Measuring ATP Concentration In another embodiment, the present invention provides methods for measuring the concentration of ATP in a solution. In some embodiments of the present invention, the above methods include the steps of (a) contacting a split Luc/HEK293 cell expressing the P2Y receptor with a solution and (b) measuring luciferase activity in the cell. The P2Y receptor is a cell surface receptor and seven-membrane G protein-coupled receptor (GPCR) whose endogenous ligands are extracellular purine nucleotides (ATP, ADP), pyrimidine nucleotides (UTP, UDP), sugar nucleotides, etc. In other embodiments of the present invention, the P2Y receptor is P2Y1 (catalog number U42029), P2Y2 (catalog number U07225), P2Y4 (catalog number X91852), P2Y6 (catalog number X97058), P2Y11 (catalog number AF030335), P2Y12 (part number AJ320495), P2Y13 (part number AF295368) or P2Y14 (part number D13626). In some embodiments of the present invention, the P2Y receptor is P2Y11 (catalog number AF030335). Luc/HEK293 split cells are recombinant cells produced using split-luciferase technology owned by ProbeX (Misawa et al., Anal. Chem., 82, 2010, 25522560) and express the following two proteins: (i) a fusion protein, in in which the C-terminal fragment of two fragments of a halved luciferase molecule is attached to the C-terminus of the P2Y receptor, and (ii) a fusion protein in which the N-terminal fragment of two fragments of a halved luciferase molecule is attached to an N- end of beta-arrestin (catalog number NM_004313).

When ATP is added to cleaved Luc/HEK293 cells expressing the P2Y receptor, interaction between the P2Y receptor and beta-arrestin occurs in close proximity to the cell membrane, and as a result, fragments of cleaved luciferase bind to restore the active luciferase molecule inside the cell. Luciferase activity can be measured using a luciferase substrate such as, for example, luciferin. In some embodiments of the present invention, the methods described above further include the step of contacting the cell with a solution containing a luciferase substrate. The solution containing the luciferase substrate may contact the cell before step (a) or after step (a).

In some embodiments of the present invention, the solution for which the ATP concentration is measured may be an in vitro solution or a body fluid in tissue in vivo. In other embodiments of the present invention, the body fluid may be selected from the group consisting of blood, lymph fluid, tissue fluid (interstitial fluid, interstitial fluid, interstitial fluid), body cavity fluid (serous cavity fluid, pleural fluid, ascites fluid, pericardial fluid ), cerebrospinal fluid, joint fluid (synovial fluid) and intraocular fluid. In some embodiments of the present invention, the body fluid is an intercellular fluid. In another embodiment of the present invention, the tissue may be healthy tissue/normal tissue and may be diseased tissue (eg, tumor tissue). In one embodiment of the present invention, when the concentration of ATP in body fluid in tissue in vivo is measured by the methods described above, step (i) may be the step of transplanting a cleaved Luc/HEK293 cell expressing the P2Y receptor into the tissue in vivo.

In some embodiments of the present invention, luciferase activity can be measured by detecting the light emitted when the luciferase reacts with its substrate, such as luciferin. In case the solution is an in vitro solution, such luminescence can be measured using a luminescence detection device such as a plate reader. In case the solution is a body fluid in tissue in vivo, the luminescence can be measured using an in vivo luminescence imaging apparatus or the like. Measurement-based quantification can be accomplished by methods known in the art, for example, by measuring solutions containing a known concentration of ATP and constructing a calibration curve showing the correlation between ATP concentration and luminescence intensity.

V. Screening methods.

(1) A method for screening an antigen binding domain or an antigen binding molecule that has a molecular compound dependent antigen binding activity using multivalent antigens.

In one embodiment, the present invention provides a method for highly efficient

- 98 046075 active and high-precision screening, identification and production of an antigen-binding domain or an antigen-binding molecule that has molecular compound-dependent antigen-binding activity, using a fusion molecule obtained by fusing two or more molecules (two or more units) of an antigen with one molecule ( one unit) of the fusion partner molecule.

In one embodiment of the present invention, in the method of the present invention, a method of screening for an antigen binding domain or an antigen binding molecule that has a molecular compound dependent antigen binding activity includes:

(a) contacting, in the presence of a small molecule compound, an antigen binding domain or an antigen binding molecule or a library of antigen binding domains or antigen binding molecules with a fusion molecule in which two or more units of an antigen are fused to one unit of a fusion partner, (b) placing an antigen binding domain or an antigen binding molecule associated with the antigen in the fusion molecule in step (a), in the absence or presence of a low concentration of the compound, and (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b).

In one embodiment of the present invention, in the method of the present invention, the fusion partner molecule to which two or more antigen molecules (two or more units) are fused is a dimeric Fc region.

In one embodiment of the present invention, in the method of the present invention, the Fc region includes a first Fc subunit and a second Fc subunit, and one antigen is fused to both the first and second Fc subunit.

In one embodiment of the present invention, in the method of the present invention, one of the antigens is fused to the N-terminus of both the first and second Fc subunits.

In one embodiment of the present invention, in the method of the present invention, the library of antigen binding domains or antigen binding molecules is a phage library.

In one embodiment of the present invention, in the method of the present invention, the phages included in the phage library are phages having two or more antigen binding domains or antigen binding molecules on their surface.

In one embodiment of the present invention, in the method of the present invention, the phages included in the phage library are phages having a defect in the helper phage-derived pIII gene.

In one embodiment of the present invention, in the method of the present invention, the antigen may be any antigen as described above, and examples may include the various types of antigens described in section IV. Compositions and methods (antigen-binding molecules, the binding activity of which to antigens varies depending on the concentration of the low-molecular compound), B. Antigen. In one embodiment of the present invention, the antigen may preferably be membrane-type antigens (eg, co-stimulatory molecules such as CD137, CTLA4, CD40, OX40, RANK, GITR and ICOS).

In one embodiment of the present invention, in the method of the present invention, the fusion of an Fc region (Fc subunit) and an antigen can be produced by a conventional method using genetic recombination technology as described above.

In one embodiment of the present invention, the method of the present invention provides as a small molecule compound various compounds as described above (for example, compounds specific for tumor tissue, such as nucleosides having a purine ring structure, such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine, and commercially available ADP-beta-S (Sigma); amino acids such as alanine, glutamic acid and aspartic acid; amino acid metabolites such as kynurenine, anthranilic acid acid, 3-hydroxykynurenine and kynurenic acid; arachidonic acid metabolites such as prostaglandin E2; primary metabolites of the glycolytic pathway or Krebs cycle such as lactic acid, succinic acid and citric acid; and nicotinamide metabolites such as 1-methylnicotinamide) may be used .

In one embodiment, the method of the present invention involves contacting an antigen (e.g., a fusion molecule of an Fc region and an antigen) with a phage library of antigen-binding domains or antigen-binding molecules, isolating the antigen-binding domain or antigen-binding molecule associated with the antigen, analyzing the isolated antigen binding domain or antigen binding molecule in the presence or absence of a small molecule compound can be carried out in accordance with the method described above and the method described below in the examples, referring to known methods in the field of technology to which the present invention relates.

In one embodiment of the present invention, the method of the present invention

- 99 046075 may be the method described in example 2.

(2) A method for screening an antigen binding domain or an antigen binding molecule that has antigen binding activity dependent on two or more different small molecules.

In one embodiment, the present disclosure provides a method of screening for an antigen binding domain or antigen binding molecule that has antigen binding activity dependent on two or more different small molecule compounds.

In one embodiment of the present invention, the method of the present invention includes:

(a) contacting the antigen binding domain or antigen binding molecule or library of antigen binding domains or antigen binding molecules with the antigen in the presence of a first small molecule compound, (b) placing the antigen binding domain or antigen binding molecule associated with the antigen in step (a), in the absence or in in the presence of a low concentration of the first compound, (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b), (d) contacting the antigen binding domain or antigen binding molecule isolated in step (c) with the antigen in the presence of a low molecular weight compound, (e) placing the antigen binding domain or antigen binding molecule associated with the antigen in step (c) in the absence or presence of a low concentration of a second compound, and (e) isolating the antigen binding domain or antigen binding molecule dissociated in step (e), wherein the method does not include between steps (c) and (d) amplification of the gene encoding the antigen binding domain or antigen binding molecule isolated in step (c).

In one embodiment of the present invention, in the method of the present invention, the library of antigen binding domains or antigen binding molecules is a phage library.

In one embodiment of the present invention, in the method of the present invention, the antigen can be any antigen as described above, preferably a membrane type antigen (eg, co-stimulatory molecules such as CD137, CTLA4, CD40, OX40, RANK, GITR and ICOS).

In one embodiment of the present invention, various compounds described above can be used as small molecular compounds in the method of the present invention, for example, compounds specific for tumor tissue, such as nucleosides having a purine ring structure, such as adenosine (ADO), adenosine triphosphate ( ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and inosine; amino acids such as alanine, glutamic acid and aspartic acid; amino acid metabolites such as kynurenine, anthranilic acid, 3hydroxykynurenine and kynurenic acid; arachidonic acid metabolites such as prostaglandin E2; primary metabolites of the glycolytic pathway or Krebs cycle, such as lactic acid, succinic acid and citric acid; and nicotinamide metabolites such as 1-methylnicotinamide.

In one embodiment of the present invention, in the method of the present invention, the first small molecule compound and the second small molecule compound may be any two types of different compounds selected from the above-mentioned various small molecule compounds.

In one embodiment of the present invention, the method of the present invention contacts an antigen with a phage library of antigen binding domains or antigen binding molecules, isolates the antigen binding domain or antigen binding molecule associated with the antigen, analyzes the isolated antigen binding domain, binding domain or antigen binding molecule in the presence or absence of a first small molecule compounds, and analysis in the presence or absence of a second small molecule compound can be carried out in accordance with the method described above and the method described below in the examples, referring to known methods in the field to which the present invention relates.

In one embodiment of the present invention, the method of the present invention may be the method described in Example 9.

(3) A method for screening an antigen binding domain or an antigen binding molecule that has a small molecule dependent antigen binding activity using a naïve library.

In one aspect, the present invention provides a method for screening an antigen binding domain or antigen binding molecule that has small molecule dependent antigen binding activity using a naïve library containing a plurality of phages presenting antigen binding domains or antigen binding molecules (e.g., Fab domains) of various human antibody sequences, derived from lymphocytes

- 100 046075 people (for example, human PBMCs) who did not receive antigen stimulation.

In one embodiment of the present invention, the method of the present invention includes:

(a) contacting a naïve library of antigen binding domains or antigen binding molecules with an antigen in the presence of a small molecule compound, (b) placing an antigen binding domain or antigen binding molecule coupled to the antigen in step (a) in the absence or presence of a low concentration of the compound, and (c) isolating the antigen binding domain or antigen binding molecule dissociated in step (b).

In one embodiment of the present invention, in the method of the present invention, the naive library is a phage library containing phages presenting two or more antigen binding domains or antigen binding molecules on their surface.

In one embodiment of the present invention, in the method of the present invention, the naïve library is a library containing phages having a defect in the helper phage-derived pIII gene.

In one embodiment of the present invention, in the method of the present invention, a naive library is a library containing phages obtained by increasing the expression of an antigen binding domain or antigen binding molecule using a small molecule additive that increases the level of expression from a promoter that regulates the expression of the antigen binding domain or antigen binding molecule.

In one embodiment of the present invention, in the method of the present invention, the low molecular weight additive is isopropyl-β-thiogalactopyranoside (IPTG, isopropyl-βthiogalactopyranoside) or arabinose.

In one embodiment of the present invention, in the method of the present invention, the antigen can be any antigen as described above, preferably a membrane type antigen (eg, a co-stimulatory molecule such as CD137, CTLA4, CD40, OX40, RANK, GITR or ICOS).

In one embodiment of the present invention, the method of the present invention may use various compounds as described above as the small molecule compound (e.g., tumor tissue specific compounds such as nucleosides having a purine ring structure such as adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and inosine; amino acids such as alanine, glutamic acid and aspartic acid; amino acid metabolites such as kynurenine, anthranilic acid, 3-hydroxykynurenine and kynurenic acid, arachidonic acid metabolites such such as prostaglandin E2; primary metabolites of the glycolytic pathway or Krebs cycle such as lactic acid, succinic acid and citric acid, and nicotinamide metabolites such as 1methylnicotinamide).

In one embodiment of the present invention, in the method of the present invention, contacting an antigen with a phage library of antigen binding domains or antigen binding molecules, isolating the antigen binding domain or antigen binding molecule associated with the antigen, analyzing the isolated antigen binding domain or antigen binding molecule in the presence or absence of a small molecule compound may be carried out in accordance with the method described above and the method described below in the examples, with reference to well-known methods in the field of technology to which the present invention relates.

In one embodiment of the present invention, the method of the present invention may be the method described in Example 10.

Examples

Below are examples of methods and compositions of the present invention. Given the above description of the invention, it is obvious that various other embodiments may be implemented.

Example 1. Antigen production.

(1-1) Preparation of the extracellular region of human CD137.

The extracellular domain of human CD137 (also called hCD137) is obtained by methods known to those skilled in the art. In particular, a downstream gene fragment encoding the extracellular region of human CD137, a gene fragment encoding a histidine tag, and a gene fragment encoding a specific sequence to which biotin is added (AviTag sequence, SEQ ID NO: 86) are linked. A gene fragment encoding a protein in which the extracellular region of human CD137, a histidine tag and Avitag (human CD137 or hCD137HisBAP, SEQ ID NO: 87) is linked is included in an animal cell expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At the same time, a plasmid vector containing a gene expressing EBNA1 (SEQ ID NO: 88) is transfected. Cells transfected with genes according to the above

- 101 046075 procedure, cultured at 37°C, in an atmosphere of 8% CO ₂ ; and the protein of interest (extracellular region of human CD137) is secreted into the culture supernatant. This cell culture medium is filtered through a 0.22 μm filter to obtain the culture supernatant. The culture supernatant is applied to a HisTrap-HP column (GE Healthcare) and the extracellular region of human CD137 is bound to the column.

The extracellular region of human CD137 is eluted with a solution of 20 mM sodium phosphate, 500 mM sodium chloride and 500 mM imidazole, pH 7.5. The aggregates are then removed by gel filtration chromatography using a Superdex 200 26/600 column (GE Healthcare) to obtain purified extracellular region of human CD137. Human CD137 concentrations were calculated using the method of Race et al. based on amino acid sequence, excluding the signal sequence deleted from SEQ ID NO: 87 (C. N. Race, et al. Protein Science, 4, 1995, 2411-2423).

(1-2) Preparation of the extracellular domain of human CD137 (hCD137 (cleaved FXa)).

The extracellular domain of human CD137 (also called hCD137 (FXa protease-cleaved)) is prepared in a manner known to those skilled in the art. Specifically, a gene fragment encoding a factor Xa cleavage sequence and a gene fragment encoding an antibody constant region are joined downstream of a gene fragment encoding the extracellular region of human CD137. A gene fragment encoding a protein in which the human CD137 extracellular region, the FactorXa protease cleavable sequence, and the antibody constant region are linked (hCD137-F-Fc, SEQ ID NO: 89) is included in an animal expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At the same time, a plasmid vector containing the gene encoding EBNA1 (SEQ ID NO: 88) is transfected. Cells transfected with genes according to the above procedures are cultured at 37°C in an atmosphere of 8% CO ₂ , while hCD137-F-Fc is secreted into the culture supernatant. The cell culture medium is filtered through a 0.22 μm filter and the culture supernatant is collected.

Cell culture supernatants are applied to a Protein A column (MabSelect SuRe, GE Healthcare). hCD137-F-Fc binds to the column and is then eluted with 50 mM acetic acid. After neutralizing the eluate with 1 M Tris-HCl, pH 8.0, the hCD137-F-Fc solvent was replaced with a solution of 20 mM Tris, 100 mM sodium chloride, 2 mM calcium chloride, pH 8.0. Factor Xa protease (NEW ENGLAND BioLabs, catalog number P8010L) is then added and hCD137-F-Fc is digested. A protease inhibitor (product DNS-GGACK, Calbiochem, catalog number 251700) is then added to stop the reaction. 5 M sodium chloride is added to the solution in which the protease acts, and the entire volume of the prepared solution is applied to a HiTrap Benzamidine column (GE Healthcare, catalog number 17-5143-01). The columnated solution is then applied to a Protein A column (Mab Select SuRe, GE Healthcare) and the fractions passed through the column are collected. From the skipped fractions, aggregates were removed by gel filtration chromatography using Superdex 200 Increase, 10/30 (GE Healthcare, catalog number 28990944), yielding purified extracellular region of human CD137.

(1-3) Preparation of biotinylated human Fc-fusion CD137.

Biotinylated human Fc-fusion CD137 (also called biotinylated hCD137-Fc, or bio-hCD137-Fc, or hCD137-Fc-Bio) is prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding the antibody constant region and a gene fragment encoding a specific sequence to which biotin is added (AviTag sequence, SEQ ID NO: 86) are attached downstream of the gene fragment encoding the extracellular region of human CD137. A gene fragment encoding a protein (Fc fused to human CD137, SEQ ID NO: 90) in which the extracellular region of human CD137, the antibody constant region and Avitag are linked is included in an animal expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). Simultaneously, the gene expressing EBNA1 (SEQ ID NO: 88) and the gene expressing biotin ligase (BirA, SEQ ID NO: 91) are transfected, and biotin is added to biotinylate the Fc fused to human CD137. Cells transfected with genes according to the above procedures are cultured at 37°C in an atmosphere of 8% CO ₂ ; the protein of interest (a biotinylated human Fc CD137 fusion) is secreted into the culture supernatant. The cell culture medium is filtered through a 0.22 μm filter and the culture supernatant is collected.

The culture supernatant is applied to a Protein A column (MabSelect SuRe, GE Healthcare) and biotinylated human Fc fusion CD137 is bound to the column. Biotinylated human Fc-fusion CD137 was eluted with 50 mM acetic acid. The aggregates are then removed by gel filtration chromatography using Superdex 200, 26/600 (GE Healthcare) to obtain purified biotinylated human Fc fusion CD137.

(1-4) Preparation of human Fc-fusion CD137.

Human Fc-fusion CD137 (also called hCD137-Fc) is produced by methods known to those skilled in the art. In particular, the gene fragment encoding the anti-antibody constant region

- 102 046075 la, and a gene fragment encoding a specific sequence to which biotin is added (AviTag sequence, SEQ ID NO: 86) is attached downstream of the gene fragment encoding the extracellular region of human CD137. Gene fragments encoding a protein (fusion to human CD137 Fc, SEQ ID NO: 90) in which the extracellular region of human CD137, the antibody constant region and Avitag are included in an animal expression vector. The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). At the same time, the gene expressing EBNA1 (SEQ ID NO: 88) is transfected. Cells transfected with genes according to the above procedures are cultured at 37°C in an atmosphere of 8% CO ₂ ; the protein of interest (fused to human Fc CD137) is secreted into the culture supernatant. The cell culture medium is filtered through a 0.22 μm filter and the culture supernatant is collected.

The culture supernatant is applied to a Protein A column (MabSelect SuRe, GE Healthcare) and human Fc fusion CD137 is bound to the column. The human Fc-fusion CD137 was eluted with 50 mM acetic acid. The aggregates are then removed by gel filtration chromatography using Superdex 200, 26/600 (GE Healthcare) to obtain purified human Fc fusion CD137.

(1-5) Preparation of biotinylated monkey Fc-fusion CD137.

Biotinylated monkey Fc CD137 fusion (also called cyCD137-Fc-BAP) is prepared by methods known to those skilled in the art. Specifically, a gene fragment encoding the antibody constant region and a gene fragment encoding a specific sequence to which biotin is added (AviTag sequence, SEQ ID NO: 86) are attached downstream of the gene fragment encoding the extracellular region of monkey CD137. A gene fragment encoding a protein in which the simian CD137 extracellular region, the antibody constant region, and Avitag are linked (fused to simian CD137 Fc, SEQ ID NO: 92) is included in an animal expression vector.

The constructed plasmid vector was transfected into FreeStyle293 cells (Invitrogen) using 293-fectin (Invitrogen). The gene expressing EBNA1 (SEQ ID NO: 88) and the gene expressing biotin ligase (BirA, SEQ ID NO: 91) are simultaneously transfected; biotin is added to provide a biotin label for the monkey CD137 Fc fusion. Cells transfected with genes according to the above procedures are cultured at 37°C in an atmosphere of 8% CO ₂ ; the protein of interest (a biotinylated monkey Fc CD137 fusion) is secreted into the culture supernatant. The cell culture medium is filtered through a 0.22 μm filter and the culture supernatant is collected.

The culture supernatant is applied to a protein A column (MabSelect SuRe, GE Healthcare) and biotinylated monkey Fc-fusion CD137 is bound to the column. Biotinylated monkey Fc CD137 fusion was eluted with 50 mM acetic acid. Aggregates were then removed by gel filtration chromatography using Superdex 200, 26/600 (GE Healthcare) to obtain purified biotinylated monkey Fc CD137 fusion.

Example 2. Preparation of ATP-dependent anti-CD137 antibodies.

(2-1) Generation of antibodies with small-molecule-dependent antigen-binding activity (small-molecule-switched antibodies) from a rationally designed library using ATP (1).

(2-1-1) Panning.

Antibodies that exhibit antigen binding activity in the presence of adenosine triphosphate (adenosine 5'-triphosphate; ATP) were obtained from a rationally designed antibody phage display library created in the previous patent WO 2015/083764. It should be noted that antibodies with small molecule dependent antigen binding activity (antigen, e.g. CD137) may be referred to as switchable antibodies or small molecule switchable antibodies, and antibodies with antigen binding activity (eg CD137 antigen) dependent on ATP may be referred to as switchable antibodies or ATP-switched antibodies. To produce antibodies, phages are collected that present antibodies that exhibit binding activity in the presence of ATP to the antigen captured on the beads. Phages are then collected from the eluates eluted from the beads in the absence of ATP.

Phages are obtained by the usual method from the bacterium E. coli carrying a phagemid constructed by the phage display method. Specifically, E. coli carrying the constructed phagemid vector is infected with phage Μ13ΚΟ7ΔρΙΙΙ (designated hyperphage) (PROGEN Biotechnik), and the phages are collected from supernatants cultured overnight at 30°C. A phage library suspension is prepared by diluting the phage population with Tris-Buffered Saline (TBS), which is pelleted by adding 2.5 M NaCl/10% PEG to the E. coli culture in which the phage is being produced. BSA is then added to the phage library suspension to a final concentration of 4%. Panning is carried out using antigen immobilized on magnetic beads. Sera-Mag NeutrAvidin beads (Thermo Fisher Scientific) or Dynabeads M-280 StreptAvidin (Life Technologies) are used as magnetic beads. The antigen used is biotinylated adenosine triphosphate (adenosine 5'-triphosphate; ATP) from Jena Bioscience, or hCD137-Fc-Bio (SEQ ID NO: 90) or bio-hCD137 (SEQ ID NO: 89), which are biotinylated from hCD137 (FXa cleavage),

- 103 046075 using pre-measured NHS-PEO4-6uoTUHa without weighing (PIERCE) obtained in examples 1-2 and 1-3.

Panning is performed to efficiently obtain a small molecule that can act as a switch in cancer tissue-dependent switchable antibodies. In particular, panning to enrich antibodies that bind to antigen in the presence of ATP, which is a small molecule, and do not bind to antigen in the absence of ATP, is performed with reference to the methods shown in the previous patent WO 2015/083764. In the first round, for all biotinylated hCD137 (bio-hCD137), hCD137-Fc-Bio, and biotinylated ATP (bio-ATP), panning is performed using a method in which solid-phase immobilization of biotinylated antigens onto magnetic beads (referred to as the solid-phase bead-based method) is preceded by adding the prepared phage library suspension. For Bio-ATP, panning to enrich antibodies that can bind to an antigen (Bio-ATP) in the absence of ATP as a small molecule compound is performed with reference to the methods described in the previous patent WO 2015/083764 noted above. For hCD137-Fc-Bio, 4 nM non-biotinylated human IgG1 Fc region is added to remove antibodies that bind to the Fc region. The collected phage is added to E. coli strain ER2738 to infect the E. coli phage, then the collected E. coli cells are infected with hyperphage and the phage is collected from the supernatant after overnight culture at 30°C.

From Round 2 onwards, panning is performed on only biotinylated hCD137 (bio-hCD137) and hCD137-Fc-Bio to enrich for antibodies that bind antigen in the presence of ATP and do not bind antigen in the absence of ATP, using a solid-phase bead method, referring to the method shown in the previous patent WO 2015/083764. For hCD137-Fc-Bio, 4 nM non-biotinylated human IgG1 Fc region is added in both cases to remove antibodies that bind to the Fc region. This panning is repeated up to round 5 to enrich antibody sequences of interest.

(2-1-2) Evaluation of binding activity in the presence and absence of ATP using the phage enzyme-linked immunosorbent assay (ELISA) method.

From individual E. coli colonies obtained by the methods described above, the phage culture supernatant is collected in the usual manner (Methods Mol Biol., 178, 2002, 133-145). NucleoFast 96 (MACHEREY-NAGEL) is used to ultrafiltrate the collected culture supernatant. The eluate is removed by centrifugation (4500 g, 45 min) of a NucleoFast 96 plate, into which 100 μl of the culture supernatant was added to each well. NucleoFast 96 is washed again by centrifugation (4500 g for 30 min), for which 100 μl of H2O is added to each well. Finally, 100 μl of TBS is added, and the phage suspension contained in the supernatants in the wells of the NucleoFast 96 plate, which were left at room temperature for 5 min, is recovered.

Purified phages supplemented with TBS or ATP/TBS are subjected to enzyme-linked immunosorbent assay (ELISA) using the following procedure. Streptavidin-coated 384-well microplates (Greiner) are coated overnight with 10 μl of TBS buffer containing biotinylated antigens (bio-hCD137, hCD137-Fc-Bio and bio-Fc) prepared in Example 1. After removing the biotinylated antigens, that do not bind to the plate, by washing each well of the plate with Tris-buffered saline with Tween-20 (TBST), the wells are blocked with 80 μl of 2% skim milk TBS for 1 hour or more. The 2% skim milk-TBS is removed with TBST washes and then the antibody presenting phages are allowed to bind to the biotinylated antigen present in each well in the absence and presence of ATP, leaving the plates with the prepared purified phages added to each well for 1 hour at room temperature. HRP-conjugated anti-M13 antibody (GE Healthcare, catalog no. 27-9421-01) diluted in TBS or ATP/TBS is added to each well washed with TBST or ATP/TBST and the plate is incubated for one hour. After washing with TBST or ATP/TBST, the color development of the solution in each well to which a single TMB solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm.

As a result, several antibodies were identified with altered binding activity to biohCD137 or hCD137-Fc-Bio in the presence and absence of ATP.

The results of phage ELISA using clones after 4 and 5 rounds of panning are shown in Table. 9.

In the present invention, clones with an absorbance of 0.2 or more in the presence of ATP and an absorbance S/N ratio greater than 2 in the presence/absence of antigen are defined as positive clones. In addition, among the positive clones, clones with an S/N absorption ratio greater than 2 times in the presence/absence of ATP are considered clones with ATP-dependent antigen-binding activity (switchable clones).

- 104 046075

Table 9

Antigen used in panning Biotinylated hCD137 hCD137- Fc-Bio Round 4 5 4 5 Number of clones subjected to ELISA 384 384 384 384 Number of positive clones (ATP uptake + > 0.2; antigen +/- ratio > 2.0) 31 4 125 169 Number of clones switched (ATP+/ratio > 2) 12 2 118 164

(2-1-3) Sequence analysis of switchable antibodies whose antigen-binding activity changes depending on the presence/absence of ATP.

The nucleotide sequences of genes amplified using specific primers pBAD-F, G1seq-R, from clones with ATP-dependent antigen-binding activity (switch clones) are analyzed based on the results of phage ELISA. The analysis yields nucleotide sequences of clones that are found to bind to human CD137 in the presence of ATP, but not to human CD137 in the absence of ATP.

(2-2) Generation of antibodies that bind to antigens in the presence of a small molecule from a rationally designed library using ATP (2).

(2-2-1) Panning.

Antibodies that exhibit antigen binding activity in the presence of ATP were obtained from the rationally designed antibody phage display library constructed in the previous patent WO 2015/083764. To obtain antibodies, phages presenting antibodies that exhibit binding activity in the presence of ATP to antigens are collected, followed by the isolation of phages from the eluate eluted from beads in the absence of ATP.

Phages are obtained by the usual method from the bacterium E. coli carrying a phagemid constructed by the phage display method. In particular, E. coli carrying the constructed phagemid vector is infected with phage M13KO7TC (WO 2015046554 A1) or M13ΚΟ7ΔρΙΙΙ (designated hyperphage) (Progen Biotechnik), and the phages are isolated from supernatants cultured overnight at 30°C. A phage library suspension is prepared by diluting the phage population with Tris-Buffered Saline (TBS), which is pelleted by adding 2.5 M NaCl/10% PEG to the E. coli culture in which the phage is being produced. BSA is then added to the phage library suspension to a final concentration of 4%. Panning is carried out using antigens immobilized on magnetic beads. The magnetic beads used are NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated), NeutrAvidin beads (TAMAGAWA SEIKI) or Dynabeads MyOne StreptAvidin T1 (Thermo Fisher Scientific). The antigen used is hCD137Fc-Bio or bio-hCD137, obtained in example 1.

Panning is performed to efficiently produce antibodies switched by a small molecule that can act as a switch in cancerous tissues. In particular, panning to enrich for antibodies that bind to antigen in the presence of a small molecule of adenosine triphosphate (adenosine 5'-triphosphate; ATP) and do not bind to antigen in the absence of ATP is performed with reference to the methods described in the previous patent WO 2015/083764 . For 6uo-HCD137, a method is used in which a prepared phage library suspension is added after pre-solid-phase immobilization of biotinylated antigens on magnetic beads (the so-called solid-phase bead method), and a method in which magnetic beads are added after mixing a prepared phage library suspension with pre-biotinylated antigens (the so-called liquid-phase method). For hCD137-Fc-Bio, 4 nM non-biotinylated human IgG1 Fc region is added to remove antibodies that bind to the Fc region, and panning is performed using a liquid phase method only. The collected phage is added to E. coli strain ER2738 to infect the E. coli phage, then the collected E. coli cells are infected with M13KO7TC (WO 2015046554 A1) or Μ13ΚΟ7ΔρΙΙΙ (hyperphage) (Progen Biotechnik) and after culturing overnight at 30°C the phage collected from the supernatant. This type of panning is repeated until round 5.

Example (2-2-2). Assessment of binding activity in the presence and absence of ATP using phage ELISA.

From individual E. coli colonies obtained after each round by the methods described above, phage culture supernatants are collected using the usual method (Methods Mol. Biol., 178, 2002, 133-145). NucleoFast 96 (MACHEREY-NAGEL) is used for ultrafiltration of collected culture supernatants. The eluate is removed by centrifugation (4500 g, 45 min) of NucleoFast 96 plates, into which 100 μl of culture supernatant was added to each well. NucleoFast 96 is washed again by centrifugation (4500 g for 30 min), for which 100 μl of H ₂ O is added to each

- 105 046075 I’m blowing a hole. Finally, 100 μl of TBS is added, and the phage suspension contained in the supernatants in the wells of the NucleoFast 96 plate, which were left at room temperature for 5 min, is removed.

Purified phages supplemented with TBS or ATP/TBS are subjected to enzyme-linked immunosorbent assay (ELISA) using the following procedure. StreptaWell 96 microtiter plates (Roche) are immobilized overnight with 100 μl of TBS containing biotinylated antigens (hCD137-Fc-Bio or bio-hCD137) obtained in Example 1. After removing biotinylated antigens that are not bound to the plate by washing each well of the plate with Tris-buffered saline (TBST), the wells are blocked with 250 μl of 2% skim milk-TBS for 1 h or more. The 2% skim milk-TBS was removed, after which the antibody-presenting phages bound to the biotinylated antigen present in each well in the absence and presence of ATP, leaving the plates with the prepared purified phages added to each well at 37°C for 1 h. HRP-conjugated anti-M13 antibody (GE Healthcare, catalog no. 27-9421-01) diluted in TBS or ATP/TBS is added to each well washed with TBST or ATP/TBST and the plate is incubated for one hour. After washing with TBST or ATP/TBST, the color development of the solution in each well to which a single TMB solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm.

As a result, several antibodies were identified with altered binding activity to biohCD137 or hCD137-Fc-Bio in the presence and absence of ATP.

As an example, the results of phage ELISA using clones after 4 and 5 rounds of panning are shown in Table. 10.

In the present invention, clones with an absorbance of 0.2 or more in the presence of ATP and an absorbance S/N ratio greater than 2 in the presence/absence of antigen are defined as positive clones. In addition, among the positive clones, clones with an S/N absorption ratio greater than 2 times in the presence/absence of ATP are considered clones with ATP-dependent antigen-binding activity (switchable clones).

Table 10

Antigen used in panning hCD137Fc-Bio hCD137-FcBio Biotinylation of hCD137 Biotinylated hCD137 Panning method Liquid phase Liquid phase Liquid phase Solid phase on granules Helper phage used in panning Μ13ΚΟ7Δ рШ Μ13ΚΟ7Δρ III and M13KO7TS Μ13ΚΟ7ΔρΙΙΙ Μ13ΚΟ7ΔρΙΠ Number of clones subjected to ELISA 384 96 96 96 Number of positive clones (ATP absorption + > 0.2; antigen +/- ratio > 2.0) 383 79 41 29 Number of switched clones (ATP+/- ratio > 2) 375 77 40 29

(2-2-3) Analysis of the sequence of switchable antibodies, the antigen-binding activity of which is changed by the presence/absence of ATP.

The nucleotide sequences of genes amplified from clones with ATP-dependent antigen-binding activity (switched clones) based on phage ELISA results are analyzed using specific primers pBAD-F, G1seq-R. The analysis resulted in nucleotide sequences of clones that were found to bind to human CD137 in the presence of ATP, but not to human CD137 in the absence of ATP.

(2-3) Selection of switchable antibodies.

Seventeen samples were selected from clones that were found to have ATP-dependent antigen-binding activity from the assays in Examples 2-1-3 and 2-23; clone names were reassigned as described in Table. eleven.

- 106 046075

Table 11

Phage designation Antibody variable region clone designation 1 dBBATkl4-4_003 dBBAT007 2 dBBATkl4-4_089 dBBAT013 3 dBBATkl7-4_002 dBBAT015 4 dBBATkl7-4_009 dBBAT017 5 dBBATkl7-4_075 dBBAT019 6 dBBATk05-5_052 dBBAT021 7 dBBATk05-5_076 dBBAT025 8 dBBATkl2-5_038 dBBAT029 9 dBBATkl2-5_048 dBBAT031 10 dBBATkl4-5_012 dBBAT037 AND dBBATkl4-5_053 dBBAT042 12 dBBAHkl4SF-3_016 dBBAT053 13 dBBAHkl4SF-3_032 dBBAT056 14 dBBAHkl4FF-4_028 dBBAT091 15 dBBAHkl4FS-5_074 dBBAT112 16 dBBAHkl4MS-5_034 dBBAT118 17 dBBAHkl4MS-5_093 dBBAT119

(2-4) Expression and purification of switchable antibodies whose antigen-binding activity is altered by the presence/absence of ATP.

Genes encoding the variable regions of the antibodies described in table. 11, which are obtained from a rationally designed phage library, are inserted into human IgG1/Lambda plasmids for expression in animals. Antibodies are expressed using the methods described below. The prepared plasmids are introduced by lipofection into the FreeStyle 293-F cell-derived cell line obtained from a human fetal kidney (Invitrogen), which is suspended in FreeStyle 293 expression medium (Invitrogen) at a cell density of 1.33 x 10 ⁶ cells/ml, obtained the suspension is sown 3 ml into each well of 6-well plates. Antibodies are purified using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) from culture supernatants cultured for four days in a CO ₂ incubator (37°C, 8% CO ₂ , 90 rpm) by a method known to those skilled in the art. areas. Spectrophotometers are used to measure the absorbance of purified antibody solutions at 280 nm. From the obtained measurement results, the concentrations of purified antibodies were calculated using extinction coefficients calculated by the PACE method (Protein Science, 4, 1995, 2411-2423).

(2-5) Evaluation of anti-CD137 antibodies identified by phage display.

(2-5-1) Expression and purification of switchable antibodies that bind antigens depending on the presence or absence of ATP and its metabolites.

Genes encoding the variable regions of antibodies obtained from a rationally constructed human phage library are inserted into animal expression plasmids having a heavy chain constant region fused to a gene encoding Gly and Lys downstream of a modified human IgG1 (P253) (SEQ ID NO: 93 ), and a light chain constant region, the Lamlib lambda chain (SEQ ID NO: 63). Designations and serial numbers of clones are listed in table. 12.

- 107 046075

Table 12

Research clones

Clones SEQ ID NO of heavy chain (full length) SEQ ID NO light chain (full length) 1 dBBAT007-P253 94 FROM 2 dBBAT013-P253 95 114 3 dBBAT015-P253 96 115 4 dBBAT017-P253 97 116 5 dBBAT019-P253 98 117 6 dBBAT025-P253 99 118 7 dBBAT029-P253 100 119 8 dBBAT031-P253 100 119 9 dBBAT037-P253 101 120 10 dBBAT042-P253 102 121 AND dBBAT053-P253 103 122 12 dBBAT056-P253 104 123 13 dBBAT021-P253 105 124 14 dBBAT091-P253 106 125 15 dBBATl 12-P253 107 126 16 dBBATl 18-P253 108 127 17 dBBATl 19-P253 109 128 18 dBBAT121-P253 110 129 19 dBBAT122-P253 111 130 20 dBBAT134-P253 112 131

Once expressed, the antibodies are purified by methods known to those skilled in the art. Spectrophotometers are used to measure the optical density of purified antibody solutions at 280 nm. From the established values, the concentrations of purified antibodies are calculated using extinction coefficients calculated by the PACE method (Protein Science, 4, 1995, 2411-2423).

(2-5-2) Assessment by surface plasmon resonance of the effect of ATP, ADP and AMP on the binding of human CD137.

Biacore T200 (GE Healthcare) is used to analyze the antigen-antibody interaction between anti-CD137 antibodies and hCD137 (FXa cleavage). Ahtu-CD137 antibodies are captured on a CM3 sensor chip (GE Healthcare) on which Protein G (CALBIOCHEM) has been immobilized in appropriate amounts using the amine coupling method, and hCD137 (FXa cleavage) obtained in Example 1-2 can react interact. 20 mM ACES, 150 mM NaCl, 2 mM MgC12, 0.05% Tween20 (pH 7.4) and 10 mM glycine-HCl (pH 1.5) are used as the running buffer.

After capturing anti-CD137 antibodies suspended in TBS, 500 nM hCD137 (FXa digestion) was injected into each flow cell at a flow rate of 10 μL/min for three minutes. This three-minute period serves as the binding phase for hCD137 (FXa cleavage), and after the end of the binding phase, a two-minute period of switching to the running buffer serves as the dissociation phase for hCD137 (FXa cleavage). After completion of the dissociation phase, the regenerating solution is administered at a rate of 30 μl/min for 30 s. Above is a cycle for measuring the binding activity of anti-CD137 antibodies. The binding amount of hCD137 (cleaved FXa) that interacts with the anti-CD137 antibody in the binding phase is controlled by the amount of antibody captured. Biacore T200 Evaluation Software Version 2.0 is used to display the binding amount (RU) per 1RU of captured ligand, and the values of the amount of antibody captured and the amount of antigen bound are obtained. The amount of bound antigen is shown in Table. 13. Since the amount of antigen bound reflects binding activity, small molecule dependence can be said to be recognized when the value in the presence of the small molecule compound (ATP, ADP or AMP) is higher than the value without the small molecule compound. In particular, the greater the difference, the greater the dependence on the small molecule compound.

Table 13

Clone Low molecular weight compound, concentration ATP 1 mM ATP 100 mM ADP 100 mM ADP 10 mM AMP 1 mM AMP 100 mM No low molecular weight compound dBBAT007-P253 49.2 29.5 89.0 70.0 28.5 16/5 3.6 dBBAT013-P253 26.2 8.1 77.3 48.5 13.8 6.6 2.7 dBBAT015-Р253 8.3 3.1 41.8 17.9 I,9 5.5 1.5 dBBAT017-P253 9.8 5.0 39.7 18.8 6.8 4.8 4.4

- 108 046075

dBBAT019- P253 3.8 3.0 25.7 7.4 11.1 4.6 3.7 dBBAT021-P253 9.2 2.4 56.6 19.6 5.7 3.0 2.9 dBBAT025-P253 28.5 10.0 89.1 55.7 16.4 6.1 4.4 dBBAT029-P253 10.9 6.9 53.3 38.5 17.4 11.4 3.7 dBBAT031-P253 8.4 5.0 47.8 33.3 14.8 8.3 2.8 dBBAT037-P253 27.5 15.9 72.1 62.9 21.7 13.8 3.8 dBBAT042-P253 11.7 4.3 58.7 19.1 7.8 4.7 2.9 dBBAT053- P253 15.0 8.4 46.9 40.4 9.6 6.9 1.9 dBBAT056-P253 20.3 13.4 59.4 50.7 12.8 9.7 3.7 dBBAT118-P253 27.7 14.1 76.9 62.9 14.9 8.7 3.3 dBBAT121-P253 11.5 4.8 42.3 24.7 6.1 2.6 1.9 dBBAT122-P253 11.5 4.7 55.5 19.2 5.7 4.1 3.3 dBBAT134-P253 5.2 4.4 29.3 16.2 3.7 4.5 3.0 dBBAT091-P253 6.2 3.0 33.0 20.0 2.9 3.3 1.5 dBBAT112-P253 12.6 6.3 43.0 27.4 6.5 4.8 3.7 dBBAT119-P253 45.7 12.9 112.5 67.2 58.6 26.9 3.3

(2-5-3) Evaluation of binding activity to monkey CD137 antigen.

The resulting antibodies were assessed for binding to monkey CD137 by ELISA. The cyCD137-Fc-BAP obtained in Example 1 was immobilized on Streptawell microtiter plates. After removing unbound antigen from the plates by washing each well of the plates with wash buffer, the wells are blocked with 150 μl of blocking buffer (TBS with 2% BSA) for 1 hour or more. The blocking buffer is removed from each well and 100 μl of purified antibodies diluted in TBS with a final concentration of 1 mM ADP or TBS with a final concentration of 1 mM ADP is added to each well. The plate into which the antibody was added was shaken at 600 rpm for 1 hour. AP-conjugated anti-human lambda antibodies (BETHYL), diluted in TBS with a final concentration of 1 mM ADP, were added to each well after washing with wash buffer (TBS with 0.1% Tween20) containing a final concentration of 1 mM ADP. After incubation for one hour and washing with wash buffer with a final concentration of 1 mM ATP, BluePhos phosphate substrate (KPL) is added. Color development is measured by absorbance at 600 nm. The rate of increase in absorption at an antibody concentration of 0 μg/ml is shown in table. 14. Samples with a concentration-dependent increase in absorption coefficient can be said to be associated with monkey CD137.

Table 14

Absorption coefficient

CD 137 monkeys dBBAT007 dBBAT013 dBBAT015 dBBAT017 dBBAT019 dBBAT021 dBBAT025 dBBAT029 dBBAT037 dBBAT042 Antibody concentration (µg/ml) 10 30.5 43.2 42.9 25.6 40.5 40.5 72.0 50.3 32.5 41.8 2.5 30.2 42.4 32.4 8.7 40.3 14.0 67.6 42.7 12.7 40.0 0.625 29.2 33.3 14.4 3.1 39.1 4.6 49.7 26.7 4.6 31.3 0.156 22.0 15.3 3.6 1.4 23.7 1.7 20.7 9.7 1.9 12.2 0.039 0.7 4.2 1.5 1.2 8.2 1.2 4.5 2.0 1.3 2.9 0.010 3.8 1.5 1.1 1.2 2.5 0.9 1.6 1.1 1.1 1.3 0 1.0 ι,ο 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 CD 137 monkeys dBBAT053 dBBAT056 dBBAT091 dBBAT112 dBBAT118 dBBAT119 dBBAT121 dBBAT122 dBBAT134 Antibody concentration (µg/ml) 10 17.9 23.1 15.1 19.8 35.4 55.3 26.0 31.9 33.9 2.5 6.9 10.7 5.5 8.1 30.6 56.6 16.4 16.7 16.8 0.625 2.3 3.6 1.7 2.8 18.1 57.2 5.9 5.7 5.8 0.156 1.0 1.3 0.7 1.0 7.1 47.7 1.3 1.9 2.2 0.039 0.7 0.8 0.5 0.6 1.8 26.6 0.6 1.0 1.2 0.010 0.8 1.2 0.6 0.6 1.0 10.2 0.6 0.9 1.0 0 ι,ο ι,ο 1.0 1.0 1.0 1.0 1.0 1.0 1.0

(2-5-4) Evaluation of CD137 agonist activity using Jurkat cells.

The GloResponse™ NF-kappa B-Luc2/4-1BB Jurkat cell line (Promega) is used for

- 109 046075 measurements of antibody activity in vitro. To each well of 384-well plates, add 10 μl of FcyRIIB CHO-K1 cells (Promega), prepared at a concentration of 2x10 ⁶ /ml in assay medium (99% RPMI, 1% PBS). Then, 10 μl of an antibody solution containing ADP, or an antibody solution containing ATP, or an antibody solution not containing ATP or ADP, is added to each well. Then 10 μl of the GloResponseTM NF-kB-Luc2/4-1BB Jurkat cell line, prepared at a concentration of 2x10 ⁶ /ml in assay medium (99% RPMI, 1% PBS), is added to each well. The final concentration of ADP is 50 μM; the final ATP concentration is 50 µM. The plates are left for six hours at 37°C in a 5% CO ₂ incubator, then 15 minutes at room temperature and 30 μl of Bio-Glo reagent is added to each well. The Bio-Glo Luciferase Assay System (buffer and substrate) is used for the Bio-Glo reagent. Subsequently, the luminescence of each well is measured using a plate reader. The luminescence value of each well divided by the luminescence value of the well without added antibodies represents the luminescence fold and serves as an indicator for assessing the activity of each antibody.

The results obtained are shown in Fig. 1 and 2. FIG. 1 and 2 confirm that all antibodies except NS1-P253, which is not a switch antibody, exhibit human CD137 agonist activity in an ATP- and ADP-dependent manner.

(2-6) Evaluation of CD137 agonistic activity in vitro of switchable panned antibodies using human T cells.

(2-6-1) Propagation of human T-cell cultures.

Human peripheral blood mononuclear cells isolated from blood samples of healthy volunteers are used. 50 ml of blood is mixed with 0.5 ml of heparin and further diluted with 50 ml of PBS. Human peripheral blood mononuclear cells are isolated in two steps, described below. In the first step, Ficoll-Paque PLUS (GE Healthcare) is added to Leucosep tubes (Greiner Bio-one) and centrifuged at 1000xg for one minute at room temperature, followed by the addition of blood diluted with PBS and centrifuged at room temperature at 400xg in for 30 minutes. In the second step, the buffy coat is collected from the tubes after centrifugation and then washed with 60 ml of PBS (Wako). A human T cell activation/expansion kit (MACS Miltenyi biotec) is then used to expand the T cell cultures.

(2-6-2) Evaluation of CD137 agonist activity in vitro using human T cells.

2 x 10 ⁴ T cells derived from peripheral blood mononuclear cells and 2 x ^{10 4} REC-1 cells are suspended in 100 μl of RPMI1640 medium containing 30 U/ml human IL-2 (SIGMA), 10 ng/ml PMA (SIGMA) , 0.5 μg/ml ionomycin, 500 μM ADP-beta-S (Sigma) and 10% FST (Sigma), seeded in 96-well flat-bottom multiwell plates (Corning) with control antibody IC17HdK-hIgG1/ IC17L-k0, NS1-P253, which is an antibody with ATP-independent binding activity against human CD137 (referred to in all examples herein as a non-switchable antibody or a non-switchable anti-CD137 antibody), or any of the clones described in Table 1. 12. NS1-P253, IC17HdK-hIgG1/IC17L-k0 and clones described in table. 12, evaluated at a concentration of 10 μg/ml. IFN-γ production is also assessed in ADP-beta^-minus medium. The plates are shaken and then left at 37°C for 72 hours in an incubator under 5% CO2. The culture supernatants are then collected and the amount of IFN-γ contained in the culture supernatants is determined using the Human IFN-γ ELISA Development Kit (PeproTech). The ELISA method is carried out in accordance with the manufacturer's instructions (PeproTech). Absorbance measurements are carried out using an EnVision instrument (PerkinElmer).

The results are shown in Fig. 3.

It has been confirmed that dBBAT007-P253, dBBAT013-P253, dBBAT015-P253, dBBAT019-P253, dBBAT021-P253, dBBAT025-P253, dBBAT031-P253, dBBAT042-P253, dBBAT056-P253, dBBAT118-P253 , dBBAT121-P253, dBBAT122- P253 and dBBATl 19-P253 exhibit agonistic activity against human CD137 in an ADP-beta^-dependent manner. ADP-beta-S is an analogue of ADP that is less susceptible to hydrolysis than ADP. This raises the possibility that these human hhtu-CD137 switch antibodies exhibit agonistic activity against human CD137 dependent on a small molecule such as ATP, ADP or AMP.

(2-6-3) Evaluation of CD137 agonist activity in vitro using human T cells (2).

2x10 ⁴ human peripheral blood mononuclear cells derived from T cells and 2x10 ⁴ REC-1 cells were suspended in 100 μl of RPMI1640 medium containing 30 U/ml human IL-2 (SIGMA), 10 ng/ml PMA (SIGMA ), 0.5 μg/ml ionomycin, 500 μM ADP-beta-S (Sigma) and 10% FST (Sigma), and seeded in 96-well flat-bottom plates (Corning), or CNS1-P253 (non-switchable antibody ), or with dBBAT119-P253. NS1-P253 and dBBAT119-P253 were evaluated at concentrations of 10, 2, 0.4, 0.08, 0.016, 0.0032, and 0.00064 μg/mL. The plates are shaken and left at 37°C for 72 hours in an incubator under 5% CO2. The culture supernatants were then collected and the amount of IFN-γ contained in the culture supernatants was determined using a human IFN-γ ELISA kit (PeproTech). Absorbance measurements are carried out using

- 110 046075

EnVision (PerkinElmer).

The results are presented in Fig. 4.

dBBAT119-P253 was confirmed to exhibit agonistic activity against human CD137 in the presence of ADP-beta-S. This raises the possibility that dBBAT119-P253 exhibits agonistic activity against human CD137 in a small molecule-dependent manner such as ATP, ADP, or AMP.

Example 3: Enhancing the binding activity of antibodies that bind antigens in the presence of small molecules using rationally designed light and heavy chain libraries.

(3-1) Library generation to enhance binding activity using a rationally designed light chain library.

For an antibody library containing a large number of antibodies collected in Example 2-2-1 with ATP-dependent antigen-binding activity, enhancement of the binding activity is accomplished by re-librarianship of the antibody light chains.

The light chain and heavy chain regions of the rationally designed antibody phage display library constructed in the previous patent WO 2015/083764 are used to construct an antibody light chain library and an antibody heavy chain library to enhance binding activity. They are introduced into the above light chain library or into the light chain or heavy chain region of the phagemid vector library assembled in Example 2-2-1 and introduced into E. coli strain ER2738 by electroporation.

(3-2) Enhancing the binding activity of antibodies with ATP-dependent antigen-binding activity using a rationally designed library.

Phages are produced in the usual manner from E. coli carrying an engineered phage display phagemid. Specifically, an E. coli strain carrying the constructed phagemid vector is infected with M13KO7TC (WO 2015/046554) or Μ13ΚΟ7ΔρΙΙΙ (hyperphage) (PROGEN Biotechnik), and the phages are collected from the supernatants after overnight cultivation at 30°C. A phage library suspension is prepared by diluting the phage population with TBS, precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture in which the phage was produced. BSA is then added to the phage library solution to a final concentration of 4%. Panning is carried out using antigen immobilized on magnetic beads. NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated), NeutrAvidin beads (TAMAGAWA SEIKI) or Dynabeads MyOne StreptAvidin T1 (Thermo Fisher Scientific) are used as magnetic beads. Biotinylated hCD137-Fc is used as an antigen.

Panning is carried out to efficiently produce small molecule switchable antibodies that depend on a small molecule that can act as a switch in cancerous tissues. In particular, panning to enrich antibodies that bind to antigens in the presence of adenosine triphosphate (adenosine-5'-triphosphate; ATP), a small molecule, and does not bind to antigens in the absence of ATP, is performed with reference to the methods set forth in the previous WO 2015 patent. /083764.

(3-3) Assessment of binding activity in the presence and absence of ATP using phage ELISA.

From individual E. coli colonies obtained after each round by the methods described above, phage culture supernatants are collected using the usual method (Methods Mol. Biol., 178, 2002, 133-145).

Binding activity to human CD137 in the presence and absence of ATP was then confirmed by phage ELISA methods described in Example 2-2-2.

The results are presented in Fig. 5.

A number of clones were obtained that were found to have ATP-dependent antigen-binding activity with an S/N uptake ratio greater than 2 in the presence/absence of antigen and an S/N uptake ratio greater than 2 in the presence/absence of ATP (switched clones).

Example 4. Production of modified anti-CD137 antibodies and evaluation of their activity.

(4-1) Increased binding activity by changing dBBAT119H-P253/dBBAT119LLamLib.

The heavy chain variable region, dBAT119H, and light chain variable region, dBAT119L, anti-CD137 antibody variants (clone name: dBBAT119H-P253/dBAT119L-LamLib) obtained in Example 2-4 are prepared by methods known to those skilled in the art, e.g. PCR. For the heavy chain variable region, variants are created in which D10 (referring to aspartic acid (Asp) at position 10 (Kabat numbering) and G17 (referring to glycine (Gly) at position 17 (Kabat numbering)) dBBAT119H are replaced with glycine ( Gly) and serine (Ser), respectively, yielding dBBAT119H010; and N99 (for asparagine (Asn) at position 99 (Kabat numbering)), M100a (for methionine (Met) at position 100a (Kabat numbering)) and N100b (for asparagine (Asn) at position 100b (numbering by Kabat) )) dBBAT119H010 are replaced by other amino acids. For the light chain variable region, variants have been created in which F87 (related to phenylalanium

- 111 046075 well (Phe) at position 87 (Kabat numbering)) dBBAT119L is replaced by tyrosine (Tyr) to form dBBAT119L010; and D27b (referring to aspartate (Asp) at position 27b (Kabat numbering), N31 (referring to asparagine (Asn) at position 31 (Kabat numbering)) and D94 (referring to aspartate (Asp) at position 94 (Kabat numbering) according to Kabat))) in dBBAT119L010 are replaced by other amino acids.

For heavy chain variable region variants, binding activity to human CD137 is measured using a BiacoreT200 Surface Plasmon Resonance Analyzer (GE Healthcare). Antibodies are captured by reacting purified variants with immobilized protein G (CALBIOCHEM) on a CM3 S series sensor chip (GE Healthcare). A solution of human CD137 (FXa digest) with ATP added or a solution of human CD137 (FXa digest) without ATP is then reacted in the presence of ATP and in the absence of ATP to evaluate the binding activity of the variants towards human CD137 (FXa digest). Measurements are carried out at 25°C using a running buffer containing 20 mM ACES, 150 mM NaCl, 0.05% Tween20 and 2 mM MgCl ₂ , pH 7.4. The measurement results show that the L100a variant (a variant in which methionine (Met) at position 100a (Kabat numbering) is replaced by leucine (Leu)) has increased binding activity to human CD137 only in the presence of ATP (Fig. 6). The heavy chain variable region of the L100a variant is dBATk119H024 (SEQ ID NO: 132). The binding amount of human CD137 is calibrated against the captured amount of each variant (1000 RU).

For light chain variable region variants, binding activity to human CD137 (FXa cleavage) was determined using BiacoreT200 under conditions similar to those described above. The measurement results show that the E94 variant (a variant in which aspartate (Asp) at position 94 (Kabat numbering) is replaced by glutamate (Glu)) has increased binding activity only to human CD137 in the presence of ATP (Fig. 6). The light chain variable region of the E94 variant is dBBATk119L020 (SEQ ID NO: 133).

Such variants combining these heavy chain and light chain variants are abbreviated as dBBATk119H024-P253/dBATk119L020-LamLib (heavy chain variable region SEQ ID NO: 132; light chain variable region SEQ ID NO: 133; heavy chain constant region SEQ ID NO: 133 : 93 and light chain constant region SEQ ID NO: 63).

Antibodies in the present invention are designated according to the following rule: (heavy chain variable region) - (heavy chain constant region)/(light chain variable region) - (light chain constant region).

For example, the antibody name dBBAT119H-P253/dBAT119L-LamLib means that it consists of an antibody heavy chain variable region - dBAT119H, an antibody heavy chain constant region - P253, an antibody light chain variable region - dBBAT119L, and a LamLib light chain constant region.

(4-2) Evaluation of the CD137 agonist activity of modified human anti-CD137 antibodies in vitro using human T cells.

(4-2-1) Propagation of human T-cell cultures.

Human T cells are expanded and cultured as described in Examples 2-6-1.

(4-2-2) Evaluation of CD137 agonist activity in vitro using human T cells.

The methods described in Example 2-6-2 evaluate IFN-γ production in the presence of NS1-P253 (non-switchable antibody), dBBAT119H-P253/dBAT119L-LamLib, dBBATk119H024P253/dBATk119L020-LamLib or control antibody, IC17HdK-hIgG1/IC17 L-k0 at 10, 2, 0.4, 0.08 and 0.016 μg/ml, respectively. The production of IFN-γ in ADP-beta^-minus medium is also assessed.

Both results are shown in Fig. 7.

The modified antibody dBBATk119H024-P253/dBBATk119L020-LamLib exhibits enhanced ADP-beta^-dependent agonist activity compared to dBBAT119H-P253/dBBAT119LLamLib. This raises the possibility that dBBATk119H024-P253/dBATk119L020-LamLib exhibits stronger ATP-, ADP-, and AMP-dependent agonist activity against human CD137 compared to dBBAT119H-P253/dBBAT119L-LamLib.

Example 5 Additional Modifications of Anti-CD137 Antibodies (5-1) Search for modifications that increase binding activity by making complex modifications.

To obtain high quality anti-CD137 antibodies, amino acid modifications are clearly introduced into the heavy chain variable region, dBBATk119H024, and the light chain variable region, dBBATk119L020, of the anti-CD137 antibodies obtained in Examples 4-1. Using methods known to those skilled in the art, such as PCR, variants were created in which each of all amino acid residues comprising the dBBATk119H024 and dBBATk119L020 CDRs were replaced with all 18 amino acids except cysteine. Measurements of approximately 1200 variants produced to bind human CD137 are performed using Biacore4000. Antibodies are captured by reacting culture supernatants of variants with immobilized protein G (CALBIOCHEM) on a CM3 S series sensor chip (GE Healthcare). Then, a solution of human CD137 with the addition of small molecules (ATP) or a solution of human CD137 without the addition of small molecules is subjected to

- 112 046075 interaction in the presence of a small molecule or in the absence of a small molecule to evaluate the binding activity of antibodies against human CD137. Measurements are carried out at 25°C using a CaCl ₂ -supplemented running buffer containing 20 mM ACES, 150 mM NaCl, 0.02% Tween20 and 2 mM MgCl ₂ , pH 7.4.

(5-2) Increased ATP binding.

The antibody heavy chain gene A002-P253 (SEQ ID NO: 134) was created by combining the modifications identified in Example 5-1, which increase binding activity to human CD137 in the presence of a small molecule, in the dBBATk119H024-P253 gene having dBBATk119H024 (SEQ ID NO : 132) as the heavy chain variable region, and P253 (SEQ ID NO: 93) as the heavy chain constant region, which is obtained by introducing the S267E/L328F modification and removing the C-terminal Gly and Lys from human IgG1. The B040-Lamlib antibody light chain gene (SEQ ID NO: 135) is also created by combining the modifications found in Example 5-1, which increase binding activity to human CD137 in the presence of a small molecule, into the dBBATk119L020-Lamlib antibody light chain having dBBATk119L020 ( SEQ ID NO: 133) as the variable region light chain human λ Lamlib (SEQ ID NO: 63) as the constant region light chain. These genes are combined to express and purify antibodies by methods known to those skilled in the art to produce the anti-CD137 antibody of interest, A002-P253/B040-Lamlib. The heavy chain variable region of A002-P253/B040-Lamlib is A002 (SEQ ID NO: 136), the light chain variable region is B040 (SEQ ID NO: 137), the heavy chain constant region is P253 (SEQ ID NO: 93), and the light chain constant region is the human λ Lamlib chain (SEQ ID NO: 63).

For the heavy chain variable region A002-P253/B040-Lamlib obtained in this section, various variants are created in which the 53rd, 54th or 55th amino acids of the Kabat numbering are replaced with other amino acids in order to increase the level of ATP binding activity . Table 15 shows amino acid modifications (Kabat numbering) from A002 in the variable regions of the developed antibodies.

Table 15

Heavy chain variable region Amino acid changes from A002 (Kabat numbering) A002 — A146 S54A/N55S A159 R53S/N55S A160 R53T/N55S A161 R53Q/N55S A162 R53K/N55S A163 R53H/N55S A164 R53S/N55T A165 R53T/N55T A166 R53Q/N55T A167 R53K/N55T A168 R53H/N55T A169 R53S/N55H A170 R53T/N55H A171 R53Q/N55H

The binding activity of the produced variants towards ATP and human CD137 was assessed using Biacore T200.

ATP binding activity measurements are performed at 37°C using running buffer containing 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ and 0.05% Tween20. First, antibodies are captured by reacting antibody solutions in running buffer with Sure Protein A (GE Healthcare) immobilized on a CM3 Series S sensor chip (GE Healthcare). The binding activity of the antibodies is then assessed by interaction with an ATP solution prepared in a running buffer. The chip was regenerated using 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and measurements were made by repeated antibody capture. The amount of ATP binding for each antibody is calculated by adjusting the amount of ATP binding when injected at 100 nM by the amount of antibody captured on the chip surface as the amount of ATP per unit amount of antibody.

Measurement of binding activity towards human CD137 is carried out at 37°C using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ and 0.05% Tween20 as running buffer. First, antibodies are captured by reacting antibody solutions prepared in running buffer with Sure Protein A (GE Healthcare) immobilized on a CM3 Series S sensor chip (GE Healthcare). Human CD137 binding activity is then assessed by reacting with a solution of human CD137 supplemented with 100 μM small molecule ATP.

- 113 046075

For the human CD137 antigen, hCD137-HisBAP obtained in Example (1-1) is used, and measurements are carried out at an antigen concentration of 0, 15,625, 62.5, 250 or 1000 nM. The chip was regenerated using 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and measurements were made by repeated antibody capture. The dissociation constant (KD) of the corresponding antibodies for human CD137 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the association rate constant ka (L/mol/sec) and the dissociation rate constant kd (1/sec) are calculated by globally fitting the sensorgrams obtained by measuring with the 1:1 Langmuir binding model and the dissociation constant KD (mol/L ) are calculated from these values.

In table Figure 16 presents the results of such measurements.

Table 16

Binding assay for human ATP and CD137

Antibody ATP binding Binding parameters to human CD 137 (ATP = 100 µM) ka kd KD A002-P253/B040-Lamlib -0.0001 3.94E+04 2.89E-03 7.35E-08 A146-P253/B040-Lamlib -0.0001 7.80E+04 2.32E-02 2.97E-07 A159-P253/B040-Lamlib 0.0013 8.21E+04 3.83E-02 4.66E-07 A160-P253/B040-Lamlib -0.0001 9.59E+03 2.46E-02 2.56E-06 A161-P253/B040-Lamlib 0.0010 l,87E+04 2.35E-02 l,25E-06 A162-P253/B040-Lamlib 0.0002 l,47E+05 3.17E-02 2.16E-07 A163-P253/B040-Lamlib 0.0016 5.60E+04 l,36E-02 2.43E-07 A164-P253/B040-Lamlib 0.0017 9.81E+04 4.38E-02 4.46E-07 A165-P253/B040-Lamlib 0.0002 1D4E+04 l,96E-02 l,72E-06 A166-P253/B040-Lamlib 0.0011 l,21E+04 l,92E-02 l,59E-06 A167-P253/B040-Lamlib 0.0001 8.45E+04 3.96E-02 4.69E-07 A168-P253/B040-Lamlib 0.0017 7.01E+04 l,81E-02 2.58E-07 A169-P253/B040-Lamlib 0.0011 1D5E+04 5.88E-03 5.11E-O7 A170-P253/B040-Lamlib 0.0000 5DZE+OZ 5.36E-03 l,04E-06 A171-P253/B040-Lamlib 0.0012 4.93E+03 5.24E-03 l,06E-06

As shown in table. 16, variants in which the 53rd, 54th or 55th amino acids (Kabat numbering) of the heavy chain variable region are replaced by other amino acids have increased ATP-binding activity compared to the A002-P253/B040-Lamlib antibody before modification, for with the exception of A146-P253/B040-Lamlib and A160-P253/B040-Lamlib. Comparison of the association rate constant ka (l/mol/sec) also shows that the association rate constant for human CD137 is increased for antibodies A146-P253/B040-Lαmlib, A159-P253/B040-Lamlib, A162-P253/B040-Lamlib, A163 -P253/BO4OLamlib, A164-P253/B040-Lamlib, A167-P253/B040-Lamlib and A168-P253/B040-Lamlib are among the antibodies rated above.

(5-3) Increasing binding activity by introducing complex modifications.

To create better anti-CD137 antibodies, the modifications identified in Example 5-1, which increase the binding activity of human CD137 in the presence of a small molecule and reduce binding to human CD137 in conditions where the small molecule is absent, and the modifications identified in Examples 5- 2, which increase ATP binding activity and increase the association rate constant for human CD137, were combined to produce antibodies against human CD137 that exhibit a better profile. Antibody heavy chain genes were developed that combine the modifications found in Examples 5-1 and 5-2 in the A002-G1T3 antibody heavy chain gene having the sequence A002 (SEQ ID NO: 136) as the heavy chain variable region and the sequence G1T3 (SEQ ID NO: 138) as a heavy chain constant region, which was obtained by introducing the modification K214R/Q419E and removing the C-terminal Gly and Lys from human IgG1. Antibody light chain genes are prepared by combining the modifications found in Example 5-1 in the B040-Lamlib antibody light chain having the sequence B040 (SEQ ID NO: 137) as the light chain variable region and the human Lamlib λ chain as the constant region light chain.

The antibody heavy chain gene 20H4.9-P253 having the heavy chain variable region 20H4.9 (SEQ ID NO: 139) of the existing anti-CD137 antibody described in US 8137667 and P253 (SEQ ID NO: 93) is used as an object for comparison. as a heavy chain constant region; and an antibody light chain gene that combines the light chain variable region of 20H4.9LC (SEQ ID NO: 140) with the human k chain k0 (SEQ ID NO: 141) as the light chain constant region. As another subject of comparison, the antibody heavy chain gene MOR-7480.1H-P253 having the heavy chain variable region of MOR-7480.1H (SEQ ID NO: 142), which constitutes the existing anti-CD137MOR7480.1 antibody described in US 8337850, and P253 (SEQ ID NO: 93) as the heavy chain constant region; and a MOR-7480.1L-lam antibody light chain combining the MOR-7480.1L light chain variable region (SEQ ID NO: 143) with the human λ chain lam (SEQ ID NO: 63) as a constant region

- 114 046075 light chain (note: both human Lamlib λ chain and human lam have the same amino acid sequence (SEQ ID NO: 63)).

These genes are combined for antibody expression and purification using methods known to those skilled in the art to produce anti-CD137 antibodies of interest. Table 17 is a list of sequence numbers of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and hypervariable region (hypervariable region; also called HVR or CDR) of the developed antibodies.

Table 17

Amino acid sequences of heavy chains, light chains and their hypervariable regions (sequences are designated by numbers (ID))______

Antibody Variable region Constant region Hypervariable region (HVR) Heavy chain Light chain Heavy chain Light chain H1 H2 NZ L1 L2 L3 A375-G1T3/B167-Lamlib 43 54 138 63 7 8 17 21 26 27 A372-GlT3/B040-Lamlib 44 55 138 63 7 9 17 22 26 27 A356-GlT3/B040-Lamlib 45 55 138 63 7 10 17 22 26 27 A486-G1T3/B167-Lamlib 46 54 138 63 7 eleven 18 21 26 27 A487-G1T3/B167-Lamlib 47 54 138 63 7 8 18 21 26 27 A488-G1T3/B226-Lamlib 48 56 138 63 7 12 18 21 26 28 A489-G1T3/B223-Lamlib 49 57 138 63 7 13 18 21 26 29 A548-G1T3/B376-Lamlib 50 58 138 63 7 14 19 23 26 27 A551-G1T3/B256-Lamlib 51 59 138 63 7 15 20 24 26 27 A551-G1T3/B379-Lamlib 51 60 138 63 7 15 20 25 26 27 A555-G1T3/B379-Lamlib 52 60 138 63 7 16 20 25 26 27 A548-G1T3/B256-Lamlib 50 59 138 63 7 14 19 24 26 27 A549-G1T3/B167-Lamlib 53 54 138 63 7 14 17 21 26 27

Binding of the designed variants to ATP and human CD137 was assessed using Biacore T200. Human CD137 binding measurements were performed at 37°C using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ and 0.05% Tween20 as running buffer. First, 250-400 RU of antibodies are captured by reacting antibody solutions prepared in running buffer with Sure Protein A (GE Healthcare) immobilized on a CM3 Series S sensor chip (GE Healthcare). Binding activity to human CD137 in the presence or absence of ATP is then assessed by reacting with a solution of human CD137 prepared in running buffer supplemented with the desired concentration of ATP or with a solution of human CD137 prepared in running buffer without ATP. For the human CD137 antigen, hCD137HisBAP obtained in Example (1-1) is used, and KD value measurements are carried out at antigen concentrations of 0, 15,625, 62.5, 250 and 1000 nM. To estimate the bound amount, measurements are taken at 0 and 1000 nM antigen concentrations. The chip was regenerated with 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and measurements were made by multiple antibody capture. The dissociation constant of the corresponding antibodies for human CD137 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the association rate constant ka (l/mol/s) and the dissociation rate constant kd (1/s) are calculated by globally fitting the sensorgrams obtained by measuring with the 1:1 Langmuir binding model and the dissociation constant KD (mol/l ) are calculated from these values.

In table Figure 18 presents the results of such measurements.

- 115 046075

Table 18

Analysis of the binding of modified antibodies to human CD137

Antibody name Linking to human CD 137 Without ATP ATP 1 µM ATP 10 µM ATP 100 µM ADP 10 µM AMP 10 µM 20H4.9-P253/20H4.9LCк0 0.280 0.283 0.283 0.283 0.287 0.287 MOR-7480.1H- P253/MOR-7480.1L-Iam 0.260 0.261 0.261 0.261 0.266 0.266 A375-G1T3/B167-Lamlib 0.053 0.179 0.255 0.275 0.280 0.276 A372-GlT3/B040-Lamlib 0.017 0.135 0.242 0.269 0.284 0.245 A356-GlT3/B040-Lamlib 0.008 0.083 0.211 0.262 0.278 0.255 A486-G1T3/B167-Lamlib 0.128 0.260 0.282 0.292 0.294 0.289 A487-G1T3/B167-Lamlib 0.110 0.229 0.270 0.282 0.283 0.279 A488-G1T3/B226-Lamlib 0.042 0.135 0.232 0.268 0.281 0.261 A489-G1T3/B223-Lamlib 0.004 0.095 0.214 0.257 0.276 0.261 A548-G1T3/B376-Lamlib 0.012 0.149 0.245 0.270 0.270 0.262 A551-G1T3/B256-Lamlib 0.006 0.133 0.254 0.285 0.285 0.275 A551-G1T3/B379-Lamlib 0.014 0.173 0.271 0.290 0.291 0.283 A555-G1T3/B379-Lamlib 0.0017 0.180 0.267 0.282 0.282 0.274 A548-G1T3/B256-Lamlib 0.009 0.156 0.257 0.284 0.287 0.276 A549-G1T3/B167-Lamlib 0.048 0.209 0.261 0.277 0.279 0.272

Antibody name KD (M) for CD 137 person Without ATP 1 µM ATP 10 µM ΑΤΦ 100 µM ATP 10 µM ADP 10 µM AMP 20H4.9-P253/20H4.9LCк0 2.68E08 2D7E08 2.03E08 2.04E-08 l,90E08 2D7E-08 MOR-7480.1H- P253/MOR-7480.1L-Iam 8.40E08 8.40E08 7.50E08 6.31E-08 7.30E08 8.46E-08 A375-G1T3/B167-Lamlib ND 4.43E07 6D7E08 l,35E-08 5.4E-09 8.91E-09 A372-GlT3/B040-Lamlib ND 1D4E06 1.58E- 07 5.25E-08 9.36E09 l,47E-07 A356-GlT3/B040-Lamlib ND ND 4.90E07 1D9E-07 3.31E08 l,34E-07 A486-G1T3/B167-Lamlib 1.04E06* 7.30E08 l,08E08 3.22E-09 2.48E09 4.33E-09 A487-G1T3/B167-Lamlib 1.43E- 1.63E- 2.70E- 7.33E-09 3.01E- 3.86E-09 06* 07 08 09 A488-G1T3/B226-Lamlib ND 4.24E07 1.33E07 5.77E-08 l,60E08 6.80E-08 A489-G1T3/B223-Lamlib ND 4.42E06 3.45E07 l,59E-07 5.45E08 l,05E-07 A548-G1T3/B376-Lamlib ND 6.75E07 7.69E08 l,53E-08 1.55E- 08 3.71E-08 A551-G1T3/B256-Lamlib ND 1.23E06 1.44E07 2.91E-08 2.81E08 6D2E-08 A551-G1T3/B379-Lamlib ND 5.59E07 7D7E08 l,58E-08 1.51E- 08 3.25E-08 A555-G1T3/B379-Lamlib ND 4.77E07 5.84E08 l,35E-08 1.31E08 2.75E-08 A548-G1T3/B256-Lamlib ND 9.31E07 l,08E07 2.63E-08 2D4E- 08 4.61E-08 A549-G1T3/B167-Lamlib ND 2.04E07 2.83E08 6D2E-09 ZAZEOO l,35E-08

ND (no data) - activity is too low to determine the KD value ^KD value is determined using the steady state model

The binding values for human CD137 are in the table. 18 above indicate the amount of human CD137 binding per unit amount of antibody when human CD137 is allowed to interact at 1000 nM at each ATP concentration above, and KD(M) for human CD137 indicates the dissociation constant of human CD137 at each ATP concentration. KD values marked with * in the table were calculated using the steady state model. The amount of binding of all designed variants in the presence of 10 μM ATP is greater than in the presence of 1 μM ATP, and even greater in the presence of 100 μM, suggesting that they bind to human CD137 in an ATP concentration-dependent manner. On the other hand, the comparison objects, 20H4.9P253/20H4.9LC-k0 and MOR-7480.1H-P253/MOR-7480.1L-lam, do not exhibit binding to human CD137 in an ATP concentration-dependent manner.

(5-4) In vitro assessment of ATP-dependent agonist activity against CD137 modified

- 116 046075 bathed antibodies against human CD137 using the 4-1BB Jurkat reporter gene assay.

The GloResponse™ NF-kB-Luc2/4-1BB Jurkat cell line (Promega, CS196004) was used to measure in vitro activity of the developed variants. To each well of 96-well plates, add 200 μl of a suspension of FcyRIIB CHO-K1 cells (Promega), prepared at a concentration of 5x10 ⁴ /ml in the medium, and leave overnight at 37°C in an incubator in an atmosphere of 5% CO ₂ . CHO culture medium (90% Ham's F12, 10% PBS) is used as a nutrient medium. Then, after all the medium is removed, 25 μl of a suspension of the GloResponse™ NF-kB-Luc2/4-1BB Jurkat cell line with a concentration of 2x10 ⁶ /ml in the study medium (99% RPMI, 1% PBS) is added to each hole. Then add 25 μl of each antibody solution diluted with assay medium so that the final concentration is 0, 0.001, 0.01, 0.1, 1 and 10 μg/ml; and finally, 25 μL of ATP solution diluted with the assay medium was added so that the final concentration became 0 and 250 μM. The plates are left for 6 hours at 37°C in an incubator under an atmosphere of 5% CO2 and for 15 minutes at room temperature; 75 µl of Bio-Glo reagent is added to each well. For the BioGlo reagent, use the Bio-Glo Luciferase Assay System (buffer and substrate). Subsequently, the luminescence of each well is measured using a plate reader. The luminescence value of each well divided by the luminescence value of the well without added antibody is the relative light unit (fold induction) and serves as an indicator for assessing the CD137 agonistic activity of each antibody.

The results are shown in Fig. 8.

(5-5) In vitro assessment of ATP-dependent CD137 agonist activity of modified anti-human CD137 antibodies using human peripheral blood mononuclear cells.

(5-5-1) Isolation of human peripheral blood mononuclear cells Human peripheral blood mononuclear cells (PBMCs) are isolated from blood samples of healthy volunteers in the Applicant's company using the technology described in Example 2-6-1. Subsequently, the cells are diluted in medium (5% human serum (SIGMA), 95% AIM-V (Thermo Fischer Scientific) to a cell density of 5x10 ⁶ /ml.

(5-5-2) Evaluation of CD137 agonist activity using human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells are brought to a density of 5x10 ⁶ /ml and seeded into 96-well flat-bottom plates (Corning) with 100 μl per well. Then add 50 μl of a solution with a concentration of 0.04 μg/ml of an antibody against human CD3ε (BD, clone SP34) and 20 μg/ml of an antibody against human CD28 (BD, clone: CD28.2), diluted in the medium. The plates are shaken and left in an incubator in an atmosphere of 5% CO2 at 37°C for 6 hours. Then 25 μl of medium with 2 mM ATP (SIGMA) or medium without ATP and 25 μl of each antibody at a concentration of 40 μg are added to each well. /ml, and the plates are shaken before leaving in an incubator at 5% CO2 at 37°C for 18 hours. A portion of the culture supernatant is then collected, and the culture supernatant is used to calculate the IL-2 content of the Human IL-2 culture supernatant DuoSet ELISA kit (R&D systems) or Human IL-2 ELISA Set (BD Biosciences). After collecting the culture supernatant, the plates are again left in the incubator under 5% CO2 at 37°C for 24 hours. A portion of the culture supernatant is then collected and the amount of IFN-γ contained in the culture supernatant is determined using the Human IFN-γ DuoSet ELISA kit (R&D systems) or Human IFN-γ ELISA Development Kit (PeproTech). The ELISA assay is performed primarily according to the protocol of the kit supplier. For the Human IL-2 DuoSet ELISA kit (R&D systems) and the Human IFN-γ DuoSet ELISA kit (R&D systems), color appearance and cessation of color development is performed in accordance with protocols using a substrate solution (R&D systems) containing H ₂ O ₂ and tetramethylbenzidine, and 1N H2SO4 (Wako). For the Human IL-2 ELISA Set (BD Biosciences), color development is stopped using 1N H2SO4 (Wako). The IFN-γ ELISA Development Kit (PeproTech) uses a solution of TMB chromogen (Thermo Fischer Scientific) and 1N H ₂ SO ₄ (Wako) to develop and stop coloring. Absorbance measurements are then taken using an EnVision reader (PerkinElmer).

The results are described in detail in Example 5-5-3 and in the following description.

(5-5-3) Assess the enhancement of agonistic activity of the heavy chain constant region by increasing binding activity to the Fcy receptor.

In vitro, using human peripheral blood monocytes as described in Examples 5-5-1 and 5-5-2, the effect of using P587, MY518, TT14 and TT16, which differently increase the FcYRIIb-binding activity of the heavy chain constant region, is assessed by agonistic activity against CD137.

The antibodies evaluated are presented in table. 19.

Obtaining antibodies listed in table. 19, described in Example 7-1. Compared to the amounts of IL-2 and IFN-γ in the culture supernatant when the negative control antibody (IC17HdK

- 117 046075

MY518/IC17L-k0, IC17HdK-G4d/IC17L-k0) is added in the presence of 250 μM ATP if the addition of antibody increases the amount of IL-2 in the culture supernatant by 1.05-fold or more and the amount of IFN-γ by 1.15-fold or Moreover, the antibody was found to demonstrate agonistic activity against CD137. Measurement of agonist activity using human peripheral blood mononuclear cells (PBMCs) may differ among blood donors. In this regard, it was determined that antibodies that do not exhibit CD137 agonistic activity in some or more than half of human PBMCs isolated from multiple donors can be regarded as not exhibiting CD137 agonistic activity, even if they meet the standards agonistic activity for another part of human PBMC.

Antibodies considered to exhibit agonistic activity against CD137 in the presence of 250 μM ATP are shown in table. 19. This indicates that agonistic activity against CD137 was enhanced by combining TT16 with P587, which increases binding activity to the FcYRIIb heavy chain constant region (FIGS. 9 and 10). The agonist activity is believed to be enhanced by increased binding activity to cells expressing FcYRIIb, which is the cross-linking scaffold required for the antibody to exhibit agonist activity to cells expressing CD137 (see Protein Eng Des Sel, 26 (10), 2013, 589-598, doi:10.1093/protein).

Table 19

Antibody of interest Antibody with established activity in the presence of ATP A375-MY518/В167-LamLib А375-Р587/В167-LamLib A356-MY518/B040-LamLib A356-P587/B040-LamLib А375-Р587/В167-LamLib A375-TT16/B 167-LamLib A356-P587/B040-LamLib A356-TT16/B040-LamLib A375-TT16/B167-LamLib A356-TT16/B040-LamLib A375-TT14/B167-LamLib

(5-5-4) Assessment of ATP-dependent agonist activity against CD137 variable regions.

The ATP-dependent CD137 agonist activity of each variable region by combining the heavy chain constant regions of P587 and P253 with different variable regions was assessed in vitro using human peripheral blood monocytes as described in Examples 5-5-1 and 5-5-2.

The antibodies evaluated are presented in table. 20. The production of antibodies listed in Table 20 is described in Example 7-1. Compared to the amounts of IL-2 and IFN-γ in the culture supernatant when a negative control antibody (IC17HdK-P253/IC17L-k0, IC17HdK-P587/IC17L-k0) is added in the presence of 250 μM ATP, and when the addition of antibody increases the amount of IL -2 in the culture supernatant by 1.05 times or more and the amount of IFN-γ by 1.15 times or more, it is concluded that the antibody exhibits agonistic activity against CD137. Measurement of agonist activity using human peripheral blood mononuclear cells (PBMCs) may differ from donor to donor depending on the blood sample. In this regard, it has been determined that antibodies that do not exhibit CD137 agonist activity in some or more than half of human PBMCs isolated from multiple donors can be regarded as not exhibiting CD137 agonist activity, even if they meet the standards for agonist activity in another parts of human ICPC. Antibodies assessed to exhibit agonistic activity against CD137 in the presence of 250 μM ATP are presented in table. 20 (Fig. 11, Fig. 12, Fig. 13, Fig. 14 and Fig. 15).

In addition, among the antibodies assessed to exhibit CD137 agonistic activity in the presence of 250 μM ATP, antibodies whose fold change relative to the group in which the negative control antibody was added (IC17HdK-P253/IC17L-k0, IC17HdKP587/IC17L-k0) relative to the amount of IL-2 and IFN-γ in the culture supernatant in the absence of ATP, less than a fold change compared to the negative control in conditions in the presence of 250 μM ATP, have reduced agonistic activity against CD137 in the absence of ATP. Antibodies that exhibit CD137 agonistic activity in the presence of 250 μM ATP and that are assessed to have CD137 agonistic activity in the absence of ATP are presented in Table 1. 20 (Fig. 11, Fig. 12, Fig. 13, Fig. 14 and Fig. 15). These antibodies were found to exhibit ATP-dependent agonist activity against CD137.

Based on the above, it is confirmed that for the variable regions of the heavy chain variable region/light chain variable region combination (A375/B167), (A356/B040), (A372/B040), (A486/B167), (A488/B226), ( A489/B223), (A551/B256), (A548/B256) and (A551/B379) exhibit agonistic activity against CD137 in an ATP-dependent manner.

- 118 046075

Table 20

Antibody of interest Antibody active in ATP Antibody with reduced activity without ATP A372-P253/B040-LamLib A372-P253/B040-LamLib A372-P253/B040-LamLib А486-Р253/В167-Lam Lib A486-P253/B167-LamLib А486-Р253/В167-Lam Lib A488-P253/B226-LamLib A488-P253/B226-LamLib A488-P253/B226-Lam Lib A489-P253/B223-LamLib A489-P253/B223-LamLib A489-P253/B223-Lam Lib A551-P587/B256-LamLib A551-P587/B256-LamLib A551-P587/B256-Lam Lib A375-P587/B167-Lam Lib A375-P587/B167-LamLib A375-P587/B167-LamLib A548-P587/B256-Lam Lib A548-P587/B256-LamLib A548-P587/B256-Lam Lib A356-P587/B040-Lam Lib A356-P587/B040-LamLib A356-P587/B040-Lam Lib A551-P587/B379-LamLib A551-P587/B379-LamLib A551-P587/B379-LamLib

(5-5-5) Assess the enhancing effect on CD137 agonist activity of combined changes that increase the pI of the heavy chain constant region with a heavy chain constant region with increased Fcy receptor binding activity.

The effect on CD137 agonistic activity of introduced various amino acid modifications that increase the pI of the heavy chain constant region into a heavy chain constant region with various increased FcyRIIb-binding activity was assessed by in vitro assessment using human peripheral blood monocytes as described in Examples 5-5 -1 and 5-5-2. Amino acid changes that increase pI are introduced into the heavy chain constant regions of TT16, MY518 and TT14 and thus into the heavy chain constant regions TT16+P343R/D413K (SCF028), TT16+Q311R/P343R (SCF033), MY518+P343R/ D413K (SCF025), MY518+Q311R/P343R (SCF030), TT14+P343R/D413K (SCF027) and TT14+Q311R/P343R (SCF032). The names of the antibodies evaluated that had amino acid changes that increase the pI, and the names of the corresponding antibodies before the amino acid changes that increased the pI were introduced, are shown in Table 1. 21. Production of antibodies listed in table. 21 is described in Example 7-2.

With regard to assessing the enhancement of agonist activity by introducing amino acid modifications that increase the pI, when the addition of an antibody increases the amount of IL-2 by 1.04 times or more and the amount of IFN-γ by 1.1 times or more in the culture supernatant, relative to groups of antibodies containing heavy chain constant region, which does not contain these amino acid modifications, A375-TT16/B167-LamLib, A375-MY518/B167-LamLib and A375-TT14/B167-LamLib, the agonistic activity against CD137 was found to be enhanced. Compared to antibodies containing a heavy chain constant region without pI-increasing amino acid modifications, antibodies containing a heavy chain constant region in which amino acid modifications have been shown to have increased agonistic activity against CD137 in the presence of 250 μM ATP are shown in Table 1. 22 (Fig. 10).

This indicates that regardless of the extent to which the FcyRIIb binding activity of the heavy chain constant region is increased, introduction of modifications that increase the pI of the heavy chain constant region enhances the CD137 agonistic activity of the anti-human CD137 antibody. It has been proposed that: the combination of modifications that increase the pI of the heavy chain constant region with modifications that increase the binding of the FcyRIIb heavy chain constant region to the heavy chain constant region enhances interaction with the negatively charged FcyRIIb-expressing cell surface; immune complexes of antibodies or antigen-related antibodies come closer to the surface of cells expressing FcyRIIb; this further increases binding to cells expressing FcyRIIb, which are the cross-linking scaffolds required for the antibodies to exhibit agonistic activity against cells expressing CD137; and thus the agonistic activity was further enhanced.

Table 21

Name of the heavy chain constant region assessed into which amino acid changes are introduced that increase pl Name of the corresponding heavy chain constant region before the introduction of amino acid changes that increase pl Introduction of change A375-SCF028/B167-LamLib AZ 7 5 -TT16/B167 -LamLib P343R/D413K A375-SCF033/B167-LamLib AZ 7 5 -TT16/B167 -LamLib Q311R/P343R A375-SCF025/B167-LamLib A375-MY518/B167-LamLib P343R/D413K A375-SCF030/B167-LamLib A375-MY518/B167-LamLib Q311R/P343R A375-SCF027/B167-LamLib AZ 7 5 -TT 14/B 167 -LamLib P343R/D413K A375-SCF032/B167-LamLib AZ 7 5 -TT 14/B 167 -LamLib Q311R/P343R

- 119 046075 ________________________________________________________________ Table 22

Antibodies identified as having increased agonistic ________________________________ activity________________________________ A375-SCF028/B167-LamLib ________________________A375-SCF033/B167-LamLib________________________ ________________________A375-SCF025/B167-LamLib________________________ ________________________A375-SCF030/B167-LamLib________________________ ________________________A375-SCF027/B167-LamLib________________________ ________________________A375-SCF032/B167-LamLib________________________ (5-5-6) Assess the effect on agonist activity by introducing modifications that increase the pI of the heavy chain constant region.

The effect on CD137 agonistic activity of the introduction of various amino acid modifications that increase the pI of the heavy chain constant regions of MY201aPh and MY518 was assessed in vitro using human peripheral blood monocytes as described in Examples 5-5-1 and 5-5-2. The heavy chain constant regions of MY518, MY518a, and MY201aPh are introduced with amino acid modifications that increase the pI; thus, MY518+P343R/D413K (SCF025), MY518+Q311R/P343R(SCF030), MY518+P343R(SCF039) were created,

MY518+D413K (SCF040), MY518a+Q311R (SCF060a), MY201aPh+P343R(SCF041aPh),

MY201aPh+P343R/D413K (SCF043aPh), MY201aPh+Q311R (SCF056aPh), MY201aPh+Q311R/P343Rb (SCF057aPh) and MY201aPh+Q311R/D413K (SCF059aPh). The names of the antibodies in question, in which amino acid changes have been made that increase the pI, and the names of the antibodies before the amino acid changes have been made, which increase the corresponding pI value, are shown in table. 23. Production of antibodies listed in table. 23 is described in Example 7-2.

Regarding the assessment of enhancement of agonist activity by introducing amino acid modifications that increase the pI, when the addition of antibody increases the amount of IL-2 by 1.04-fold or more and the amount of IFN-γ by 1.1-fold or more in the culture supernatant, relative to the group in which added antibodies containing a heavy chain constant region that does not contain such amino acid modifications, A375-MY518a/B167-LamLib and A375-MY201aPh/B167-LamLib, were confirmed to have improved agonistic activity against CD137. Compared to antibodies containing a heavy chain constant region without pI-increasing amino acid modifications, antibodies containing a heavy chain constant region in which amino acid changes were shown to have increased agonistic activity against CD137 in the presence of 250 μM ATP are shown in Table. 24 (Figs. 16 and 17).

Table 23

Name of the heavy chain constant region assessed into which amino acid changes are introduced that increase pl The name of the corresponding heavy chain constant region before the introduction of amino acid changes that increase p! Introduction of changes pl A375-SCF025/B 167-LamLib A375-MY518/B 167-LamLib P343R/D413K A375-SCF030/B 167-LamLib A375-MY518/B 167-LamLib Q311R/P343R A375-SCF039/B 167-LamLib A375-MY518/B 167-LamLib P343R A375-SCF040/B 167-LamLib A375-MY518/B 167-LamLib D413K A375-SCF060a/B 167-LamLib A375-MY518a/B 167-LamLib Q311R A375-SCF041a/B 167-LamLib A375-MY201aPh/B 167-LamLib P343R A375-SCF043a/B 167-LamLib A375-MY201aPh/B 167-LamLib P343R/D413K A375-SCF056a/B 167-LamLib A375-MY201aPh/B 167-LamLib Q311R A375-SCF057a/B 167-LamLib A375-MY201aPh/B 167-LamLib Q311R/P343R A375-SCF059a/B 167-LamLib A375-MY201aPh/B 167-LamLib Q311R/P343R/D413K

- 120 046075

Table 24

Antibodies identified as having increased I agonist activity A375-SCF025/B167-LamLib_____________

A375-SCF030/B167-Laml_ib______________

A375-SCF039/B167-Laml_ib______________

A375-SCF040/B167-Laml_ib______________

A375-SCF060a/B167-Laml_ib____________

A375-SCF041aPh/B167-LamLib__________

A375-SCF043aPh/B167-LamLib__________

A375-SCF056aPh/B167-LamLib__________

A375-SCF057aPh/B167-Laml_ib__________

A375-SCF059aPh/B167-Laml_ib__________ (5-5-7) Evaluation of the ATP-dependent CD137 agonist activity of anti-human CD137 antibodies that combine engineered variable regions and constant regions.

The ATP-dependent CD137 agonist activity of anti-human CD137 antibodies prepared by combining the variable regions described above with heavy chain constant regions that increase the pI was assessed in vitro using human peripheral blood monocytes as described in Examples 5-5-1 and 5-5 -2. The antibodies studied are presented in table. 25.

Compared with the amounts of IL-2 and IFN-γ in the culture supernatant with the addition of a negative control antibody (IC17HdK-MY518a/IC17L-k0, IC17HdK-MY201aPh/IC17L-k0, IC17HdKMY201/IC17L-k0, IC17HdK-G4d/IC17L-k0, IC17HdK-MY518/IC17L-k0, or IC17HdK-TT16/IC17L-k0) in the presence of 250 μM ATP, when the addition of antibody increases the amount of IL-2 in the culture supernatant by 1.05-fold or more and the amount of IFN-γ by 1.15 times or more, the antibody is assessed to demonstrate agonistic activity against CD137. Measurement of agonist activity using human peripheral blood mononuclear cells (PBMCs) may vary from donor to donor per blood sample. In this regard, it has been determined that antibodies that do not exhibit agonist activity against CD137 in some or more than half of human PBMCs isolated from multiple donors can be regarded as not exhibiting CD137 agonist activity, even if they met the standards for agonists for another part of human PBMCs. . Antibodies found to exhibit CD137 agonistic activity in the presence of 250 μM ATP are shown in Table 25 (FIG. 16, FIG. 17, FIG. 18, FIG. 19, FIG. 20, FIG. 21, FIG. 22 and Fig. 23).

Additionally, among the antibodies found to exhibit agonistic activity against CD137 in the presence of 250 μM ATP, antibodies whose fold change compared to the group in which the negative control antibody was added (IC17HdK-MY518a/IC17L-k0, IC17HdK- MY201aPh/IC 17L-k0, IC17HdK-MY201/IC17L-k0, IC17HdK-G4d/IC17L-k0, IC17HdKMY518/IC17L-k0, or IC17HdK-TT16/IC17L-k0), relative to the amounts of IL-2 and IFN-γ in the supernatant cultures in the absence of ATP less than a fold change relative to the negative control in the presence of 250 μM ATP were found to exhibit reduced agonistic activity against CD137 in the absence of ATP. Antibodies found to have reduced agonist activity against CD137 in the absence of ATP are shown in Table 1. 25 (Fig. 16-23). These antibodies have been shown to exhibit ATP-dependent agonist activity against CD137.

- 121 046075

Table 25

Antibody of interest Antibody active in ATP Antibody with reduced activity without ATP А375-М Y518/В167-Lam Lib A375-SCF025/B167-Lam Lib A375-SCF025/B167-Lam Lib A375-SCF025/B167-Lam Lib A375-SCF030/B167-Lam Lib A375-SCF030/B167-Lam Lib A375-SCF030/B167-Lam Lib A375-SCF039/B167-Lam Lib A375-SCF039/B167-Lam Lib A375-SCF039/B167-Lam Lib A375-SCF040/B167-Lam Lib A375-SCF040/B167-Lam Lib A375-SCF040/B167-Lam Lib A375-MY518a/B167-LamLib A375-M Y518a/B 167-LamLib A375-MY518a/B167-LamLib A375-SCF060a/B167-LamLib A375-SCF060a/B 167-Lam Lib A375-SCF060a/B167-Lam Lib A375-SCF041 aPh/B167-LamLib A375-SCF041aPh/B167-LamLib A375-MY201 aPh/B167-Lam Lib A375-SCF043aPh/B167-LamLib A375-SCF043aPh/B167-LamLib A375-SCF041aPh/B167-LamLib A375-SCF056aPh/B167-LamLib A375-SCF056aPh/B167-LamLib A375-SCF043aPh/B167-LamLib A375-SCF057aPh/B167-LamLib A375-SCF057aPh/B167-LamLib A375-SCF056aPh/B167-LamLib A375-SCF059aPh/B167-LamLib A551-SCF041a/B256-LamLib A375-SCF057aPh/B167-LamLib A551-SCF041 a/B256-Lam Lib A551-SCF057a/B256-LamLib A375-SCF059aPh/B167-LamLib A551-SCF057a/B256-LamLib A551-SCF059a/B256-LamLib A551-SCF041 a/B256-Lam Lib A551-SCF059a/B256-LamLib A551-SCF057aPh/B256-LamLib A551-SCF056a/B256-Lam Lib A551-SCF057aPh/B256-LamLib A551-SCF025a/B256-Lam Lib A551-SCF057a/B256-Lam Lib A551-SCF059aPh/B256-LamLib A551-SCF039a/B256-LamLib A551-SCF059a/B256-Lam Lib A551-SCF025a/B256-Lam Lib A551-SCF043a/B256-Lam Lib A551-SCF056aPh/B256-LamLib A551-SCF039a/B256-LamLib A551-SCF041 aPh/B256-LamLib A551-SCF057aPh/B256-LamLib A551-MY201a/B256-LamLib A551-SCF043aPh/B256-LamLib A551-SCF059aPh/B256-LamLib A551-SCF043a/B256-Lam Lib A551-SCF039a/B379-LamLib A551-MY518/B256-LamLib A551-SCF041aPh/B256-LamLib A551-SCF041a/B379-LamLib A551-SCF025a/B256-Lam Lib A551-SCF043aPh/B256-Lam Lib A551-SCF043a/B379-LamLib A551-SCF039a/B256-Lam Lib A551-SCF039a/B379-LamLib A551-SCF041 aPh/B379-LamLib A551-MY201a/B256-LamLib A551-SCF041 a/B379-Lam Lib A551-SCF043aPh/B379-LamLib A551-SCF043a/B256-Lam Lib A551-SCF043a/B379-Lam Lib A551-SCF060a/B379-LamLib A551 -Μ Y201 aPh/B256-Lam Lib A551-SCF041aPh/B379-LamLib A551-SCF057a/B379-LamLib A551-SCF041aPh/B256-LamLib A551-SCF043aPh/B379-LamLib A551-SCF059a/B379-LamLib A551-SCF043aPh/B256-LamLib A551-SCF060a/B379-LamLib A551-SCF056aPh/B379-LamLib A551-MY518a/B379-LamLib A551-SCF057a/B379-LamLib A551-SCF057aPh/B379-LamLib A551-SCF039a/B379-Lam Lib A551-SCF059a/B379-LamLib A551-SCF059aPh/B379-LamLib A551-SCF041 a/B379-Lam Lib A551-SCF056aPh/B379-LamLib A375-TT16/B 167-Lam Lib A551-SCF043a/B379-Lam Lib A551-SCF057aPh/B379-LamLib A356-TT16/B040-LamLib A551-SCF041aPh/B379-LamLib A551-SCF059aPh/B379-LamLib A551-TT16/B379-Lam Lib A551-SCF043aPh/B379-LamLib A375-TT16/B167-Lam Lib A551-SCF060a/B379-Lam Lib A356-TT16/B040-LamLib A551-SCF056a/B379-Lam Lib A551-TT16/B379-LamLib A551-SCF057a/B379-Lam Lib A551-SCF059a/B379-Lam Lib A551-SCF056aPh/B379-LamLib A551-SCF057aPh/B379-LamLib A551-SCF059aPh/B379-LamLib A375-TT16/B167-LamLib A356-TT16/B040-LamLib A551-TT16/B379-LamLib

Example 6: Human CD137 knockin mice as test for administration of switchable anti-human CD137 antibodies.

(6-1) Development of antibodies for human CD137 knockin mice administration studies.

Switchable and nonswitchable anti-human CD137 antibodies containing a mouse constant region were developed for administration studies in human CD137 knockin mice. In particular, non-switchable antibodies against human CD137 are obtained (20H4.9-mIgG1/20H4.9LC-mk0 abbreviation: NS1-mIgG1, 20H4.9-MB110/20H4.9LC-mk0 abbreviation: NS1-MB110, 20H4.9-MB492/20H4 .9LCmk0 abbreviation: NS1-MB492, MOR-7480.1H-MB110/MOR-7480.1L-ml0r abbreviation: NS2-MB110, MOR-7480.1H-MB492/MOR-7480.1L-ml0r abbreviation: NS2-MB492) and switchable antibodies against Human CD137 (A375-mIgG1/B167-ml0r, A372-mIgG1/B040-ml0r, A372-MB110/B040-ml0r, A372MB492/B040-ml0r, A356-MB110/B040-ml0r, A486-MB492/B167-ml0r, A48 8 -MB492/B226-ml0r, A489MB492/B223-ml0r, A551-mIgG1/B256-ml0r, A551-MB110/B379-ml0r and A548-mIgG1/B256-ml0r).

For the antibody heavy chains NS1-mIgG1, NS1-MB110 and NS1-MB492, the antibody heavy chain genes are designed by combining the heavy chain variable region 20H4.9 (SEQ ID NO: 139) with any of the sequences:

(i) mIgG1, the mouse IgG1 heavy chain constant region (SEQ ID NO: 144), (ii) MB110, described in WO 2014030750 (SEQ ID NO: 145), and

- 122 046075 (ill) MB492, described in WO 2014030750 (SEQ ID NO: 146), as a heavy chain constant region. That is, the genes 20H4.9-mIgG1, 20H4.9MB110 and 20H4.9-MB492 were developed.

For antibody light chains NS1-mIgG1, NS1-MB110 and NS1-MB492, the 20H4.9LC-mk0 antibody light chain gene is developed by combining the 20H4.9LC light chain variable region (SEQ ID NO: 140) with the anti-mouse mk0 chain (SEQ ID NO : 147) as the light chain constant region. By combining these heavy chain and light chain genes, each antibody is expressed and purified by methods known to those skilled in the art.

For antibody heavy chains NS2-MB110 and NS2-MB492, antibody heavy chain genes were created by combining the heavy chain variable region of MOR-7480.1H (SEQ ID NO: 142) with one of the sequences:

(i) MB110 (SEQ ID NO: 145), or (ii) MB492 (SEQ ID NO: 146), as the heavy chain constant region. That is, the genes MOR-7480.1H-MB110 and MOR7480.1H-MB492 were created.

For the light chains of antibodies NS2-MB110 and NS2-MB492, the antibody light chain gene MOR-7480.1L-ml0r is prepared by combining the light chain variable region of MOR-7480.1L (SEQ ID NO: 143) with the mouse λ chain ml0r (SEQ ID NO: 148 ) as the light chain constant region. By combining these heavy chain and light chain genes, each antibody is expressed and purified by methods known to those skilled in the art.

For anti-CD137 switchable antibodies, the antibody heavy chain and antibody light chain genes listed in Table 1 are obtained. 26 and 27; each antibody is expressed and purified by combining these genes by methods known to those skilled in the art.

Table 26

Heavy chain anti-CD137 switchable antibodies having a mouse constant region

Full length heavy chain А375mlgGl Variable region A375 (SEQ ID NO: 43) Constant region mlgGl (SEQ ID NO: 144) Full length heavy chain А372mlgGl Variable region A372 (SEQ ID NO: 44) Constant region mlgGl (SEQ ID NO: 144) Full length heavy chain A372mvio Variable region A372 (SEQ ID NO: 44) Constant region MB110 (SEQ ID NO: 145) Full length heavy chain A372- MV492 Variable region A372 (SEQ ID NO: 44) Constant region MB492 (SEQ ID NO: 146) Full length heavy chain A356- MV110 Variable region A356 (SEQ ID NO: 45) Constant region MB110 (SEQ ID NO: 145) Full length heavy chain A486- MB492 Variable region A486 (SEQ ID NO: 46) Constant region MB492 (SEQ ID NO: 146)

- 123 046075

Full length heavy chain A488- MV492 Variable region A488 (SEQ ID NO: 48) Constant region МВ492 (SEQ ID NO: 146) Full length heavy chain A489- MV492 Variable region A489 (SEQ ID NO: 49) Constant region MB492 (SEQ ID NO: 146) Full length heavy chain А548mlgGl Variable region A548 (SEQ ID NO: 50) Constant region mlgGl (SEQ ID NO: 144) Full length heavy chain А551mlgGl Variable region A551 (SEQ ID NO: 51) Constant region mlgGl (SEQ ID NO: 144) Full length heavy chain A551- MV110 Variable region A551 (SEQ ID NO: 51) Constant region MB110 (SEQ ID NO: 145)

Table 27

Light chains of aHTu-CD137 switchable antibodies having a mouse constant region

Lightweight full length chain В040mlOr Variable region B040 (SEQ ID NO: 55) Constant region mlOr (SEQ ID NO: 148) Lightweight full length chain В167mlOr Variable region B167 (SEQ ID NO: 54) Constant region mlOr (SEQ ID NO: 148) Lightweight full length chain В226mlOr Variable region B226 (SEQ ID NO: 56) Constant region mlOr (SEQ ID NO: 148) Lightweight full length chain В223mlOr Variable region B223 (SEQ ID NO: 57) Constant region mlOr (SEQ ID NO: 148) Lightweight full length chain B256mlOr Variable region B256 (SEQ ID NO: 59) Constant region mlOr (SEQ ID NO: 148) Lightweight full length chain B379mlOr Variable region B379 (SEQ ID NO: 60) Constant region mlOr (SEQ ID NO: 148)

(6-2) Evaluation of binding activity of anti-human CD137 antibodies prepared for human CD137 knockin mice administration study.

The binding activity of the non-switchable anti-human CD137 antibody and the switchable anti-CD137 antibody prepared in Example 6-1 to human CD137 was assessed. Human CD137 binding measurements were performed at 37°C using 20 mM ACES (pH 7.4), 150 mM NaCl, 2 mM MgCl ₂ and 0.05% Tween20 as running buffer. First, antibodies are captured using reacting antibody solutions prepared in running buffer with rabbit anti-mouse IgG (Thermo Scientific) on a Series S Sensor Chip CM5 (GE Healthcare). Binding activity to human CD137 is then assessed in the presence of ATP and in the absence of ATP by reacting with a solution of human CD137 in running buffer without ATP. For the human CD137 antigen, hCD137 (FXa cleaved) obtained in Example (1-2) is used, and measurements are carried out at antigen concentrations of 0, 15,625, 62.5, 250 and 1000 nM. The chip was regenerated using 25 mM NaOH and 10 mM glycine-HCl (pH 1.5), and measurements were made by repeated antibody capture.

The dissociation constant (KD) of the corresponding antibodies against human CD137 was calculated using Biacore T200 Evaluation Software 2.0. In particular, con

- 124 046075 The association rate constant ka (l/mol/s) and the dissociation rate constant kd (1/s) are calculated by globally fitting the sensorgrams obtained by measuring with the 1:1 Langmuir binding model and the dissociation constant KD (mol/l ) are calculated from these values.

In table Figure 28 presents the results of such measurements.

Table 28

Human CD137 Binding Assay by Anti-CD137 Mouse Constant Region Antibodies

Antibody Κ _Ό (M) for CD 137 person Without ATP ATP=1 µM ATP=10 µM ATP=100 µM NSl-mlgGl 6D7E-08 5.62E-08 5.44E-08 5.36E-08 NS1-MB110 6.10E-08 5.65E-08 5.39E-08 5D9E-08 NS1-MB492 6.16E-08 5.49E-08 5.35E-08 5.04E-08 NS2-MB110 l,88E-07 l,44E-07 l,02E-07 l,04E-07 NS2-MB492 2.16E-07 l,70E-07 1D2E-07 1D2E-07 A375-mIgGl/ ND 3.43E-07 1D4E-07 2.92E-08 B167-ml0r A372-mIgGl/ B040-ml0r ND 4.78E-07 2.59E-07 l,08E-07 A372-MV110/ B040-ml0r ND 6.11E-07 2.64E-07 1D2E-07 A372-MV492/ B040-ml0r ND 7.82E-07 2.51E-07 l,04E-07 A356-MV110/ B040-ml0r ND l,01E-06 4.10E-07 l,68E-07 A486-MV492/ B167-ml0r ND l,31E-07 2.70E-08 7.67E-09 A488-MV492/ B226-ml0r ND HO l,36E-07 8.25E-08 A489-MV492/ B223-ml0r ND l,62E-06 3.58E-07 l,54E-07 A551-mIgGl/ B256-ml0r ND 7.55E-07 2.31E-07 6.22E-08 А551-МВ110/ B379-ml0r ND 5D2E-07 l,43E-07 3.51E-08 A548-mIgGl/ B256-ml0r ND 8.61E-07 2.19E-07 5.58E-08

ND (no data) - activity is too low to determine the KD value NA - not determined

Both the non-switchable anti-human CD137 antibody and the above-derived switchable anti-human CD137 antibody, which contain a mouse constant region, were confirmed to bind to human CD137. All switchable anti-human CD137 antibodies were also shown to bind to human CD137 in an ATP concentration-dependent manner. On the other hand, non-switchable antibodies did not show such ATP concentration-dependent binding to human CD137, and binding was almost equal at all ATP concentrations.

(6-3) Evaluation of the kinetics of modified antibodies in the plasma of human CD137 knockin mice.

(6-3-1) Generation of human CD137 knockin mice.

By introducing a human CD137 gene replacement vector into mouse embryonic stem cells (ES cells), a human CD137 knockin mouse is generated in which the mouse CD137 gene is replaced with a human CD137 gene.

This mouse is designated hCD137 KI.

(6-3-2) Measurement of the concentration of anti-human CD137 antibody in the plasma of the hCD137KI mouse model.

After generating the hCD137KI mouse in Example 6-3-1, the corresponding antibodies were administered intravenously as a single dose of the hCD137KI mouse, as shown in Table 1. 29. In table. 29 antibodies NS1-mIgG1, NS1MB110 and NS1-MB492 are all non-switchable antibodies, while the others are switchable antibodies. Blood is collected several times from 5 minutes to 28 days after administration. The resulting blood is centrifuged to separate the plasma. Plasma is stored in a freezer at a temperature below 20°C until measurement.

- 125 046075

Table 29

Details of groups for the plasma kinetics study

Group Number of animals Pharmaceutical agent Dose (mg/kg) 1 3 NSl-mlgGl 7.5, single injection 2 3 NS1-MB110 7.5, single injection 3 3 NS1-MB492 7.5, single injection 4 3 A375-mIgGl/B167-ml0r 7.5, single injection 5 3 AZ 56-MV110/V040-sh10g 7.5, single injection 6 3 A372-mIgGl/B040-ml0r 7.5, single injection 7 3 AZ 72-MV110/V040-sh10g 7.5, single injection 8 3 A372-MV492/V040-sh10g 7.5, single injection 9 3 А486-МВ492/В167-mlOr 7.5, single injection 10 3 A488-MB492/B226-ml0r 7.5, single injection AND 3 А489-МВ492/В223 -mlOr 7.5, single injection 12 3 A548-mIgGl/B256-ml0r 7.5, single injection 13 3 A55 l-m!gGl/B256-ml0r 7.5, single injection 14 3 A551-MB110/B379-ml0r 7.5, single injection

The plasma concentration of each switched antibody is measured by electrochemiluminescence (ECL, electrochemiluminescence). Specifically, hCD137 (Sino Biological Inc.) is diluted in PBS (-) and added to a MULTI-ARRAY 96-well plate (Meso Scale Diagnostics, LLC). Add hCD137 to the plate and shake at room temperature for 1 hour, hCD137 is immobilized in the plate. A PBS solution containing 1% BSA and 0.05% Tween 20 is then added to block and shaken at room temperature for 1 hour. Calibration curves for individual switch antibodies are plotted at plasma concentrations of 64, 32, 16, 8, 4, 2 and 1 ng/ml. After adding a PBS solution containing 3 mM ADP, 1% BSA and 0.05% Tween 20 to the hCD137-immobilized plate, two times the volume of plasma samples is diluted in a PBS solution containing 1% BSA and 0.05% Tween20, as well as the samples calibration curve. After shaking the plate at room temperature for 1 hour, biotinylated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc.) was added. Additionally, after shaking the plate at room temperature for 1 hour, streptavidin-labeled SULFO-TAG (Meso Scale Diagnostics, LLCs) was added. In addition, after shaking the plate at room temperature for 1 hour, add Read buffer T (Meso Scale Diagnostics, LLCs), diluted twice with a PBS solution containing 2 mM ADP, 1% BSA and 0.05% Tween 20. Measurement of antibody concentrations in the plasma of hCD137KI mice is carried out by detection using SULFO-TAG on a SECTOR Imager (Meso Scale Diagnostics, LLC). Calculation of individual antibody concentrations in mouse plasma is carried out using the SOFTmax PRO program (Molecular Devices).

The concentration of nonswitchable antibodies in plasma is measured by electrochemiluminescence (ECL). Specifically, hCD137 (Sino Biological Inc.) is diluted in PBS (-) and added to a 96-well MULTI-ARRAY plate (Meso Scale Diagnostics, LLC). hCD137 is added to the plate and shaken at room temperature for one hour to immobilize hCD137 in the plate. A PBS solution containing 1% BSA and 0.05% Tween 20 is then added to block and shaken at room temperature for one hour. Calibration curves for individual nonswitchable antibodies were constructed at plasma concentrations of 32, 16, 8, 4, 2, 1, and 0.5 ng/mL. Plasma samples diluted in PBS solution containing 1% BSA, 0.05% Tween 20, as well as calibration curve samples, are added to the plate with immobilized hCD137. After shaking the plate at room temperature for one hour, biotinylated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc.) is added. Additionally, after shaking the plate at room temperature for one hour, SULFO-TAG Labeled Streptavidin (Meso Scale Diagnostics, LLC) is added. Reading buffer T (Meso Scale Diagnostics, LLCs), diluted twofold, was added. Measurement of antibody concentrations in the plasma of hCD137KI mice is performed similarly to that described above for switchable antibodies.

The results are shown in Fig. 24, 25 and 26.

In fig. Figure 24 shows the dynamics of average concentrations in mouse plasma of hCD137 KI antibodies having mIgG1 as an Fc (NS1-mIgG1 as a non-switchable antibody and A375-mIgG1/B167-ml0r, A372-mIgG1/B040-ml0r, A548-mIgG1/B256-ml0r or A551-mIgG1/B256-ml0r as a switchable antibody) among the antibodies shown in Table. 29. In FIG. Figure 25 shows the dynamics of average concentrations in mouse plasma of hCD137 KI antibodies having MB110 as an Fc (NS1-MB110 as a non-switchable antibody and A356-MB110/B040-ml0r, A372-MB110/B040-ml0r, and A551-MB110/B379- ml0r as switchable antibodies). In fig. Figure 26 shows the dynamics of average concentrations in mouse plasma of CD137 KI antibodies having MB492 as an Fc (NS1-MB492 as a non-switchable antibody and A372MB492/B040-ml0r, A486-MB492/B167-ml0r, A488-MB492/B226-ml0r or A489- MB492/B223-ml0r as a switchable antibody). In fig. 26, as for A488-MV492/B226-ml0r, a sharp decrease in

- 126 046075 plasma concentration time suspected to be caused by ADA was confirmed in one subject, and the average was taken from two subjects.

In the hCD137 KI mouse with either Fc, the switching antibody exhibits slower clearance from plasma than the nonswitching antibody. It is believed that this is due to the fact that, compared to the switch antibody, the non-switch antibody binds to CD137 expressed in the body and is eliminated more quickly in the plasma. It is also suggested that the concentration of extracellular ATP in normal tissue is so low that the switch antibodies used in this study did not bind to human CD137. These results suggest that binding to systemically expressed antigen can be reduced by switchable antibodies, and thus an antibody with improved blood kinetics can be obtained.

(6-4) Movement of markers of drug efficacy and systemic antibody response to CD137 in a syngeneic tumor cell transplantation model using hCD137KI mice.

(6-4-1) Generation of cell lines and mouse model of syngeneic tumor transplantation, as well as a method for assessing the antitumor effect and systemic response.

Cells from the National Cancer Institute-licensed MC38 mouse colorectal cancer cell line and hCD137 KI mice described in Example 6-3-1 were used for various studies. The model is formed when the tumor volume reaches approximately 50-300 mm ³ by subcutaneous implantation of the MC38 cell line into the peritoneal cavity of mice. After establishing the model, mice transplanted with the MC38 cell line are grouped and then administered with various anti-CD137 antibodies.

To assess the antitumor effect, tumor volume is measured 1-2 times a week. Tumor volume is calculated using the following formula:

Tumor volume (mm) = length (mm) x width (mm) x width (mm)/2

By removing the liver and spleen or lymph nodes at an appropriate time after antibody administration, systemic responses are assessed by organ weight, cell counts in lymphocyte fractions, or T-cell analysis using liquid cytometry (FCM). In evaluating these measures, the subject's switch antibody was found to suppress systemic responses and/or T cell activation in non-tumor tissues (eg, lymph nodes, spleen, and liver) when the systemic response rate was low compared to the same amount of non-switch antibody (control). anti-human CD137 antibody without ATP-dependent binding activity against human CD137).

The antibodies obtained in Example 6-1 (antibody production for the human CD137 knockin mouse test) are used in various tests.

(6-4-2) Obtaining various organs from mice transplanted with the MC38 cell line and preparing lymphocyte fractions.

The mass of collected spleens and lymph nodes is measured, as well as cell counting of lymphocyte fractions. Lymphocyte fractions obtained using a mouse liver dissociation kit (Milteny Biotec) were also used to assess liver activation. Lymphocyte fractions from erythrocyte lysis are used to assess activation in the spleen, and crushed lymphocyte fractions are used to assess activation in lymph nodes.

(6-4-3) FCM analysis using lymphocyte fractions from various organs.

To assess marker activation by FCM, granzyme B expression or ICOS expression on CD8α-positive T cells or the percentage of CD8α-positive T cells among CD45-positive cells are used. For this reason, fluorescently labeled antibodies against granzyme B, PD-1, ICOS, CD8a and CD45 are used in the FCM assay. Measurements are performed using a BD LSRFortessa™ X-20 (BD Biosciences).

(6-4-4) Studies of the medicinal efficacy (antitumor effect) of the antibody A375mIgG1/B167-ml0r.

After the formation of a tumor in mice as a result of transplantation of MC38 cancer cells in examples 6-4-1, the solvent and A375-mIgG1/B167-ml0r are administered in the doses shown in table. 30. Antibodies are administered twice through the tail vein on the day of group formation and four days after that. PBS containing 0.05% Tween-20 is used as a solvent.

Table 30

Detailed information on efficacy study arms A375-mIgG1/B167-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - On the day the group is formed and on the fourth day after the group is formed 2 5 A375-mIgGl/ 22.5 On the day of group formation and on the fourth

- 127 046075

В167-ш10г day after group formation 3 5 A375-mIgGl/ B167-ml0r 7.5 On the day the group is formed and on the fourth day after the group is formed 4 5 A375-mIgGl/ B167-ml0r 2.5 On the day the group is formed and on the fourth day after the group is formed 5 5 A375-mIgGl/ B167-ml0r 0.83 On the day the group is formed and on the fourth day after the group is formed 6 5 A375-mIgGl/ B167-ml0r 0.28 On the day the group is formed and on the fourth day after the group is formed

The antitumor effect in each group is assessed by tumor volume, calculated as described in example 6-4-1. The result confirms the dose-dependent antitumor effect of the antibody A375-mIgG1/B167-ml0r (Fig. 27).

(6-4-5) Antibody administration and sampling and FCM analysis to evaluate systemic effects of A375-mIgG1/B167-ml0r.

After establishing the MC38 transplantation mouse model in Example 6-4-1, vehicle, NS1-mIgG1 (non-switchable antibody) and A375-mIgG1/B167-ml0r were administered as shown in Table. 31. Antibody injections are administered twice into the tail vein on the day of group formation and three days after group formation. PBS containing 0.05% Tween-20 is used as a solvent.

Five days after the initial administration, the liver, lymph nodes and spleen are collected as shown in Example 6-4-2 (collection of various organs from a mouse transplanted with the MC38 cell line and obtaining lymphocyte fractions). In addition, for the spleen and lymph nodes, organs from three mice were pooled and FCM analysis was performed as shown in Example 6-4-3 (FCM analysis using lymphocyte fractions from different organs).

Table 31

Detailed information by group for assessing systemic responses with antibody A375-mIgG1/B167-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Day of sampling 1 3 Solvent - On the day the group is formed and on the third day after the group is formed Five days after the group was formed 2 3 NSl-mlgGl 0.06 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 3 3 NSl-mlgGl 0.3 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 4 3 NSl-mlgGl 1.5 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 5 3 NSl-mlgGl 7.5 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 6 3 NSl-mlgGl 37.5 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 7 3 A375-mIgGl/ B167-ml0r 1.5 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 8 3 A375-mIgGl/ B167-ml0r 7.5 On the day the group is formed and on the third day after the group is formed Five days after the group was formed 9 3 A375-mIgGl/ B167-ml0r 37.5 On the day the group is formed and on the third day after the group is formed Five days after the group was formed

Systemic effects from the administration of antibodies are assessed. As a result of assessing the weight of the organs of the lymph nodes and spleens, an increase in the weight of these organs was established when NS1-mIgG1 was administered at a dose of 0.3 mg/kg and more pronounced organ hypertrophy at 1.5 mg/kg. kg and 7.5 mg/kg. On the other hand, organ enlargement was not observed with A375-mIgG1/B167-ml0r at any of the doses of 1.5 mg/kg, 7.5 mg/kg, or 37.5 mg/kg (FIG. 28).

In addition, as a result of assessing T cell activation using FCM assessment, an increase in the expression of all markers PD-1, ICOS and granzyme B was observed at 0.3 mg/kg after administration of NS1-mIgG1. On the other hand, when A375-mIgG1/B167-ml0r was administered, a significant inhibition of the expression of all markers PD-1, ICOS and granzyme B was observed at doses of 1.5 mg/kg, 7.5 mg/kg and 37.5 mg/kg ( Fig. 29 and Fig. 30). Because the results for A375-mIgG1/B167-ml0r showed that organ weight correlated with markers of T cell activation in the lymph nodes and spleen, organ weight is used primarily as an indicator of immune cell activation in the lymph nodes and spleen in subsequent antibody assessments.

As a result of assessing the activation of T cells in the liver, an increase in the expression of PD-1 and granzyme B was observed when NS1-mIgG1 was administered at a dose of 0.3 mg/kg. On the other hand, when A375-mIgG1/B167ml0r was administered, expressions were suppressed at all doses of 1.5 mg/kg, 7.5 mg/kg and 37.5 mg/kg (Fig. 31).

Thus, the switching antibody inhibits T cell activation in the lymph nodes, spleen, and liver compared with the same amount of nonswitching antibody.

(6-4-6) Study of the medicinal efficacy (antitumor effect) of the antibody A356MB110/B040-ml0r.

After the formation of the mouse model after MC38 transplantation in example 6-4-1, the solvent and A356-MVN()/B040-t10g are administered in the doses shown in table. 32. The antibody is administered twice into the tail vein on the day of group formation and three days after the group is formed. PBS containing 0.05% Tween-20 is used as a solvent.

Table 32

Detailed information about the effectiveness study groups A356-MV110/V040-t10g

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - On the day the group is formed and on the third day after the group is formed 2 5 А356МВ110/В040mlOr 7.5 On the day the group is formed and on the third day after the group is formed 3 5 А356МВ110/В040mlOr 2.5 On the day the group is formed and on the third day after the group is formed

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. The results confirm the dose-dependent antitumor effect of A356MB110/B040-t10g (Fig. 32).

(6-4-7) Antibody administration and sampling, as well as FCM analysis to assess the systemic effects of A3 56-MB110/B040-t10g.

After establishing the MC38 transplant mouse model in Example 6-4-1, vehicle, NS2MB110 (non-switchable antibody) and A356-MB110/B040-t10g were administered as shown in Table. 33. Antibodies are administered twice into the tail vein on the day of group formation and three days after group formation. PBS containing 0.05% Tween-20 is used as a solvent.

Seven days after the first administration, the liver, lymph nodes and spleen were collected as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and preparation of lymphocyte fractions). For the spleen and lymph nodes, only the weight of the organs is measured.

- 129 046075

Table 33

Detailed information on groups for assessing systemic responses to antibody A356-MB110/B040-ml0r

Group | Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Day of sampling 1 3 Solvent - On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 2 3 NS2-MB110 7.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 3 3 NS2-MB110 2.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 4 3 NS2-MB110 0.3 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 5 3 A356mvpo/ B040-ml0r 7.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 6 3 A356mvpo/ B040-ml0r 2.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed

The systemic effects of the administered antibodies are assessed. The result of assessing the mass of organs of the lymph nodes and spleen with the administration of NS2-MB110 and A356-MB110/B040-ml0r shows that hypertrophy is also observed with the administration of NS2-MB110 at a dose of 2.5 mg/kg. On the other hand, at the time of administration of A356-MB110/B040-ml0r, lymphadenopathy was suppressed even at 7.5 mg/kg, and splenomegaly was suppressed at 2.5 mg/kg (Fig. 33).

In addition, the result of assessing the activation of T cells in the liver shows that an increase in the expression of PD-1 in CD8α-positive cells is also observed when NS2-MB110 is administered at a dose of 0.3 mg/kg. Meanwhile, when A356-MB'1'10/B040-ml0r was administered, the expression of PD-1 was suppressed at 7.5 mg/kg and significantly suppressed at 2.5 mg/kg. A slight increase in ICOS expression in CD8α-positive cells was also observed when the NS2-MB11O antibody was administered at a dose of 0.3 mg/kg. On the other hand, solvent expression was observed to be suppressed at 2.5 mg/kg upon administration of A356-MB'1'10/B040-ml0r (FIG. 34).

Thus, the switchable antibody inhibits T cell activation in the lymph nodes, spleen, and liver compared to the nonswitchable antibody.

(6-4-8) Study of the medicinal effectiveness (antitumor effect) of the antibody A372mIgG1/B040-ml0r.

After establishing the MC38 transplant mouse model in Example 6-4-1, vehicle and A372-mIgG1/B040-ml0r were administered at the doses shown in Table 1. 34. The antibody is administered four times into the tail vein on the day of group formation and 4 days, 7 days and 10 days after group formation. PBS containing 0.05% Tween-20 is used as a solvent.

Table 34

Detailed information on efficacy study groups A372-mIgG1/B040-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - On the day the group is formed and on the fourth, seventh and tenth days after the group is formed 2 5 A372-mIgGl/ B040-ml0r 7.5 On the day the group is formed and on the fourth, seventh and tenth days after the group is formed

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. The results confirm the antitumor effect of the administration of the antibody A372mIgG1/B040-ml0r (Fig. 35).

(6-4-9) Antibody administration and sampling and FCM analysis to evaluate systemic effects of A372-mIgG1/B040-ml0r.

After establishing the MC38 transplant mouse model in Example 6-4-1, vehicle and A372-mIgG1/B040-ml0r were administered as shown in Table. 35. The antibody is administered three times into the tail vein on the day of group formation and 3 days and 6 days after group formation. PBS containing 0.05% Tween-20 is used as a solvent.

Seven days after the first injection, the liver, lymph nodes and spleen are collected as

- 130 046075 is shown in example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and preparation of lymphocyte fractions). For the spleen, only the organ mass is measured; For lymph nodes, only cell counts are performed for lymphocyte fractions obtained from lymphocytes pooled from three mice.

Table 35

Detailed information on systemic response study groups using antibody A372-mIgG1/B040-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/k G) Introduction day Day of sampling 1 3 Solvent - On the day the group is formed and on the third and sixth days after the group is formed Seven days after the group was formed 2 3 AZ 72mlgGl/ B040-ml0r 7.5 On the day the group is formed and on the third and sixth days after the group is formed Seven days after the group was formed

Systemic effects from the administration of antibodies are assessed. As a result of measuring the number of cells in the lymph nodes and the weight of the spleens, it was confirmed that there was no increase in the number of cells in the lymph nodes and an increase in the weight of the spleen with the administration of A372-mIgG1/B040-ml0r, and that the weight of this organ was equivalent to that in the vehicle group (Fig. 36).

As a result of the assessment of T cell activation in the liver, granzyme B expression levels were shown to be similar to those in the vehicle group when A372-mIgG1/B040-ml0r was administered (FIG. 37).

Thus, the switch antibody was confirmed to inhibit T cell activation in lymph nodes, spleen and liver.

(6-4-10) Study of the medicinal efficacy (antitumor effect) of the antibody A372-MB110/B040-ml0r.

After creating a mouse model with cancer transplantation MC38 in example 6-4-1, the solvent and A372MB110/B040-ml0r are administered in the doses presented in table. 36. Antibodies are administered twice into the tail vein on the day of group formation and four days after that. PBS containing 0.05% Tween-20 is used as a solvent.

Table 36

Detailed information on efficacy study arms A372-mIgG1/B040-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - On the day the group is formed and on the third day after the group is formed 2 5 A372-MV110/ В040-ш10г 7.5 On the day the group is formed and on the third day after the group is formed

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. The results confirm the antitumor effect of A372-mIgG1/B040-ml0r (Fig. 38).

(6-4-11) Antibody administration and sampling and FCM analysis to evaluate systemic effects of Α372-ΜΒ110/Β040-^0γ

After establishing the MC38 cancer transplant mouse model in Example 6-4-1, vehicle, NS2MB110 (non-switchable antibody) and A372-MB110/B040-ml0r were administered at the doses shown in Table. 37. Antibodies are administered twice into the tail vein, namely on the day the group is formed and three days after that. PBS containing 0.05% Tween-20 is used as a solvent.

Seven days after the initial administration, the liver, lymph nodes and spleen are collected as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and obtaining lymphocyte fractions). For the spleen and lymph nodes, only the organ mass is measured.

- 131 046075

Table 37

Detailed information in groups for assessing systemic responses with antibody A372-MB110/B040-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Day of sampling 1 3 Solvent - On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 2 3 NS2mvpo 7.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 3 3 NS2mvpo 2.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 4 3 NS2mvpo 0.3 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 5 3 A372mvpo/ В040mlOr 7.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed 6 3 A372mvpo/ В040mlOr 2.5 On the day the group is formed and on the third day after the group is formed Seven days after the group was formed

Systemic effects from the administration of antibodies are assessed. As a result of measuring the weight of lymph nodes and spleens, it was confirmed that the weight of these organs increases with the administration of NS2-MB110 at doses of 2.5 mg/kg and 7.5 mg/kg. On the other hand, inhibition of organomegaly was observed at both doses of 2.5 mg/kg and 7.5 mg/kg with the introduction of A372-MB110/B040-ml0r (Fig. 39).

As a result of assessing T cell activation in the liver, an increase in PD-1 expression was observed upon administration of NS2-MB110. This was also observed upon administration of A372-MB110/B040-ml0r (FIG. 40).

Thus, the switchable antibody inhibits T cell activation in the lymph nodes and spleen, unlike the nonswitchable antibody.

(6-4-12) Study of the medicinal efficacy (antitumor effect) of the antibody A372-MB492/B040-ml0r.

After creating a mouse model with cancer transplantation MC38 in example 6-4-1, the solvent and A372MB110/B040-ml0r are administered in the doses presented in table. 38. Antibodies are administered twice into the tail vein on the day the group is formed and three days after that. PBS containing 0.05% Tween-20 is used as a solvent.

Table 38

Detailed information on efficacy study groups A372-MB492/B040-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Day of administration 1 5 Solvent - On the day the group is formed and on the third day after the group is formed 2 5 A372-MV492/ В040-ш10г 1.5 On the day the group is formed and on the third day after the group is formed 3 5 A372-MV492/ В040-ш10г 3.0 On the day the group is formed and on the third day after the group is formed 4 5 A372-MV492/ В040-ш10г 7.5 On the day the group is formed and on the third day after the group is formed

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. As a result, the antitumor effect of the antibody A372-MB492/B040ml0r is observed for all doses (Fig. 41).

(6-4-13) Antibody administration and sampling and FCM analysis to evaluate systemic effects of antibody A372-MB492/B040-ml0r.

After obtaining the MC38 cancer transplant mouse model in Example 6-4-1, solvent, NS1MB492 (non-switchable antibody) and A372-MB492/B040-mYr were administered at the doses shown in

- 132 046075 table. 39. Antibodies are administered twice into the tail vein, namely on the day the group is formed and three days after that. PBS containing 0.05% Tween-20 is used as a solvent.

Eight days after the initial administration, the liver, lymph nodes and spleen are collected as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and obtaining lymphocyte fractions). The mass of the selected spleens is measured, and lymphocytes in fractions from the lymph nodes are counted.

Table 39

System Response Study Group Details A372-MB492/B040-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Sample collection day 1 3 Solvent - On the day the group is formed and three days after the group is formed 8 days after the formation of the group 2 3 NS1-MB492 7.5 On the day the group is formed and three days after the group is formed 8 days after the formation of the group 3 3 A372-MV492/ B040-ml0r 1.5 On the day the group is formed and three days after the group is formed 8 days after the formation of the group 4 3 A372-MV492/ B040-ml0r 3.0 On the day the group is formed and three days after the group is formed 8 days after the formation of the group 5 3 A372-MV492/ B040-ml0r 7.5 On the day the group is formed and three days after the group is formed 8 days after the formation of the group

The systemic effects of the administered antibodies are assessed. An increase in spleen mass was observed upon administration of NS1-MB492 and an increase in cells in the lymphocyte fractions upon administration of NS1-MB492. On the other hand, when A372-MB492/B040-ml0r was administered at doses of 1.5 mg/kg, 3.0 mg/kg and 7.5 mg/kg, inhibition of organomegaly was observed compared to the administration of NS1-MB492 (Fig. 42) .

As a result of assessing T cell activation in the liver, an increase in granzyme B expression was observed with the administration of NS1-MB492 and A372-MB492/B040-ml0r compared to the administration of vehicle (Fig. 43).

Thus, the switched antibody inhibits T cell activation in the lymph nodes and spleen compared to the non-switched antibody.

(6-4-14) Study of the medicinal efficacy (antitumor effect) of antibodies A486MB492/B167-ml0r and A488-MB492/B226-ml0r.

After creating the MC38 cancer transplant mouse model in Example 6-4-1, the solvent and antibodies A486-MB492/B167-ml0r and A488-MB492/B226-ml0r were administered in the doses shown in Table. 40. The formation of groups is carried out 10 days after cell transplantation, and the introduction of antibodies is carried out into the tail vein 11, 15, 18 and 22 days after cell transplantation, a total of four times.

Table 40

Detailed information on drug efficacy for antibodies A486-MB492/B167-ml0r and A488-MB492/B226-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - 11, 15, 18 and 22 days after transplantation 2 5 А486-МВ492/В167mlOr 7.5 11, 15, 18 and 22 days after transplantation 3 5 А488-МВ492/В226mlOr 7.5 11, 15, 18 and 22 days after transplantation

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. The results confirm the antitumor effect of A486-MB492/B167-ml0r and A488-MB492/B226-ml0r (Fig. 44).

(6-4-15) Antibody administration and sampling, and FCM analysis to evaluate the systemic effects of antibodies A486-MB492/B167-ml0r and A488-MB492/B226-ml0r.

After establishing the MC38 cancer transplant mouse model in Example 6-4-1, solvent, NS1MB492 (non-switchable antibody), L486-MB492/B167-t10g and A488-MB492/B226-ml0r were administered at the doses shown in Table. 41.

- 133 046075

The formation of groups is carried out 10 days after cell transplantation, and the introduction of antibodies is carried out into the tail vein 11, 15, 18 and 22 days after cell transplantation, a total of four times. PBS containing 0.05% Tween-20 is used as a solvent.

Liver, lymph nodes and spleen were collected 13 days after initial administration as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and preparation of lymphocyte fractions). The mass of the spleens is measured, and lymphocytes are counted for fractions from the lymph nodes.

Table 41

Details of the Systemic Antibody Response Study Groups

A486-MB492/B167-ml0r and Α488-ΜΒ492/Β226^0γ

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Sampling day 1 3 Solvent - 11, 15, 18 and 22 days after transplantation 24 days after transplantation (13 days after the start of administration) 2 3 NS1-MB492 7.5 11, 15, 18 and 22 days after transplantation 24 days after transplantation (13 days after the start of administration) 3 3 A486-MV492/ B167-ml0r 7.5 11, 15, 18 and 22 days after transplantation 24 days after transplantation (13 days after the start of administration) 4 3 A488-MV492/ B226-ml0r 7.5 11, 15, 18 and 22 days after transplantation 24 days after transplantation (13 days after the start of administration)

By measuring the number of cells in lymphocyte fractions from lymph nodes and measuring spleen weight, an increase in the number of lymphocytes and spleen weight was observed upon administration of NS1-MB492. On the other hand, it was observed that the increase in lymphocyte count and spleen weight were suppressed by administration of A486-MB492/B167-ml0r and A488-MB492/B226-ml0r compared with those by administration of NS1-MB492 (FIG. 45).

As a result of assessing T cell activation in the liver, an increase in the percentage of CD8α-positive cells was observed with the administration of NS1-MB492, A486-MB492/B167-ml0r and A488-MB492/B226-ml0r compared to the administration of vehicle (Fig. 46).

Based on the above, it is confirmed that, compared with the non-switchable antibody, the switchable antibody inhibits the activation of T cells in the lymph nodes and spleen.

(6-4-16) Study of the medicinal efficacy (antitumor effect) of the antibody A489-MB492/B223-ml0r.

After creating the MC38 cancer transplant mouse model in Example 6-4-1, the solvent and antibody A489-MB492/B223-ml0r were administered in the doses shown in Table. 42. Antibody administration is carried out four times through the tail vein on the next day, four, seven and ten days after group formation. PBS containing 0.05% Tween-20 is used as a solvent.

Table 42 Detailed study of drug efficacy in groups with the introduction of the antibody A489-MB492/B223-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - The next day after the formation of the group, on the fourth, seventh and tenth days after the formation of the group 2 5 A489-MV492/ B223-ml0r 7.5 The next day after the formation of the group, on the fourth, seventh and tenth days after the formation of the group

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. The results confirm the antitumor effect of the 7.5 mg/kg dose (Fig. 47).

(6-4-17) Antibody administration and sampling and FCM analysis to evaluate systemic effects of antibody A489-MB492/B223-ml0r.

After establishing the MC38 cancer transplant mouse model in Example 6-4-1, vehicle, NS1MB492 (non-switchable antibody) and A489-MB492/B223-ml0r were administered at the doses shown in Table. 43. Antibodies are administered through the tail vein on the day of group formation, as well as 3 days and 6 days after group formation, a total of three times. As a solvent we use

- 134 046075 nyat FSB containing 0.05% Tween-20.

Seven days after the initial administration, the liver, lymph nodes and spleen are collected as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and preparation of lymphocyte fractions). In the spleens and lymph nodes, only the cell number is measured only in the lymphocyte fractions.

Table 43

Details of the Systemic Antibody Response Study Groups

A489-MB492/B223-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Sampling day 1 3 Solvent - On the day the group is formed, on the third and sixth days after the group is formed Seventh day after group formation 2 3 NS1-MB492 7.5 On the day the group is formed, on the third and sixth days after the group is formed Seventh day after group formation 3 3 A489-MV492/ B223-ml0r 7.5 On the day the group is formed, on the third and sixth days after the group is formed Seventh day after group formation

As a result of measuring the number of cells in the lymphocyte fractions from the lymph nodes and spleen, an increase in the number of cells was observed upon administration of NS1-MB492. On the other hand, suppression of the increase in the number of lymphocytes was observed with the administration of A489-MB492/B223-ml0r compared with that with the administration of NS1-MB492 (Fig. 48).

As a result of assessing the activation of T cells in the liver, an increase in the percentage of CD8a-positive cells was observed with the introduction of NS1-MB492 and A489-MB492/B223-ml0r compared to the solvent (Fig. 49).

Based on the above, it is confirmed that, compared with the non-switchable antibody, the switchable antibody inhibits the activation of T cells in the lymph nodes and spleen.

(6-4-18) Study of the medicinal efficacy (antitumor effect) of antibodies A548mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r.

After creating the MC38 cancer transplant mouse model in Example 6-4-1, the solvent and antibodies A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r are administered in the doses presented in table. 44. The antibody is administered through the tail vein twice: on the day of group formation and three days later. PBS containing 0.05% Tween-20 is used as a solvent.

Table 44

Detailed information from the study on the drug efficacy groups of antibodies A548-mIgG1/B256-ml0r and Α551^§Ο1/Β256^0ι

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 5 Solvent - On the day the group is formed and three days later 2 5 A548-mIgGl/ B256-ml0r 7.5 On the day the group is formed and three days later 3 5 A548-mIgGl/ B256-ml0r 37.5 On the day the group is formed and three days later 4 4 A548-mIgGl/ B256-ml0r 100 On the day the group is formed and three days later 5 5 A5 51-mlgGl/ B256-ml0r 7.5 On the day the group is formed and three days later 6 5 A5 51-mlgGl/ B256-ml0r 37.5 On the day the group is formed and three days later 7 4 A5 51-mlgGl/ B256-ml0r 100 On the day the group is formed and three days later

The antitumor effect in each group is assessed by tumor volume according to the calculations described in example 6-4-1. The results confirm the antitumor effect of administration of A548mIgG1/B256-ml0r at doses of 37.5 mg/kg and 100 mg/kg. A551-mIgG1/B256-ml0r also exhibited a significant antitumor effect at a dose of 100 mg/kg (Fig. 50).

(6-4-19) Antibody administration and sampling, and FCM analysis to assess systemic effects

- 135 046075 antibodies A548-mIgG1/B256 and A551-mIgG1/B256

After establishing the MC38 cancer transplant mouse model in Example 6-4-1, vehicle, NS1mIgG1 (non-switchable antibody), A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r were administered at the doses shown in Table. 45. Antibodies are administered through the tail vein on the day the group is formed, and also 3 days after the group is formed, a total of two times. PBS containing 0.05% Tween-20 is used as a solvent.

Seven days after the initial administration, the liver, lymph nodes and spleen are collected as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and preparation of lymphocyte fractions). In samples of spleens and lymph nodes, only organ mass is determined.

Table 45

Detailed information from the study by groups of systemic responses to the use of antibodies A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Sampling day 1 3 Solvent - On the day the group is formed and three days later Five days after the group was formed 2 3 NSl-mlgGl 0.3 On the day the group is formed and three days later Five days after the group was formed 3 3 NSl-mlgGl 1.5 On the day the group is formed and three days later Five days after the group was formed 4 3 NSl-mlgGl 7.5 On the day the group is formed and three days later Five days after the group was formed 5 3 A548-mIgGl/ B256-ml0r 1.5 On the day the group is formed and three days later Five days after the group was formed 6 3 A548-mIgGl/ B256-ml0r 7.5 On the day the group is formed and three days later Five days after the group was formed 7 3 A548-mIgGl/ B256-ml0r 37.5 On the day the group is formed and three days later Five days after the group was formed 8 3 A548-mIgGl/ B256-ml0r 100 On the day the group is formed and three days later Five days after the group was formed 9 3 A551-mIgGl/ B256-ml0r 1.5 On the day the group is formed and three days later Five days after the group was formed 10 3 A551-mIgGl/ B256-ml0r 7.5 On the day the group is formed and three days later Five days after the group was formed eleven 3 A551-mIgGl/ B256-ml0r 37.5 On the day the group is formed and three days later Five days after the group was formed 12 3 A551-mIgGl/ B256-ml0r 100 On the day the group is formed and three days later Five days after the group was formed

As a result of assessing the organ weight of the lymph nodes and spleen, an increase in organ weight is observed when NS1-mIgG1 is administered at a dose of 0.3 mg/kg, 1.5 mg/kg and 7.5 mg/kg. On the other hand, organ hypertrophy is not observed when A548-mIgG1/B256-ml0r or A551-mIgG1/B256-ml0r is administered at any dose of 1.5 mg/kg, 7.5 mg/kg, 37.5 mg/kg and 100 mg/kg (Fig. 51).

As a result of assessing the activation of T cells in the liver, an increase in the expression of both granzyme B and PD-1 was observed when NS1-mIgG1 was administered at a dose of 0.3 mg/kg. On the other hand, the expression is suppressed when A548-mIgG1/B256-ml0r and A551-mIgG1/B256-ml0r are administered at a dose of 1.5 mg/kg, 7.5 mg/kg, 37.5 mg/kg and 100 mg/kg. kg (Fig. 52).

Thus, compared to a non-switchable antibody, a switchable antibody inhibits T cell activation in the lymph nodes, spleen and liver.

(6-4-20) Study of the medicinal effectiveness (antitumor effect) of the antibody A5 51 -MV 110/B3 79-ml0r.

After creating the mouse model with MC38 transplantation in example 6-4-1, the solvent and antibody A551-MB110/B379-ml0r are administered in the doses indicated in table. 46. Antibodies are injected twice into the tail vein and three days after group formation. PBS with 0.05% Tween-20 is used as a solvent.

- 136 046075

Table 46

Detailed information on antibody drug efficacy study groups

A551-MB110/B379-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day 1 6 Solvent - On the day the group is formed and three days later 2 5 A551-MV110/ B379-ml0r 2.5 On the day the group is formed and three days later 3 6 A551-MV110/ B379-ml0r 7.5 On the day the group is formed and three days later 4 6 A551-MV110/ B379-ml0r 22.5 On the day the group is formed and three days later

The antitumor effect in each group is assessed by the tumor volume calculated as described in example 6-4-1. As a result, the antitumor effect was confirmed for each dosage (Fig. 53).

(6-4-21) Antibody administration and sampling, and FCM analysis to assess systemic effects

After establishing the MC38 transplant mouse model in Example 6-4-1, vehicle was administered, NS1mIgG1 (non-switchable antibody) and A551-MB110/B379-ml0r were administered as shown in Table. 47. The antibody is administered twice into the tail vein on the day the group is formed and 3 days after the group is formed. PBS containing 0.05% Tween-20 is used as a solvent.

Livers, lymph nodes and spleens are collected 5 days after initial administration as shown in Example 6-4-2 (collection of various organs from mice transplanted with the MC38 cell line and preparation of lymphocyte fractions) and FCM analysis is performed as shown in Example 6- 4-3 (FCM analysis using lymphocyte fractions from various organs).

Table 47

Detailed information on groups for assessing systemic responses with antibody A551-MB110/B379-ml0r

Group Number of animals Pharmaceutical agent Dose (mg/kg) Introduction day Sampling day 1 3 Solvent - On the day the group is formed and three days after the group is formed Five days after the group was formed 2 3 NSl-mlgGl 7.5 On the day the group is formed and three days after the group is formed Five days after the group was formed 3 3 А551-МВ110/ B379-ml0r 0.83 On the day the group is formed and three days after the group is formed Five days after the group was formed 4 3 А551-МВ110/ B379-ml0r 2.5 On the day the group is formed and three days after the group is formed Five days after the group was formed 5 3 А551-МВ110/ B379-ml0r 7.5 On the day the group is formed and three days after the group is formed Five days after the group was formed 6 3 А551-МВ110/ B379-ml0r 22.5 On the day the group is formed and three days after the group is formed Five days after the group was formed

As a result of assessing the organ mass of the lymph nodes and spleen, an increase in organ mass was observed when NS1-mIgG1 was administered at a dose of 7.5 mg/kg. On the other hand, organ enlargement was not observed when A551-MB110/B379-ml0r was administered at any dose, namely 0.83 mg/kg, 2.5 mg/kg, 7.5 mg/kg and 22.5 mg/kg. (Fig. 54). In addition, when assessing the activation of T cells in the spleen using the FCM method, the increase in activation markers (PD-1, ICOS, granzyme B) observed with the administration of NS1-mIgG1 was not confirmed with the administration of A551-MB110/B379-mYr at any dose ( Fig. 55).

When assessing T cell activation in the liver, an increase in the expression of both PD-1 and ICOS was observed when NS1-mIgG1 was administered at a dose of 7.5 mg/kg. On the other hand, suppression of expression at the solvent level when A551-MB110/B379-ml0r is administered at doses of 0.83 mg/kg and 2.5 mg/kg, and even greater suppression of expression than from NS1-mIgG1 at dose 7 .5 mg/kg (Fig. 56).

Thus, compared to a non-switchable antibody, a switchable antibody inhibits

- 137 046075 activation of T cells in the lymph nodes, spleen and liver.

Example 7. Preparation of a modified Fc capable of enhancing agonistic activity.

(7-1) Preparation of variants with increased FcyR-binding activity.

TT14 heavy chain constant region (SEQ ID NO: 149), which increases FcyRIIb binding activity as described in WO 2017104783 (heavy chain constant region containing T250V/T307P as amino acid modifications, and L234Y/P238D/V264I/A330K as amino acid modifications to increase FcγRIIb-binding activity in the Fc region containing SEQ ID NO: 182 (both modified positions are given in accordance with the EU numbering)), the TT16 region (SEQ ID NO: 150) containing the amino acid mutation G237D introduced into the heavy chain constant region TT11, which increases FcγRIIb binding activity as described in WO 2017104783 (heavy chain constant region containing amino acid modifications T250V/T307P and L234Y/P238D/A330K as modifications to increase FcyRIIb binding activity in the Fc region containing SEQ ID NO: 182 (both modified positions are given in accordance with the EU numbering)), the P587 heavy chain constant region (SEQ ID NO: 151), which increases FcyRIIb-binding activity, as described in WO 2014163101; and a P253 sequence (SEQ ID NO: 93), which is a precursor variant of the heavy chain constant region with increased binding to FcyRIIb, combined with the heavy chain variable region of the switch antibody (A375; A372; A356; A486; A488; A489; A548; and A551) or with the heavy chain variable region of a negative control antibody (IC17HdK (SEQ ID NO: 152)). As a result, genes were created with the following combinations of each heavy chain constant region and variable region.

Table 48

Full length heavy chain Heavy chain variable region (SEQ ID NO) Heavy Chain Constant Region (SEQ ID NO) A375-TT14 AZ75 (SEQ ID NO 43) TT14 (SEQ ID NO: 149) A375-TT16 AZ75 (SEQ ID NO 43) TT16 (SEQ ID NO: 150) А375-Р587 A375 (SEQ ID NO 43) P587 (SEQ ID NO: 151) А372-Р253 A3 72 (SEQ ID NO 44) P253 (SEQ ID NO: 93) A356-TT16 A3 56 (SEQ ID NO 45) TT16 (SEQ ID NO: 150) А356-Р587 A3 56 (SEQ ID NO 45) P587 (SEQ ID NO: 151) А486-Р253 A486 (SEQ ID NO 46) P253 (SEQ ID NO 93) А488-Р253 A488 (SEQ ID NO 48) P253 (SEQ ID NO 93) А489-Р253 A489 (SEQ ID NO 49) P253 (SEQ ID NO 93) А548-Р587 A548 (SEQ ID NO 50) P587 (SEQ ID NO: 151) A551-TT16 A551 (SEQ ID NO 51) TT16 (SEQ ID NO: 150) A551-P587 A551 (SEQ ID NO 51) P587 (SEQ ID NO: 151) IC17HdK-TT16 IC17HdK (SEQ ID NO 152) TT16 (SEQ ID NO: 150) IC17HdK-P587 IC17HdK (SEQ ID NO 152) P587 (SEQ ID NO: 151) IC17HdK-P253 IC17HdK (SEQ ID NO 152) P253 (SEQ ID NO: 93)

The switchable antibody heavy chain variable region (A375, A356 and A551) or the negative control antibody heavy chain variable region (IC17HdK (SEQ ID NO: 152)) is combined with the heavy chain constant region of MY201 (SEQ ID NO: 153) (heavy chain constant region chain containing G236N/H268D/A330K as amino acid modifications that increase binding activity to FcyRIIb in the Fc region comprising the amino acid sequence SEQ ID NO: 182 (all modified positions are indicated by EU numbering)) or MY518 (SEQ ID NO: 154) (heavy chain constant region containing L235W/G236N/H268D/Q295L/K326T/A330K as amino acid modifications that increase binding activity to FcyRIIb in the Fc region containing the amino acid sequence SEQ ID NO: 182 (all modified positions are indicated by EU numbering) , which are described in WO 2017104783 and have increased binding activity to FcyR, the heavy chain constant region of MY201a (SEQ ID NO: 155) or MY518a (SEQ ID NO: 156), in which the K214R modification is introduced into MY201 or MY518, or the heavy chain constant region chain MY201aPh (SEQ ID NO: 157), in which the L235W modification is introduced into MY201a. As a result, genes were created with combinations of each constant region and variable region of the heavy chain, presented below in table. 49.

- 138 046075

Table 49

Full length heavy chain Heavy chain variable region (SEQ ID NO) Heavy Chain Constant Region (SEQ ID NO) A375-MY201aPh A375 (SEQ ID NO 43) MY201aPh (SEQ ID NO: 157) A375-MY518 A375 (SEQ ID NO 43) MY518 (SEQ ID NO: 154) A375-MY518a A375 (SEQ ID NO 43) MY518a (SEQ ID NO: 156) A356-MY518 A356 (SEQ ID NO 45) MY518 (SEQ ID NO: 154) A551-MY201a A551 (SEQ ID NO 51) MY201a SEQ ID NO: 155) A551-MY201aPh A551 (SEQ ID NO 51) MY201aPh (SEQ ID NO: 157) A551-MY518 A551 (SEQ ID NO 51) MY518 (SEQ ID NO: 154) A551-MY518a A551 (SEQ ID NO 51) MY518a (SEQ ID NO: 156) IC17HdK-MY201 IC17HdK (SEQ ID NO: 152) MY201 (SEQ ID NO: 153) IC17HdK- MY201aPh IC17HdK (SEQ ID NO: 152) MY201aPh (SEQ ID NO: 157) IC17HdK-MY518 IC17HdK (SEQ ID NO: 152) MY518 (SEQ ID NO: 154) IC17HdK-MY518a IC17HdK (SEQ ID NO: 152) MY518a (SEQ ID NO: 156)

In table 50 shows that the antibodies of interest are expressed and purified by methods known to those skilled in the art by combining the above antibody heavy chain genes with a switchable antibody light chain gene (B040-Lamlib, B167-Lamlib, B226Lamlib, B223-Lamlib, B256- Lamlib and B379-Lamlib prepared in Examples 5-2 and 5-3) or the negative control antibody light chain genome (IC17L-k0, prepared by combining the light chain variable region IC17L (SEQ ID NO: 158) with the human k chain (SEQ ID NO: 141) as the light chain constant region).

Table 50

Variants with increased FcYR-binding activity

Variable region (heavy chain/light chain) Heavy chain constant region Light chain constant region Antibody designation А375/В167 MY201aPh Lamlib A375-MY201aPh/B167-Lamlib MY518 Lamlib A375-MY518/B167-Lamlib MY518a Lamlib A3 75 -MY518a/B 167 -Lamlib P587 Lamlib A375-P587/B167-Lamlib TT14 Lamlib A375-TT14/B167-Lamlib TT16 Lamlib A375-TT16/B167-Lamlib А372/В040 P253 Lamlib A372-P253/B040-Lamlib А356/В040 MY518 Lamlib A356-MY518/B040-Lamlib P587 Lamlib A356-P587/B040-Lamlib TT16 Lamlib A356-TT16/B040-Lamlib А486/В167 P253 Lamlib A486-P253/B 167-Lamlib А488/В226 P253 Lamlib A488-P253/B226-Lamlib А489/В223 P253 Lamlib A489-P253/B223-Lamlib А548/В256 P587 Lamlib A548-P587/B256-Lamlib A551/B256 MY201a Lamlib A5 51 -Μ Y2 01 a/B 2 5 6 -Lamlib MY201aPh Lamlib A551-MY201 aPh/B256-Lamlib MY518 Lamlib A5 51-MY518/B 25 6 -Lamlib P587 Lamlib A551-P587/B256-Lamlib А551/В379 MY518a Lamlib A5 5 l-MY518a/B 379 -Lamlib P587 Lamlib A551-P587/B379-Lamlib TT16 Lamlib A5 51 -TT16/B 3 79 -Lamlib IC17HdK/IC17L MY201 kO IC17HdK-MY201/IC17L-k0 MY201aPh kO IC17HdK-MY201aPh/IC17L-k0 MY518 kO IC17HdK-MY518/IC17L-kO MY518a kO IC17HdK-MY518a/IC17L-kO P253 kO IC17HdK-P253/IC17L-kO P587 kO IC17HdK-P587/IC17L-kO TT16 kO IC17HdK-TT16/IC17L-kO

(7-2) Obtaining variants with increased pI by amino acid modification of the constant region.

The heavy chain constant region is prepared by combining the heavy chain constant region with increased FcYR binding activity obtained in Example 7-1 with Q311R, P343R and D413K, which are amino acid mutations that increase pI without significantly changing FcYR binding activity, as described in WO 2017046994.

In particular, heavy chain constant region genes with amino acid mutations are obtained,

- 139 046075 introduced into each of the antibody heavy chain constant region genes, as shown below.

Introduction of P343R/D413K into MY518 (SEQ ID NO: 154): SCF025 (SEQ ID NO: 64)

Introduction of P343R/D413K into MY518a (SEQ ID NO: 156): SCF025a (SEQ ID NO: 65)

Introduction of P343R/D413K into TT14 (SEQ ID NO: 149): SCF027 (SEQ ID NO: 66)

Introduction of P343R/D413K into TT16 (SEQ ID NO: 150): SCF028 (SEQ ID NO: 67)

Introduction of Q311R/P343R into MY518 (SEQ ID NO: 154): SCF030 (SEQ ID NO: 68)

Introduction of Q311R/P343R into TT14 (SEQ ID NO: 149): SCF032 (SEQ ID NO: 69)

Introduction of Q311R/P343R into TT16 (SEQ ID NO: 150): SCF033 (SEQ ID NO: 70)

Introduction of P343R into MY518 (SEQ ID NO: 154): SCF039 (SEQ ID NO: 71)

Introduction of P343R into MY518a (SEQ ID NO: 156): SCF039a (SEQ ID NO: 72)

Introduction of D413K into MY518 (SEQ ID NO: 154): SCF040 (SEQ ID NO: 73)

Introduction of P343R into MY201a (SEQ ID NO: 155): SCF041a (SEQ ID NO: 74)

Introduction of P343R into MY201aPh (SEQ ID NO: 157): SCF041aPh (SEQ ID NO: 75)

Introduction of D413K in MY201a (SEQ ID NO: 155): SCF042a (SEQ ID NO: 76)

Introduction of P343R/D413K into MY201a (SEQ ID NO: 155): SCF043a (SEQ ID NO: 77)

Introduction of P343R/D413K into MY201aPh (SEQ ID NO: 157): SCF043aPh (SEQ ID NO: 78)

Introduction of Q311R into MY201a (SEQ ID NO: 155): SCF056a (SEQ ID NO: 79)

Introduction of Q311R into MY201aPh (SEQ ID NO: 157): SCF056aPh (SEQ ID NO: 80)

Introduction of Q311R/P343R into MY201a (SEQ ID NO: 155): SCF057a (SEQ ID NO: 81)

Introduction of Q311R/P343R into MY201aPh (SEQ ID NO: 157): SCF057aPh (SEQ ID NO: 82)

Introduction of Q311R/D413K into MY201a (SEQ ID NO: 155): SCF059a (SEQ ID NO: 83)

Introduction of Q311R/D413K into MY201aPh (SEQ ID NO: 157): SCF059aPh (SEQ ID NO: 84)

Introduction of Q311R into MY518a (SEQ ID NO: 156): SCF060a (SEQ ID NO: 85)

Antibody heavy chain genes are prepared as shown in Table. 51 below by combining the heavy chain constant region gene obtained in the present invention with the heavy chain variable region gene A375 (SEQ ID NO: 43) or A551 (SEQ ID NO: 51).

Table 51

Full length heavy chain Heavy chain variable region (SEQ ID NO) Heavy Chain Constant Region (SEQ ID NO) A375-SCF025 A375 (SEQ ID NO: 43) SCF025 (SEQ ID NO: 64) A375-SCF027 A375 (SEQ ID NO: 43) SCF027 (SEQ ID NO: 66) A375-SCF028 A375 (SEQ ID NO: 43) SCF028 (SEQ ID NO: 67) A375-SCF030 A375 (SEQ ID NO: 43) SCF030 (SEQ ID NO: 68) A375-SCF032 A375 (SEQ ID NO: 43) SCF032 (SEQ ID NO: 69) A375-SCF033 A375 (SEQ ID NO: 43) SCF033 (SEQ ID NO: 70) A375-SCF039 A375 (SEQ ID NO: 43) SCF039 (SEQ ID NO: 71) A375-SCF040 A375 (SEQ ID NO: 43) SCF040 (SEQ ID NO: 73) A375-SCF041aPh A375 (SEQ ID NO: 43) SCF041aPh (SEQ ID NO: 75) A375-SCF043aPh A375 (SEQ ID NO: 43) SCF043aPh (SEQ ID NO: 78) A375-SCF056aPh A375 (SEQ ID NO: 43) SCF056aPh (SEQ ID NO: 80) A375-SCF057aPh A375 (SEQ ID NO: 43) SCF057aPh (SEQ ID NO: 82) A375-SCF059aPh A375 (SEQ ID NO: 43) SCF059aPh (SEQ ID NO: 84) A375-SCF060a A375 (SEQ ID NO: 43) SCF060a (SEQ ID NO: 85) A551-SCF025a A551 (SEQ ID NO: 51) SCF025a (SEQ ID NO: 65) A551-SCF039a A551 (SEQ ID NO: 51) SCF039a (SEQ ID NO: 72) A551-SCF041a A551 (SEQ ID NO: 51) SCF041a (SEQ ID NO: 74) A551-SCF041aPh A551 (SEQ ID NO: 51) SCF041aPh (SEQ ID NO: 75) A551-SCF043a A551 (SEQ ID NO: 51) SCF043a (SEQ ID NO: 77) A551-SCF043aPh A551 (SEQ ID NO: 51) SCF043aPh (SEQ ID NO: 78) A551-SCF056a A551 (SEQ ID NO: 51) SCF056a (SEQ ID NO: 79) A551-SCF056aPh A551 (SEQ ID NO: 51) SCF056aPh (SEQ ID NO: 80) A551-SCF057a A551 (SEQ ID NO: 51) SCF057a (SEQ ID NO: 81) A551-SCF057aPh A551 (SEQ ID NO: 51) SCF057aPh (SEQ ID NO: 82) A551-SCF059a A551 (SEQ ID NO: 51) SCF059a (SEQ ID NO: 83) A551-SCF059aPh A551 (SEQ ID NO: 51) SCF059aPh (SEQ ID NO: 84) A551-SCF060a A551 (SEQ ID NO: 51) SCF060a (SEQ ID NO: 85)

In addition, by combining these antibody heavy chain genes with the B167-Lamlib, B256Lamlib and B379-Lamlib genes obtained in Example 5-3 as antibody light chain genes, the desired antibody was expressed and purified as shown in Table. 52 by methods known to those skilled in the art.

- 140 046075

Table 52

Variable region (heavy chain/light chain) Constant region heavy chain Light chain constant region Antibody name A375 / B167 (A375 SEQ ID NO: 43) (B167 SEQ ID NO: 54) SCFO25 (SEQ ID NO: 64) Lamlib (SEQ ID NO: 63) A375-SCF025/B167-LamLib SCF027 (SEQ ID NO: 66) Lamlib (SEQ ID NO: 63) A375SCFO27/B167-LamLib SCF028 (SEQ ID NO; 67) Lamlib (SEQ ID NO: 63) A375-SCFO28/B167LamLib SCF030 (SEQ ID NO:68) Lamlib (SEQIDNO: 63) A375-SCF030/B167LamLib SCF032 (SEQ ID NO: 69) Lamlib (SEQ ID NO: 63) A375-SCFO32/B167-LamLib SCF033 (SEQ ID NO:70) Lamlib (SEQ ID NO: 63) A375-SCFO33/B167-LamLib SCF039 (SEQ ID NO:71) Lamlib (SEQ ID NO: 63) A375-S CFO3 9/B167-LamLib SCF040 (SEQ ID NO:73) Lamlib (SEQIDNO: 63) A375-SCF040iB167-LamLib SCF041aPh (SEQ ID NO: 75) Lamlib (SEQIDNO: 63) A375-SCFO41aPh/B 167-Lamlib SCF043aPh (SEQ ID NO: 78) Lamlib (SEQ ID NO: 63) A375-SCFO43aPh/B 167-LamLib SCF056aPh (SEQ ID NO: 80) Lamlib (SEQ ID NO: 63) A375-SCFO56aPh/B 167-Lamlib SCF057aPh (SEQ ID NO: 82) Lamlib (SEQIDNO: 63) A375-SCFO57aPh/B167LamIib SCF059aPh (SEQ ID NO: 84) Lamlib (SEQ ID NO: 63) A375SCFO59aPh/B167LamLib SCF060a (SEQ ID NO: 85) Lamlib (SEQ ID NO: 63) A375SCF060a/B167-LamIib A551/B256 (A551 SEQ ID NO: 51) (B256 SEQ ID NO: 59) SCF025a (SEQ ID NO: 6B) Lamlib (SEQ ID NO: 63) A551SCF025a/B256-LamLib SCF039a (SEQ ID NO: 72) Lamlib (SEQ ID NO: 63) A551-SCF039a/B256-LamIib SCF041a (SEQ ID NO: 74) Lamlib (SEQ ID NO: 63) A551-SCFO41a/B256-LamLib SCF041aPh (SEQ ID NO: 75) Lamlib (SEQ ID NO: 63) A551-SCF041aPh/B256LamLib SCF043a (SEQ ID NO: 77) Lamlib (SEQ ID NO: 63) A551-SCF043a/B256-LamIib SCF043aPh (SEQ ID NO: 78) Lamlib (SEQ ID NO: 63) A551-SCFO43aPh/B256LamLib SCF056a (SEQ ID NO: 79) Lamlib (SEQIDNO: 63) A551-SCFO56a/B256-LamLib SCFOSSaPh (SEQ ID NO: 80) Lamlib (SEQ ID NO: 63) A551-SCF056aPh/B256-Lamlib SCF057a (SEQ ID NO: 81) Lamlib (SEQ ID NO: 63) A551-SCF057a/B256-LamLib SCF057aPh (SEQ ID NO: 82) Lamlib (SEQ ID NO: 63) A551-SCF057aPh/B256LamLib SCF059a (SEQ ID NO: 83) Lamlib (SEQ ID NO: 63) A551-SCF059a/B256-LamLib SCF059aPh (SEQ ID NO: 84) Lamlib (SEQ ID NO: 63) A551-SCFO59aPh/B256LamLib A551/B379 (A551 SEQ ID NO: 51) (B379 SEQ ID NO: 60) SCF039a (SEQ ID NO: 72) Lamlib (SEQ ID NO: 63) A551SCF039a/B379-LamLib SCF041a (SEQ ID NO: 74) Lamlib (SEQ ID NO: 63) A551SCF041a/B379-LamLib SCF041aPh (SEQ ID NO:75) Lamlib (SEQ ID NO: 63) A551 SCF041a?h/B379 LamLib SCF043a (SEQ ID NO: 77) Lamlib (SEQ ID NO; 63) A551-SCF043a/B379-LamLib SCF043aPh (SEQ ID NO:78) Lamlib (SEQIDNO: 63) A551-SCFO43aPh/B379-LamLib SCF056a (SEQ ID NO: 79) Lamlib (SEQ ID NO: 63} A551SCF056a/B379-Lamlib SCF056aPh (SEQ ID NO: 80) Lamlib (SEQIDNO: 63} A551-SCF056aPh/B379LamIib SCF057a (SEQ ID NO:81) Lamlib (SEQ ID NO: 63} A551-SCF057a/B379-LamIib SCF057aPh (SEQ ID NO:82) Lamlib (SEQ ID NO: 63) A551SCF057aPh/B379-LamIib SCF059a (SEQ ID NO:83) Lamlib (SEQ ID NO: 63) A551-SCF059a/B379-LamLib SCF059aPh (SEQ ID NO: 84) Lamlib (SEQ ID NO: 63} A551-SCF059aPh/B379-LamLib SCF060a (SEQ ID NO:85) Lamlib (SEQ ID NO: 63} A551-SCF060a/B379-LamLib

(7-3) Development of control antibodies to compare agonist activity.

Antibodies are obtained (IC17HdK-MY201/IC17L-k0; IC17HdK-MY201aPh/IC17L-k0; IC17HdKMY518/IC17L-k0; IC17HdK-MY518a/IC17L-k0; IC17HdK-P253/IC17L-k0; IC17HdK-P58 7/IC17L-k0;IC17HdK -TT16/IC17L-k0) with the heavy chain variable region IC17HdK (SEQ ID NO: 152), the preparation of which is described in Example 7-1 as a control antibody to compare the agonistic active

- 141 046075 details. In addition, the antibody of interest (IC17HdK-G4d/IC17L-k0) is expressed and purified by methods known in the art by combining the IC17L-k0 antibody light chain gene (SEQ ID NO: 158, variable region; SEQ ID NO: 141, constant region) with the antibody heavy chain gene IC17HdK-G4d, which is obtained by combining the heavy chain variable region gene IC17HdK (SEQ ID NO: 152) with the G4d gene (SEQ ID NO: 159) from which Gly and Lys at the C-terminus have been removed human IgG4 heavy chain constant region. These control antibodies were used in Example 5.

(7-4) Assessment of human Fcy receptor binding of variants with increased pI by amino acid modification of the constant region.

Biacore T200 software (GE Healthcare) is used to evaluate binding activity to the corresponding human Fcy receptor (hereinafter referred to as FcyR). The measurement is performed at 25°C using a buffer containing 50 mM phosphate, 150 mM NaCl, 0.05 w/v% - P20, pH 7.4. Approximately 1000 RU of antibody is captured on a sensor chip immobilized with Capture Select™ Human Fab-lambda Kinetics Biotin Conjugate (Thermo Fisher Scientific) as the ligand capture molecule. Human FcyR is diluted to 8 nM for FcYRIa and 1000 nM for other FcyR in measurement buffer, after which it is reacted with captured antibody. The FcyR binding activity of each antibody was assessed by calculating the FcyR binding amount (RU) per unit amount of antibody using Biacore T200 Evaluation Software 2.0.

The extracellular domain of FcyR is obtained by the methods described below. First, gene synthesis of the extracellular domain of FcyR is carried out by a method known to those skilled in the art. In this case, the FcyR sequences are obtained based on the data registered by NCBI. In particular, the FcyRI sequence is obtained based on the sequence from the NCBI database NM_000566.3, the FcYRIIa sequence based on the sequence from the NCBI database NM_001136219.1, the FcYRIIb sequence based on the sequence from the NCBI database NM_004001.3 and the FcYRIIIa sequence based on the sequence from the database NCBI data NM_001127593.1; and its tag had a His tag added to their C-termini. The polymorphic site of FcYRIIa is obtained with reference to the publication J. Exp. Med., 172, 1990, 19-25; and the FcYRIIIa polymorphic site are obtained with reference to the publication of J. Clin. Invest., 100, 1997, 1059-1070. The resulting gene fragments are inserted into an animal cell expression vector to produce expression vectors. The resulting expression vectors are transiently introduced into FreeStyle293 cells derived from human embryonic kidney cells (Invitrogen), after which the proteins of interest are expressed. Culture supernatants were collected, passed through a 0.22 μm pore filter, and purified in essentially four steps described below. The first step is performed using cation exchange column chromatography (SP Sepharose FF), the second step using affinity column chromatography (HisTrap HP) for His tag, the third step using gel filtration column chromatography (Superdex200), and the fourth step using using sterilizing filtration. However, for FcyRI, the first step is anion exchange column chromatography with Q Sepharose FF. Purified protein concentrations were calculated by measuring adsorption at 280 nm using a spectrophotometer, and adsorption coefficients were calculated from the obtained values using the methods of PACE et al. (Protein Science, 4, 1995, 2411-2423).

Table 53 shows the binding amount per unit amount of antibody, as well as the amount relative to the binding amount of A375-G1T3/B167-Lamlib. All antibodies in the table. 53 show a higher binding amount to human FcYRIIb per unit amount of antibody than A375G1T3/B167-Lamlib, and the relative value is 2.85-14.26, while the binding amount of A375-G1T3/B167-Lamlib to human FcYRIIb is set to be 1.0. The amount of human FcYRIIb binding per unit amount of antibody and the relative amounts bound were similar in the presence or absence of amino acid modifications to increase the pI of the constant region. In addition, the binding amount of antibodies containing the L235W modification to hFcgRIa is reduced compared to that for A375-TT14/B167-Lamlib.

- 142 046075

Table 53

Binding activity of each human FcyR

Abbreviation Binding amount per 1RU of antibody (RU) hFcgRIa hFcgRIlaH hFcgRIIaR hFcgRIIb hFcgRIIIaF hFcgRIIIaV A375-G1T3/B167-Lamlib 0.0813 0.0567 0.0463 0.0110 0.0213 0.0700 A375-MY201a/B167-Lamlib 0.0396 0.0534 0.0338 0.0401 0.0007 0.0024 A375-MY201aPh/B167-Lamlib 0.0040 0.0328 0.0335 0.0313 0.0007 0.0011 A375-MY518/B167-Lannlib 0-0020 0.0466 0.0460 0.0482 0.0009 0.0015 A375-MY518g/B167-Lamlib 0.0033 0.0479 0.0470 0.0489 0.0006 0.0009 A375-TT11/B167-Laml ib 0.0075 0.0020 0.0041 0.0477 -0.0009 -0.0010 A375-TT14/B167-Laml ib 0.0045 0.0042 0.0067 0.0805 0.0000 0.0003 A375-TT16/B167-Laml ib 0.0034 0.0126 0.0359 0.1090 0.0001 -0.0002 A375-P587/B167-Laml ib 0.0962 0.0029 0.0371 0.1427 0.0001 0.0001 A375-P253/B167-Laml ib 0.0937 0.0497 0.1678 0.1563 0.0056 0.0019 A375-SCF025/B167Lamlib 0.0022 0.0514 0.0519 0.0512 0.0012 0.0014 A375-SCF025a/B167-Lamli b 0.0021 0.0514 0.0518 0.0511 0.0009 0.0016 A375-SCF027/B167-Lamlib 0.0044 0.0031 0.0058 0.0766 -0.0005 -0.0006 A375-SCF028/B167-Lamlib 0.0027 0.0101 0.0299 0.0995 -0.0009 -0.0010 A375-SCF030/B167-Lamlib 0.0020 0.0533 0.0532 0.0532 0.0017 0.0020 A375-SCF032/B167Lamlib 0.0049 0.0030 -0.0004 0.0782 -0.0007 -0.0007 A375-SCF033/B167-Lamlib 0-0033 0.0103 0.0255 0.0979 0.0000 0.0002 A375-SCF039/B167-Lamlib 0.0018 0.0535 0.0542 0.0531 0.0008 0.0013 A375-SCF039a/B167-Lamli b 0.0018 0.0542 0.0554 0.0540 0.0012 0.0013 A375-SCF040/B167-Lamlib 0.0016 0.0454 0.0446 0.0465 0.0010 0.0013 A375-SCF041a/B167-Lamlib 0.0446 0.0571 0.0376 0.0433 0.0009 0.0022 A375-SCF042a/B167-Lamli b 0.0396 0.0522 0.0327 0.0393 0.0003 0.0014 A375-SCF043a/B167-Lamli b 0.0429 0.0552 0.0361 0.0419 0.0011 0.0025 A375-SCF056a/B167-Lamli b 0.0395 0.0529 0.0331 0.0399 0.0011 0.0023 A375-SCF057a/B167-Lamli b 0.0405 0.0525 0.0367 0.0412 0.0013 0.0025 A375-SCF059a/B167-Lamli b 0.0380 0.0502 0.0319 0.0358 0.0010 0.0021 A375-SCF060a/B167-Lamli b 0.0021 0.0463 0.0459 0.0472 0.0011 0.0008 A375-SCF041aPh/B167-Lamlib 0.0013 0.0371 0.0371 0.0350 0.0007 0.0005 A375-SCF043aPtVB167-Lamlib 0.0011 0.0349 0.0349 0.0339 0.0001 0.0001 A375-SCF056aPh/B167-Lamlib 0.0019 0.0314 0.0299 0.0292 0.0010 0.0010 A375-SCF057aPh/B167-Lamlib 0.0021 0.0365 0.0364 0.0345 -0.0003 0.0005 A375-SCF059aPh/B167-Lamlib 0.0089 0.0332 0.0319 0.0291 0.0003 0.0028

- 143 046075

Abbreviation. J Relative bound quantity 1 hFcgRIa hFcgRIIaH hFcgRIIaR PFcgRlIb hFcgRIIIaF hFcgRIIiaV A375-G1T3/B167-Lamlib 1.00 1.00 1.00 1.00 1.00 1.00 A375-MY201a/B167-Lamlib 0.49 0.94 0.73 3.66 0.04 0.03 A375-MY201aPh/B167-Lamlib 0.05 0.58 0.72 2.85 0.03 0.02 A375-MY518/B167-Lamli b 0.02 0.82 0.99 4.40 0.04 0.02 Α375-ΜΫ518g/B167-Lamlib 0.04 0.84 1.01 4.46 0.03 0.01 A375-TT11/B167-Lamlib 0.09 0.03 0.09 4.35 -0.04 -0.01 A375-TT14/B167-Lamlib 0.05 0.07 0.14 7.34 0.00 0.00 A375-TT16/B167-Lamlib. 0.04 0.22 0.77 9.94 0.00 0.00 A375-P587/B167-Lamlib. 1.18 0.05 0.80 13.02 0.00 0.00 A375-P253/B167-Lamlib 1.15 0.88 3.62 14.26 0.26 0.03 A375-SCF025/B167-Lamlib 0.03 0.91 1.12 4.67 0.06 0.02 A375-SCFQ253/B167-Lamlib 0.03 0.91 1.12 4.67 0.04 0.02 A375-SCF027/B167-Lam 1 i b 0.05 0.06 0.12 6.99 -0.02 -0.01 A375-SCF028/B167-Lamlib 0.03 0.18 0.64 9.08 -0.04 -0.01 A375-SCF030/B167-Lamlib 0.02 0.94 1.15 4.85 0.08 0.03 A375-SCFO32/B167Lamlib 0.06 0.05 -0.01 7.14 -0.03 -0.01 A375-SCF033/B167-Lamlib' 0.04 0.18 0.55 8.94 0.00 0.00 A375-SCFO39/B167-Lamlib. 0.02 0.94 1.17 4.85 0.04 0.02 A375-SCF039a/B167-Lamli b 0.02 0.96 1.20 4.93 0.06 0.02 A375-SCF040/B167-Lamlib 1 0.02 0.80 0.96 4.24 0.05 0.02 A375-SCF041ai/B167-Lamlib : 0.55 1.01 0.81 3.95 0.04 0.03 A375-SCFQ42a/B167-Lamlib 0.49 0.92 0.71 3.59 0.01 0.02 A375-SCF043a/B167-Lamlib i 0.53 0.97 0.78 3.82 0.05 0.04 A375-SCF0563/B167-Lamiib । 0.49 0.93 0.71 3.64 0.05 0.03 A375-SCFO57a/B167-Lamlib i 0.50 0.93 0.79 3.76 0.06 0.04 A375-SCF059a/B167-Lamlib 0.47 0.88 0.69 3.27 0.05 0.03 A375-SCF060a/B167-Lamlib 1 0.03 0.82 0.99 4.31 0.05 0.01 A375- SCF04 laPh/B 167- Lamli b 0.02 0.65 0.80 3.20 0.03 0.01 A375-SCF043aPtVB167-Lamlib 0-01 0.62 0.75 3.10 0.00 0.00 A375-SCFO5 6aPh/B 167- Lamli b 0.02 0.55 0.64 2.67 0.05 0.01 A375-SCF057aPtVB167-Lamlib 0.03 0.64 0.79 3.15 -0.02 0.01 A375-SCF059aPtVB167-Lamlib 0.11 0.59 0.69 2.66 0.01 0.04

(7-5) Assessment of human FcRn binding in variants with increased pI by amino acid modification of the constant region.

Biacore T200 software (GE Healthcare) was used to assess human FcRn binding activity. Measurements are performed at 25°C using a buffer containing 50 mM phosphate, 150 mM NaCl, 0.05 w/v% - P20, pH 6.0. The human FcRn proteins used for this determination are prepared according to the procedure described in WO 2010107110. Approximately 400 RU of antibody is captured using the immobilized CaptureSelect™ Human Fab-lambda Kinetics Biotin Conjugate sensor chip (Thermo Fisher Scientific) as the capture molecule ligand followed by binding of human FcRn diluted in measurement buffer. The FcRn binding activity of the respective antibodies is assessed by calculating the KD (mol/L) using a steady-state model using Biacore T200 Evaluation Software 2.0. KD values were similar in the presence or absence of amino acid modifications to increase the pI of the constant region. In table 54 shows the KD value (mol/l) of the corresponding antibodies to human FcRn.

- 144 046075

Table 54

Abbreviation K _D (mol/l) A375-G1T3/B167-Lamlib 1.27E-06 A375-MY201a/B167-Lamlib 1.19E-06 A375-MY201aPh/B167-Lamlib 1.18E-06 A375-MY518/B167-Lamlib 1.27E-06 A375-MY518a/B167-Lamlib 1.32E-06 A375-TT11/B167-Lamlib 1.56E-06 A375-TT14/B167-Lamlib 1.77E-06 A375-TT16/B167-Lamlib 1.51E-06 A375-P587/B167-Lamlib 1.70E-06 A375-P253/B167-Lamlib 1.42E-06 A375-SCF025/B167-Lamlib 1.10E-06 A375-SCF025a/B167-Lamlib 1.13E-06 A375-SCF027/B167-Lamlib 1.33E-06 A375-SCF028/B167-Lamlib 1.27E-06 A375-SCF030/B167-Lamlib 8.63E-07 A375-SCF032/B167-Lamlib 9.66E-07 A375-SCF033/B167-Lamlib 8.74E-07 A375-SCF039/B167-Lamlib 1.09E-06 A375-SCF039a/B167-Lamlib 1.11E-06 A375-SCF040/B167-Lamlib 1.46E-06 A375-SCF041a/B167-Lamlib 1.10E-06 A375-SCF042a/B167-Lamlib 1.49E-06 A375-SCF043a/B167-Lamlib 1.18E-06 A375-SCF056a/B167-Lamlib 9.56E-07 A375-SCF057a/B167-Lamlib 9.28E-07 A375-SCF059a/B167-Lamlib 1.03E-06 A375-SCF060a/B167-Lamlib 1.01E-06 A375-SCF041aPh/B167-Lamlib 1.13E-06 A375-SCF043aPh/B167-Lamlib 1.21E-06 A375-SCF056aPh/B167-Lamlib 1.00E-06 A375-SCF057aPh/B167-Lamlib 9.09E-07 A375-SCF059aPh/B167-Lamlib 1.05E-06

Example 8. Preparation of kynurenine (Kup)-dependent antibodies against CD137.

(8-1) Generation of antibodies with kynurenine-dependent binding activity against human CD137 from a rationally designed antibody library.

Antibodies exhibiting binding activity to human CD137 in the presence of kynurenine and no binding activity in the absence of kynurenine are derived from a rationally designed phage display antibody library (kynurenine library) to generate kynurenine-switchable antibodies using anti-kynurenine panning that are constructed as described in WO 2015 /083764. To obtain biotinylated antigens bound by phages in the presence of kynurenine, beads are captured, and phages presenting antibodies that exhibit antigen binding activity in the presence of kynurenine are recovered. Phages are then collected from the eluates obtained from the beads.

Phages are produced in the usual manner from E. coli cells carrying a phagemid constructed by the phage display method. In particular, E. coli cells carrying the constructed phagemid vector are infected with phage Μ13ΚΟ7ΔρΙΙΙ (designated hyperphage) (PROGEN Biotechnik) and after culturing overnight at 30°C, the phages are isolated from the supernatants. A phage library solution is prepared by diluting the phage population precipitated by adding 2.5 M NaCl/10% PEG to an E. coli culture in which phage production is carried out using Tris Buffered Saline (TBS). BSA is then added to the phage library solution to a final concentration of 4%. Panning is carried out using antigen immobilized on magnetic beads. The magnetic beads used were Sera-Mag SpeedBeads coated with NeutrAvidin (Thermo Fisher Scientific), FG NeutrAvidin beads (Tamakawa Seminator), or Dynabeads MyOne Streptavidin T1 (Life Technologies). Sera-Mag SpeedBeads NeutrAvidin-coated (Thermo Fisher Scientific), FG NeutrAvidin beads (Tamakawa Seminator), or Dynabeads MyOne Streptavidin T1 (Life Technologies) are used as magnetic beads. The antigen used is biotinylated hCD137-Fc (hCD137-Fc-Bio), obtained as described in example 1-3 (preparation of biotinylated human CD137 Fc-fusion).

- 145 046075

Panning is performed to efficiently produce small molecule switchable antibodies that rely on a small molecule that may play a switching role in cancerous tissues. In particular, panning to enrich for antibodies that bind antigens in the presence of kynurenine, which is a small molecule, and that do not bind antigens in the absence of kynurenine, is performed with reference to the methods described in the previous patent WO 2015/083764. In Round 1, panning is performed on hCD137-Fc-Bio or biotinylated kynurenines (bio-Kyn) (compound 028 (represented by [Formula 28]), 029 (represented by [Formula 29]) and 036 (represented by [Formula 36]), as described in WO 2015/083764). Panning for hCD137-Fc-Bio is performed by adding a phage solution to an antigen, followed by isolating the phage bound to the antigen using magnetic beads; and panning for bio-Kyn is performed in such a way that the prepared phage library solution is added after the first immobilization of the biotinylated antigen on the magnetic beads. For Bio-Kyn, panning to enrich antibodies capable of binding to antigen in the absence of small molecule kynurenine is performed with reference to the methods described in the aforementioned patent WO 2015/083764. For hCD137-Fc-Bio, 4 nmol of non-biotinylated human IgG1 Fc region is added to remove antibodies that bind to the Fc region. Reconstituted phages are added to E. coli strain ER2738 to infect E. coli phages, then the isolated E. coli cells are infected with hyperphages and phages are isolated from the supernatants after overnight culture at 30°C.

After the second round of panning to enrich for antibodies that bind to antigens in the presence of kynurenine and do not bind to antigens in the absence of kynurenine, is performed on hCD137Fc-Bio with reference to the methods shown in the previous patent WO 2015/083764. In both cases, 4 nM non-biotinylated human IgG1 Fc region is added to remove antibodies that bind to the Fc region of hCD137-Fc-Bio. Similar panning is repeated until the fifth round to enrich antibody sequences of interest.

(8-2) Generation of antibodies that bind hCD137 in the presence of kynurenine from an antibody library. (8-2-1) Preparation of hCD137p12-FX-Fc-Bio.

The preparation of hCD137p12-FX-Fc-Bio is carried out by a method known to those skilled in the art. In particular, a gene fragment encoding a factor Xa cleavage sequence and a gene fragment encoding an antibody constant region are fused downstream of a gene fragment encoding a human CD137 extracellular domain region. A gene fragment encoding a protein (hCD137p12-FX-Fc-Bio, SEQ ID NO: 160) in which the extracellular portion of human CD137, the factor Xa cleavage sequence and the antibody constant region are linked is included in an animal expression vector. The constructed plasmid vector was introduced into FreeStyle293 cells (Invitrogen) using 293-Fectin (Invitrogen). At the same time, a plasmid vector containing genes expressing BirA and EBNA1 (SEQ ID NO: 88) is introduced. Biotin is added to the culture. Cells transduced with genes according to the above procedures are cultured at 37°C, 8% CO ₂ , and hCD137p12-FX-Fc-Bio is secreted into the culture supernatant. This culture supernatant is collected. Protein A is used to purify hCD137p12-FX-Fc-Bio from the culture supernatant.

By using a rationally designed library, it is believed that by panning antibodies that exhibit antigen-binding activity only in the presence of a small molecule, small molecule-switchable antibodies that exhibit antigen-binding activity in the presence of small molecules can be obtained. That is, from a kynurenine library generated by the method described in international publication WO 2015/083764, phages presenting antibodies that exhibit binding activity in the presence of kynurenine to hCD137 captured on beads were recovered. In the present production method, biotinylated hCD137-Fc (hCD137-Fc-Bio) prepared as described in Example 1-3 (Preparation of biotin-labeled human CD137 Fc-fusion) or hCD137p12-FX-Fc-Bio are used as antigens.

An E. coli bacterium carrying a phagemid vector for phage display, constructed as described in international publication WO 2015/083764, is infected with Μ13ΚΟ7ΔρΙΙΙ (hyperphage) (Progen Biotechnik), and phages are isolated from the supernatants after overnight cultivation at 30°C. A polyvalent antibody display phage library solution is prepared by diluting the TBS population of phages precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture in which the phage was propagated. BSA is then added to the phage library solution to a final concentration of 4%. Panning is carried out with antigen immobilized on magnetic beads. FG NeutrAvidin beads (Tamakawa Seminator) or Dynabeads MyOne Streptavidin T1 (Life Technologies) are used as magnetic beads.

In the first round of panning, phages capable of binding to the antigen are enriched in the presence of kynurenine. Specifically, the phage library is contacted with antigen and kynurenine for one hour at room temperature by adding 0.8 ml of the prepared phage library solution, at a final concentration of 500 μM kynurenine and 4 nmol of non-biotinylated human IgG1 Fc region (hFC) with 125 pM biotin-labeled antigen. Add BSA-blocked magnetic beads (FG NeutrAvidin beads) and allow antigen-phage complexes to bind to

- 146 046075 magnetic beads for 15 minutes at room temperature. The beads are washed twice with 0.5 ml kynurenine/TBST and once with kynurenine/TBS. Then 0.5 ml of trypsin solution with a concentration of 1 mg/ml is added to the mixture. After suspending the mixture at room temperature for 15 min, the phage solution is released from the beads, which are immediately separated using a magnetic platform. Reconstituted phages are added to 20 ml of E. coli strain ER2738 in logarithmic growth phase (OD600 0.4-0.7). E. coli is infected with phages by culturing E. coli at 37°C for one hour with gentle agitation. Infected E. coli cells are seeded into 225 mm x 225 mm dishes. Libraries of phage presenting monovalent antibodies are then prepared by infecting disseminated E. coli cultures with phage M13KO7TC (WO 2015046554 A1) (helper phage) (Takara) and phages are collected from supernatants cultured overnight at 30°C.

For the second round of panning, the phage library is contacted with antigen and kynurenine for one hour at room temperature by adding 0.8 ml of the prepared phage library solution to a final concentration of 500 μM kynurenine and 4 nM biotin-unlabeled human IgG1 Fc region along with 40 pM biotin-labeled antigen . Add BSA-blocked magnetic beads (Dynabeads MyOne Streptavidin T1) and allow antigen-phage complexes to bind to the magnetic beads for 15 min at room temperature. The beads are washed twice with 0.5 ml kynurenine/TBST and once with kynurenine/TBS. Then 0.5 ml of trypsin solution with a concentration of 1 mg/ml is added to the mixture. After suspending the mixture at room temperature for 15 min, the phage solution is released from the beads, which are immediately separated using a magnetic platform. A phage library solution is then prepared for monovalent antibody display in the same manner as in the first round described above.

For round 3 panning, the phage library is contacted with antigen and kynurenine for one hour at room temperature by adding 0.8 ml of the prepared phage library solution, with a final concentration of 500 μM kynurenine and 4 nM biotin-unlabeled human IgG1 Fc region along with 10 pM biotin-labeled antigen. Add BSA-blocked magnetic beads (FG NeutrAvidin beads) and allow the antigen-phage complexes to bind to the magnetic beads for 15 minutes at room temperature. The granules are washed three times with 0.5 ml kynurenine/TBST and twice with kynurenine/TBS. Then 0.5 ml of trypsin solution with a concentration of 1 mg/ml is added to the mixture. After suspending the mixture at room temperature for 15 min, the phage solution was isolated from the beads, which were immediately separated using a magnetic platform. A monovalent antibody phage display library solution is then prepared in the same manner as in the first round described above. During the 4th round, panning is carried out under conditions similar to the conditions of the 3rd round of panning. However, Dynabeads MyOne Streptavidin T1 are used as magnetic beads.

(8-3) Generation of antibodies that bind to human CD137 in the presence of kynurenine from antibody libraries using SS-biotinylated antigens and negative selection methods.

(8-3-1) Preparation of hCD137p12-FX-Fc.

hCD137p12-FX-Fc is prepared in a manner known to those skilled in the art. Specifically, a gene fragment encoding a factor Xa cleavage sequence and a gene fragment encoding an antibody constant region are fused downstream of a gene fragment encoding a human CD137 extracellular domain region. A gene fragment encoding a protein that links the extracellular portion of human CD137, the FactorXa cleavage sequence, and the antibody constant region (hCD137p12-FX-Fc, SEQ ID NO: 160) is included in an animal expression vector. The constructed plasmid vector was introduced into FreeStyle293 cells (Invitrogen) using 293fectin (Invitrogen). At the same time, a plasmid vector containing genes expressing EBNA1 (SEQ ID NO: 88) is introduced. Cells transduced with genes according to the above procedures are cultured at 37°C, 8% CO ₂ , and hCD137p12-FX-Fc-Bio is secreted into the culture supernatant. Protein A is used to purify hCD137p12-FX-Fc-Bio from this cell culture supernatant.

(8-3-2) Generation of antibodies that bind human CD137 in the presence of kynurenine from an antibody library using SS-biotinylated antigens and a negative selection method.

In the production of small molecule switchable antibodies that exhibit antigen binding activity in the presence of a small molecule, it was thought that after phage display, antibodies that bind to the antigen using magnetic beads are captured using the antigen biotinylated (SS-biotinylated) through a disulfide bonded linker (SS-linked ), only phage display antigen binding antibody can be eluted from the beads using dithiothreitol (DTT) or a similar compound to cleave the SS bonds under reducing conditions. A kynurenine library constructed as described in international publication WO 2015/083764 is screened for antibodies that exhibit antigen binding activity in the presence of kynurenine. For screening, a phage antibody display library is first contacted with biotin-labeled antigen magnetic beads in the absence of kynurenine, and phages displaying antibodies that have antigen-binding activity even in the absence of kynurenine are removed. Subsequent screening for antibodies with antigen-binding

- 147 046075 activity is carried out only in the presence of kynurenine, similarly carrying out panning in the presence of kynurenine. In this method, biotin-labeled antigen (SS-biotinylated antigen) is used as the antigen according to the attached protocols using hCD137Fc prepared as described in Example 1-4 or hCD137p12-FX-Fc prepared in (8-3-1) , and Sulfo-NHS-SS-Biotinylation EZ-Link kit (Thermo Fisher Scientific). It should be noted that hCD137Fc-Bio obtained as described in Example 1-3 or hCD137p12-FX-Fc-Bio obtained in (8-2-1) is used for negative selection.

E. coli cells carrying a phagemid phage display vector constructed as described in WO 2015/083764 are infected with Μ13ΚΟ7ΔρΙΙΙ (hyperphage) (PROGEN Biotechnik), and after overnight cultivation at 30°C, phages are isolated from the supernatants. Phage display libraries of polyvalent antibodies are prepared by diluting the TBS population of phages precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture in which the phage was produced. BSA is then added to the phage library solution to a final concentration of 4%. Panning is carried out with antigen immobilized on magnetic beads. The magnetic beads used are Sera-Mag SpeedBeads NeutrAvidin-coated (Thermo Fisher Scientific), FG NeutrAvidin beads (Tamakawa Seminator), or Dynabeads MyOne Streptavidin T1 (Life Technologies).

In the first round of panning, phages capable of binding to the antigen in the presence of kynurenine are enriched using a negative selection method. Specifically, 800 pmol of hCD137-Fc-Bio or hCD137pl2-FX-Fc-Bio is added to Sera-Mag SpeedBeads coated with skim milk-blocked neutravidin that has been preincubated with streptavidin (Roche) and allowed to bind for 15 min at room temperature. The beads are washed three times with TBST and 0.8 ml of BSA-blocked phage library solution is added to allow binding for one hour at room temperature. By separating the beads on a magnetic platform, antigens and phages that have not bound to the beads are collected.

225 pM SS-biotinylated hCD137-Fc or 450 pM SS-biotinylated hCD137p12FX-Fc-Bio was then added to FG NeutrAvidin beads blocked with skim milk that had been incubated with streptavidin (Roche) and allowed to bind for 15 minutes at room temperature. The beads are washed three times with TBST, and the isolated phages are added to a final concentration of 0.2 mM kynurenine and 4 nM hFc; the phage library is brought into contact with the antigen, as well as kynurenine, for one hour at room temperature. The beads are washed twice with 0.5 ml kynurenine/TBST (0.2 mM kynurenine, 0.1% Tween20, TBS, pH 7.4) and once with kynurenine/TBS (0.2 mM kynurenine, TBS, pH 7.4) . 500 μl of TBS solution containing a final concentration of 25 mM DTT is then added and after five minutes of stirring at room temperature, phages are isolated from the separated beads using a magnetic platform. Trypsin is then added to the mixtures to a final concentration of 1 mg/ml. The mixture is stirred at room temperature for 15 min before the phages are recovered from the separated beads using a magnetic platform. Reconstituted phages are added to 20 ml of E. coli strain ER2738 in logarithmic growth phase (OD600 0.4-0.7). E. coli is infected with phages by culturing E. coli at 37°C for one hour with gentle agitation. Infected E. coli cells are plated in 225 mm x 225 mm dishes. A polyvalent antibody-displaying phage library is then prepared by infecting disseminated E. coli cultures with hyperphages and recovering phages from the supernatants after overnight culture at 30°C.

For the second round of panning, 400 pM hCD137-Fc-Bio or hCD137p12-FX-Fc-Bio is added to Sera-Mag SpeedBeads NeutrAvidin beads coated with neutral avidin, blocked with skim milk, pre-incubated with streptavidin (Roche), and allowed to bind for 15 minutes at room temperature. 0.4 ml of BSA-blocked phage library solution was added to the beads washed three times with TBST and allowed to bind for one hour at room temperature. By separating the beads using a magnetic platform, antigen and phages that are not bound to the beads are recovered. The recovered phages were again incubated with Sera-Mag SpeedBeads NeutrAvidin coated with neutral avidin, which had been incubated with 400 pM hCD137-Fc-Bio or hCD137p12-FX-Fc-Bio in a similar procedure, followed by antigens and phages that did not bind to the beads. collected on a magnetic platform.

56.3 pM SS-biotinylated hCD137-Fc or 113 pM SS-biotinylated hCD137p12-FXFc was added to FG NeutrAvidin beads blocked with skim milk that had been pre-incubated with streptavidin (Roche) and allowed to bind for 15 min at room temperature . The beads are washed three times with TBST, and 0.2 mM kynurenine and 4 nM non-biotinylated human IgG1 Fc region are added along with the isolated phages to a final concentration; the phage library is brought into contact with the antigen, as well as kynurenine, for one hour at room temperature. The beads are washed three times with 0.5 ml kynurenine/TBST (0.2 mM kynurenine, 0.1% Tween20, TBS, pH 7.4) and twice with kynurenine/TBS (0.2 mM kynurenine, TBS, pH 7.4). Then add 500 μl of TBS solution containing a final concentration of 25 mM DTT, and after 5 min mix

- 148 046075 at room temperature, phages are isolated from separated granules using a magnetic platform. A multivalent antibody display phage library solution is then prepared in the same manner as in the first round described above.

For the third round of panning, 200 pM hCD137-Fc-Bio or hCD137p12-FX-Fc-Bio is added to Sera-Mag SpeedBeads NeutrAvidin-coated beads blocked with Sera-Mag SpeedBeads skim milk pre-incubated with streptavidin (Roche) and fed ability to bind at room temperature for 15 minutes. The beads are washed three times with TBST and 0.2 ml of BSA-blocked phage library solution is added and allowed to bind for one hour at room temperature. By separating the granules using a magnetic platform, antigens and phages that are not bound to the granules are removed. The recovered phages are again incubated with Sera-Mag SpeedBeads NeutrAvidin, which were incubated with 200 pM hCD137-Fc-Bio or hCD137p12-FX-Fc-Bio in a similar procedure, then antigens and phages that did not bind to the beads are recovered using a magnetic platform.

12.5 pM SS-biotinylated hCD137-Fc or 25 pmol SS-biotinylated hCD137p12-FXFc was added to FG NeutrAvidin NeutrAvidin skim milk-blocked beads that had been incubated with streptavidin (Roche) and allowed to bind at room temperature for 15 min. . The beads are washed three times with TBST, and 0.2 mM kynurenine and 4 nM non-biotinylated human IgG1 Fc region are added along with the isolated phages to a final concentration; the phage library is brought into contact with the antigen, as well as kynurenine, for one hour at room temperature.

The beads are washed five times with 0.5 ml kynurenine/TBST (0.2 mM kynurenine, 0.1% Tween20, TBS, pH 7.4) and five times with kynurenine/TBS (0.2 mM kynurenine, TBS, pH 7.4 ). Then, 500 μl of TBS solution containing a final concentration of 25 mM DTT is added, and after 5 min of stirring at room temperature, phages are isolated from the separated beads using a magnetic platform. A multivalent antibody display phage library solution is then prepared in the same manner as in the first round described above. During the fourth round, panning is carried out under conditions similar to the conditions of the third round of panning. However, Dynabeads MyOne Streptavidin T1 beads are used as magnetic beads instead of FG beads NeutrAvidin beads. For the fifth round, panning is carried out under conditions close to those of the third round.

(8-4) Evaluation of kynurenine-dependent anti-CD137 antibodies identified by phage display.

(8-4-1) Expression and purification of switchable antibodies that change their binding to antigens due to the presence/absence of kynurenine.

The genes encoding the variable regions of antibodies derived from the kynurenine libraries described in the present invention are inserted into animal expression plasmids having human IgG1 or modified human IgG1 (P253) and kappa chains. Clone designations and serial numbers are listed in table. 55.

Table 55

Clone analysis results

Designation clone SEQ ID NO of heavy chain (full length) SEQ ID NO light chain (full length) Example dBBK026-P253 161 169 8-1 dBBK051-P253 162 170 8-1 dBBK054-P253 163 171 8-1 dBBK060-P253 164 172 8-1 dBBK070-P253 165 173 8-1 dBBK101-P253 166 174 8-2 dBBK102-P253 167 175 8-2 dBBK143-P253 168 176 8-2

Antibodies are expressed and purified using methods known to those skilled in the art. Spectrophotometers were used to measure the absorbance of the purified antibody solution at 280 nm. From the obtained measured values, the concentrations of purified antibodies are calculated using extinction coefficients calculated by the PACE method (Protein Science, 4, 1995, 2411-2423).

(8-4-2) Evaluation of the effect of kynurenine on human CD137 binding using surface plasmon resonance technique.

Biacore T200 software (GE Healthcare) was used to evaluate the binding activity interaction between the anti-CD137 antibody and hCD137-His-BAP (SEQ ID NO: 87). CM3 sensor chip (GE Healthcare), immobilized with appropriate amounts of protein A by

- 149 046075 immobilization at the amino group, captured by aHTu-CD137 antibody to interact with hCD137His-BAP obtained in example 1-1. TBS is used as the running buffer, and 10 mM glycineHCl (pH 1.5) and 25 mM NaOH is used as the regeneration solution.

After capturing the anti-CD137 antibody suspended in TBS, 500 nM hCD137-His-BAP was injected into each flow cell at a flow rate of 10 μL/min for 3 min. This 3-minute period is the binding phase for hCD137-His-BAP, and after completion of the binding phase, there is a 5-minute period of switching to the running buffer for the dissociation phase of hCD137-His-BAP. After completion of the dissociation phase, the regenerating solution is injected at a rate of 30 μl/min for 10 s. The above description is a cycle for measuring the binding activity of anti-CD137 antibodies. The binding amount of hCD137-His-BAP that interacted with the anti-CD137 antibody in the binding phase was analyzed using Biacore T200 Evaluation Software Version 2.0, as shown in Table. 56.

Table 56

Amount of antigen bound in the presence of kynurenine

Clone designation No low molecular weight compound 500 µM kynurenine dBBK026-P253 18.2 60 dBBK047-P253 -2 10.4 dBBK051-P253 -2.3 thirty dBBK054-P253 -2.2 3.6 dBBK060-P253 2.8 38.5 dBBK070-P253 -1.4 2.6 dBBK101-P253 -3.6 21.2 dBBK102-P253 -2.5 9.9 dBBK143-P253 -1.9 44.3

(8-4-3) Evaluation of the binding activity of the resulting antibodies towards parts of the CD137 sequences.

(8-4-3-1) Production of antigens with parts of CD137 sequences.

Antigens with portions of the CD137 sequences are produced by methods known to those skilled in the art. Specifically, a gene fragment encoding the antibody constant region was linked downstream of a gene fragment encoding part of the extracellular region of human CD137. A gene fragment encoding a protein (Table 57) in which part of the extracellular domain of human CD137 and the antibody constant region are linked is included in an animal expression vector. The constructed plasmid vector is introduced into Expi293 cells (Invitrogen) using ExpiFectamine293 (Invitrogen), and the protein of interest is secreted into the culture supernatant. Protein A is used to purify the protein of interest from the culture supernatant.

Table 57

Antigens with partial CD137 sequences

Designation of an antigen having a partial sequence of CD 137 SEQ ID NO hCD137.1-F-Gld 177 hCD137.1.2-F.Gld 178 hCD137.2.4-F.Gld 179 hCD137.2.3-F.Gld 180 hCD137.3.4-F.Gld 181

(8-4-3-2) Binding confirmation.

Whether the resulting antibodies bind to antigens having a partial sequence of CD137 is assessed by ELISA. First, the various antigens obtained in Example 1 or Example 8-4-3-1 are immobilized on Maxisorp 384 microtiter plates. After removing unbound antigens from the plates by washing each well with wash buffer, the wells are blocked with 50 μl of blocking buffer (TBS containing BlockAce) in for one hour or more. To each of the wells from which the blocking buffer has been removed, add 10 μl of purified antibodies prepared at a concentration of 10 μg/ml in TBS with a final concentration of 500 μM L-kynurenine or TBS with a final concentration of 500 μM kynurenine, and the plate is left alone. hour. AP-conjugated anti-human kappa antibodies (BIOSOURCE), diluted in TBS with a final concentration of 500 μM kynurenine, are added to each well after washing with wash buffer containing a final concentration of 500 μM kynurenine (TBS with 0.1% Tween20). After 1 hour of incubation, BluePhos phosphate substrate (KPL) is added after washing with wash buffer containing 500 µM kynurenine. The color development of the solution in each well is measured by absorbance at 620 nm after the color development reaction of the solution in each well is stopped by adding BluePhos stop solution. The absorption coefficient is shown by setting the values in wells with antibody concentrations from 0 to 1 μg/ml. Based on binding with an absorbance coefficient greater than or equal to 2, it is assumed that the clones evaluated include antibodies that exhibit different binding properties.

(8-5) In vitro assessment of the activity of the resulting antibodies The GloResponseTM NF-kBLuc2/4-1BB Jurkat cell line (Promega) was used to measure the activity of antibodies in vitro. Add 10 μl of FcyRIIB CHO-K1 cells, prepared at a concentration of 2x10 ⁶ /ml with assay medium (99% RPMI, 1% PBS), to each well of 384-well plates. Antibody solutions with or without kynurenine are added to each well in 10 μl. Then, 10 μl of the GloResponseTM NF-kB-Luc2/4-1BB Jurkat cell line, prepared at a concentration of 2x10 ⁶ /ml in assay medium (99% RPMI, 1% PBS), was added to each well. The final concentration of kynurenine is 500 µM. The plates are left for six hours at 37°C in a 5% CO ₂ incubator, then incubated for 15 minutes at room temperature and 30 μl of Bio-Glo reagent added to each well. For the Bio-Glo reagent, use the Bio-Glo Luciferase Assay System (buffer and substrate). Subsequently, the luminescence of each well is measured using a plate reader. The luminescence value of each well divided by the luminescence value of the well without added antibody represents the luminescence fold and serves as an indicator for assessing the activity of each antibody.

The results obtained are shown in Fig. 57.

The luminescence fold was higher when the concentration of L-kynurenine was 500 μM than when it was 0 μM, indicating that agonist activity varied depending on the concentration or presence of kynurenine.

Example 9. Preparation of ATP- and AMP-dependent CD137-binding antibodies by double round selection.

(9-1) Obtaining antibodies that bind to antigens in the presence of ATP and AMP, respectively, from a rationally designed library by double-round selection.

Antibodies have been produced that exhibit antigen binding activity in the presence of a particular small molecule, but antibodies that exhibit antigen binding activity in the presence of a variety of different small molecules have never been reported. One way to address this problem is to consider alternating panning, which changes the small molecule used for each round of panning, such as ATP and AMP. However, in this case, there is a problem that the selection pressure is applied to the small molecule in each round, and thus the selection pressure cannot be applied equally to both small molecules at the same time. To solve this problem, a double round selection method was implemented. The double round selection method is a procedure in which two rounds of panning are performed on the same antigen in one round of panning (J Mol Biol., 226(3), 1992, 889896). In the present invention, for example, in the case of the phage display method, one round refers to the step of isolating phages from E. coli to re-infect E. coli with phages after panning; and in the service

- 151 046075 In the case of the ribosomal display method, one round refers to the stage from in vitro transcription to gene amplification with the PCR method. Although a double round of selection has been reported for one type of antigen, there are no reports of the method being used to generate antibodies that are dependent on small molecules, and accordingly, there are no reports of using the method to generate antibodies that exhibit dependence on several separate different small molecules. By modifying the double-round selection method and applying selective pressure on both ATP and AMP in a single round, the present inventors believe that it is possible to obtain antibodies that exhibit antigen-binding activity in an ATP- and AMP-dependent manner more efficiently.

Antibodies exhibiting antigen binding activity in the presence of ATP and AMP are obtained from a rationally designed antibody phage display library created in the previous patent WO 2015/083764. Panning conditions are shown in Table. 59. Conditions 1 and 2 are applied to the normal panning condition and the above variable panning condition; while condition 3 is applied to the above double round selection method. In the present invention, a double round of selection is carried out in round 2 and round 3. Biotinylated hCD137-Fc is used as the antigen.

In table 59 ATP denotes panning using ATP, AMP denotes panning using AMP, and the designation Dual denotes panning using a double round of selection.

Table 59

Condition Round 1 Round 2 Round 3 1 ATP ATP ATP 2 ATP AMF ATP 3 ATP Double Double

Phages are produced in the usual manner from E. coli cells carrying an engineered phage display phagemid. Specifically, an E. coli strain carrying the constructed phagemid vector is infected with phages M13KO7TC (WO 2015046554 A1) or Μ13ΚΟ7ΔρΙΙΙ (hyperphage) (PROGEN Biotechnik), and the phages are isolated from the supernatants after overnight cultivation at 30°C. Phage antibody display libraries are prepared by diluting TBS, a population of phages precipitated by adding 2.5 M NaCl/10% PEG to the E. coli culture from which the phages were produced.

BSF is then added to the phage library solution to a final concentration of 4%. Panning is carried out using antigen immobilized on magnetic beads. NeutrAvidin beads (TAMAGAWA SEIKI) or Dynabeads MyOne StreptAvidin T1 (Thermo Fisher Scientific) are used as magnetic beads.

Biotinylated hCD137-Fc and ATP at a final concentration of 1 mM are added to the prepared phage library solution and the reaction is carried out at room temperature for 60 min. The blocked magnetic beads are added to the phage and antigen reaction solution and they react at room temperature for 15 min. The beads are washed two or three times with TBS/0.1% Tween20 buffer containing 1 mM ATP and once or twice with TBS containing 1 mM ATP. The beads to which TBS is subsequently added are suspended at room temperature and the phage solution is recovered from the beads separated using a magnetic platform. After repeating this procedure twice, the eluted phage solution is mixed. Trypsin is added to the resulting phage solution at a final concentration of 1 mg/ml. Reconstituted phages are added to 20 ml of E. coli strain ER2738 in logarithmic growth phase (OD600 0.4-0.7). The E. coli culture is infected with phages by culturing the E. coli for one hour at 37°C with agitation. E. coli is sown in 225 mm x 225 mm dishes. E. coli is infected with M13KO7TC (WO 2015046554 A1) or Μ13ΚΟ7ΔρΙΙΙ (called hyperphage) (PROGEN Biotechnik) and cultured overnight at 30°C; phages are isolated from supernatants. Under condition 1, this procedure is repeated three times. Under condition 2, after the above procedure was performed once, ATP is replaced with AMP and the above procedure was performed again (round 2), and then the above procedure was performed again with ATP (round 3).

On the other hand, according to condition 3, after the above procedure is performed once, a double round of selection is performed in rounds 2 and 3. Specifically, biotinylated hCD137-Fc and AMP at a final concentration are first added to the prepared phage library solution 1 mM and they react at room temperature for 60 min. The blocked magnetic beads are added to the phage and antigen reaction solution and they react at room temperature for 15 min. The beads are washed two or three times with TBS/0.1% Tween20 buffer containing 1 mM AMP and once or twice with TBS buffer containing 1 mM AMP. The beads to which TBS is subsequently added are suspended at room temperature and the phage solution is recovered from the beads separated using a magnetic platform. After repeating this procedure twice, the eluted phage solution is mixed. In addition, biotinylated hCD137-Fc and ATP at a final concentration of 1 mM were then added to the reconstituted phage library solution and reacted at room temperature for 60 min. The blocked magnetic beads are added to the phage and antigen reaction solution and they react at room temperature for 15 min.

- 152 046075

The beads are washed two or three times with TBS/0.1% Tween20 buffer containing 1 mM ATP and once or twice with TBS containing 1 mM ATP. The beads to which TBS is subsequently added are suspended at room temperature and the phage solution is recovered from the beads separated using a magnetic platform. After repeating this procedure twice, the eluted phage solution is mixed. Trypsin is added to the resulting phage solution at a final concentration of 1 mg/ml. The recovered phages are added to 20 ml of a suspension of E. coli strain ER2738 in logarithmic growth phase (OD600 0.4-0.7). E. coli is infected with phages by culturing E. coli for one hour at 37°C with agitation. E. coli is sown in 225 mm x 225 mm dishes. E. coli is infected with phages M13KO7TC (WO 2015046554 A1) or Μ13ΚΟ7ΔρΙΙΙ (called hyperphage) (PROGEN Biotechnik) and cultured overnight at 30°C; phages are isolated from supernatants.

(9-2) Evaluation of binding activity in the presence and absence of ATP or AMP by phage ELISA.

From individual E. coli colonies obtained by the methods described above, supernatants of phage-containing cultures are collected using the usual method (Methods Mol. Biol., 178, 2002, 133-145). NucleoFast 96 (MACHEREY-NAGEL) is used to ultrafiltrate the collected culture supernatant. The duct is removed by centrifugation (4500 g, 45 min) with NucleoFast 96, into which 100 μl of culture supernatant is added to each well. NucleoFast 96, in which 100 μl of H2O is added to each well, is washed again by centrifugation (4500 g, 30 min). Finally, 100 μl of TBS was added and the phage solution contained in the supernatants of 96-well NucleoFast plates, which were left to stand at room temperature for 5 min, was collected.

TBS, 1 mM AMP/TBS or 1 mM ATP/TBS are added to the purified phages, which are then subjected to ELISA using the procedure described below. Streptavidin-coated 384-well microplates (Greiner, 781990) are coated overnight with 100 μl of TBS containing biotinylated hCD137-Fc prepared in Example 1. After removing unbound antigens from the plates by washing each well with TBST, the wells are blocked with 250 μl of 0.4% Block Ace, 1% BSA, 0.02% Tween, 0.05% ProClin 300-TBS for one hour or more. Antibody presenting phages are then allowed to bind to the antigen present in each well in the absence and presence of ATP or AMP by incubating the prepared purified phage plates added to each well at 37°C for one hour. Each well is washed with TBST, 1 mM ATP/TBST or 1 mM AMP/TBST and supplemented with HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS, 1 mM AMP/TBS or 1 mM ATP/TBS and incubated for one hour. After washing with TBST, 1 mM ATP/TBST or 1 mM AMP/TBST, the color of the solution in each well to which a single TMB solution (ZYMED) was added was stopped by adding sulfuric acid, and then the development of color was measured by absorbance at 450 nm.

The results of phage ELISA are presented in table. 60.

In the present invention, clones with an absorbance ratio S/N (signal to noise) greater than 2 in the presence of ATP (the ratio of absorbance in the presence of antigen to absorbance in the absence of antigen) were scored as positive for binding, and clones with an absorbance ratio of 3 or higher in the presence of /absence of ATP or AMP were regarded as clones with antigen binding activity dependent on ATP or AMP (switched clones). As a result, for hCD137-Fc, most clones obtained under condition 1 show binding in the presence of ATP, but do not show binding in the presence of AMP. Among the clones that show binding in the presence of ATP and in the absence of binding in the absence of ATP obtained under condition 2, there are several antibodies that show binding even in the presence of AMP, but the percentage is 50% or less. On the other hand, under condition 3, which is a double selection, 65% or more of the clones that show binding in the presence of ATP and no binding in the absence of ATP are able to show binding even in the presence of AMP.

For the resulting clones, the amplified heavy chain genes were sequenced using the specific primers pBAD-F and G1seq-R.

As a result, condition 3, in which a double round of selection was performed, produced antibody heavy chain sequences showing both ATP- and AMP-dependent human CD137 binding that was 3 times greater than condition 1, which was performed with only ATP, and about 1.5 times more than condition 2, in which ATP and AMP are used alternately. This indicates that double round-robin selection is a more valuable approach to obtaining antibodies that exhibit antigen-binding activity dependent on multiple individual small molecules.

- 153 046075

Table 60

Condition 1 Condition 2 Condition 3 Number of clones subjected to ELISA 96 96 96 Number of positive clones (signal to noise ratio > 2, ATP) 69 88 91 Number of ATP-switched clones (ATP ratio +/- >= 3) 65 84 82 Number of ATP-, AMP-switched clones (ratio of ATP +/- >= 3 and AMP +/- >= 3) 13 40 54 Proportion of ATP-, AMP-switched clones 20.0% 47.6% 65.9% Number of sequences of ATP-, AMP-switched clones (ratio of ATP +/- >= 3 and AMP +/- >= 3) 13 25 38

Example 10. Preparation of ATP-dependent antibodies against mouse CTLA4 from a naive library.

(10-1) Generate antibodies that bind to mouse CTLA4 in the presence of small molecules from a naïve human antibody library using bead panning.

A human antibody phage display library, consisting of a variety of phages presenting the Fab domains of various human antibody sequences, was constructed according to methods known to those skilled in the art using poly(A) RNA derived from human PBMCs, commercially available poly(A) ) human RNA, or such as a template.

From the generated naïve human antibody phage display library, antibodies exhibiting binding activity to the extracellular domain of mouse CTLA4 (mCTLA4) in the presence of small molecules are screened. That is, phages are collected that present antibodies that exhibit binding activity to mCTLA4 captured beads in the presence of small molecules. Phages are isolated from phage bead-eluted phages in the absence of small molecules. Biotinylated mCTLA4 (mCTLA4-His-Biotin) is used as the antigen in this method. mCTLA4-His-Biotin is obtained by biotinylation (PIERCE, catalog number 21329) mCTLA4-His (Sino Biologics Inc. 50503-M08H, catalog number NP_033973.2) by the amine coupling method, which is carried out by fusion of the His tag with the extracellular mCTLA4 domain.

Phages obtained from E. coli cells carrying engineered phage display phagemids are purified by known methods. The phage library solution is then dialyzed against TBS. Panning is carried out with antigen immobilized on magnetic beads. The magnetic beads used are neutravidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin) or streptavidin-coated beads (Dynabeads M-280 Streptavidin).

To efficiently produce small molecule switching antibodies that depend on a small molecule capable of playing a switching role in cancer tissues, panning to enrich antibodies that bind antigens in the presence of adenosine triphosphate (adenosine 5'-triphosphate; ATP) and ATP metabolites that do not bind antigens in the absence of ATP, is carried out with reference to the methods described in the previous patent WO 2013/180200.

In particular, biotinylated mCTLA4 and ATP, as well as ATP metabolites, are added to the prepared phage library solution at a final concentration of 1 mM to carry out the reaction at room temperature for 60 min. The blocked magnetic beads are added to the phage and antigen reaction solution and they react at room temperature for 15 min. The beads are washed two or three times with TBS/0.1% Tween20 buffer containing 1 mM ATP and once or twice with TBS containing 1 mM ATP. The beads to which TBS is subsequently added are suspended at room temperature and the phage solution is recovered from the beads separated using a magnetic platform. Trypsin is then added to the beads at a final concentration of 1 mg/ml in round 1 and TBS in round 2 onwards and suspended at room temperature; phage solutions are recovered from beads separated using a magnetic platform. If TBS is used, this procedure is repeated twice, after which the eluted phage solution is mixed and trypsin is added to the resulting phage solution at a final concentration of 1 mg/ml. The extracted phages are added to 10 ml of E. coli strain ER2738 in logarithmic growth phase (OD600 0.4-0.7). E. coli is infected with phages by culturing E. coli for one hour at 37°C with agitation. E. coli is sown in 225 mm x 225 mm dishes. E. coli is infected with M13KO7TC (WO 2015046554 A1) or Μ13ΚΟ7ΔρΙΙΙ (called hyperphage) (PROGEN Biotechnik) and cultured overnight at 30°C; phages are isolated from supernatants. During cultivation, a 100 μM IPTG addition condition and a no IPTG addition condition were used to induce the expression of Fab genes from the lac promoter. This procedure is repeated four times.

(10-2) Assessment of binding activity in the presence and absence of small molecules by phage enzyme-linked immunosorbent assay (ELISA).

- 154 046075

Phage culture supernatants were collected from individual E. coli colonies obtained in Example 10-1 using the conventional method (Methods Mol. Biol., 178, 2002, 133-145). NucleoFast 96 (MACHEREY-NAGEL) is used to ultrafiltrate the collected culture supernatants. 100 μl of each culture supernatant is added to each well of a NucleoFast 96 plate and centrifuged at 4500 g for 45 min to remove the duct. 100 µl of H2O is added to each well and washed again by centrifugation (4500 g, 30 min). Finally, 100 μl of TBS is added and left at room temperature for 5 minutes, then the phage solution is isolated from the supernatants.

TBS is added to purified phages, which are then subjected to ELISA analysis using the procedure described below. StreptaWell 96 microtiter plates (Roche) are coated overnight with 100 μl of TBS containing mCTLA4-His-Biotin. After removing unbound mCTLA4-His-Biotin from the plates by washing each well with TBST, the wells are blocked with 250 μl of 2% skim milk-TBS for over one hour. Antibody-presenting phages are then allowed to bind to the mCTLA4-His-Biotin present in each well in the absence and presence of ATP by removing 2% skim milk-TBS, followed by storing the prepared purified phage plates added to each well at room temperature for one hour. Each well is washed with TBST or ATP/TBST and supplemented with HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS or ATP/TBS and incubated for one hour. After washing with TBST or ATP/TBST, the color development of the solution in each well to which a separate TMB solution (ZYMED) was added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm. The results confirm that several antibodies bind to mCTLA4 only in the presence of ATP.

The results of the analysis using the phage ELISA method are presented in table. 61.

In the present invention, clones with absorbance greater than 0.2 in the presence of ATP are considered positive, and clones with absorbance greater than 2 in the presence/absence of ATP are assessed as clones with ATP-dependent antigen-binding activity (switched clones).

Table 61

Helper phage used in panning M13KO7 TS M13KO7 TS M13KO7r III IPTG concentration during culture for panning 0 µM 100 µM 0 µM Number of clones analyzed by ELISA 96 96 96 Number of positive clones (absorbance > 0.2) 20 41 62 Number of switched clones (SM+/- ratio > 2) 0 12 16

The above invention has been described in detail with examples and illustrations in order to facilitate a clear understanding, and the above description and illustrations should not be construed as limiting the scope of the present invention. The substance of all patents and scientific literature cited in the present invention is incorporated herein by reference.

Example 11. In vitro assessment of ATP- and ADP-dependent CD137 agonist activity of modified anti-human CD137 antibodies using the 4-1BB Jurkat reporter gene assay.

The GloResponse™ NF-κB Luc2/4-1BB Jurkat cell line (Promega, CS196004) was used to apply the in vitro activity of the variants obtained in Example 7-2 (Table 52). Add 10 μl of each FcyRIIB CHO-K1 cell (Promega) prepared at a concentration of 4x105/ml in assay medium (99% RPMI, 1% PBS) to each well of 384-well plates. Then, 10 μl of an antibody solution containing ADP, or an antibody solution containing ATP, or an antibody solution without ATP or without ADP, is added to each well. Then 10 μl of the GloResponseTM NF-kB-Luc2/4-1BB Jurkat cell line, prepared at a concentration of 2x10 ⁶ /ml in assay medium (99% RPMI, 1% PBS), is added to each well. The final concentration of ADP is 10 µM, the final concentration of ATP is 10 µM. The plates are left for 6 hours at 37°C in a 5% CO2 incubator and 15 minutes at room temperature; 30 µl of Bio-Glo reagent is added to each well. For the Bio-Glo reagent, use the Bio-Glo Luciferase Assay System (buffer and substrate). The amount of luminescence in each well is then measured using a plate reader. The luminescence value of each well divided by the luminescence value of the well without the addition of antibody is determined as the relative light unit (fold induction), which serves as an indicator for assessing the CD137 agonistic activity of each antibody.

The results are presented in Fig. 58. In FIG. 58 provide confirmation that A375-P587/B167-LamLib, A551-P587/B256-LamLib, A551-P587/B379-LamLib, A375-SCF041aPh/B167-LamLib, A551-SCF041aPh/B256LamLib, A551-SCF041 aPh/B379-LamLib , A375-SCF043aPh/B167-LamLib, A551-SCF043aPh/B256-LamLib, A551-SCF043aPh/B379-LamLib, A375-SCF057aPh/B167-LamLib, A551-SCF057aPh/B256-LamLib and A5 51SCF057aPh/B379-LamLib exhibit agonistic activity in against human CD137 ATP- and ADP

- 155 046075 in a dependent manner.

Example 12 Measurement of extracellular ATP levels at tumor sites.

An ATP-switched antibody binds to a target molecule in the presence of ATP and exhibits drug efficacy. The assessment of extracellular ATP levels in areas of tumor tissue is carried out using P2Y11 split Luc/HEK293 cells. Cells are produced using ProbeX using the company's split-luciferase technology. The cells express C-terminal luciferase protein associated with the purinergic receptor P2Y11 (accession number AF030335) with ATP as a ligand, and N-terminal luciferase protein associated with β-arrestin-2 isoform (accession number NM_004313). Active luciferase is produced by combining two luciferase fragments that are separately present in cells by stimulation with ATP, and luminescence can be produced using luciferin as a substrate.

(12-1) ATP sensitivity of P2Y11-cleaved Luc/HEK293 cells.

P2Y11 split Luc/HEK293 cells were cultured and maintained in Dulbecco's Modified Eagle's Medium high glucose: D5796/Sigma-Aldrich containing 10% fetal calf serum, 0.8 mg/ml G418 (Geneticin™) and 0.05 mg/ml phleomycin D1 (Zeocin™) for passages. P2Y11 split Luc/HEK293 cells are plated in a 96-well microplate (CLEAR™, WHITE, CELLSTAR™/Greiner Bio-One) at 30,000 cells/well, cultured overnight and then the culture solution is withdrawn from each well and replaced with 0. 18 ml of culture solution containing 0.5 mg/ml luciferin (VivoGlo™ Luciferin, In Vivo Grade/Promega). To achieve final concentrations of 0, 0.125, 0.25, 0.5, 1.0 and 2.0 mM, add 0.02 ml of adenosine, AMP, ADP or ATP solution and measure luminescence values on an EnVision plate reader (Perkin Elmer ) after 30 min of incubation in a CO ₂ incubator (37°C, 5% CO ₂ atmosphere).

The results are shown in Fig. 59. P2Y11 split Luc/HEK293 cells increase their luminescence in an ATP-specific manner depending on the ligand concentration.

(12-2) Assessment of the concentration of extracellular ATP in areas of tumor tissue.

P2Y11 split Luc/HEK293 cells were separated from culture flasks using enzyme-free cell dissociation buffer and PBS (Thermo Fisher Scientific), washed with HBSS, and then a cell suspension in HBSS was prepared, which was used to measure extracellular ATP levels in vivo.

(12-2-1) Establishment of an in vivo ATP calibration curve.

0.2 ml of cell suspension containing a fixed concentration of ATP, 1 mg/ml luciferin (VivoGlo, Promega) and 1.0 x 107 cells/ml (P2Y11 split Luc/HEK293 cells) is transplanted subcutaneously into the ventral region of C3H/HeN mice, and after 20 min, fluorescence imaging is measured under isoflurane anesthesia using an IVIS Spectrum CT in vivo imaging device. The resulting images were analyzed using Living Image Software, and an ATP calibration curve was generated under in vivo measurement conditions based on the calculated luminescence values and added ATP concentrations.

In fig. Figure 60 shows the results. P2Y11 split Luc/HEK293 cells exhibit ATP concentration-dependent luminescence even in vivo when transplanted subcutaneously into C3H/HeN mice; these results can be converted into a calibration curve to estimate in vivo ATP levels.

(12-2-2) Assessment of the level of extracellular ATP in areas of tumor tissue of 0.2 ml of cell suspension containing 1 mg/ml luciferin (VivoGlo, Promega) and 1.0x107 cells/ml (P2Y11 split Luc/HEK293 cells), transplanted subcutaneously into the tumor site of FM3A tumor-bearing mice (tumor volume 200-300 mm ³ ) and fluorescence imaging measured after 20 min under isoflurane anesthesia using an IVIS Spectrum CT in vivo imaging device. The resulting images are analyzed using Living Image Software, and the ATP level is calculated from the luminescence value of the tumor site using an in vivo ATP calibration curve, which is considered as the extracellular ATP level in the tumor site.

The results of the luminescence imaging measurements are shown in FIG. 61, and the levels of extracellular ATP at the tumor site, calculated from the ATP calibration curve, are shown in Table. 62. The level of luminescence in the tumor tissue area (mouse with FM3A tumor) is higher than in the background (normal area), and the level of extracellular ATP in the tumor tissue area in the FM3A transplantation model is calculated as the average value of 1.31 mM on the prepared in vivo calibration curve ATP.

Table 62

Mice with FM3A tumor 1 2 3 Mean ± SD Extracellular ATP (mM) 0.81 1.26 1.86 1.31 ± 0.53

Example 13. ATP-dependent antitumor activity of anti-hIL6R antibodies with ATP-dependent binding properties.

(13-1) Production of anti-hIL6R antibodies having ATP-dependent binding properties.

Referring to the methods described in WO 2015/083764 and WO 2013/180200, the resulting antibodies were

- 156 046075 are modified to produce antibodies against human interleukin 6 receptor (hIL6R), which have ATP-dependent binding properties. Heavy chains and light chains are combined as shown in Table. 63, for the expression and purification of anti-hIL6R antibodies by a method known to those skilled in the art.

Table 63

Combinations of heavy chains and light chains of antibodies

Antibody name VH CH VL C.L. MRAH-G4T1/ MRAL-k0 MRAH SEQ ID NO: 190 G4T1 SEQ ID NO: 198 MRAL SEQ ID NO: 191 kO SEQ ID NO: 199 H0002-G4T1/ L1058-laml H0002 SEQ ID NO: 192 L1058 SEQ ID NO: 193 laml SEQ ID NO: 189 H0041-G4T1/ L1088-laml H0041 SEQ ID NO: 194 L1088 SEQ ID NO: 195 H0052-G4T1/ L1083-laml H0052 SEQ ID NO: 196 L1083 SEQ ID NO: 197

hIL6R (SEQ ID NO: 200) is genetically synthesized as an antigen and inserted into a plasmid for expression in animals, then it is introduced into CHO cells, and thus, as a result of cloning, a stable expression line (hIL6R-CHO) is obtained that constitutively expresses antigen. The antigenic protein is expressed and purified using the methods described below. The hIL6R-CHO cell line is suspended at an appropriate cell density, seeded and cultured in flasks, and the antigen is purified from the culture supernatant in a manner known to those skilled in the art. Spectrophotometers are used to measure the absorbance of a purified antigen solution at 280 nm. Purified antigen concentrations are calculated from the measured values using absorption coefficients calculated by the PACE method (Protein Science 4, 1995, 2411-2423).

(13-2) Calculation of ATP concentration-dependent KD values of anti-hIL6R antibodies.

Affinity analysis for hIL6R in the presence of each of the ATP concentrations of 0, 1, 10 and 100 μM was carried out using Biacore T200 using the antibodies described in table. 63. Measurements are carried out at a flow rate of 10 µl/min, reaction time 1 min, dissociation time 1 min and one cycle kinetics at 37°C. A buffer of 20 mM ACES, 150 mM NaCl, 2 mM MgCl ₂ and 0.05% Tween20, pH 7.4, containing various concentrations (0, 1, 10 and 100 μM) of ATP was used. The optional Biacore Evaluation Software from Biacore T200 is used for analysis and the 1:1 Langmuir binding model is used for the proportionality model. The results of the analysis are presented in table. 64 and in FIG. 62.

Table 64

Calculation of KD (M) values of anti-hIL6R antibodies depending on the ATP concentration (M)

Antibody ATP concentration (µM) 0 1 10 100 MRAH-G4T1/ Average 1.70E-08 1.88E-08 1.79E-08 1.80E-08 MRAL-kO SD 3.81E-09 4.07E-09 4.08E-09 4.00E-09 H0002-G4T1/ Average No data* No data* No data* 1D6E-06 L1058-laml SD No data* No data* No data* 4.23E-07 H0041-G4T1/ Average No data* 2.38E-06 3.20E-07 4.83E-08 L1088-laml SD No data* 6.00E-07 7.45E-08 1D6E-08 H0052-G4T1/ Average No data* 1.45E-06 5.05E-08 8.31E-09 L1083-laml SD No data* 1D7E-06 Ts41E-08 2.84E-09

*Samples with commented kinetic constants cannot be clearly determined in the Biacore assay and are considered rejected and the results are shown as no data.

The results show that the KD for hIL6R MRAH-G4T1/MRAL-k0 is independent of the ATP concentration in the assay, but the KD for hIL6R H0002-G4T1/L1058-lam1, H0041-G4T1/L1088-lam1 and H0052G4T1/L1083-lam1 becomes smaller with higher concentration of ATP in the assay. That is, the higher the concentration of ATP in the assay, the stronger the binding activity becomes. The results confirm that H0002-G4T1/L1058-lam1, H0041-G4T1/L1088-lam1 hH0052-G4T1/L1083-lam1 have ATP-dependent hIL6R-binding activity.

In the following, an antibody whose antigen-binding activity does not depend on the ATP concentration, such as MRAH-G4T1/MRAL-k0, can be called a non-switchable antibody, and an antibody whose antigen-binding activity depends on the ATP concentration, such as H0002-G4T1/L1058lam1, H0041-G4T1 /L1088-lam1 and H0052-G4T1/L1083-lam1, can be designated as switchable antibody.

(13-3) Calculation of ADP and AMP concentration-dependent KD values of anti-hIL6R antibodies.

- 157 046075

Then the antibodies described in table. 63 was used to assay hIL6R affinity in the presence of 10 and 100 μM ADP or AMP instead of ATP using Biacore T200 according to the conditions described above. The results of the measurement analysis using ADF are shown in Table. 65 and in FIG. 63, and the results of the measurement analysis using AMF are shown in table. 66 and in FIG. 64. Antibodies with a response of 10 RU or less at the time of measurement are considered not analyzed and are indicated in the table as not available.

Table 65

Calculation of KD (M) values of anti-hIL6R antibodies depending on the concentration of ADP (M)

Antibody ADP concentration (µM) 10 100 MRAH-G4T 1/MRAL-lam 1 5.64E-09 5.96E-09 H0002-G4Tl/L1058-laml No data No data H0041-G4Tl/L1088-laml 1, OZE-05 2.96E-07 H0052-G4Tl/L1083-laml 5.81E-08 1.01E-08

Table 66

Calculation of KD (M) values of anti-hIL6R antibodies depending on the concentration of AMP (M)

Antibody AMP concentration (µM) 10 100 MRAH-G4T 1/MRAL-lam 1 5,ZZE-09 5.46E-09 H0002-G4Tl/L1058-laml No data No data H0041-G4Tl/L1088-laml No data 1.81E-06 H0052-G4Tl/L1083-laml 6.64E-06 1D8E-07

The results show that the KD for hIL6R MRAH-G4T1/MRAL-k0 is not affected by ADP and AMP concentrations in the assay, but as with ATP, higher ADP or AMP concentrations in the assay are accompanied by lower KD values for hIL6R H0002-G4T1/ L1058-lam1, H0041-G4T1/L1088lam1 and H0052-G4T1/L1083-lam1, i.e. they have stronger binding activity. The results confirm that H0002-G4T1/L1058-lam1, #041^4^/11088-^1 and H0052-G4T1/L1083-lam1 have ADP- and AMP-dependent binding activity towards hIL6R.

(13-4) Assessment of ATP concentration-dependent ADCC activity of anti-hIL6R antibodies.

Antibodies are obtained by a method known to specialists in this field, as shown in table. 67, by changing the heavy chain constant regions of the above antibodies to mFa55 (SEQ ID NO: 184), in which ADCC activity is enhanced by adding mIgG2a modifications that enhance FcyRIV binding, and by changing the light chain constant region from a human chain to a mouse chain. In addition, KLH-mFa55 (IC17Hdk-mFa55/IC17L-mk1) was prepared as a negative control.

Table 67

Combinations of heavy chains and light chains of antibodies

Antibody VH CH VL C.L. IC17HdK-mFa55/ IC17L-mkl IC17HdK SEQ ID NO: 187 mFa55 SEQ ID NO: 184 IC17L SEQ ID NO: 188 mkl SEQ ID NO: 185 MRAH-mFa55/ MRAL-mkO MRAH SEQ ID NO: 190 MRAL SEQ ID NO: 191 mkO SEQ ID NO: 211 H0002-mFa55/ L1058-ml0 H0002 SEQ ID NO: 192 L1058 SEQ ID NO: 193 mlO SEQ ID NO: 212 H0041-mFa55/ L1088-ml0 H0041 SEQ ID NO: 194 L1088 SEQ ID NO: 195 H0052-mFa55/ L1083-ml0 H0052 SEQ ID NO: 196 L1083 SEQ ID NO: 197

These antibodies are used to evaluate ADCC against Hepa1-6 (hIL6R-Hepa1-6), a mouse hepatoma line that expresses full-length hIL6R (SEQ ID NO: 213), obtained by methods known to those skilled in the art, described below.

For the measurements below, mFcyRIV ADCC Reporter Bioassay, Core Kit (Promega) is used. Add 100 μl of hIL6R-Hepa1-6, prepared in a medium with a cell concentration of 2x10 ⁵ /ml, to each well of a 96-well plate, and leave the plate at 37°C for 21 hours. 90% DMEM, 10% PBS, 600 µg geneticin and 500 µg zeocin. After centrifuging the plate for five minutes at 200 x g, 4°C, the supernatant from each well is removed. A solution of antibodies described in table. 67 diluted in assay buffer to a final concentration of 0.2 µg/ml, ATP solution diluted in assay buffer to final concentrations of 0, 1, 10 and 100 µM, and 25 µl of a solution prepared by adding 0.69 ml of kit auxiliary effector cells to 3.82 ml of assay buffer, mixed to a total volume of 75 µl, the mixtures were added to each well and allowed to stand at 37°C for six hours. As a buffer for ana

- 158 046075 lyse use 1.5 ml of low IgG booster serum from the kit, which is added to 36 ml of RPMI 1640 medium. The plate is then left at room temperature for 15 minutes and 75 µl of Bio-Glo reagent is added to each well. For the Bio-Glo reagent, use the Bio-glo Luciferase Assay System (buffer and substrate). The luminescence in each well is then measured using a plate reader. The luminescence value in each well divided by the luminescence value in the well without the addition of antibodies is an indicator of the fold induction, and it served as an indicator for assessing the ADCC of each antibody.

The results obtained are shown in Fig. 65. In this figure, the induction factor is designated as a relative light unit (RLU, relative light unit).

The results show that the ADCC of the non-switchable antibody MRAH-mFa55/MRAL-mk0 does not affect hIL6R-expressing cells depending on the ATP concentration, but the ADCC of the switchable antibodies H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 and H0052-mFa55 /L1083-ml0 becomes stronger at higher ATP concentrations in the assay. The results confirm that H0002mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 have ATP-dependent cytotoxic activity against hIL6R.

(13-5) Evaluation of Antitumor Activity of Anti-hIL6R Antibodies in Vivo After confirming the ADCC of each antibody, a test was performed to evaluate the antitumor activity of these antibodies in vivo in a tumor-bearing hIL6R-Hepa1-6 C57BL/6 mouse model according to the methods below.

Anti-asialo GM1 antibody (Wako Pure Chemical Industries) was dissolved in 1 ml of distilled water (Otsuka Pharmaceutical Factory), and then diluted with 4 ml of PBS. The anti-asialo GM1 antibody thus obtained was administered intraperitoneally to 100 C57BL/6 mice at a dose of 0.1 ml per mouse. Every other day, a mixed solution is prepared, to which an equal volume of Matrigel Matrix (Corning) is added, and added to a cell suspension of hIL6R-Hepa1-6 with 1x10 ⁸ /ml in Hanks' balanced salt solution (Sigma). The mixed solution is injected subcutaneously into 100 C57BL/6 mice at a dose of 0.2 ml per mouse and cells are implanted. The tumor diameter of the mice was measured with a caliper, and the tumor volume was determined as (major axis) x (minor axis) x (minor axis) x (1/2). The measurement of tumor diameter is carried out by an individual who is unaware of the composition of the drug solution administered to each mouse. Tumor diameter and body weight of 100 of the above C57BL/6 mice were measured on day 11 after cell transplantation. Based on the obtained values, mice are randomized and divided into a total of five groups: four groups of 10 animals each and one group of 8 animals; In each group, animals are injected through the tail vein with a solution of the antibodies shown in Table. 67. In a group of 8 animals, animals receive H0002-mFa55/L1058-ml0. The dose (ml) of the administered antibody solution is (0.01 (ml/g)) x (body weight (g) of each mouse). Tumor diameter and body weight of the C57BL/6 mice grouped above were measured in the same manner at days 14, 18, 21, and 25 posttransplantation. On the 18th day after transplantation, solutions of antibodies shown in table. 67 is administered to the animals in the respective group via the tail vein at a dosage determined using the same calculation formula as described above based on body weight measurements. The average tumor volume in each antibody treatment group on each measurement day was calculated and is an indicator to evaluate the in vivo antitumor activity of each antibody.

The results obtained are shown in Fig. 66.

Among the switch antibodies for which ATP-dependent ADCC has been confirmed, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 have been confirmed to exhibit antitumor activity against hIL6R-expressing cells to the same extent as MRAH-mFa55 /MRAL- mk0, which is not a non-switchable antibody according to the above results. The results indicate that extracellular ATP in the vicinity of tumors was present at a concentration level at which H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 function in a similar manner to MRAH-mFa55/MRAL-mk0.

Example 14. Evaluation of the systemic effects of anti-hIL6R antibodies having ATP-dependent binding properties.

To evaluate in normal tissues the effects of anti-hIL6R antibodies having ATP-dependent binding properties, normal mice (C57BL/6) and transgenic mice with systemic overexpression of hIL6R (hIL6R-tgm) (Proc Natl Acad Sci USA, 92 (11), 1995, 4862- 4866) is used to assess the kinetics of antibodies in plasma. For this assessment, the antibodies described in Table. 67 were administered to normal mice (C57BL/6) and hIL6R-tgm transgenic mice and their plasma kinetics were compared. In hIL6R-tgm, conventional antibodies that bind to hIL6R have been reported to disappear through membrane-type hIL6R as a result of binding to systemically expressed membrane-type hIL6R (Nature Biotechnology, 28, 2010, 1203-1207).

mg/ml MRAH-mFa55/MRAL-mk0, H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 or H0052mFa55/L1083-ml0 was administered intravenously to normal and hIL6R-tgm mice at a single dose of 10 ml/kg. From normal mice, blood was collected at 5 min, 7, 24, 48, 72 and 168 h after administration in all groups: MRAH-mFa55/MRAL-mk0 group, H0002-mFa55/L1058-ml0 group, H0041-mFa55/L1088- group ml0 and

- 159 046075 group H0052-mFa55/L1083-ml0. In hIL6R-tgm mice, blood was collected at 5 min, 7, 24, 48, 52, 55, 72, and 168 h post-administration in the MRAH-mFa55/MRAL-mk0 group and at 5 min, 7, 24, 48, 72. and 168 hours after administration in the H0002-mFa55/L1058-ml0 group, the H0041-mFa55/L1088-ml0 group and the H0052mFa55/L1083-ml0 group. The resulting blood was quickly centrifuged at 4°C at 12,000 rpm for 10 min; the plasma is separated. Plasma is stored in a freezer set at -20°C or lower until measurement is performed.

The concentration of each antibody in plasma is measured by electrochemiluminescence (ECL, electrochemiluminescence). The hIL6R solution is added to the MULTI-ARRAY PR plate (Meso Scale Diagnostics, LLCs), left to stand at 37°C for one hour, and hIL6R is immobilized on the plate. For the calibration curve, MRAH-mFa55/MRAL-mk0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 are obtained at plasma concentrations of 8, 4, 2, 1, 0.5, 0.25 and 0.125 µg/ ml; and H0002-mFa55/L1058-ml0 are obtained at plasma concentrations of 64, 32, 16, 8, 4, 2 and 1 μg/ml. After adding 1 M Tris-HCl, pH 8.0, containing 0.05% Tween20 and 1% BSA to the hIL6R-immobilized plate, plasma samples diluted 250-fold in 0.3 M acetic acid, and samples The calibration curve is quickly added, and then the ATP solution is added and mixed. After the plate was allowed to stand for one hour at room temperature, biotin-labeled anti-mouse IgG antibody (Southern Biotechnology Associates, Inc) was added and left at room temperature for one hour. SULFO-TAG Labeled Streptavidin (Meso Scale Diagnostics, LLC) is then added and left at room temperature for one hour, after which Reading Buffer T (Meso Scale Diagnostics, LLC) is added and the reaction is measured using a SECTOR Imager 6000 Electrochemical Luminescence Reader. (Meso Scale Diagnostics, LLC). The SOFTmax PRO program (Molecular Devices) was used to calculate the concentration of each antibody in mouse plasma.

The concentration of soluble antigen in plasma is measured by the ECL method. Mouse plasma samples diluted 50-fold are mixed with SULFO-TAG (Meso Scale Diagnostics, LLC)-labeled anti-human IL6R antibody (R&D Systems), biotin-labeled anti-human IL6R antibody (R&D Systems), and excess anti-IL6R antibody. human tocilizumab (the product is prepared by the inventors themselves) and the mixture is incubated overnight at 37°C. After adding the mixture to a standard 96-well streptavidin-coated blocking plate (Meso Scale Diagnostics, LLC), followed by the addition of readout buffer, the reaction is measured on a SECTOR Imager 6000 (Meso Scale Diagnostics, LLC). SOFTmax PRO software (Molecular Devices) is used to calculate soluble antigen concentrations in mouse plasma.

The results of comparison of the blood levels of MRAH-mFa55/MRAL-mk0, H0002-mFa55/L1058-ml0, H0041mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 in normal and hIL6R-tgm mice are presented in Fig. 67, fig. 68, fig. 69 and fig. 70, respectively.

The nonswitchable antibody MRAH-mFa55/MRAL-mk0 is cleared more rapidly in hIL6R-tgm mice that systemically express hIL6R than in normal mice (FIG. 67). This result appears to be caused by the fact that the non-switchable antibody binds to the membrane-type hIL6R expressed in normal tissues throughout the body and disappears through hIL6R.

On the other hand, for the switch antibodies H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 in hIL6R-tgm transgenic mice, no dramatic decrease in antibody concentrations was observed compared to normal mice. The results indicate that, in contrast to the result with the non-switched antibody, the switched antibodies do not bind to systemically expressed membrane-type hIL6R and do not cause hIL6R-mediated elimination.

Compared to the switch antibodies H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0, only the H0052-mFa55/L1083-ml0 antibody, which exhibits stronger binding at low ATP concentrations, results in accelerated loss of antibody in hIL6R-tgm compared to normal mice.

These results suggest that extracellular ATP concentrations in normal tissues are insufficient to switch antibodies to hIL6R binding.

Plasma concentrations of soluble hIL6R in hIL6R-tgm mice are then compared after administration of the corresponding antibodies. The results are shown in Fig. 71.

Compared with soluble hIL6R concentrations in mice receiving the isotype control IC17HdK-mFa55/IC17L-mk1, soluble hIL6R concentrations in mice receiving the nonswitchable antibody, MRAH-mFa55/MRAL-mk0, were observed to accumulate. This is thought to be caused by the non-switchable antibody binding to the soluble form of hIL6R, interfering with normal metabolic pathways.

On the other hand, mice treated with the switchable antibodies H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 do not exhibit significant antigen accumulation similar to that observed in the case of administration of a non-switchable antibody. When comparing the switchable antibodies H0002-mFa55/L1058-ml0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0, ten

- 160 046075 dent in antigen accumulation for antibodies demonstrating stronger binding at low ATP concentrations.

These results suggest that extracellular ATP concentrations in normal tissues and blood are low to switch antibodies to membrane binding and soluble forms of hIL6R.

Example 15: Simultaneous assessment of antitumor effects and systemic effects of anti-hIL6R antibodies with ATP-dependent binding properties.

Antitumor effects and systemic effects are simultaneously assessed to determine the systemic effects of anti-L6R antibodies having ATP-dependent binding properties in the presence of a tumor. For this evaluation, the hIL6R-tgm model with transplanted hIL6RHepa1-6 is used and the antibodies described in Table 1 are administered. 67.

Anti-asialo GM1 antibody (Wako Pure Chemical Industries) was dissolved in 1 ml of distilled water (Otsuka Pharmaceutical Factory) and then diluted with 4 ml of PBS. The anti-asialo GM1 antibody thus obtained was administered intraperitoneally to 83 hIL6R-tgm mice at a dose of 0.1 ml per mouse. Every other day, a mixed solution is prepared, to which the same volume of Matrigel Matrix (Corning) is added to the hIL6R-Hepa1-6 cell suspension suspended to a titer of 1x10 ⁸ /ml in Hanks' balanced salt solution (Sigma). The mixed solution was injected subcutaneously into 83 hIL6R-tgm transgenic mice at a dose of 0.2 ml per mouse, resulting in cell implantation. The tumor diameter of mice is measured with a caliper, and the tumor volume is determined as:

(major axis) x (minor axis) x (minor axis) x (1/2)

The measurement of tumor diameter is carried out by an individual who is unaware of the composition of the drug solution administered to each mouse. Tumor diameter and body weight of the 83 above hIL6R-tgm mice were measured on day 10 after cell transplantation. Based on the obtained values, mice are randomized, which are divided into a total of four groups of 10 animals, in each group the animals are injected through the tail vein with the appropriate antibody solution from among the antibodies shown in table. 67, with the exception of H0052-mFa55/L1083-ml0. The dose (ml) of the administered antibody solution is (0.01 (ml/g)) x (body weight (g) of each mouse). Subsequently, tumor diameters and body weights of hIL6R-tgm grouped as above were measured in the same manner on days 13, 17 and 20 after transplantation. On the 17th day after transplantation, solutions of the four antibodies shown in Table. 67, with the exception of H0052-mFa55/L1083-ml0, are administered to animals in the corresponding group through the tail vein at a dosage determined using the same calculation formula as described above, based on body weight measurements. The average tumor volume in each antibody treatment group and on each measurement day was calculated, which is an indicator to evaluate the in vivo antitumor activity for each antibody.

In the above experiments, blood was collected from hIL6R-tgm mice divided into the above groups under isoflurane anesthesia at one hour, 1, 2, 3 and 7 days after the initial administration of antibody solutions. Blood samples are transferred into a series of eight individually numbered tubes and kept on ice. After a series of eight tubes are centrifuged at 1900 x g for 10 min at 4°C, the centrifugation supernatant is recovered as a plasma component and transferred to another series of eight individually numbered tubes, which are stored at -30°C.

The plasma concentration of each antibody is measured by electrochemiluminescence (ECL). The hIL6R solution is added to the MULTI-ARRAY PR plate (Meso Scale Diagnostics, LLC) and left at 37°C for one hour, hIL6R is immobilized on the plate. For the calibration curve, solutions of MRAH-mFa55/MRAL-mk0 and H0041-mFa55/L1088-ml0 in plasma are prepared with concentrations of 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/ml; H0002-mFa55/L1058-ml0 was prepared at plasma concentrations of 64, 32, 16, 8, 4, 2, and 1 μg/ml. After adding 1 M Tris-HCl, pH 8.0, containing 0.05% Tween20 and 1% BSA to the hIL6R-immobilized plate, plasma samples are diluted 250-fold in 0.3 M acetic acid and quickly added samples for the calibration curve and the ATP solution and mix. After the plate was left for one hour at room temperature, biotin-labeled anti-mouse IgG antibody (Southern Biotechnology Associates, Inc) was added and allowed to stand at room temperature for one hour. Streptavidin-labeled SULFO-TAG (Meso Scale Diagnostics, LLC) is subsequently added and the plate is left at room temperature for one hour followed by the addition of Reading Buffer T (Meso Scale Diagnostics, LLC) and the reaction is measured using a SECTOR Imager 6000 (Meso Scale Diagnostics, LLC). SOFTmax PRO (Molecular Devices) is used to calculate antibody levels in mouse plasma.

Similarly, anti-asialo GM1 antibody (Wako Pure Chemical Industries) was dissolved in 1 ml of distilled water (Otsuka Pharmaceutical Factory) and then diluted with 4 ml of PBS. The anti-asialo GM1 antibody thus obtained was administered intraperitoneally into 91 hIL6R-tgm mice at a dose of 0.1 ml per mouse. Every other day, a mixed solution is obtained in which the same volume of Matrigel Matrix (Corning) is added to the hIL6R-Hepa1-6 cell suspension, suspended to 1x 108 cells/ml

- 161 046075 in Hanks' balanced salt solution (Sigma). The mixed solution was injected subcutaneously into 91 hIL6R-tgm mice at a dose of 0.2 ml per mouse and the cells were implanted. The tumor diameter of the mice was measured with a caliper, and the tumor volume was determined as:

(major axis) x (minor axis) x (minor axis) x (1/2)

The tumor diameter is measured by an individual who is unaware of the composition of the drug solution administered to each mouse. Tumor diameter and body weight of 91 above hIL6R-tgm mice were measured on day 10 after cell transplantation. Based on the obtained values, mice are randomized, which are divided into a total of four groups of 6 animals, in each group the animals are injected through the tail vein with the appropriate antibody solution from among the antibodies shown in table. 67, with the exception of H0052-mFa55/L1083-ml0. The dose (ml) of the administered antibody solution is (0.01 (ml/g)) x (body weight (g) of each mouse). Subsequently, tumor diameters and body weights of hIL6R-tgm mice grouped as above were measured in the same manner at days 13, 17 and 20 post-transplantation. On the 17th day after transplantation, four antibody solutions from the antibody solutions presented in Table. 67, with the exception of H0002-mFa55/L1058-ml0, are administered to mice of the respective groups through the tail vein at a dosage determined by the calculation formula described above based on measured body weight data. The average tumor volume in each antibody treatment group on each measurement day was calculated, and the in vivo antitumor activity of each antibody was assessed based on the data obtained.

In the above experiments, blood is collected by collecting a blood sample from the orbital sinus of hIL6R-tgm mice divided into the above groups under isoflurane anesthesia at one hour, 3 and 7 days after the initial administration of antibody solutions. The collected blood is transferred into a series of eight individually numbered tubes and left on ice. After centrifuging a series of eight individually numbered tubes at 1900xg for 10 min at 4°C, the supernatant is obtained as a plasma component and transferred to another series of eight individually numbered tubes and stored at -30°C.

The plasma concentration of each antibody is measured by electrochemiluminescence (ECL). The hIL6R solution is added to the MULTI-ARRAY PR plate (Meso Scale Diagnostics, LLC) and left at 37°C for one hour, whereby hIL6R is immobilized on the plate. For the calibration curve, prepare MRAH-mFa55/MRAL-mk0, Н0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 in plasma with concentrations of 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg/ ml. After adding 1 M Tris-HCl, pH 8.0, containing 0.05% Tween20 and 1% BSA to the hIL6R-immobilized plate, plasma samples are diluted 250-fold in 0.3 M acetic acid and quickly added samples for the calibration curve and then the ATP solution and mix. After the plate was left for one hour at room temperature, biotin-labeled anti-mouse IgG antibody (Southern Biotechnology Associates, Inc) was added and allowed to stand at room temperature for one hour. Streptavidin-labeled SULFO-TAG (Meso Scale Diagnostics, LLC) is subsequently added and the plate is left at room temperature for one hour followed by the addition of Reading Buffer T (Meso Scale Diagnostics, LLC) and the reaction is measured using a SECTOR Imager 6000 (Meso Scale Diagnostics, LLC). SOFTmax PRO (Molecular Devices) is used to calculate antibody levels in mouse plasma.

The concentration of soluble antigen in plasma is measured by electrochemiluminescence (ECL). Mouse plasma samples, diluted 50-fold, were mixed with SULFO-TAG (Meso Scale Diagnostics), LLQ-labeled anti-human IL6R antibody (R&D Systems), biotin-labeled anti-human IL6R antibody (R&D Systems), and excess anti-IL6R antibody. human tolizumam (prepared by the present inventors), the mixture is incubated overnight at 37° C. After adding the mixture to a standard 96-well streptavidin-coated blocked plate (Meso Scale Diagnostics, LLC), followed by the addition of readout buffer, the reaction measured on a SECTOR Imager 6000 (Meso Scale Diagnostics, LLC) SOFTmax PRO software (Molecular Devices) was used to calculate soluble antigen concentrations in mouse plasma.

First, the antitumor effects, plasma antibody concentrations, and plasma antigen concentrations of IC17HdK-mFa55/IC17L-mk1, MRAH-mFa55/MRAL-mk0, H0002-mFa55/L1058-ml0, and H0041-mFa55/L1088-ml0 were compared. The results are shown in Figures 72, 73 and 74, respectively.

In the present invention, compared with the isotype control group IC17HdKmFa55/IC17L-mk1, the groups treated with MRAH-mFa55/MRAL-mk0 and H0002-mFa55/L1058-ml0 do not show higher tumor growth inhibition (TGI) scores. On the other hand, a higher TGI was observed in the group treated with the H0041-mFa55/L1088-ml0 switch antibody (FIG. 72). Weaker drug efficacy was observed in the group of mice treated with MRAHmFa55/MRAL-mk0 compared to that of H0041-mFa55/L1088-ml0, suggesting that MRAH-mFa55/MRAL-mk0 is cleared faster than H0041-mFa55/L1088-ml0 , and it cannot maintain a concentration sufficient to demonstrate the effectiveness of the drug (Fig. 73).

- 162 046075

Also, accumulation of soluble hIL6R was not observed in the H0002-mFa55/L1058ml0 treated group. In the H0041-mFa55/L1083-ml0-treated group, soluble IL6R accumulation occurs to a lesser extent than in the MRAH-mFa55/MRAL-mk0-treated group (FIG. 74).

Antitumor effects, plasma antibody concentrations, and plasma antigen concentrations of IC17HdK-mFa55/IC17L-mk1, MRAH-mFa55/MRAL-mk0, H0041-mFa55/L1088-ml0, and H0052-mFa55/L1083-ml0 were then compared. The results are shown in Fig. 75, 76 and 77 respectively.

In the present study, compared with the group treated with the isotype control IC17HdK-mFa55/IC17L-mk1, the groups treated with MRAH-mFa55/MRAL-mk0, H0041-mFa55/L1088-ml0 and H0052-mFa55/L1083-ml0 show higher rates tumor growth inhibition (TGI) (Fig. 75). On the other hand, when comparing their plasma concentrations, MRAH-mFa55/MRAL-mk0 shows faster clearance than H0052-mFa55/L1083-ml0 and a lower blood concentration (FIG. 76).

In addition, compared to MRAH-mFa55/MRAL-mk0, H0052-mFa55/L1083-ml0 only accumulates less soluble IL6R (Fig. 77).

These results suggest that in tumor-bearing mice, switchable antibodies have similar or stronger antitumor effects than nonswitchable antibodies; however, unlike nonswitchable antibodies, switchable antibodies do not bind to hIL6R in normal tissues. The results show that even if cancer was present, extracellular ATP concentrations in normal tissues and blood are not elevated enough to stimulate the binding of switched antibodies.

Example 16. ATP-dependent neutralizing activity of anti-PD1 antibodies having ATP-dependent binding properties

Heavy chains and light chains are combined as shown in Table. 68, for the expression and purification of anti-PD-1 antibodies by methods known to those skilled in the art.

Table 68

Combinations of heavy chains and light chains of antibodies

Antibody VH CH VL C.L. mPDlF2VH-mF18/ mPDlF2VL-mkl mPDlF2VH SEQ ID NO: 201 mF18 SEQ ID NO: 203 mPDlF2VL SEQ ID NO: 202 mkl SEQ ID NO: 185 H5029-mFa31/ L3021-ml0 H5029 SEQ ID NO: 204 mFa31 SEQ ID NO: 208 L3021 SEQ ID NO: 205 mlO SEQ ID NO: 212 H5041-mFa31/ L3023-ml0 H5041 SEQ ID NO: 206 L3023 SEQ ID NO: 207

Neutralizing activity of the antibodies described in table. 68, PDL1-PD1 interaction was assessed according to the methods below.

Initially, the mPD1-G1CH2 (SEQ ID NO: 209) and mPDL1-G1dCH2His (SEQ ID NO: 210) antigens were genetically synthesized and inserted into plasmids for expression in animals. Antigenic proteins are expressed and purified using the methods described below. The resulting plasmids are introduced by lipofection into the FreeStyle 293-F line (Invitrogen), obtained from human fetal kidney cells (Invitrogen), suspended in FreeStyle 293 expression medium (Invitrogen) at the appropriate density, and seeded in flasks. Antigens are purified from culture supernatants of cells cultured in a CO ₂ incubator (37°C, 8% CO ₂ , 125 rpm) for four days by a method known to those skilled in the art. Spectrophotometers are used to measure the absorbance of purified antigen solutions at 280 nm. Concentrations of purified antigens are calculated using absorption coefficients calculated by the PACE method from the measured values obtained (Protein Science 4, 1995, 2411-2423).

mPD1-G1CH2 (SEQ ID NO: 209), which is an Fc-fusion of mouse PD1, is diluted to 5 μg/ml (55 nM) with a solution of 0.1 M NaHCO ₃ , 0.05% NaN ₃ , and 100 μl diluted solution added to a 96-well plate, left overnight at 4°C and immobilized on the surface of the plate. After washing the wells three times with PBS and 0.1% Tween20, 250 μl of BSA solution diluted to 2% PBS was added to each well to block the plate surfaces. Then wash three times. mPDL1-G1dCH2His (SEQ ID NO: 210), which is mouse PDL1 fused to an Fc antibody and a His tag, is diluted in PBS to a final concentration of 55 nM; solution of antibodies described in table. 68, diluted to final concentrations of 6.25, 1.56, 0.390, 0.0977, 0.0061, and 0 μg/ml; and ATP solution diluted to final concentrations of 0, 1, 10, and 100 μM are mixed to a total amount of 100 μL. The mixture is then added to each well and left at 37°C for one hour. Each well is then washed three times with 0.1% Tween20 and PBS, which are prepared with the same concentration of ATP as the ATP concentration in the solutions added to each well. Anti-His-tag mAb-HRP-Direct (MBL Life Science) is diluted 10,000-fold in blocking buffer to contain the same ATP concentration as the ATP concentration in the solutions added to each well, of which 100 µl is added to each well and left at 37°C for one hour. Then wash each well three times

- 163 046075 contain 0.1% Tween20 and PBS, which are prepared with the same ATP concentration as the ATP concentration in the solutions added to each well. Then, 100 μl of TMB solution was added to each well, and the plate was left at 37°C for one hour, and 50 μl of 1 M H ₂ SO ₄ was added to each well to stop the reaction. The absorbance of each well was then determined at 450 nm using a microplate absorbance reader (Wako Sunrise).

The amount of absorbance in an antibody-free well under the same ATP concentration conditions is set as 100% PD1-PDL1 binding, and the extent to which the rate of binding is reduced by the addition of antibody is assessed. The results are shown in Fig. 78 and 79.

The results show that the neutralizing activity of mPD1F2VH-mF18/mPD1F2VL-mk1 upon PD1/PDL-1 interaction is not affected by ATP concentrations, whereas the neutralizing activity of H5029-mFa31/L3021-ml0 and H5041-mFa31/L3021-ml0 becomes stronger at higher ATP concentrations in analyses. The results confirmed that H5029-mFa31/L3021-ml0 and H5041-mFa31/L3021-ml0 have ATP-dependent binding activity and neutralization activity.

The Luciferase Assay System (Promega) was used to measure neutralizing activity in vitro. For the effector cell, hPDL1-CHO auxiliary cells are used for the PD-1/PD-L1 blockade bioassay, Core Kit (Promega). JurkatNFAT-Luc2-mPD1 cells obtained by the authors of the present invention are used as the target cell. First, the extracellular mPD1 and intracellular hPD1 fusion protein gene (SEQ ID NO: 214) is introduced into Jurkat-NFAT-Luc2 cells (Promega) by a method known to those skilled in the art to obtain Jurkat-NFAT-Luc2mPD1 cells. The interaction of hPDL1 on hPDL1-CHO with the fusion protein of extracellular mPD1 and intracellular hPDl on Jurkat-NFAT-Luc2-mPDl is used as a PD-1/PDL-1 interaction to evaluate neutralizing activity in vitro.

To each well of a 96-well plate, add 100 μl of the hPDL1CHO accessory kit, which is prepared to a cell concentration of 4 x 10 ⁵ /ml in the medium, and the plate is left overnight at 37°C. For the culture medium, 90% Ham's F-12, 10% PBS, 250 μg of geneticin and 200 μg of hygromycin were used. Supernatants are removed from each well; 20 µl of a solution of antibodies described in table. 68, diluted with assay buffer to a final concentration of 7.5 μg/ml, mixed with 20 μl of AMP solution diluted with assay buffer to final concentrations of 0, 1, 10 and 100 μM, and 40 μl of NFAT-Luc2-hPD1- cell solution mPD1-Jurkat prepared with assay buffer to achieve a cell count of 5 x 10 ⁴ /well. The mixture is added to each well and left at 37°C for four hours. 98% RPMI 1640 and 2% FST are used as assay buffer. The plates are then left at room temperature for 10 minutes and 80 µl of BioGlo reagent is added to each well. For the Bio-Glo reagent, use the Bio-glo Luciferase Assay System (buffer and substrate). The luminescence in each well is then measured using a plate reader.

The amount of luminescence in each well at each AMP concentration divided by the amount of luminescence in the antibody-free well represents the fold induction and is an indication of the activity of each antibody in neutralizing the PD-1/PDL-1 interaction. The results are shown in Fig. 80. In the figure, the induction factor is designated as relative light unit (RLU).

ATP is tested in the same way as AMP, and 100 ml of hPDL1-CHO, prepared at a concentration of 4x 105/ml in medium, is added to each well of a 96-well plate, and the plate is left at 37°C overnight. The nutrient medium used is 90% Ham's F-12, 10% PBS, 250 μg of geneticin and 200 μg of hygromycin. Supernatants are removed from each well by aspiration; 20 µl of a solution of antibodies described in table. 68, diluted with assay buffer to a final concentration of 10 μg/ml, mixed with 20 μl of ATP solution diluted with assay buffer to final concentrations of 0, 12.5 and 125 μM, and 40 μl of NFAT-Luc2-hPD1-mPD1- cell suspension Jurkat, prepared with assay buffer to achieve a cell count of 5 x 10 ⁴ /well. The mixture is added to each well and left at 37°C for four hours. 98% RPMI 1640 and 2% FST are used as assay buffer. The plates are then left at room temperature for 10 minutes and 80 µl of BioGlo reagent is added to each well. For the Bio-Glo reagent, use the Bio-glo Luciferase Assay System (buffer and substrate). The luminescence in each well is then measured using a plate reader.

The luminescence value in each well at each AMP concentration divided by the luminescence value in the antibody-free well represents the fold induction and serves as a metric for assessing the activity of each antibody in neutralizing the PD1/PDL-1 interaction. The results are shown in Fig. 81. In the figure, the induction factor is designated as relative luminous unit (RLU).

Example 17. ATP-dependent ADCC-mediated antitumor activity of anti-PD1 antibodies having ATP-dependent binding properties.

Heavy chains and light chains are combined as shown in Table. 69, for the expression and purification of anti-PD-1 antibodies by methods known to those skilled in the art.

- 164 046075

Table 69

Combinations of heavy chains and light chains of antibodies

Antibody VH CH VL C.L. IC17HdK-mFa55/ IC17L-mkl IC17HdK SEQ ID NO: 187 mFa55 SEQ ID NO: 184 IC17L SEQ ID NO: 188 mkl SEQ ID NO: 185 mPDlF2VH-mFa55/ mPDlF2VL-mkl mPDlF2VH SEQ ID NO: 201 mPDlF2VL SEQ ID NO: 202 H5041-mFa55/ L3023-ml0 H5041 SEQ ID NO: 206 L3023 SEQ ID NO: 207 mlO SEQ ID NO: 212

The heavy chain constant regions of these antibodies are modified to mFa55, which enhances ADCC activity by adding modifications that enhance FcyRIV binding to mIgG2a. Therefore, it is considered possible to exert ADCC activity on cells expressing PD-1 and to remove cells expressing PD-1. Antibodies are used to assess antitumor activity in vivo and to assess the elimination of cells expressing PD-1, in accordance with the methods below.

The mouse colorectal cancer line Colon38 (NCI) was implanted at 5x10 ⁶ with Matrigel (Corning) subcutaneously into the right abdomen of C57BL/6J mice (Japan Charles River) to form solid tumors. On the fourteenth day after implantation, IC17HdK-mFa55/IC17L-mk1 was administered intravenously (IV) at a dose of 15 mg/kg, mPD1F2VH-mFa55/mPD1F2VL-mk1 at a dose of 1.5, 5 and 15 mg/kg and H5041-mFa55/ L3023-ml0 at a dose of 25.5 and 100 mg/kg, respectively (n=7 per group). In addition, on day 17 after transplantation, mice in the H5041-mFa55/L3023-ml0 group were administered the same antibodies intravenously (IV) at doses of 25.5 and 100 mg/kg, respectively. As a result, the antitumor effect was observed in all groups of antibody administration, with the exception of the negative control group (Fig. 82).

Elimination of PD1-expressing cells in tumor tissue after administration of appropriate antibodies is assessed using liquid cytometry. Solid Colon38 tumors are allowed to form as described above, and on day 14 after implantation, IC17HdK-mFa55/IC17L-mk1 (isotype control) is administered intravenously (IV) at a dose of 15 mg/kg, mPD1F2VH-mFa55/mPD1F2VL- mk1 at a dose of 1.5, 5 and 15 mg/kg, and H5041-mFa55/L3023-ml0 at a dose of 25.5 and 100 mg/kg, respectively (n=3 per group). Spleen and tumor tissues from each mouse were collected three days after antibody administration. The spleens are crushed with tweezers, 10% PBS-supplemented RPMI 1640 medium is added and centrifuged at 400 x g for 5 min, after which the supernatant is removed. Add 2 ml of lysis buffer ACK (Thermo Fisher Scientific) and leave at room temperature for 2.5 minutes, the hemolyzed sample is used as spleen cells for liquid cytometry analysis. The tumor tissue is minced with scissors followed by the addition of enzymes according to the protocol of the Tumor Dissociation Kit (Miltenyi) and then minced using a MACS soft dissociator (Miltenyi Biotec). With the addition of 10% PBS-supplemented RPMI 1640 medium, it is centrifuged at 400 x g for 5 minutes, the supernatant is removed and used for liquid cytometry analysis as tumor cells. Spleen cells and tumor cells are transferred to a V-bottom plate (Corning), centrifuged at 400 x g for 5 minutes, and the supernatant is removed. Cells are resuspended in 100 μl of FcR blocking reagent (Miltenyi Biotec), diluted 10 times in PBS (FACS buffer) containing 1% FST and 2 mM EDTA (Sigma). After incubating the cells at room temperature for 10 min, 0.4 µl BUV737 Anti-Mouse CD3e (BD), 0.1 µl Zombie Aqua (Biolegend), 0.4 µl PerCP/Cy5.5 Anti-Mouse CD8a ( Biolegend), 0.4 µl PE/Cy7 Anti-Mouse CD4 (Biolegend), 0.2 µl APCR700 anti-mouse CD45 (BD), 0.4 µl FITC anti-human/mouse/rat CD278 (Biolegend), 0.4 μl of anti-mouse CD25 APC/FireTM 750 (Biolegend) and 0.4 μl of anti-mouse CD279 APC (Biolegend), which has already been confirmed not to compete with each of the administered PD1 antibodies, are added to each well, and FACS buffer is added at 20 μl/well. After 30 min of incubation at 4°C, add 100 μl of FACS buffer, centrifuge at 400 xg for 5 min, and remove the supernatant. According to the protocol from the Foxp3/transcription factor staining buffer kit (eBioscience), fixation/permeabilization concentrate and fixation/permeabilization diluent were mixed and 200 μl of the mixture was added to each well. After incubation at 4°C for 30 min and centrifugation at 400 x g for 5 min, the supernatant was removed. Permeabilization buffer is added in an amount of 200 μl and after centrifugation at 400xg for 5 minutes, the supernatant is removed. This washing procedure is repeated again. eFluor450 AntiMouse/Rat Foxp3 (eBioscience) in an amount of 0.4 μl and PE Anti-Mouse CD152 (BD) in a concentration of 0.4 μl are added to each well, FACS buffer is added in an amount of 20 μl/well. After incubation at 4°C for 30 min, 100 μl of FACS buffer was added and after centrifugation at 400 x g for 5 min, the supernatant was removed. Permeabilization buffer is added to each well at 200 μl, and after centrifugation at 400 x g for 5 min, the supernatant is removed. Samples are resuspended in 200 μl FACS buffer and measured in a FACS Fortessa liquid cytometer (BD). For

- 165 046075 expression analysis using FlowJo software. CD4-positive cells passing through the cell population are analyzed to analyze the expression of Foxp3 and PD1. The level of PD1 expression in the CD4 ⁺ Foxp3 − cell population is calculated from fluorescence intensity. As a result, the expression level of PD1 in both spleen cells and tumor cells tended to decrease in groups in which mice were administered 5 and 15 mg/kg mPD1F2VH-mFa55/mPD1F2VL-mk1, but not in groups in which mice were administered 25. 5 and 100 mg/kg H5041-mFa55/L3023-ml0, where the expression level of PD1 tended to decrease only in tumor cells (Fig. 83A and B). All switch antibodies that bind to PD1 in an ATP-dependent manner were confirmed to exhibit drug efficacy against the tumor while systemic response (downregulation of PD1 expression) did not occur, and switch antibodies were confirmed to be able to exhibit site-specific activity tumors.

Example 18. Analysis of the crystal structure of hIL6R-binding antibodies having ATP-dependent binding properties.

X-ray diffraction analysis of the ATP complex, the extracellular domain of hIL6R (shIL6R) and the Fab fragment of the antibody H0041L1088 binding to the human IL6 receptor (hIL6R) (ATP switch) obtained in Example 13 was carried out.

(18-1) Preparation of full-length antibody H0041L1088.

Preparation and purification of full-length antibodies H0041L1088 (VH: SEQ ID NO: 194; CH: SEQ ID NO: 217, VL: SEQ ID NO: 195, CL: SEQ ID NO: 189), having the human IgG1 format, is performed by methods known specialists in this field.

(18-2) Preparation of Fab H0041L1088 fragments.

Full length antibody sample H0041L1088 is fragmented into Fab and Fc using papain (SIGMA-ALDRICH, 10108014001) at 35°C for approximately 18 hours and then purified on a HiTrap SP HP 1 ml column (GE Healthcare) and HiTrap MabSelect SuRe 1 ml (GE Healthcare), and SEC purification using HiLoad 16/600 Superdex 200 pg (GE Healthcare) to obtain Fab samples.

(18-3) Preparation of shIL6R.

Based on the amino acid sequence of UniProtKB: P08887 (IL6RA_HUMAN) (SEQ ID NO: 213), its domains 2 and 3 (amino acids 111-320) were genetically generated and included in expression vectors. For gene synthesis, a secretion expression signal sequence is added at the N-terminus, a FLAG tag + Hisx8 tag is added at the C-terminus for purification, and a C193S modification was introduced to eliminate the imperfection of Cys-Cys pairs. which results from the removal of domain 1. Protein expression using the Expi293TM expression system (Thermo Fisher Scientific) is performed in the presence of kifunensin (Santa Cruz Biotechnology) using the resulting expression plasmids and the expression culture supernatant containing the protein of interest. From this sample, shIL6R was prepared by affinity purification using a 5 mL cOmplete™ His-Tag Purification column (SIGMA-ALDRICH) and SEC purification using a HiLoad 16/600 Superdex 200 pg (GE Healthcare).

(18-4) Preparation of the H0041L1088 Fab-shIL6R-ATP ternary complex.

Endoglycosidase F1 (EndoF1) is added to the shIL6R sample to cleave the N-type sugar chain and enterokinase (SIGMA-ALDRICH, 11334115001) to cleave the Hisx8 tag, left to stand at room temperature for one day and then run-through on a HisTrap EXCEL column. volume 1 ml (GE Healthcare) and SEC purification Superdex 200 Increase 10/300 GL (GE Healthcare). After addition of Fab H0041L1088 sample, the resulting purified fraction was enriched by ultrafiltration and SEC purification using Superdex 200 Increase 10/300 GL (GE Healthcare) using 20 mM HEPES, pH 7.3, 100 mM NaCl, 0.5 mM ATP buffer for the preparation of complex (complex) samples. Complex samples for crystallization are obtained by ultrafiltration enrichment of the resulting purified fractions.

(18-5) Preparation of crystals of the H0041L1088 Fab-shIL6R-ATP ternary complex.

Sessile drop vapor diffusion crystallization was performed at 21°C using complex tank crystallization samples in 60 mM Tris, pH 7.5, 12.0% w/v. polyethylene glycol 1500 (polyethylene glycol 1500), 60 mM ammonium sulfate; Crystals suitable for X-ray diffraction analysis were obtained.

(18-6) Measurement of X-ray diffraction data from crystals of the FabshIL6R-ATP H0041L1088 ternary complex and determination of crystal structures.

The resulting crystals are immersed in a solution of 68 mM Tris, pH 7.5, 13.6% w/v. polyethylene glycol 1500 (polyethylene glycol 1500), 68 mM ammonium sulfate, 14.6% ethylene glycol and 0.375 mM ATP, and then frozen in liquid nitrogen. X-ray diffraction data are measured in the High Energy Accelerator Research Organization radiation light facility, Photon Factory BL-17A. During measurements, the crystals were continuously placed in a nitrogen stream at -178°C to maintain a frozen state. Received

- 166 046075 diffracted images are processed using autoPROC ((Acta Cryst. D67, 2011, 293-302) and diffraction intensity data is obtained with a resolution of up to 2.76 Angstroms.

Using the obtained X-ray diffraction intensity data, the parent structure is determined by molecular displacement using Phaser (J. Appl. Cryst., 40, 2007', 658-674), using the crystal structures of the known Fab and shIL6R PDB. ID=1N26 as search models. Subsequently, building and refining the model using coot (Acta Cryst. D66, 2010, 486-501) and refmac5 (Acta Cryst. D67, 2011, 355-367), as well as phenix.refine (Acta Cryst. D68, 2012, 352- 367) are repeated, which leads to the final clarification of the coordinates. Crystallographic statistics are presented in table. 70. It should be noted that among the crystal structure coordinates, the amino acid residue numbers in Fab are given according to the Kabat numbering scheme, and the amino acid residue numbers in the antigen, shIL6R, are given to correspond to the amino acid residue numbers in UniProtKB: P08887 (IL6RA_HUMAN). Although there are two complexes in the asymmetric units of this crystal, a specialized dimerization called domain shuffling is observed in domain 2 of shIL6R. Such dimers are not found in the crystal structures of shIL6R PDB ID=1N26, making them unique to the shIL6R constructs used in the present invention.

Table 70

<Measurement data> Measurement wavelength (Angstrom) 0.98 Number of crystals measured 1 Space group S222 Cell constant a b c (Angstrom) 103.043 185.612 152.085 α β γ (°) 90.00 90.00 90.00 Number of complexes in an asymmetric block 2 Resolution (Angstrom) 90.09 - 2.757 (2.86 - 2.76) Number of observed reflections/ Number of unique reflections 508114/38008 Reservation 13.37 (13.66) Completeness (%) 99.99 (100.00) Diffraction intensity Signal to noise ratio (S/N) 18.9 (3.1) R absorption 0.106 (0.868) <Clarification> R working/R free 0.1923/0.2656 Number of atoms 9244 Except water molecules 9181 water molecules 63 RMSD (Root Mean Square Deviation) from Theoretical Value Communication distance (Angstrom) 0.008 Connection angle(°) 1.05 According to Ramachandran's map Preferred Area (%) 95.95 Allowed area (%) 3.72 Forbidden area (%) 0.34 Values in parentheses are values at extreme resolution.

(18-7) Interaction between H0041L1088 and ATP.

In fig. 84, ATP is recognized primarily by the antibody heavy chain. Specifically, the adenine ring portion of ATP is recognized by each side chain of the antibody heavy chain CDR1: T33, CDR3: Y95, L98, N100B, and W100C, and each of the main chains of G96, L100A, and W100C. Specifically, hydrogen bonds are formed between the backbone carbonyl oxygen of G96 and L100A and the NH2 at position 6 of NH ₂ ATP, as well as between the NH group of the backbone amide W100C and the N position 1 of ATP; and interactions such as CH-pi (π) and π-π are formed between the side chains of Y95, L98 and W100C and the adenine ring residue. The antibody reliably recognizes a fragment of the adenine ring of ATP. The ribose moiety is recognized by van der Waals interactions with the corresponding side chains of the heavy chain CDR1: T33 and CDR2: H56 and Y58. The triphosphate moiety is recognized by the heavy chain CDR2 side chains: S52, S52A, Y55, and H56, as well as the main chain, S52A and Q53. In particular, the side chain of S52 and the side chain of S52A, as well as the main chain NH group and the main chain NH group of Q53 form a dense network of hydrogen bonds with the triphosphor group moiety, which play a crucial role in the recognition of triphosphor group moieties.

- 167 046075 (18-8) Interaction between H0041L1088 and hIL6R.

Based on the present crystal structure, amino acid residues of shIL6R containing one or more non-hydrogen atoms located at distances within 4.2 Angstroms from Fab or ATP H0041L1088 were selected as epitope residues, and they are shown in the amino acid sequence of shIL6R. (Fig. 85). Details of the interactions of these epitope residues with the antibody are shown in FIG. 86 and 87. These epitope residues form van der Waals interactions, hydrogen bonds, electrostatic interactions, etc. with light chain CDR1 residues: D27B, G28, D29, A31 and Y32, and CDR3: R91, S92, P93, G94 and P95, and heavy chain CDR2: H56 and Y58 and CDR3: L98, Y99, N100B and W100C antibodies; and they are tightly bound to the antibody.

(18-9) ATP-Dependent Antigen-Binding Mechanism Additionally, upon binding of Fab-shIL6R H0041L1088, as shown in FIG. 86 and 87, a large intermolecular contact is formed between F298 shIL6R and ATP, which binds to Fab H0041L1088. Because this interaction is lost in the ATP-unbound state, it is proposed that this direct interaction between the antigen and ATP that binds to Fab H0041L1088 is the primary driver of ATP-dependent binding. Structurally, ATP is believed to contribute to the structural stabilization of heavy chain CDR3 through hydrogen bonding and van der Waals interactions with heavy chain CDR3 residues. It is believed that stabilization of the heavy chain CDR3 structure in its antigen-bound form by ATP binding results in a significant enhancement of the direct interaction of the heavy chain CDR3:L98 or Y99 with shIL6R, thereby contributing to ATP-dependent binding.

Example 19. Enhancement of agonistic activity by modified anti-CD137 antibodies. (19-1) Obtaining antibodies for evaluation.

Regarding the case where various amino acid modifications to increase pI were introduced into the heavy chain constant region with increased FcYRIIb binding activity, the effect of such amino acid modifications on CD137 agonist activity and on plasma kinetics and drug efficacy in hCD137KI/mFcYR2bKO/hFcYR2bTg #90 is assessed. First, as described in Example 7-1, Example 7-2 and Example 7-3, A375-MY201aPh/B167-Lamlib, A375-SCF041aPh/B167Lamlib, A375-SCF057aPh/B167-Lamlib and IC17HdK-MY201aPh/ are prepared. IC17L-k0.

(19-2) In vitro assessment of the ATP-dependent CD137 agonist activity of modified anti-human CD137 antibodies using the 4-1BB Jurkat reporter gene assay.

Regarding the case where various amino acid modifications to increase pI are introduced into the constant region of the heavy chain with increased binding activity to FcYRIIb, in order to evaluate the effect of such amino acid modifications on the activity of the CD137 agonist, the activity of the CD137 agonist A375-MY201aPh/B167-Lamlib, A375-SCF041aPh/ is assessed. B167-Lamlib, A375-SCF057aPh/B167-Lamlib and IC17HdK-MY201aPh/IC17L-k0.

The GloResponseTM NF-kB-Luc2/4-1BB Jurkat cell line (Promega, CS196004) was used to measure antibody activity in vitro. 200 μl of FcyRIIB CHO-K1 cells (Promega), prepared at a concentration of 5x10 ⁴ /ml with culture medium, are added to each well of a 96-well plate, and the plate is left overnight in an incubator under an atmosphere of 5% CO2 at 37°C. CHO culture medium (90% Ham's F12, 10% PBS) is used as a nutrient medium. All culture medium is then removed by suction, after which 25 µl of GloResponseTM NF-kB-Luc2/4-1BB Jurkat cell line, prepared at a concentration of 2x10 ⁶ cells/ml with assay medium (99% RPMI, 1% PBS) is added to each well . Then add 25 μl of each of the antibody solutions diluted in the assay medium to final concentrations of 0, 0.001, 0.01, 0.1, 1 and 10 μg/ml. Then add 25 μl of ATP solution diluted with analytical medium to a final concentration of 250 μM. The plate is left for 6 hours in an incubator under 5% CO2 at 37°C and then left for 15 minutes at room temperature, and 75 µl of Bio-Glo reagent is added to each well. For the Bio-Glo reagent, use the Bio-Glo Luciferase Assay System (buffer and substrate). The relative light unit of each well is then determined using a plate reader. The amount of luminescence in each well divided by the amount of luminescence in the well without added antibody is determined as a relative light unit and serves as an indicator for assessing the CD137 agonist activity of each antibody.

The results are shown in Fig. 88 The results show that the agonist activity of CD137 is increased by introducing various amino acid modifications to increase the pI.

(19-3) Pharmacokinetic study in mice of modified anti-human CD137 antibodies.

(19-3-1) Generation of hCD137KI/mFcYR2bKO/hFcYR2bTg#90 mouse.

First, a human CD137 knockin mouse is generated in which the mouse CD137 gene is replaced with a human CD137 gene by introducing a human CD137 gene replacement vector into mouse embryonic stem cells (ES cells) together with a zinc finger nuclease (ZFN) that targets mouse CD137. ZFN mRNA targeting the mouse Fcgr2b gene is then microinjected into fertilized mouse eggs, and Fcgr2b knockout mice are generated by selecting mice that have been injected

- 168 046075 mutation to the target site. In addition, a BAC vector in which the human FCGR2B gene is cloned is microinjected into fertilized mouse eggs, and by selecting from mice derived from these cells those in which the genomic region of the human FCGR2B gene has been introduced, a human FCGR2B transgenic mouse is obtained ( Iwayanagi et al., J Immunol, 195, 2015, 3198-3205).

By crossing mice of the three above strains, a transgenic mouse with human Fcgr2b CD137 knockin and human FCGR2B knockout is obtained. This mouse is designated hCD137KI/mFcyR2bKO/hFcyR2bTg#90 mouse.

(19-3-2) Measurement of the concentration of anti-human CD137 antibody in the plasma of the hCD137KI/mFcyR2bKO/hFcyR2bTg#90 mouse model

The corresponding anti-human CD137 antibodies were administered intravenously as a single dose to hCD137KI/mFcyR2bKO/hFcyR2bTg#90 mice, as shown in Table 1. 71. Blood is collected several times over a period of time from 5 minutes to 28 days after administration. The resulting blood is centrifuged to separate the plasma. Plasma is stored in a freezer at a temperature below -20°C until measurement.

Table 71

Group Number of animals Pharmaceutical agent Dose (mg/kg) 1 3 A375-MY201aPh/ B167-Lamlib 7.5 Single administration 2 3 A375-SCF041aPh/ B167-Lamlib 7.5 Single administration

The plasma concentration of each anti-human CD137 antibody is measured by electrochemiluminescence (ECL, electrochemiluminescence). hCD137 (Sino Biological Inc.) is diluted with PBS () and added to a 96-well MULTI-ARRAY plate (Meso Scale Diagnostics, LLC). The plate containing hCD137 is shaken for 1 hour at room temperature, and hCD137 is immobilized on the plate. PBS solution containing 1 mM ADP, 1% BSA, and 0.05% Tween-20 is then added to block, and the plate is shaken for 1 h at room temperature. Calibration curves for the corresponding anti-human CD137 antibodies were generated at plasma concentrations of 640, 320, 160, 80, 40, 20, and 10 ng/mL. Plasma samples diluted with PBS containing 1 mM ADP, 1% BSA and 0.05% Tween-20 and samples for the calibration curve are added to the hCD137 immobilized plate. The plate is then shaken for 1 hour at room temperature, and then the antibody described in WO 2019112027, which specifically recognizes the constant regions of anti-human CD137 antibodies, is added as a secondary antibody. The plate is shaken for an additional 1 hour at room temperature and then SULFO-TAG-labeled goat anti-rabbit antibodies (Meso Scale Diagnostics, LLC) are added. The plate is additionally shaken for 1 hour at room temperature and then SULFO-TAG-labeled goat anti-rabbit antibodies (Meso Scale Diagnostics, LLC), diluted twofold, are added to it and 1 mM ADP is added. The concentration of each antibody in mouse plasma is measured by detecting SULFO-TAG using a SECTOR Imager (Meso Scale Diagnostics, LLC). The concentration of each antibody in mouse plasma was calculated using SOFTmax PRO (Molecular Devices).

The results are shown in Fig. 89. A375-SCF041aPh/B167-Lamlib shows faster elimination than A375-MY201aPh/B167-Lamlib. This is believed to be due to the fact that the A375-SCF041aPh/B167-Lamlib heavy chain constant region is introduced with amino acid modifications that increase the pI.

(19-4) Evaluation of the drug efficacy of modified anti-human CD137 antibodies in mice.

(19-4-1) Generation of cell lines and mouse model transplanted with a syngeneic tumor line.

Clone LLC1/OVA/GPC3 of the C5 cell line (LLC1/OVA/GPC3) was used, obtained by introducing expression plasmids of chicken ovalbumin (OVA) and human glypican-3 (GPC3) into mouse lung cancer cells obtained from cells of the LLC1 cell line [LL /2 (designation: LLC1), distributor: ATCC company, catalog number: CRL-1642]. The hCD137KI/mFcyR2bKO/hFcyR2bTg#90 mouse (11 weeks old, female) described above in (19-3-1) was used. The LLC1/OVA/GPC3 cell line was maintained and passaged in RPMI1640 medium (Sigma-Aldrich, Co. LLC) containing 9.8% fetal bovine serum (Sigma-Aldrich, Co. LLC), 0.44 mg/ml G418 (Nacalai Tesque, Inc.) and 0.88 mg/ml Zeocin (Thermo Fisher Scientific, Inc.). The LLC1/OVA/GPC3 cell line was transplanted subcutaneously into the peritoneal cavity of a mouse, and the model was considered to be established when the tumor volume reached approximately 250-500 mm ³ . Once the model is established, mice transplanted with the LLC1/OVA/GPC3 cell line are grouped and then administered vehicle and appropriate anti-human CD137 antibodies.

(19-4-2) Preparation and administration of pharmaceutical agents for the administration and measurement of tumors.

A375-MY201aPh/B167-Lamlib or A375-SCF041aPh/B167-Lamlib, prepared at the dosages indicated in Table 72, with PBS containing 0.05% Tween-20, is administered to the transplant model

- 169 046075 cell line LLC1/OVA/GPC3 on the 11th and 14th days after tumor transplantation. PBS containing 0.05% Tween-20 was administered to the control group. The prepared liquid for administration is injected at a dose of 10 ml/kg into the tail vein.

Table 72

Measuring antitumor effects in the LLC1/OVA/GPC3 cell line transplantation model

Group Number of animals Pharmaceutical agent Dose Method of administration Introduction day 1 5 PBS containing 0.05% Tween-20 - Tail vein 11 days and 14 days after transplantation 2 5 A375-MY201aPh/ B167-Lamlib 2.50 mg/kg Tail vein 11 days and 14 days after transplantation 3 5 A375-SCF041aPh/ B167-Lamlib 2.50 mg/kg Tail vein 11 days and 14 days after transplantation

To assess the antitumor effect, tumor volume is measured 1-2 times a week. Tumor volume is calculated using the following equation:

tumor volume (mm ³ ) = length (mm) x width (mm) x width (mm)/2

Thus, the results of Example (19-3-2) show that the change in blood concentration in mice administered A375-SCF041aPh/B167-Lamlib is less compared to that of A375MY201aPh/B167-Lamlib; however, A375-SCF041aPh/B167-Lamlib exhibited a stronger antitumor effect than A375-MY201aPh/B167-Lamlib (Fig. 90).

Based on the above results in this mouse model, the antitumor effect of human anti-CD137 antibodies is enhanced by introducing amino acid modifications that increase the pI in the heavy chain constant region.

Example 20. Enhancement of agonist activity by modified anti-human CD3 antibodies.

Enhancement of the agonist activity of the anti-CD3 antibody is explored by combining amino acid modifications to increase the pI and heavy chain constant region with increased binding activity for FcyRIIb.

SCF057aPh is used, which is obtained by introducing the amino acid modification Q311R/P343R to increase the pI to MY201aPh and MY201aPh, which are heavy chain constant regions with increased binding activity for FcyRIIb obtained in Example 7-1. In addition, G1T6 (SEQ ID NO: 223) is used as the constant region of native human IgG1. The corresponding anti-human CD3 antibodies are prepared using TR01H113 (SEQ ID NO: 224) as the heavy chain variable region; MY201aPh, SCF057aPh, or G1T6 as the heavy chain constant region; and L0011-k0 (SEQ ID NO: 225) as the light chain of an anti-human CD3 antibody. IC17HdK-MY201aPh/IC17L-k0 was used as a negative control.

Bioassay (NFAT) (Promega, CS176401) is used to activate T cells to measure in vitro activity of the antibodies produced. 200 μl of FcyRIIB CHO-K1 cells (Promega), prepared at a concentration of 5x104/ml in culture medium, are added to each well of a 96-well plate, and the plate is left overnight in an incubator under an atmosphere of 5% CO2 at 37°C. CHO culture medium (90% Ham's F12, 10% PBS) is used as a nutrient medium. Then all the culture medium is removed by suction, then 25 μl of NFAT-RE-Luc2 cell suspension prepared at a concentration of 2x10 ⁶ /ml with assay medium (99% RPMI, 1% PBS) is added to each well. Then add 25 µl of each antibody solution diluted in assay medium to final concentrations of 0, 0.0001, 0.001, 0.01, 0.1, 1 and 10 µg/ml. The plate was left for 6 hours in an incubator in an atmosphere of 5% CO ₂ at 37°C, then left for 15 minutes at room temperature, then 75 μl of Bio-Glo reagent was added to each well. For the Bio-Glo reagent, use the Bio-Glo Luciferase Assay System (buffer and substrate). The relative light unit of each well is then determined using a plate reader. The luminescence value of each well divided by the luminescence value of the well without added antibody is determined as a relative light unit and serves as an indicator for assessing the CD3 agonist activity of each antibody.

The results are shown in Fig. 91. Compared with TR01H113-G1T6/L0011-k0, which has the constant region of native human IgG1, TR01H113-MY201aPh/L0011-k0, which has increased binding to FcyRIIb, showed higher CD3 agonist activity. TR01H113-SCF057aPh/L0011-k0 with amino acid modifications that increase pI showed higher CD3 agonist activity compared to TR01H113-MY201aPh/L0011-k0 before amino acid modifications.

Industrial Application

The anti-CD137 antigen-binding molecules described and methods for their use are applicable to the development, preparation, supply and use of pharmaceuticals that have

- 170 046075 immune cell activation effect, cytotoxic effect or antitumor effect, but have little effect on non-tumor tissues such as normal tissues and cause few side effects.

Claims (8)

1. Ahtu-CD137 antibody comprising any combination of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 regions selected from (a) to (i) below: (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 8, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; (b) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 9, HVR-H3 containing the amino acid sequence of SEQ ID NO: 17, HVR-L1 containing the amino acid sequence of SEQ ID NO: 22, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; (c) HVR-H1 containing the amino acid sequence SEQ ID NO: 7, HVR-H2 containing the amino acid sequence SEQ ID NO: 10, HVR-H3 containing the amino acid sequence SEQ ID NO: 17, HVR-L1 containing the amino acid sequence SEQ ID NO: 22, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; (d) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 11, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; (e) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 12, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 28; (e) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 13, HVR-H3 containing the amino acid sequence of SEQ ID NO: 18, HVR-L1 containing the amino acid sequence of SEQ ID NO: 21, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 29; (g) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, HVR-H3 containing the amino acid sequence of SEQ ID NO: 20, HVR-L1 containing the amino acid sequence of SEQ ID NO: 24, HVR-L2 containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 27; (h) HVR-H1 containing the amino acid sequence of SEQ ID NO: 7, HVR-H2 containing the amino acid sequence of SEQ ID NO: 15, HVR-H3 containing the amino acid sequence of SEQ ID NO: 20, HVR-L1 containing the amino acid sequence of SEQ ID NO: 25, HVR-L2, containing the amino acid sequence of SEQ ID NO: 26, and HVR-L3, containing the amino acid sequence of SEQ ID NO: 27, and/or HVR-H1, containing the amino acid sequence of SEQ ID NO: 7, HVR -H2 containing the amino acid sequence SEQ ID NO: 14, HVR-NZ containing the amino acid sequence SEQ ID NO: 19, HVR-L1 containing the amino acid sequence SEQ ID NO: 24, HVR-L2 containing the amino acid sequence SEQ ID NO: 26 , and HVR-L3 containing the amino acid sequence SEQ ID NO: 27. 2. Ahtu-CD137 antibody according to claim 1, which is a human antibody, a humanized antibody or a chimeric antibody, or an antigen-binding fragment thereof. 3. Ahtu-CD137 antibody according to claim 1 or 2, comprising a heavy chain constant region containing any of the amino acid sequences of SEQ ID NO: 64-85. 4. An isolated nucleic acid encoding an anti-CD137 antibody according to any one of claims 1 to 3. 5. A vector into which the nucleic acid according to claim 4 is introduced. 6. A host cell containing the nucleic acid according to claim 4 or the vector according to claim 5. 7. A method for producing an anti-CD137 antibody, which comprises culturing a host cell according to claim 6 such that an anti-CD137 antibody is produced. - 171 046075 8. Immunoconjugate containing aHTu-CD137 agent.