Isolated antigen-binding proteins and uses thereof

Abstract

The present invention relates to an isolated antigen-binding protein comprising a CD3-binding portion, wherein said CD3-binding portion consists of an amino acid sequence having at least 95% identity to heavy chain variable regions HCDR1, HCDR2, and HCDR3, and wherein the amino acid sequence of HCDR1 is as set forth in any one of SEQ ID NOs: 1 and 2, the amino acid sequence of HCDR2 is as set forth in any one of SEQ ID NOs: 3, 4, and 5, and the amino acid sequence of HCDR3 is as set forth in SEQ ID NO: 6; and / or wherein said CD3-binding portion consists of an amino acid sequence having at least 95% identity to light chain variable regions LCDR1, LCDR2, and LCDR3, and wherein the amino acid sequence of LCDR1 is as set forth in any one of SEQ ID NOs: 7, 8, and 9, the amino acid sequence of LCDR2 is as set forth in any one of SEQ ID NOs: 11 and 12, and the amino acid sequence of LCDR3 is as set forth in any one of SEQ ID NOs: NO:13, wherein said CD3 binding moiety is capable of inducing T cell activation.

Description

[Technical Field]

[0001] This invention claims priority from Chinese patent application no. CN202211162830.5 filed on September 23, 2022, and the entire contents of the description, drawings and claims of the priority document are incorporated by reference into the specification of this invention and shall be deemed to be part of the original description of the present invention. The applicant further declares that the applicant has the right to amend the specification and claims of this invention based on the priority document.

[0002] The present application relates to the field of biopharmaceuticals, and in particular to antigen-binding proteins that contain a CD3-binding moiety. [Background technology]

[0003] Currently, bispecific antibodies and antibody fragments are being investigated as a means of recruiting cytolytic T cells to kill tumor cells. However, many clinical applications of bispecific antibodies to recruit T cells are limited by challenges such as excessive cell factor release, potential immunogenicity, and manufacturing issues. Therefore, there is a need for bispecific antibodies to recruit cytolytic T cells to kill tumor cells that exhibit low toxicity.

[0004] The human CD3 T cell antigen receptor protein complex is composed of six distinct chains: one CD3 γ chain (SwissProt P09693), one CD3 δ chain (SwissProt P04234), two CD3 ε chains (SwissProt P07766), and one CD3 ζ chain homodimer (SwissProt P20963) (εγ:ε δ:ζ ζ). This homodimer associates with the T cell receptor α and β chains. This complex plays a key role in coupling antigen recognition to several intracellular signaling pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for the immune response.

[0005] Invention Contents

[0006] The present application provides an isolated antigen-binding protein comprising a CD3 binding domain, wherein the CD3 binding domain comprises an amino acid sequence having at least 95% identity to a heavy chain variable region HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is set forth in any of SEQ ID NOs: 1 and 2, the amino acid sequence of HCDR2 is set forth in any of SEQ ID NOs: 3, 4, and 5, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO: 6, and / or;

[0007] The CD3-binding portion comprises an amino acid sequence having at least 95% identity with light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is set forth in any one of SEQ ID NOs:7, 8, and 9, the amino acid sequence of LCDR2 is set forth in any one of SEQ ID NOs:11 and 12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13, and the CD3-binding portion can induce T cell activation.

[0008] In one embodiment, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is set forth in any of SEQ ID NOs: 1 and 2, the amino acid sequence of HCDR2 is set forth in any of SEQ ID NOs: 3, 4 and 5, and the amino acid sequence of HCDR3 is set forth in any of SEQ ID NOs: 6, and / or;

[0009] The CD3-binding portion comprises light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is set forth in any one of SEQ ID NOs:7, 8, and 9, the amino acid sequence of LCDR2 is set forth in any one of SEQ ID NOs:11 and 12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13, and the CD3-binding portion can induce T cell activation.

[0010] In one embodiment, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6;

[0011] The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6.

[0012] The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6.

[0013] The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6.

[0014] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6; or

[0015] The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:6.

[0016] In one embodiment, the antigen binding protein comprises a CD3 binding portion, the CD3 binding portion comprising light chain variable regions LCDR1, LCDR2, and LCDR3, the amino acid sequence of which is set forth in SEQ ID NO:7, SEQ ID NO:11, and SEQ ID NO:13.

[0017] The amino acid sequence of LCDR1 is shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13;

[0018] The amino acid sequence of LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13;

[0019] The amino acid sequence of LCDR1 is shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13;

[0020] the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; or

[0021] The amino acid sequence of LCDR1 is shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:13.

[0022] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0023] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0024] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0025] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0026] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0027] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0028] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0029] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0030] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0031] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0032] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0033] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0034] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0035] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0036] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0037] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0038] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0039] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0040] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0041] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0042] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0043] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0044] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0045] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0046] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0047] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0048] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0049] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0050] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0051] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0052] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0053] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0054] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0055] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0056] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0057] For example, in one embodiment, the antigen binding protein comprises a CD3 binding portion, which may comprise heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6, and / or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13.

[0058] In some embodiments, the antigen-binding protein comprises a CD3-binding portion, wherein the CD3-binding portion comprises an amino acid sequence that is at least 80% identical to a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID NOs: 14 to 18, and / or the CD3-binding portion comprises an amino acid sequence that is at least 80% identical to a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in any one of SEQ ID NOs: 19 to 23.

[0059] In one embodiment, the antigen-binding protein comprises a CD3-binding portion, wherein the CD3-binding portion comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID NOs: 14 to 18, and / or the CD3-binding portion comprises a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in any one of SEQ ID NOs: 19 to 23.

[0060] In one embodiment, the antigen binding protein comprises a CD3 binding portion, wherein said CD3 binding portion comprises:

[0061] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19;

[0062] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19;

[0063] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19;

[0064] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19;

[0065] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20;

[0066] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20;

[0067] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20;

[0068] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20;

[0069] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20;

[0070] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21;

[0071] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21;

[0072] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21;

[0073] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21;

[0074] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21;

[0075] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;

[0076] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;

[0077] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;

[0078] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;

[0079] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22;

[0080] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23;

[0081] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23;

[0082] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23;

[0083] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23;

[0084] a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23;

[0085] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0086] or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39, or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 20;

[0087] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 21;

[0088] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 22;

[0089] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 23;

[0090] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0091] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39;

[0092] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 20;

[0093] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 21;

[0094] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 22;

[0095] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 23;

[0096] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0097] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39;

[0098] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 20;

[0099] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 21;

[0100] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 22;

[0101] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 23;

[0102] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0103] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39;

[0104] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0105] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39;

[0106] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0107] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39;

[0108] Or, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 40;

[0109] Alternatively, a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:18 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:39.

[0110] In certain embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

[0111] In one embodiment, the antigen binding protein comprises a CD3 binding portion and the antibody is a monoclonal antibody.

[0112] In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0113] In some embodiments, the antigen-binding fragment comprises a Fab, a Fab', an Fv fragment, a F(ab')2, a scFv, a di-scFv, and / or a dAb.

[0114] In certain embodiments, the antigen binding protein comprises a constant region from an IgA, IgG, IgD, IgE, or IgM antibody.

[0115] In one embodiment, the CD3 binding moiety comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG.

[0116] In one embodiment, the CD3 binding moiety comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3, or IgG4.

[0117] In one embodiment, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:24.

[0118] In one embodiment, the CD3 binding portion is an antibody light chain constant region, wherein the antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

[0119] In one embodiment, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:25.

[0120] In one embodiment, the CD3 binding portion comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 26-30, and the light chain comprises the amino acid sequence set forth in any of SEQ ID NOs: 31-35.

[0121] In certain embodiments, the antigen binding protein comprises a multispecific antibody.

[0122] In certain embodiments, the form of the multispecific antigen binding protein is selected from the group consisting of a diabody, a diabody, a diabody scFv, a TandAb, a trivalent binding molecule, or a tetravalent binding molecule.

[0123] In certain embodiments, the multispecific antigen-binding protein comprises binding moieties that bind to at least one tumor-associated antigen (TAA).

[0124] In one embodiment, the TAA is selected from the group consisting of EPCAM, CCR5, CD19, HER2, HER3neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, β-hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-Oacetyl-GD3, GM2, globo H, fucosyl GM1, poly SA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM (CD166), PECAM-1, CD151, MAGE-1, TROP2, IGF 1R, TGFBR2, GHRHR, GHR, IL-6R, gp130, TNFR2, OSMRp, Patched-1, Frizzled, Robol, LTpR, CD26, CD27, CD44, CD80, CD81, CD86, CD100, CXCR4, SAS, BCMA, TWEAKR / Fn14, FGFR4, EGFR1, VEGFR2, SSX1, and SSX2, carboanhydrase IX (MN / CAIX), CD44v6, sonic hedgehog (Shh), Wue-1, plasma cell antigen, (membrane-bound) IgE, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, TNFα precursor, STEAP, mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6; Selected from desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker, Mullerian inhibitory substance (MIS) receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, L6, SAS, CD63, TF antigen, cola antigen, CD7, CD79b, CD22, Igα, Igβ, gp100, MT-MMPs, F19 antigen, CO-29, and EphA2.

[0125] In certain embodiments, the TAA comprises HER2, CD20, CD79b, or CD47.

[0126] In one embodiment, the TAA binding moiety comprises an antibody heavy chain constant region, wherein the antibody heavy chain constant region comprises a constant region derived from human IgG.

[0127] In one embodiment, the TAA binding moiety comprises an antibody heavy chain constant region, wherein the antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3, or IgG4.

[0128] In one embodiment, the TAA binds to a portion of an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

[0129] In another aspect, the present application provides an isolated nucleic acid molecule encoding an antigen binding protein of the present application.

[0130] Nucleic acid molecules according to the present invention may be isolated. For example, they can be produced or synthesized by (i) in vitro amplification, such as polymerase chain reaction (PCR) amplification; (ii) production, such as cloning or recombination; (iii) purification, such as enzymatic cleavage and fractionation, such as gel electrophoresis; or (iv) synthesis, such as chemical synthesis. In certain embodiments, isolated nucleic acids are nucleic acid molecules prepared by recombinant DNA techniques.

[0131] In the present application, nucleic acids encoding antibodies and antigen-binding fragments thereof can be prepared by various methods known in the art, including, but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific procedures, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0132] In another aspect, the present application provides vectors containing the isolated nucleic acid molecules described herein. Each vector may contain one or more nucleic acid molecules. Additionally, vectors may contain other genes, for example, marker genes in the vector, which can be used to select appropriate host cells and conditions. Furthermore, vectors may further contain expression control elements that enable the correct expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence varies depending on the species or cell type, but typically includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, such as a TATA box, capping sequence, and CAAT sequence. For example, the 5' non-transcribed expression control sequence may include a promoter region containing a promoter sequence for transcription and regulation of an operably linked nucleic acid. The expression control sequence may further include an enhancer sequence or an upstream activating sequence. In the present application, suitable promoters include, for example, promoters for SP6, T3, and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV), and portions of the promoter may be fused to portions of the promoters of other cellular proteins (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) genes and may contain additional introns. One or more nucleic acid molecules described in the present invention may be operably linked to the expression control elements. Vectors may include, for example, plasmids, cosmids, viruses, bacteriophages, or other vectors commonly used in genetic engineering. For example, the vector may be an expression vector.

[0133] In another aspect, the present invention provides host cells comprising the vectors described herein. In some embodiments, each or each host cell comprises one or more nucleic acid molecules or vectors described herein. In some embodiments, each or each host cell comprises multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into host cells, such as eukaryotic cells, including cells derived from plants, fungi, and yeast cells. The vectors described herein can be introduced into host cells by methods known in the art, such as electroporation, lipofectin transfection, or lipofectamine transfection.

[0134] In another aspect, the present invention provides a method for producing an antigen binding protein according to the invention, comprising culturing a host cell according to the invention under conditions which allow expression of said antigen binding protein.

[0135] In certain embodiments, the host cell is selected from a bacterial cell, a fungal cell, a plant cell, a mammalian cell, or a virus.

[0136] In a particular embodiment, the bacterial cell is E. coli.

[0137] In certain embodiments, the fungal cell is a yeast cell.

[0138] In certain embodiments, the mammalian cells are selected from CHO, NSO, BHK, or HEK293 cells.

[0139] In certain embodiments, the cells are hybridoma cells.

[0140] In certain embodiments, the hybridoma cells are selected from a mouse, rat, or rabbit.

[0141] Alternatively, related sequences can be synthesized using artificial synthesis methods, especially when the fragment length is short. Typically, multiple small fragments are synthesized and then ligated to obtain very long fragments of the sequence. This nucleic acid molecule can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art.

[0142] The present application relates to vectors comprising the above-described suitable nucleic acid molecules and suitable promoters or control sequences. These vectors can be used to transform suitable host cells so as to express the proteins. The host cells can be prokaryotic cells, such as bacterial cells, lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. For example, animal cells can include CHO-S cells, CHO-K1 cells, and / or HEK-293 cells.

[0143] Transformation of host cells with the recombinant DNA described herein can be carried out using techniques well known in the art. The resulting transformants can be cultured using conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present invention. Depending on the host cells used, they are cultured under appropriate conditions using conventional media. Typically, the transformed host cells are cultured under conditions suitable for expression of the antigen-binding proteins of the present invention. The antigen-binding proteins of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are conventional separation and purification means well known to those skilled in the art.

[0144] The resulting monoclonal antibodies can be identified by conventional means, for example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays such as flow cytometry (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0145] In another aspect, the present application provides a pharmaceutical composition comprising an antigen binding protein described herein, an isolated nucleic acid molecule described herein, a vector described herein, and / or a host cell described herein, and optionally, a pharmaceutically acceptable vector.

[0146] Pharmaceutical compositions of the present invention may contain a safe and effective amount (e.g., 0.001-99 wt%, 0.01-90 wt%, or 0.1-80 wt%) of the antigen-binding protein described in the present invention together with a pharmaceutically acceptable vector. Such vectors may include, but are not limited to, saline, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The formulation of the drug must be tailored to the method of administration. The pharmaceutical compositions described in the present application can be made into injection forms by conventional methods using, for example, physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. The antigen-binding proteins described in the present application can also be used together with other therapeutic agents.

[0147] The antigen-binding proteins or pharmaceutical compositions described herein can be prepared, administered, and used in a manner consistent with good medical practice. Factors to consider include the specific condition being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the etiology of the symptoms, the site of drug delivery, the method of administration, and other factors known to medical scientists. Therapeutic agents need not be formulated with and / or co-administered with one or more drugs currently used to prevent or treat the condition being considered, but this is optional. The effective amount of such other drugs will depend on the amount of therapeutic agent present in the formulation, the type of condition or treatment, and other factors discussed above. These drugs can generally be used at any dose and by any route determined empirically / clinically appropriate. The dose of the antibody administered in combination therapy can be reduced compared to monotherapy. The progress of this therapy can be easily monitored by conventional techniques.

[0148] In another aspect, the present invention provides the use of an antigen binding protein according to the invention, an isolated nucleic acid molecule according to the invention, a vector according to the invention and a host cell according to the invention, and a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment of cancer.

[0149] In certain embodiments, the cancer comprises a solid tumor or a hematological tumor.

[0150] In certain embodiments, the solid tumor is prostate cancer, colorectal cancer, gastric cancer, renal clear cell carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, renal cancer, neovascular disease, renal clear cell carcinoma (CCRCC), pancreatic cancer, renal cancer, urothelial carcinoma, or liver metastatic adenocarcinoma.

[0151] In certain embodiments, the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), or blastic plasmacytoid dendritic cell tumor (BPDCN).

[0152] In another aspect, the present invention provides a method for detecting the presence of CD3 and / or a TAA in a sample using an antigen binding protein according to the invention, comprising contacting said antigen binding protein with a sample and detecting or quantitatively measuring binding of said bispecific antibody to CD3 and / or a TAA.

[0153] In another aspect, the present application provides a method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein described herein for a time sufficient to treat the cancer.

[0154] In certain embodiments, the cancer is a solid tumor or a hematological malignancy.

[0155] In certain embodiments, the solid tumor is prostate cancer, colorectal cancer, gastric cancer, renal clear cell carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, renal cancer, neovascular disease, renal clear cell carcinoma (CCRCC), pancreatic cancer, renal cancer, urothelial carcinoma, or liver metastatic adenocarcinoma.

[0156] In certain embodiments, the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), or blastic plasmacytoid dendritic cell tumor (BPDCN).

[0157] In one embodiment, the antibody is used in combination with a second therapeutic agent.

[0158] Those skilled in the art can readily ascertain other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only embodiments of the present application. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention of the present application. Accordingly, the drawings and description of the present application are illustrative and not restrictive. [Brief explanation of the drawings]

[0159] Particular features of the invention according to the present application are set forth in the appended claims. The features and advantages of the invention according to the present application can be better understood with reference to the following detailed description of the embodiments and the accompanying drawings, which are briefly described as follows:

[0160] [Figure 1] FIG. 1 is an analysis chart of anti-CD3 antibody purification.

[0161] [Figure 2] Figure 2 shows the production amount and concentration of the purified anti-CD3 antibody.

[0162] [Figure 3] Figure 3 shows the CD3E / G antigen information for ELISA.

[0163] [Figure 4] 4A to 4C show the results of CD3 antibody-binding antigen ELISA.

[0164] [Figure 5] FIG. 5 shows EC50 values ​​in CD3 antibody-binding antigen ELISA.

[0165] [Figure 6] FIG. 6 (FIG. 6-1 to FIG. 6-7) schematically shows the results of detecting the binding activity of CD3 antibody to Jurkat cells.

[0166] Specific Embodiments Hereinafter, the present invention will be described with reference to specific examples, and those skilled in the art will be able to easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0167] Definition of Terms

[0168] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, all of which are within the skill of the art.

[0169] In order to make the present invention more readily understandable, certain scientific terms are specifically defined as follows. Unless expressly defined otherwise elsewhere in this specification, scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. For definitions and terminology in the art, experts can specifically refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids. As used in this specification (including the claims), the singular form includes its corresponding plural form unless the context clearly dictates otherwise.

[0170] In this application, the term "about," when used in conjunction with a numerical value, is meant to encompass a range of numerical values ​​up to 5% below and up to 5% above the specified numerical value.

[0171] In this application, the term "and / or" should be understood to mean either one of any one option, or any two, or any two or more options in combination.

[0172] In this application, the term "CD3" generally refers to the three distinct chains, CD3ε, CD3δ, and CD3γ, that comprise the T cell receptor complex. For example, concentrating CD3 on T cells by fixation with anti-CD3 antibodies leads to T cell activation similar to T cell receptor activation but independent of TCR clonal specificity. Most anti-CD3 antibodies recognize the CD3ε chain. Unless otherwise specified, this term refers to any native CD3 from any vertebrate (including mammals such as primates (e.g., humans)) and rodents (e.g., mice and rats). This term encompasses unprocessed "full-length" CD3 and any form of CD3 or any fragment thereof generated by intracellular processing. This term also includes naturally occurring variants of CD3, such as splice variants or allelic variants. In a preferred embodiment, CD3 refers to full-length CD3 from humans and Macaca fascicularis, or a fragment thereof (e.g., a mature fragment lacking the signal peptide). In a preferred embodiment, CD3 refers to full length or a fragment thereof (eg, a mature fragment lacking the signal peptide) derived from mouse / rat.

[0173] As used herein, "percent (%) amino acid sequence identity" or simply "identity" defines the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence after aligning the amino acid sequences (introducing spaces as necessary) to achieve the maximum percent sequence identity, excluding conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods known in the art to determine percent amino acid sequence identity, for example, using commonly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

[0174] In this application, "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and lytic macromolecules (including antibodies, cell factors, and complement) from these cells or the liver to selectively damage, destroy, or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation.

[0175] As used herein, the term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal chain that typically begins with a protein-protein interaction, such as the binding of a growth factor to a receptor, resulting in the transmission of a signal from one part of a cell to another part of the cell. Typically, transmission involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that triggers signal transduction. The penultimate process typically involves a nuclear event that triggers a change in gene expression.

[0176] In this application, the terms "activity" or "biological activity" or "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CD3 activity in vivo or in vitro, IC50, the antibody's in vivo stability, and the antibody's immunogenicity. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics may be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cell factor or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from different sources, including humans, primates, or any other source.

[0177] In the present application, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion and a support or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding protein to an antigen. Antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, so long as they exhibit the desired antigen-binding activity. An "isolated antigen-binding protein" in the present application may comprise an antigen-binding portion and, optionally, a support or framework portion that allows the antigen-binding portion to adopt a conformation that promotes antigen binding.

[0178] In this application, the term "antibody" generally refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single-domain antibodies. The basic antibody structural unit is known to comprise a tetramer. Each tetramer contains two identical polypeptide chain pairs, each with one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion or fragment of each chain may contain a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion or fragment of each chain may define a constant region primarily responsible for effector function. Human light chains are generally classified as k-light chains and k-light chains. Human heavy chains are generally classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The variable and constant regions of the light and heavy chains are connected via a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. See generally, Chapter 7 of Fundamental Immunology (Paul, W., editor, 2nd edition, Raven Press, NY (1989)).

[0179] In this application, the term "isolated antibody" generally refers to a purified state of the binding compound, meaning that the molecule is substantially free of other biological molecules, e.g., nucleic acids, proteins, lipids, sugars, or other substances such as cellular debris and growth medium. The term "isolated" does not imply complete absence of such substances, or absence of water, buffers, or salts, unless such substances are present in amounts that would clearly interfere with the experimental or therapeutic use of the binding compounds described herein.

[0180] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, each antibody in the population is identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific for a single epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of antibodies directed against (or having specificities for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from an essentially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.

[0181] In this application, the term "bispecific antibody" generally refers to an artificially designed antibody that is composed of components of two different antigen-binding sites and can simultaneously bind to two different antigen-binding sites.

[0182] In this application, the term "full-length antibody" refers to an immunoglobulin molecule that typically contains four naturally occurring peptide chains: two heavy (H) chains (approximately 50-70 kDa in total length) and two light (L) chains (approximately 25 kDa in total length) linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and more conserved spacing regions called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0183] In this application, an "antigen-binding fragment" of an antibody ("parent prototypic antibody") includes a fragment or derivative of an antibody, typically comprising at least a fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent prototypic antibody that retains at least a portion of the binding specificity of the parent prototypic antibody. Antibody-binding fragments include, for example, Fab, Fab', F(ab'), and Fv fragments; bibody fragments; linear antibodies; single-chain antibody molecules such as sc-Fv; and nanobodies and multispecific antibodies formed from antibody fragments. When antigen-binding activity is expressed on a molar basis, a binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, a binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent prototypic antibody. Antigen-binding fragments of antibodies are also expected to contain conservative or non-conservative amino acid substitutions that do not appreciably alter their biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody). "Binding compound" refers to both antibodies and binding fragments thereof.

[0184] As used herein, the term "single-chain Fv" or "scFv" antibody generally refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally includes a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0185] In this application, the term "complementarity determining region (CDR)" refers to the region of an antibody that binds to an antigen. CDRs can be defined using various descriptions, such as Kabat (Wu et al., 1970, JExp Med, Vol. 132, pp. 211-250), Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., 1987, JMolBiol, Vol. 196, pp. 901-917), IMGT (Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77), AbM (Martin and Thornton, 1996, JBmol Biol, vol. 263, pp. 800-815. The correspondence between various delineations and variable region numbers has been described (see, e.g., Lefranc et al., 2003, Dev CompImmunol, vol. 27, pp. 55-77; Honegger and Pluckthun, 2001, J Mol Biol, vol. 309, pp. 657-670; International Immunogenetics (IMGT) database, web resource, http: / / www_imgt_org). To delineate CDRs, available programs such as abYsis from UCL Business PLC can be used. Unless otherwise expressly stated herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3," as used herein, include CDRs defined by any of the above methods (Kabat, Chothia, IMGT, or AbM). For example, the CDR regions of the present application can be defined using the Kabat rules.

[0186] In this application, the term "Fc" or "Fc region" or "Fc fragment" generally refers to a polypeptide consisting of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM via the hinge region. Although the resolution of the Fc fragment is variable, the heavy chain Fc fragment of human IgG generally refers to the polypeptide from A231 to its carboxyl terminus.

[0187] In this application, the term "hinge region" refers to a proline-rich, extensible, and flexible polypeptide chain that is typically located between CH1 and CH2 in an antibody. The recognized IgG hinge region is the polypeptide chain consisting of amino acid residues 216 to 230.

[0188] In this application, the term "domain antibody" refers to an immunologically functional immunoglobulin fragment that typically contains only a heavy or light chain variable region. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0189] In this application, the term "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody may also be bispecific.

[0190] In this application, a "double antibody" refers to a small antibody fragment with two antigen-binding sites, usually comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH), which is forced to pair with the complementary domains of another chain, generating two antigen-binding sites, by using a linker that is too short to allow pairing between the two domains in the same chain.

[0191] In this application, a "chimeric antibody" is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, typically from different species. Typically, the variable domains are obtained from a rodent or other antibody (the "parent antibody"), and the constant domain sequences are obtained from a human antibody, making the resulting chimeric antibody less likely to induce a poor immune response in a human subject than the parent rodent antibody.

[0192] In this application, the term "humanized antibody" generally refers to an antibody derived from a non-human (e.g., murine) immunoglobulin and is engineered to contain minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In some cases, Fv framework region (FW) residues of a human immunoglobulin are replaced by corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity.

[0193] In this application, a "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse glycosylation if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, a fully human antibody may contain rat glycosylation if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0194] In this application, an "isotype" antibody generally refers to the antibody class (e.g., IgM, IgE, IgG, such as IgG1, IgG2, or IgG4) provided by the heavy chain constant region genes. Isotype also includes modified forms of any of these classes, where modifications have been made to alter Fc function, for example, to enhance or weaken effector function or binding to Fc receptors.

[0195] In this application, the term "epitope" generally refers to the region of an antigen to which an antibody binds. Epitopes may be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0196] In this application, "affinity" or "binding affinity" generally refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for partner Y can generally be expressed by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kkon, respectively). Affinity can be measured by common methods known in the art. A specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0197] In this application, a protein or cell that "does not bind" generally means that it does not bind to the protein or cell or bind with high affinity, i.e., the KD of the binding protein or cell is 1.0×10-6 M or higher, more preferably 1.0×10-5 M or higher, more preferably 1.0×10-4 M or higher, 1.0×10-3 M or higher, more preferably 1.0×10-2 M or higher.

[0198] In this application, the term "high affinity" for an IgG antibody generally means that the KD for the antigen is 1.0 x 10 M or less, preferably 5.0 x 10 M or less, more preferably 1.0 x 10 M or less, 5.0 x 10 M or less, more preferably 1.0 x 10 M or less. For other antibody subtypes, "high affinity" binding may be different. For example, "high affinity" binding for an IgM subtype means 10 M or less, preferably 10 M or less, more preferably 10 M or less.

[0199] In this application, the term "antibody-dependent cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a cell-mediated immune defense in which immune system effector cells actively degrade cell membrane surface antigens on antibody-bound target cells, e.g., cancer cells.

[0200] In this application, the term "complement-dependent cytotoxicity" or "CDC" generally refers to the effector function of IgG and IgM antibodies that, upon binding to surface antigens, triggers the classic complement pathway, including the formation of a membrane attack complex and lysis of the target cell.

[0201] In this application, the term "nucleic acid" or "polynucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridines are substituted with pseudouridines, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). A particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orectologues, SNPs, and complementary sequences, as well as the explicitly specified sequence, unless otherwise specified. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or a number of selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0202] As used herein, the term "construct" generally refers to any recombinant polynucleotide molecule, such as a plasmid, a virus, an autonomously replicating polynucleotide molecule, a bacteriophage, a linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, and functionally operably linked, i.e., operably linked, to one or more polynucleotide molecules. Recombinant constructs typically comprise a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be induced using both heterologous and non-heterologous (i.e., endogenous) promoters.

[0203] In this application, the term "vector" generally refers to any recombinant polynucleotide construct that can be used for transformation (i.e., the introduction of heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and free mammalian vectors). After introduction into a host cell, other vectors (e.g., non-free mammalian vectors) are integrated into the genome of the host cell and are thus replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0204] In this application, "expression vector" generally refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest upon transformation, transfection, or transfer into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if necessary, to provide amplification within the host.

[0205] In this application, the terms "activation," "stimulation," and "treatment" used in connection with cells or receptors can have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context clearly dictates otherwise. A "ligand" includes natural and synthetic ligands, such as cell factors, cell factor variants, analogs, muteins, and antibody-derived binding compounds. A "ligand" also includes small molecules, such as cell factor peptidomimetics and antibody peptidomimetics. "Activation" can refer to cellular activation regulated by both internal mechanisms and external or environmental factors. A "response / reaction," such as the response of a cell, tissue, organ, or organism, includes changes in biological or physiological behavior (e.g., concentration, density, adhesion, or migration within an organism, gene expression rate, or differentiation state) that are related to activation, stimulation, or treatment, or that are related to internal mechanisms, such as genetic programming.

[0206] In this application, the term "treatment" of any disease or condition, in certain embodiments, refers to ameliorating the disease or condition (i.e., slowing, arresting, or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be discernible by the patient. In another embodiment, "treatment" refers to modulating the disease or condition physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. Unless described herein, methods for assessing treatment and / or prevention of a disease are generally known in the art.

[0207] As used herein, a "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. As used herein, the term "cyno" or "cyno" refers to a cyno monkey.

[0208] In this application, administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0209] In this application, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" generally refer to an amount of an antigen-binding protein of the invention that, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents, is effective to prevent or ameliorate one or more symptoms of a disease or condition or the progression of that disease or condition. A therapeutically effective dose refers to the amount of an antibody or antigen-binding fragment thereof that results in symptomatic improvement, e.g., an amount that treats, cures, prevents, or ameliorates an associated medical condition, or increases the rate of treatment, therapy, prevention, or improvement of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers to that ingredient alone. When administered in combination, the combined amount of active ingredients that produces a therapeutic effect refers to whether they are administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent results in at least a 10% improvement in a diagnostic criterion or parameter, usually by at least 20%; preferably at least about 30%; more preferably 40% or more, and most preferably 50% or more.

[0210] As used herein, "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers, dormant tumors, or micrometastases. Examples of cancer include, but are not limited to, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphocytic NHL, high-grade small anuclear cleaved cell NHL, bulky These include NHL, AIDS-related lymphomas, including mantle cell lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic medulloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with phakomatoses, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0211] The following examples are intended to explain the antigen-binding proteins, production methods, uses, etc. of the present application without being limited by any theory, and are not intended to limit the scope of the invention of the present application.

[0212] Example

[0213] [Example 1] Preparation of anti-CD3 monoclonal antibody

[0214] 1.1. Construction of anti-CD3 monoclonal antibody plasmid

[0215] The anti-CD3 modified sequences were fused to the IgG1 and Kappa constant regions, respectively, and then cloned into the HindIII-NotI expression box in the pTT5 vector to obtain recombinant plasmids. The constructed plasmids were sequenced, and the correct recombinant plasmids were extracted using the Qiagen Midi Plasmid Extraction Kit.

[0216] 1.2. Transient Expression of Anti-CD3 Antibodies

[0217] In this scheme, anti-CD3 antibodies are produced using the ExpiCHO expression system. Frozen ExpiCHO-S™ cells are resuscitated and cultured in ExpiCHO™ Expression Medium. Transfection can be performed after the cells have reached normal growth rate and been passaged at least twice.

[0218] The day before transfection, adjust the cell density to 3 x 106 - 4 x 106 cells / ml. The cell density increases to 7-10 x 106 cells / ml the following day.

[0219] On the day of transfection, dilute the cells to a density of 6 x 106 cells / ml with fresh ExpiCHOTME Expression Medium preheated to 37°C. Dilute the ExpiFectamine™ HO reagent and plasmid DNA (heavy chain:light chain = 1:1) separately in cold OptiPRO™ medium. Add the diluted ExpiFectamine™ HO reagent to the diluted plasmid and mix by inverting to create the ExpiFectamine™ HO / plasmid DNA mixture. After incubating at room temperature for 5 minutes, slowly add the mixture to the ExpiCHO-S cell suspension, mix, and place in a 37°C, 8% CO2 incubator for culture. One day after transfection, add 120 µL of ExpiCHOTME Enhancer and 4.8 mL of ExpiCHOTMEed to the cell suspension and continue transfection at 37°C.

[0220] Seven days after transfection, the supernatant is collected by centrifugation and used for antibody purification.

[0221] 1.3. Purification and analysis of anti-CD3 monoclonal antibodies

[0222] The anti-CD3 antibody cell-expressing supernatant obtained in the previous step is purified using Ni-NTA. After capturing the supernatant, it is washed with 20 mM imidazole in PBS pH 7.4 and eluted with 200 mM imidazole in PBS pH 7.4. After elution, the sample buffer is replaced with PBS, pH 7.4 by ultrafiltration. After obtaining the purified protein, it is analyzed using SDS-PAGE (Figure 1). The final yield passes the OD280 test, as shown in Figure 2.

[0223] The names of the obtained antibody samples are as follows: TIFF2025531360000001.tif220170

[0224] [Example 2] Analysis of binding of CD3 antibodies to CD3E and CD3G heterodimer proteins

[0225] CD3E & CD3G heterodimer protein (Figure 3) was coated onto an ELISA plate at 100 ng / well overnight at 4°C. The next day, the plate was blocked with blocking solution (3% BSA in 1x PBS) for 2 hours. After washing and draining the plate, a gradient-diluted humanized CD3 antibody was added and incubated at 37°C for 1 hour. The reaction mixture was discarded, the plate was washed and drained, and the detection antibody, THETM His Tag Antibody [HRP], diluted to 100 μL / well was added and incubated at 37°C for 1 hour. After discarding the detection antibody, the plate was washed and drained, and 100 μL / well of room-temperature TMB single-component color development solution was added and incubated at 37°C for 10 minutes. After color development, 50 μL / well of stop solution was added to stop the color reaction, and the absorbance at 450 nm was read using a microplate reader. The read data was processed and displayed using GraphPad Prism 8, as shown in Figures 4A-4C. The EC50 values ​​obtained with some of these antibodies are shown in FIG.

[0226] [Example 3] Detection of binding activity of CD3 antibody to Jurkat cells

[0227] Jurkat cells were adjusted to a concentration of 4 x 106 cells / ml with PBS. The cell suspension (50 μl / well) and various concentrations of CD3 antibody (50 μl / well) were added to a U-shaped 96-well plate to achieve final antibody concentrations of 10, 2.5, 1, 0.2, 0.04, 0.008, and 0 μg / ml. The mixture was then incubated in a refrigerator at 4°C for 1.5 hours. After incubation, the supernatant was removed by centrifugation (300 g, 5 minutes) and the wells were washed with PBS (200 μl / well). Finally, FITC-conjugated AffiniPure Rat Anti-MousegG (H+L) staining was added. Flow cytometry analysis and quantification were performed. The results are shown in Figure 6.

Claims

1. an isolated antigen binding protein, comprising a CD3 binding portion, wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity to a heavy chain variable region HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of the HCDR1 is set forth in any of SEQ ID NOs: 1 and 2, the amino acid sequence of the HCDR2 is set forth in any of SEQ ID NOs: 3, 4, and 5, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 6; and / or An isolated antigen-binding protein, wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with light chain variable regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is set forth in any of SEQ ID NOs:7, 8, and 9, the amino acid sequence of LCDR2 is set forth in any of SEQ ID NOs:11 and 12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13, and the CD3 binding portion is capable of inducing T cell activation.

2. 2. The antigen binding protein of claim 1, comprising a CD3 binding portion, wherein the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the amino acid sequence of HCDR1 is set forth in any of SEQ ID NOs: 1 and 2; the amino acid sequence of HCDR2 is set forth in any of SEQ ID NOs: 3, 4, and 5, and the amino acid sequence of HCDR3 is set forth in any of SEQ ID NOs: 6; and / or the CD3 binding portion comprises light chain variable regions LCDR1, LCDR2, and LCDR3, and the amino acid sequence of LCDR1 is set forth in any of SEQ ID NOs: 7, 8, and 9, the amino acid sequence of LCDR2 is set forth in any of SEQ ID NOs: 11 and 12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO: 13, wherein the CD3 binding portion is capable of inducing T cell activation.

3. a CD3 binding portion, said CD3 binding portion comprising heavy chain variable regions HCDR1, HCDR2 and HCDR3; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

6. or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:1, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:3, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:4, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6; or the amino acid sequence of HCDR1 is set forth in SEQ ID NO:2, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:5, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:6; 3. An antigen-binding protein according to any one of claims 1 to 2.

4. the CD3 binding portion comprises light chain variable regions LCDR1, LCDR2 and LCDR3, the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11 and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:11, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:7, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; or the amino acid sequence of LCDR1 is set forth in SEQ ID NO:9, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:12, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:13; 4. An antigen-binding protein according to any one of claims 1 to 3.

5. 5. The antigen-binding protein of claim 1, wherein the CD3 binding portion comprises an amino acid sequence having at least 80% identity to a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is set forth in any one of SEQ ID NOs: 14 to 18, and / or the CD3 binding portion comprises an amino acid sequence having at least 80% identity to a light chain variable region, and the amino acid sequence of the light chain variable region is set forth in any one of SEQ ID NOs: 19 to 23.

6. 6. The antigen-binding protein of any one of claims 1 to 5, wherein the CD3 binding portion comprises a heavy chain variable region, the amino acid sequence of which is set forth in any of SEQ ID NOs: 14 to 18, and / or the CD3 binding portion comprises a light chain variable region, the amino acid sequence of which is set forth in any of SEQ ID NOs: 19 to 23.

7. the CD3 binding moiety is a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 19; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 14 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 39; or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 37 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 20; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 21; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 22; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 36 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 40; or a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 39; 7. An antigen-binding protein according to any one of claims 1 to 6.

8. 8. The antigen-binding protein of any one of claims 1 to 7, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

9. 9. The antigen-binding protein of claim 8, wherein the antibody is a monoclonal antibody.

10. The antigen-binding protein of any one of claims 8 to 9, wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

11. 11. The antigen-binding protein of any of claims 8 to 10, wherein the antigen-binding fragment comprises a Fab, a Fab', an Fv fragment, a F(ab')2, a scfv, a di-scFv and / or a dAb.

12. 12. The antigen-binding protein of any of claims 1 to 11, wherein the antigen-binding protein comprises a constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

13. 13. The antigen-binding protein of claim 1, wherein the CD3-binding portion comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG.

14. 14. The antigen-binding protein of any of claims 1 to 13, wherein said CD3-binding portion comprises an antibody heavy chain constant region, and said antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3 or IgG4.

15. 15. The antigen-binding protein of claim 14, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

24.

16. 16. The antigen-binding protein of any one of claims 1 to 15, wherein said CD3-binding moiety is an antibody light chain constant region, and said antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

17. 17. The antigen binding protein of claim 16, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

25.

18. 18. The antigen-binding protein of any one of claims 1 to 17, wherein the CD3 binding moiety comprises an antibody heavy chain and an antibody light chain, wherein the heavy chain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26-30 and the light chain comprises the amino acid sequence set forth in SEQ ID NOs: 31-35.

19. 19. The antigen-binding protein of any one of claims 1 to 18, wherein the antigen-binding protein comprises a multispecific antibody.

20. 20. The antigen-binding protein of any one of claims 1 to 19, wherein the form of the multispecific antigen-binding protein is selected from a bispecific antibody, a bispecific biantibody, a bispecific scFv, a TandAb, a trivalent binding molecule, or a tetravalent binding molecule.

21. 21. The antigen-binding protein of any of claims 19 to 20, wherein the multispecific antigen-binding protein comprises binding moieties(s) that bind to at least one tumor-associated antigen (TAA).

22. TAA is EPCAM, CCR5, CD19, HER2, HER3neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, β-hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O acetyl-GD3, GM2, Globo H, fucosyl GM1, PolySA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM (CD166), PECAM-1, CD151, MAGE-1, TROP2, IGF1 R, TGFBR2, GHRHR, IL-6R, gp130, TNFR2, OSMRp, Patched-1, Fri zzled, Robol, LTpR, CD26, CD27, CD44, CD80, CD81, CD86, CD100, CXCR4, SAS, BCMA, TWEAKR / Fn14, FGFR4, VEGFR1, VEGFR2, SSX1, and SSX2, carboanhydrase IX (MN / CAIX), CD44v6, SonicHedgehog (Shh), Wue-1, plasma cell antigen, (membrane-bound) IgE, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, TNF-α precursor, STE AP, mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6; 22. The antigen binding protein of claim 21, wherein the antigen binding protein is selected from desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker, Mullerian inhibitory substance (MIS) receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, L6, SAS, CD63, TF antigen, cola antigen, CD7, CD79b, CD22, Igα, Igβ, gp100, MT-MMPs, F19 antigen, CO-29, and EphA2.

23. 23. The antigen binding protein of any one of claims 21 to 22, wherein said TAA comprises HER2, CD20, CD79b or CD47.

24. 24. The antigen-binding protein of any of claims 21 to 23, wherein said TAA-binding portion comprises an antibody heavy chain constant region, and said antibody heavy chain constant region comprises a constant region derived from human IgG.

25. 25. The antigen-binding protein of any one of claims 21 to 24, wherein said TAA-binding moiety comprises an antibody heavy chain constant region, wherein said antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3, or IgG4; and wherein said TAA-binding moiety comprises an antibody light chain constant region, wherein said antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

26. 26. An isolated nucleic acid molecule encoding the antigen-binding protein of any one of claims 1 to 25.

27. 27. A vector comprising the isolated nucleic acid molecule of claim 26.

28. 28. A host cell comprising the vector of claim 27.

29. A method for producing an antigen-binding protein according to any one of claims 1 to 25, comprising culturing a host cell according to claim 28 under conditions allowing expression of said antigen-binding protein.

30. 30. The method of claim 29, wherein the host cell is selected from a bacterial cell, a fungal cell, a plant cell, a mammalian cell, or a virus.

31. 31. The method of claim 30, wherein the bacterial cell is Escherichia coli, the fungal cell is a yeast cell, the mammalian cell is selected from a CHO cell, an NS0 cell, a BHK cell, or an HEK293 cell, and the cell is a hybridoma cell, and the hybridoma cell is selected from a mouse, a rat, or a rabbit.

32. 29. A pharmaceutical composition comprising an antigen binding protein according to any one of claims 1 to 25, an isolated nucleic acid molecule according to claim 26, a vector according to claim 27 and / or a host cell according to claim 28, and optionally a pharmaceutically acceptable vector.

33. 32. Use of an antigen binding protein according to any one of claims 1 to 25, an isolated nucleic acid molecule according to claim 26, a vector according to claim 27, and a host cell according to claim 28, and a pharmaceutical composition according to claim 32, in the manufacture of a medicament for use in the treatment of cancer.

34. 34. The use of claim 33, wherein the cancer comprises a solid tumor or a hematological tumor.

35. 35. The use of claim 34, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, renal clear cell carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, renal cancer, neovascular disease, renal clear cell carcinoma (CCRCC), pancreatic cancer, renal cancer, urothelial carcinoma, or liver metastatic adenocarcinoma, and the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell tumor (BPDCN).

36. 26. A method for detecting the presence of CD3 and / or TAA in a sample using the antigen-binding protein of any one of claims 1-25, the method comprising contacting the antigen-binding protein with a sample and detecting or quantifying binding of the bispecific antibody to CD3 and / or TAA.

37. 26. A method of treating cancer in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the antigen binding protein of any one of claims 1 to 25 for a time sufficient to treat the cancer.

38. 38. The method of claim 37, wherein the cancer is a solid tumor or a hematological malignancy.

39. 38. The method of claim 37, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, renal clear cell carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, renal cancer, neovascular disease, renal clear cell carcinoma (CCRCC), pancreatic cancer, renal cancer, urothelial carcinoma, or liver metastatic adenocarcinoma, and the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), or blastic plasmacytoid dendritic cell tumor (BPDCN).

40. 40. The method of any one of claims 37 to 39, wherein the antigen binding protein is used in combination with a second therapeutic agent.

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