Isolated antigen binding protein and application thereof

Abstract

More particularly, the present invention relates to an isolated antigen binding protein comprising a CD3 binding moiety wherein the CD3 binding moiety comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 2; the amino acid sequence of the HCDR2 is shown as any one of SEQ ID NO: 3, 4 and 5; the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 6, and / or the amino acid sequence of the HCDR3 is shown as SEQ ID NO: 6; the CD3 binding part comprises an amino acid sequence having at least 95% consistency with light chain variable regions LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is as shown in any one of SEQ ID NO: 7, 8 and 9; the amino acid sequence of the LCDR2 is shown as any one of SEQ ID NO: 11 and SEQ ID NO: 12; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 13, and the CD3 binding part can induce T cell activation.

Description

Isolated antigen-binding protein and its application

[0001] This application claims priority from Chinese patent application CN202211162830.5, filed on September 23, 2022. The description, drawings, and claims of this priority document are incorporated herein in their entirety and made a part of the original description of this application. The applicant further declares that the applicant reserves the right to amend the description and claims of this application based on this priority document. Technical Field

[0002] The present application relates to the field of biomedicine, and specifically to an antigen binding protein comprising a CD3 binding portion. Background Art

[0003] Currently, bispecific antibodies and antibody fragments have been explored as a means of recruiting cytolytic T cells to kill tumor cells. However, the clinical use of many bispecific antibodies that recruit T cells is limited by challenges, including excessive cytokine release, potential immunogenicity, and manufacturing issues. Therefore, there is a great need for bispecific antibodies against tumor cells that recruit cytolytic T cells to kill tumor cells, exhibit reduced toxicity, and are more amenable to manufacturing.

[0004] The human CD3 T cell antigen receptor protein complex consists of six distinct chains: a CD3γ chain (SwissProt P09693), a CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and a CD3ζ chain homodimer (SwissProt P20963) (εγ:εδ:ζζ), which associates with the T cell receptor α and β chains. This complex plays an important role in coupling antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for immune responses.

[0005] Summary of the Invention

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

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

[0008] In certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the amino acid sequence of the HCDR1 is as shown in any one of SEQ ID NOs: 1 and 2; the amino acid sequence of the HCDR2 is as shown in any one of SEQ ID NOs: 3, 4, and 5; the amino acid sequence of the HCDR3 is as shown in any one of SEQ ID NO: 6, and / or;

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

[0010] In certain embodiments, 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 as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 6;

[0011] 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: 4, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6;

[0012] 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: 5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6;

[0013] The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 2, 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;

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

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

[0016] In certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion comprises light chain variable regions LCDR1, LCDR2, and LCDR3; wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13;

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

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

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

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

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

[0022] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0023] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0024] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0025] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0026] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0027] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0028] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0029] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0030] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0031] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0032] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0033] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0034] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0035] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0036] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0037] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0038] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0039] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0040] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0041] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0042] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0043] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0044] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0045] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0046] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0047] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0048] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0049] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0050] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0051] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0052] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0053] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0054] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0055] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0056] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0057] For example, in certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion 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 as shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:12, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:13.

[0058] In certain 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 as shown in any one of SEQ ID NOs: 14-18; 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 as shown in any one of SEQ ID NOs: 19-23.

[0059] In certain embodiments, 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 as shown in any one of SEQ ID NOs: 14-18; and / or the CD3 binding portion comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 19-23.

[0060] In certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the CD3 binding portion comprises:

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

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

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

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

[0065] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 20;

[0066] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 20;

[0067] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 20;

[0068] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 20;

[0069] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 20;

[0070] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 21;

[0071] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 21;

[0072] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 21;

[0073] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 21;

[0074] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 21;

[0075] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 22;

[0076] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 22;

[0077] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 22;

[0078] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 22;

[0079] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 22;

[0080] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 23;

[0081] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 23;

[0082] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 23;

[0083] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 23;

[0084] a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 23;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0109] Or, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18, and the 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 certain embodiments, the antigen binding protein comprises a CD3 binding portion, wherein the antibody is a monoclonal antibody.

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

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

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

[0115] In certain embodiments, 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.

[0116] In certain embodiments, 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 IgG1, IgG2, IgG3 or IgG4.

[0117] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:24.

[0118] In certain embodiments, the CD3 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.

[0119] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:25.

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

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

[0122] In certain embodiments, the multispecific antigen-binding protein is in a format selected from a bispecific antibody, a bispecific diabody, a bispecific scFv, a TandAb, a trivalent binding molecule, or a tetravalent binding molecule.

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

[0124] In certain embodiments, 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, βhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-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, IGF1R, TGFB R2, 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, VEGFR1, 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, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6;desmoglein 4, E-cadherin neo-epitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, L6, SAS, CD63, TF-antigen, Cora 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 certain embodiments, the TAA binding portion comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG.

[0127] In certain embodiments, the TAA binding portion 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.

[0128] In certain embodiments, 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 the antigen-binding protein of the present application.

[0130] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0131] In the present application, nucleic acids encoding the antibodies and antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific operations, 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 N.Y., 1993.

[0132] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Furthermore, the vector may also contain expression control elements that allow for proper 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 an adjustable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence functionally linked to a nucleic acid for transcriptional control. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a certain portion of the promoter may be fused to a certain portion of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene promoters, which may or may not include other introns. One or more nucleic acid molecules described herein may be operably connected to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0133] On the other hand, the application provides a host cell comprising the vector described herein. In certain embodiments, each or each host cell comprises one or a nucleic acid molecule or vector described herein. In certain embodiments, each or each host cell comprises multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described herein. For example, the vector described herein can be introduced into the host cell, such as a eukaryotic cell, such as a cell, fungus or yeast cell from a plant. The vector described herein can be introduced into the host cell by methods known in the art, such as electroporation, lipofectin transfection, lipofectamin transfection, etc.

[0134] In another aspect, the present application provides a method for producing the antigen-binding protein described herein, wherein the method comprises culturing the host cell described herein under conditions capable of expressing the antigen-binding protein.

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

[0136] In certain embodiments, the bacterial cell is Escherichia coli.

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

[0138] In certain embodiments, the mammalian cells are selected from CHO, NS0, BHK or HEK293 cells.

[0139] In certain embodiments, the cell is a hybridoma cell.

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

[0141] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. The nucleic acid molecule can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0142] The present application also relates to vectors comprising the above-mentioned appropriate nucleic acid molecules and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The host cells can be prokaryotes, 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, CHO-K1, and / or HEK-293 cells.

[0143] The steps of transforming host cells with recombinant DNA described in this application can be carried out using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of this application. Depending on the host cell used, conventional culture medium is used to culture under appropriate conditions. Generally, the host cells obtained by transformation are cultured under conditions suitable for expression of the antigen-binding proteins of this application. The antigen-binding proteins of this application can then be purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, which are well known to those skilled in the art.

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

[0145] On the other hand, the present application provides a pharmaceutical composition comprising the antigen-binding protein described herein, the isolated nucleic acid molecule described herein, the vector described herein and / or the host cell described herein, and an optional pharmaceutically acceptable carrier.

[0146] The pharmaceutical composition of the present application may contain a safe and effective amount (such as 0.001-99wt%, 0.01-90wt%, or 0.1-80wt%) of the antigen-binding protein described in the present application and a pharmaceutically acceptable carrier. Such carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition described in the present application can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the antigen-binding protein described in the present application can also be used in conjunction with other therapeutic agents.

[0147] Antigen binding proteins or pharmaceutical compositions as described herein can be formulated, administered, and used in a manner consistent with good medical practice. Considerations in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of disease, the agent delivery site, the method of administration, and other factors known to medical practitioners. The therapeutic agent need not be formulated and / or administered simultaneously with one or more agents currently used to prevent or treat the condition under consideration. The effective amount of such other agents depends on the amount of the therapeutic agent present in the preparation, the type of disease or treatment, and other factors discussed above. These agents can generally be used empirically / clinically as any appropriate dosage and by empirically / clinically as any appropriate approach. Compared to single treatment, the dosage of the antibody used in the combination therapy can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.

[0148] On the other hand, the present application provides the antigen-binding protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the host cell described in the present application, and the pharmaceutical composition described in the present application for preparing a drug for treating 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, clear cell renal carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disease, clear cell renal cell carcinoma (CCRCC), pancreatic cancer, kidney cancer, urothelial carcinoma, or metastatic adenocarcinoma of the liver.

[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 myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0152] In another aspect, the present application provides a method for detecting the presence of CD3 and / or TAA in a sample using the antigen binding protein described herein, comprising contacting the antigen binding protein with the sample and detecting or quantifying the binding complex formed between the bispecific antibody and CD3 and / or TAA.

[0153] In another aspect, the present application provides a method for treating cancer in a subject, comprising administering a therapeutically effective amount of the antigen binding protein according to the present application to the subject in need thereof for a period of 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, clear cell renal carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disease, clear cell renal cell carcinoma (CCRCC), pancreatic cancer, kidney cancer, urothelial carcinoma, or metastatic adenocarcinoma of the liver.

[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 myeloid leukemia (CML), or blastic plasmacytoid dendritic cell neoplasm (DPDCN).

[0157] In certain embodiments, the antibody is administered in combination with a second therapeutic agent.

[0158] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0160] Figure 1 shows the purification analysis of Anti-CD3 antibodies.

[0161] Figure 2 shows the yield and concentration of purified Anti-CD3 antibodies.

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

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

[0164] FIG5 shows the EC50 values ​​of CD3 antibody binding to antigen ELISA.

[0165] FIG6 (FIGS. 6-1 to 6-7) shows a schematic diagram of the results of the detection of the binding activity of CD3 antibodies to Jurkat cells. DETAILED DESCRIPTION

[0166] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0167] Definition of terms

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

[0169] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined below. Unless otherwise clearly defined elsewhere in this article, the scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form unless otherwise clearly provided in the text.

[0170] In this application, the term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0171] In the present application, the term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0172] In this application, the term "CD3" generally refers to a part of the T cell receptor complex, which consists of three different chains CD3ε, CD3δ and CD3γ. The concentration of CD3 on T cells, such as by the fixation of anti-CD3 antibodies, leads to the activation of T cells, similar to T cell receptor-mediated activation, but independent of the specificity of the TCR clone. The vast majority of anti-CD3 antibodies recognize the CD3ε chain. The term refers to any natural CD3 from any vertebrate (including mammals such as primates (e.g., humans)) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed CD3 and any form of CD3 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD3, such as splice variants or allelic variants. In a preferred embodiment, CD3 refers to the full length or fragments thereof from humans and cynomolgus monkeys (such as mature fragments thereof lacking a signal peptide). In a preferred embodiment, CD3 refers to the full length or fragments thereof from mice / rat (such as mature fragments thereof lacking a signal peptide).

[0173] In this application, the term "percent (%) amino acid sequence identity" or simply "identity" is defined as 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 (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed to determine percent amino acid sequence identity using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0174] In this application, the term "immune response" generally refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complement) produced by the above cells or the liver, which results in the selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0175] In this application, the term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0176] In this application, the terms "activity" or "biological activity", or the terms "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, in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies 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 can 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, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0177] In the present application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen binding protein to the antigen. Examples of 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, etc., as long as they show the desired antigen-binding activity. The present application's "isolated antigen binding protein" may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the binding of the antigen binding portion to the antigen.

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

[0179] In this application, the term "isolated antibody" generally refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0180] In this application, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous antibody population and is not to 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 bind to two different antigen-binding sites simultaneously.

[0182] In this application, the term "full-length antibody" generally refers to an immunoglobulin molecule that, when naturally present, comprises four peptide chains: two heavy (H) chains (approximately 50-70 kDa when full-length) and two light (L) chains (approximately 25 kDa when full-length), interconnected 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) separated by more conserved framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins 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, the term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the 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 antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0184] As used herein, the term "single-chain Fv" or "scFv" antibody generally refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises 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)" is the region of an antibody that binds to an antigen. CDRs can be defined using various descriptors, such as Kabat (Wu et al., 1970, J Exp Med, Vol. 132, pp. 211-250) (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., 1987, J Mol Biol, Vol. 196, pp. 901-917), IMGT (Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77), and AbM (Martin and Thornton, 1996, J Bmol Biol, Vol. 263, pp. 800-815). The correspondence between various depictions and variable region numbers is described (see, for example, Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77; Honegger and Pluckthun, 2001, J Mol Biol, Vol. 309, pp. 657-670; International Immunogenetics (IMGT) database; Web resources, http: / / www_imgt_org). Available programs (such as abYsis of UCL Business PLC) can be used to depict CDRs. Unless otherwise explicitly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" 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] As used herein, the term "Fc," "Fc region," or "Fc fragment" generally refers to a polypeptide comprised of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM, connected by a hinge region. While the breakdown of the Fc fragment varies, the heavy chain Fc fragment of human IgG generally refers to the polypeptide extending from A231 to its carboxyl terminus.

[0187] In this application, the term "hinge region" generally refers to the proline-rich, easily stretchable and bendable polypeptide chain located between CH1 and CH2 in an antibody. The generally accepted IgG hinge region is the polypeptide chain consisting of amino acid residues 216 to 230.

[0188] In this application, the term "domain antibody" generally refers to an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. 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 can be bispecific.

[0190] In this application, the term "diabody" generally refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0191] In this application, the term "chimeric antibody" generally refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody ("parent antibody") such as a rodent, while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0192] In the present application, the term "humanized antibody" generally refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that is engineered to contain minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin, wherein the residues from the complementary determining region (CDR) are replaced by residues from the CDRs of non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and ability (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, the Fv framework region (FW) residues of a human immunoglobulin are replaced by corresponding residues from antibodies of non-human species with desired specificity, affinity, and ability.

[0193] In this application, the term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. 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 class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modification has been generated to alter Fc function, for example to enhance or diminish 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. An epitope can be formed by 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 a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0197] In this application, the term "not binding" to a protein or cell generally means not binding to the protein or cell, or not binding to the protein or cell 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 IgG antibodies generally refers to a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10-9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0199] As used herein, the terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells, such as cancer cells, that have antibody-bound cell surface antigens.

[0200] In this application, the term "complement-dependent cytotoxicity" or "CDC" generally refers to the effector functions of IgG and IgM antibodies, which, when bound to surface antigens, trigger the classical complement pathway, including formation of the membrane attack complex and target cell lysis.

[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-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids having similar binding properties to reference nucleic acids and analogs containing known natural nucleotides that 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 uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more 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] In this application, "construct" generally refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with the genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner. Recombinant constructs will generally comprise a polynucleotide of the present invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in the host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0203] In this application, "vector" generally refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing 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 connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0204] In this application, the term "expression vector" generally refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected, or transduced 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 desired, to provide for amplification within the host.

[0205] In this application, "activation", "stimulation" and "treatment" as used for cells or receptors may have the same meaning, e.g., a cell or receptor is activated, stimulated or treated with a ligand, unless the context otherwise or clearly dictates. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response / reaction", e.g., the response of a cell, tissue, organ or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state), where the change is related to activation, stimulation or treatment, or to internal mechanisms such as genetic programming.

[0206] In this application, the " treatment " of term any disease or illness refers to improving disease or illness (that is, slowing down or preventing or reducing at least one of the progress of disease or its clinical symptoms) in one embodiment. In another embodiment, " treatment " refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, " treatment " refers to physically (for example, the stabilization of discernible symptoms), physiologically (for example, the stabilization of physical parameters) or regulating disease or illness in these two aspects. Unless clearly described in this article, the method for the treatment and / or prevention of the assessment of disease is generally known in the art.

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

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

[0209] As used herein, "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 alone or in combination with other therapeutic agents to a cell, tissue, or subject, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount to treat, cure, prevent, or ameliorate the relevant medical condition or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40%, and most preferably by at least 50%.

[0210] As used herein, the terms "cancer" and "cancerous" generally refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, 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, as well as B-cell lymphomas (including 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 lymphoblastic NHL, high-grade small non-cleaved cell NHL, storage diseases (bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0211] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0212] Example

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

[0214] 1.1. Anti-CD3 monoclonal antibody plasmid construction

[0215] The modified Anti-CD3 sequence was fused to the IgG1 constant region and the kappa constant region, respectively, and then cloned into the HindIII-NotI expression cassette 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 protocol, anti-CD3 antibodies were produced using the ExpiCHO expression system. TM Cells were revived and cultured (ExpiCHO culture medium TM Expression Medium), and transfection can be performed after the cells have grown at a normal rate and have been passaged for more than 2 times.

[0218] The day before transfection, the cell density was adjusted to 3 × 10 6 -4×10 6 cells / ml, and the cell density grew to 7-10×10 6 cells / ml.

[0219] On the day of transfection, use fresh ExpiCHO preheated at 37°C TM Dilute the cells to a density of 6 × 10 6 cells / ml. Use cold OptiPRO TM Dilute ExpiFectamine TM CHO Reagent and plasmid DNA (heavy chain: light chain = 1:1), dilute ExpiFectamine TM Add CHO Reagent to the diluted plasmid and mix it upside down to make ExpiFectamine TM CHO / plasmid DNA mixture. After incubation at room temperature for 5 minutes, the mixture was slowly added to the ExpiCHO-S cell suspension, mixed well, and placed in a 37°C, 8% CO2 incubator for culture. One day after transfection, 120 μL of ExpiCHO was added to the cell suspension. TM Enhancer, 4.8mL ExpiCHO TM Feed and continue expression at 37°C.

[0220] Seven days after transfection, the supernatant was 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 expression supernatant obtained in the previous step was purified using Ni-NTA. After capture, the supernatant was washed with 20mM imidazole in PBS, pH 7.4, and eluted with 200mM imidazole in PBS, pH 7.4. The eluted protein was then ultrafiltered to replace the sample buffer with PBS, pH 7.4. The purified protein was analyzed using SDS-PAGE (Figure 1). The final yield was determined by OD280, as shown in Figure 2.

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

[0224] Example 2: Binding Analysis of CD3 Antibodies to CD3E & CD3G Heterodimer Protein

[0225] CD3E & CD3G Heterodimer Protein (as shown in Figure 3) was coated onto the ELISA plate at 100 ng / well at 4°C overnight. The next day, the plate was blocked with blocking solution (3% BSA in 1×PBS) for 2 hours, and the plate was washed with washing solution and drained. Then, a gradient diluted humanized CD3 antibody was added and incubated at 37°C for 1 hour. The reaction solution was discarded, the plate was washed and drained, and the diluted detection antibody was added at 100 uL / well. TM Incubate with His Tag Antibody [HRP] at 37°C for 1 hour. Discard the detection antibody, wash the plate, and drain. Add 100 μL / well of TMB single-component colorimetric solution, brought to room temperature, and incubate at 37°C for 10 minutes. After color development, add 50 μL / well of stop solution to terminate the color reaction. Read the absorbance at 450 nm on a microplate reader. Data were processed and displayed using GraphPad Prism 8, as shown in Figures 4A-4C. EC50 values ​​for some of the antibodies are shown in Figure 5.

[0226] Example 3 Detection of CD3 Antibody Binding Activity to Jurkat Cells

[0227] Adjust the concentration of Jurkat cells to 4 × 10 6cells / ml, the cell suspension (50 μl / well) and different concentrations of CD3 antibody (50 μl / well) were added to a U-shaped 96-well plate and mixed to a final concentration of 10, 2.5, 1, 0.2, 0.04, 0.008, and 0 μg / ml, respectively. The plates were incubated at 4°C for 1.5 hours. After incubation, the supernatant was removed by centrifugation (300 g, 5 minutes) and washed with PBS (200 μl / well). Finally, FITC-conjugated AffiniPure Rat Anti-Mouse IgG (H+L) was added for staining and flow cytometry analysis was 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 that is at least 95% identical to a heavy chain variable region HCDR1, HCDR2, and HCDR3, and the amino acid sequence of the HCDR1 is as shown in any one of SEQ ID NOs: 1 and 2; the amino acid sequence of the HCDR2 is as shown in any one of SEQ ID NOs: 3, 4, and 5; the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 6, and / or; The CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the light chain variable regions LCDR1, LCDR2 and LCDR3, and the amino acid sequence of LCDR1 is shown in any one of SEQ ID NOs: 7, 8 and 9; the amino acid sequence of LCDR2 is shown in any one of SEQ ID NOs: 11 and 12; and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 13, wherein the CD3 binding portion can induce T cell activation.

2. The antigen binding protein according to 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 the HCDR1 is as shown in any one of SEQ ID NOs: 1 and 2; the amino acid sequence of the HCDR2 is as shown in any one of SEQ ID NOs: 3, 4 and 5; the amino acid sequence of the HCDR3 is as shown in any one of SEQ ID NO: 6, and / or; The CD3 binding portion comprises light chain variable regions LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is shown in any one of SEQ ID NOs: 7, 8 and 9; the amino acid sequence of the LCDR2 is shown in any one of SEQ ID NOs: 11 and 12; the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 13, wherein the CD3 binding portion can induce T cell activation.

3. The antigen binding protein according to any one of claims 1 to 2, comprising 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 shown in SEQ ID NO: 1, 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; Or, 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: 4, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6; Or, 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: 5, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6; Or, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 2, 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 the HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 6; Alternatively, the amino acid sequence of the HCDR1 is shown as SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown as SEQ ID NO: 5, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO:

6.

4. The antigen binding protein according to any one of claims 1 to 3, wherein the CD3 binding portion comprises light chain variable regions LCDR1, LCDR2, and LCDR3; wherein the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 7, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 11, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 13; Or, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 13; Or, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 13; Or, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 7, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 13; Or, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 13; Alternatively, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 9, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

13.

5. The antigen binding protein according to any one of claims 1 to 4, 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 as shown 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 as shown in any one of SEQ ID NOs: 19 to 23.

6. The antigen binding protein according to any one of claims 1 to 5, wherein the CD3 binding portion comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown 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 shown in any one of SEQ ID NOs: 19 to 23.

7. The antigen binding protein according to any one of claims 1 to 6, wherein the CD3 binding portion comprises: a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 19; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 37, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 40; or, a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 37, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 39; or, a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 37, and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 37, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 37, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 37, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 38, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 40; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 38, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 39; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 38, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 38, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 38, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 38, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:36, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:40; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 36, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 39; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 36, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 20; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 36, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 21; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 36, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 22; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 36, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 23; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 40; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 39; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 40; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 16, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 39; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 40; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 39; or, a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 18, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 40; Or, the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region having the amino acid sequence shown in SEQ ID NO:

39.

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

9. The antigen binding protein according to claim 8, wherein the antibody is a monoclonal antibody.

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

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

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

13. The antigen binding protein of any one of claims 1-12, 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. The antigen binding protein according to any one of claims 1 to 13, 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 IgG1, IgG2, IgG3 or IgG4. The antigen-binding protein according to claim 14 , wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

24. 16 . The antigen binding protein according to claim 1 , wherein the CD3 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.

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

25.

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

19. The antigen binding protein of any one of claims 1-18, comprising a multispecific antibody.

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

21. The antigen binding protein of any one of claims 19-20, wherein the multispecific antigen binding protein comprises a binding moiety(ies) that binds to at least one tumor associated antigen (TAA).

22. The antigen binding protein of claim 21, wherein the TAA is selected from the group consisting of EPCAM, CCR5, CD19, HER2, HER3 neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, βhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-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, IGF1 R, 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, VEGFR1, 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, TNF-alphaprecursor, STEAP, mesothelin, A33 Antigen, Prostate Stem CellAntigen (PSCA), Ly-6;desmoglein 4, E-cadherin neo-epitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, L6, SAS, CD63, TF-antigen, Cora antigen, CD7, CD79b, CD22, Igα, Igβ, gp100, MT-MMPs, F19-antigen, CO-29 and EphA2.; 23. The antigen binding protein of any one of claims 21-22, wherein the TAA comprises HER2, CD20, CD79b, or CD47.

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

25. The antigen-binding protein of any one of claims 21-24, wherein the TAA binding portion 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; wherein the TAA binding portion comprises 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.

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

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

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

29. A method of producing the antigen binding protein of any one of claims 1 to 25, wherein the method comprises culturing the host cell of claim 28 under conditions capable of expressing the antigen binding protein.

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. 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 CHO, NS0, BHK or HEK293 cells; the cell is a hybridoma cell; The hybridoma cells are selected from mouse, rat or rabbit.

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

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

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

35. The method of claim 34, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell renal carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disease, clear cell renal carcinoma (CCRCC), pancreatic cancer, kidney cancer, urothelial carcinoma, or metastatic adenocarcinoma of the liver; wherein 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 neoplasm (DPDCN).

36. 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 to 25, comprising contacting the antigen binding protein with the sample and detecting or quantifying the bound complex formed by the bispecific antibody and CD3 and / or TAA.

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

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

39. The method of claim 37, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell renal carcinoma, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disease, clear cell renal carcinoma (CCRCC), pancreatic cancer, kidney cancer, urothelial carcinoma, or metastatic adenocarcinoma of the liver; wherein 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 neoplasm (DPDCN).

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

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