WO2025077737A1

WO2025077737A1 - An anti-cd3 activable antibody and the use thereof

Info

Publication number: WO2025077737A1
Application number: PCT/CN2024/123711
Authority: WO
Inventors: Tong Zhang; Cheng-Chi Chao; Shuang Zhang; Jinxu LIAO; Hui CHAI; Yingying XU; Sizheng GUO; Wangyue JIA; Wei Jiang; Peng Zhang
Original assignee: Biomap Suzhou Intelligent Technology Ltd
Current assignee: Biomap Suzhou Intelligent Technology Ltd
Priority date: 2023-10-11
Filing date: 2024-10-09
Publication date: 2025-04-17
Anticipated expiration: 2026-04-11

Abstract

Related to anti-CD3 antibodies, activable antibody structure, and multi-specific activable antibody structure comprising the anti-CD3 antibodies. Related to the therapeutic uses of the above, especially in the treatment of tumor.

Description

An Anti-CD3 Activable Antibody And The Use Thereof

Cross-Reference To Related Applications

This application claims the priority to the PCT Application No. PCT/CN2023/124115, filed on October 11, 2023. The content of the prior PCT application is considered as a part of the present disclosure and is incorporated herein in its entirety.

Reference To Sequence Listing

The Sequence Listing titled DCF230804WO-Sequence Listing. xml, which was created on October 9, 2023 and is 57, 369 bytes in size, is hereby incorporated by reference in its entirety.

Technical Field

The present disclosure involves cancer immunotherapy. It directs to anti-CD3 antibodies, activable antibodies comprising such, and therapeutic uses of the above, especially in the treatment, prevention, and diagnosis of conditions ameliorated by the stimulation of immune cells, such as cancer.

Background

The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art.

The non-specificity of potent new biological therapies treating conditions ameliorated by the stimulation of immune cells, such as cancer, has caused an off-target toxicity due to the broad target expression in the patients, which has limited the use of such therapies to some extent. By improving the target specificity, antibody prodrugs/activable antibodies can widen or even create a therapeutic window for the biological therapeutics that are difficult or impossible to use otherwise because of poor patient tolerance. However, with limited experience of antibody prodrugs/activable antibodies the important challenges remain, including designing the molecules to treat the conditions adequately while minimizing the off-target toxicity of the therapies, etc.

CD3 (cluster of differentiation 3) is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+ naive T cells) . It is composed of four distinct chains, which in mammals are one CD3γ chain, one CD3δ chain, and two CD3ε chains. The CD3 complex serves as a signaling module of the T cell receptor (TCR) that associates non-covalently with the antigen-binding a/b chains of the TCR. Because the direct engagement of CD3 results in the T-cell activation, CD3 is a desirable target for a variety of therapeutic and/or diagnostic applications. For instance, anti-CD3 antibodies could stimulate T cells, and has its utilization in medical treatment, such as anticancer therapies. Accordingly, there exists a need for developing antibodies and therapeutics directed to the CD3/TCR pathway with an improved off-target toxicity and/or an enhanced efficacy.

Summary of Invention

For the above-mentioned purpose, in one aspect, the present disclosure provides an antibody or antigen binding fragment thereof that specifically binds to CD3. In some embodiments, the antibody or antigen binding fragment thereof disclosed herein comprises a HCDR1 comprising SYWMH (SEQ ID NO: 9) with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising NIYPX₁SX₂X₃TNYDEX₄FKS (SEQ ID NO: 24) with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising DX₅X₆GNYYFDY (SEQ ID NO: 25) with 0, 1, 2, or 3 conservative substitutions, a LCDR1 comprising KSSQSLLNX₇RTRKX₈YLA (SEQ ID NO: 23) with 0, 1, 2, or 3 conservative substitutions, a LCDR2 comprising WASTRES (SEQ ID NO: 12) with 0, 1, 2, or 3 conservative substitutions, and a LCDR3 comprising TQSX₉X₁₀LRT (SEQ ID NO: 26) with 0, 1, 2, or 3 conservative substitutions, where X_1-10 are independently any amino acid residue. In some embodiments, X₁ is D or G. In some embodiments, X₂ is G or D. In some embodiments, X₃ is S or I. In some embodiments, X₄ is K or R. In some embodiments, X₅ is H, Q or R. In some embodiments, X₆ is Y or S. In some embodiments, X₇ is S or I. In some embodiments, X₈ is S or N. In some embodiments, X₉ is F or Y. In some embodiments, X₁₀ is I or S.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 16 or SEQ ID NO: 20, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 19 and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises HCDRs 1-3 and LCDRs1-3 selected from any one of the following (a) - (d) with 0, 1, 2, or 3 conservative substitutions: (a) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 11 and 13 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 10, 12 and 14 respectively; (b) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 16 and 17 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 15, 12 and 18 respectively; (c) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 11 and 13 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 19, 12 and 14 respectively; and (d) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 20 and 21 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 15, 12 and 22 respectively.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 13 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 10 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14 with 0, 1, 2, or 3 conservative substitutions.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 15 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 18 with 0, 1, 2, or 3 conservative substitutions.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 13 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 19 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14 with 0, 1, 2, or 3 conservative substitutions.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 21 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 15 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3, comprising a HCDR1 comprising SYWMH (SEQ ID NO: 9) with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising NIYPGSGSTNYDEKFKS (SEQ ID NO: 16) with 0, 1, 2, or 3 conservative substitutions or NIYPGSGSTNYAQKFQG (SEQ ID NO: 30) with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising DQYGNYYFDY (SEQ ID NO: 17) with 0, 1, 2, or 3 conservative substitutions, a LCDR1 comprising KSSQSLLNSRTRKNYLA (SEQ ID NO: 15) with 0, 1, 2, or 3 conservative substitutions, a LCDR2 comprising WASTRES (SEQ ID NO: 12) with 0, 1, 2, or 3 conservative substitutions, and a LCDR3 comprising TQSYILRT (SEQ ID NO: 18) with 0, 1, 2, or 3 conservative substitutions. In some embodiments, the antibody or antigen binding fragment thereof disclosed herein is humanized.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3, comprising any one of the following (a) - (f) with 0, 1, 2, or 3 conservative substitutions: (a) three CDRs of a VH as set forth in SEQ ID NO: 1 and three CDRs of a VL as set forth in SEQ ID NO: 2; (b) three CDRs of a VH as set forth in SEQ ID NO: 3 and three CDRs of a VL as set forth in SEQ ID NO: 4; (c) three CDRs of a VH as set forth in SEQ ID NO: 5 and three CDRs of a VL as set forth in SEQ ID NO: 6; (d) three CDRs of a VH as set forth in SEQ ID NO: 7 and three CDRs of a VL as set forth in SEQ ID NO: 8; (e) three CDRs of a VH as set forth in SEQ ID NO: 27 and three CDRs of a VL as set forth in SEQ ID NO: 28; (f) three CDRs of a VH as set forth in SEQ ID NO: 29 and three CDRs of a VL as set forth in SEQ ID NO: 28.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 2.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 3 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 7 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 8.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 27 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 28.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 29 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 28.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 1 and/or the VL comprises SEQ ID NO: 2. In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 3 and/or the VL comprises SEQ ID NO: 4. In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 5 and/or the VL comprises SEQ ID NO: 6. In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 7 and/or the VL comprises SEQ ID NO: 8.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 27 and/or the VL comprises SEQ ID NO: 28. In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 29 and/or the VL comprises SEQ ID NO: 28. In some embodiments, the antibody or antigen binding fragment thereof disclosed herein is humanized.

In one aspect, the present disclosure provides a multi-specific antibody or antigen binding fragment thereof comprising a first antibody or antigen binding fragment thereof (AB1) that specifically binds CD3 and at least a second antibody or antigen binding fragment thereof (AB2) that specifically binds a second target, wherein AB1 comprises any one of the antibody or antigen binding fragment thereof disclosed herein. In some embodiments, the second target is a tumor associated antigen (TAA) . In some embodiments, the second target is GPC, preferably GPC3.

In some embodiments, tumor associated antigens could be selected from the following: GPC, CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, EGFR, Ep-CAM, EphA3, Her2, Her3, ROR2, PSMA, STEAP1, FGFR2, TROP2, B7-H3, B7-H4, B7-H6, FOLR1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, SSX-2, Fibronectin, MART-2, PDL-1, VEGFR, CLAUDIN, and others.

In some embodiments, the AB2 disclosed herein comprises variable regions comprises SEQ ID NO: 55. In some embodiments, the AB2 disclosed herein comprises variable regions comprises SEQ ID NO: 58. In some embodiments, the AB2 disclosed herein comprises variable regions comprises SEQ ID NO: 59. In some embodiments, the AB2 disclosed herein comprises variable regions comprises SEQ ID NO: 58 and 59.

In another aspect, the present disclosure provides an antibody drug conjugate (ADC) comprising the antibody or antigen binding fragment thereof disclosed herein, or the multi-specific antibody or antigen binding fragment thereof disclosed herein.

In one aspect, the present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen binding fragment thereof disclosed herein, or the multi-specific antibody disclosed herein.

In another aspect, the present disclosure provides a vector comprising the isolated nucleic acid disclosed herein.

In yet another aspect, the present disclosure provides a host cell comprising the isolated nucleic acid disclosed herein, or the vector disclosed herein.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen binding fragment thereof disclosed herein, the multi-specific antibody or antigen binding fragment thereof disclosed herein, the ADC disclosed herein, the isolated nucleic acid disclosed herein, the vector disclosed herein, or the host cell disclosed herein, and a pharmaceutically acceptable carrier or excipient.

In one aspect, the present disclosure provides a method of treating or preventing a condition ameliorated by the stimulation of the immune cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof disclosed herein, the multi-specific antibody or antigen binding fragment thereof disclosed herein, the ADC disclosed herein, or the pharmaceutical composition disclosed herein.

In some embodiments of the method disclosed herein, the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, or the pharmaceutical composition disclosed herein is administered in combination with other therapeutics. In some embodiments, the other therapeutics is selected from an antibody, a chemotherapeutic agent, and a small molecular drug. In some embodiments, the other therapeutic agent is selected from, for example, a Bruton’s tyrosine kinase (BTK) inhibitor, a tyrosine kinase inhibitor (TKI) , a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a EGFR inhibitor, a VEGF inhibitor, PARP inhibitor, Her2, LAG-3, TNFR2 inhibitors.

In some embodiments of the method disclosed herein, the condition is selected from tumors, In some embodiments, the condition is selected from solid tumors, such as hepatocellular carcinoma (HCC) , gastric cancer (GC) , cholangiocarcinoma, esophageal squamous cell carcinoma (ESCC) , colorectal cancer (CRC) , thyroid cancer, pancreatic cancer, non-small cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , renal cell carcinoma (RCC) , ovarian cancer, breast cancer, uterine cancer, endometrial cancer, testicular cancer, bladder cancer, skin cancer, neuroblastoma, rhabdomyosarcoma, melanoma, osteosarcoma.

In yet another aspect, the present disclosure provides use of the antibody or antigen binding fragment thereof discloses herein, the multi-specific antibody or antigen binding fragment thereof disclosed herein, the ADC, or the pharmaceutical composition disclosed herein in the preparation of a medicament in the treatment or prevention of a condition ameliorated by the stimulation of the immune cells.

In one aspect, the present disclosure provides a blocking moiety (BM) that inhibits or weakens the binding of an antibody or antigen binding fragment disclosed herein to CD3. In some embodiments, the antibody or antigen binding fragment is the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3. In some embodiments, the BM comprises the sequence of Y₁DGY₂Y₃Y₄Y₅GY₆ITQTPYKVSIS, where Y₁-Y₆ independently are any one of the amino acid residues. In some embodiments, Y1 is Q or E.In some embodiments, Y2 is selected from Y, S or Q. In some embodiments, Y3 is E or D. In some embodiments, Y4 is E or D. In some embodiments, Y5 is M or I. In some embodiments, Y6 is G or S. In some embodiments, the BM disclosed herein comprises any one of the following (a) - (c) : (a) SEQ ID NO: 32 or SEQ ID NO: 32 with 1, 2, or 3 conservative substitutions; (b) SEQ ID NO: 33 or SEQ ID NO: 33 with 1, 2, or 3 conservative substitutions; (c) SEQ ID NO: 34 or SEQ ID NO: 34 with 1, 2, or 3 conservative substitutions. In some embodiments, the BM disclosed herein comprises any one of the SEQ ID NOs: 32-34.

In one aspect, the present disclosure provides a cleavable moiety (CM) subject to cleavage by proteases, comprising any one of the sequences of SEQ ID NOs: 36-44. In one aspect, the present disclosure provides a cleavable moiety (CM) subject to cleavage by proteases, comprising any one or more of the sequences of SEQ ID NOs: 45-53.

In one aspect, the present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the BM disclosed herein, or the CM disclosed herein.

In one aspect, the present disclosure provides an activable antibody structure, comprising an antibody or antigen binding fragment thereof (AB) ; a blocking moiety (BM) to inhibit or weaken the binding of the antibody or antigen binding fragment thereof to a target antigen when the activable antibody is in a non-cleaved state; and a cleavable moiety (CM) subject to cleavage by proteases.

In one aspect, the present disclosure provides an activable antibody structure, comprising an antibody or antigen binding fragment thereof (AB) ; a blocking moiety (BM) to inhibit or weaken the binding of the antibody or antigen binding fragment thereof to a target antigen when the activable antibody is in a non-cleaved state; and a cleavable moiety (CM) subject to cleavage by proteases. In some embodiments, the BM does not interfere or compete with the AB for binding to the target antigen when the activable antibody is in a cleaved state.

In some embodiments, the activable antibody structure disclosed herein in the non-cleaved state has a structural arrangement from N-terminus to C-terminus as follows, BM-CM-AB or AB-CM-BM.

In some embodiments, the activable antibody structure disclosed herein comprises the AB disclosed herein, and the target antigen is CD3.

In some embodiments, the activable antibody structure disclosed herein comprises the BM disclosed herein.

In some embodiments, the activable antibody structure disclosed herein comprises the CM disclosed herein.

In some embodiments, the proteases are selected from any one or more of ADAM8, ADAM9, ADAM10, ADAM I 2, ADAM I 5, ADAM17/TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, Renin, Cathepsin D, Cathepsin E, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Chymase, Cruzipain, DESCI, DPP-4, FAP, Legumain, Otubain-2, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoatase, Hepsin, HtrAl, Human Neutrophil Elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Lactoferrin, Marapsin, Matriptase-2, Meprin, MT-SPl/Matriptase, Neprilysin, NS3/4A, PACE4, Plasmin, PSMA, PSA, BMP-1, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, TMPRSS2, TMPRSS3, TMPRSS4, tPA, Thrombin, Tryptase, and uPA. In some embodiments, the proteases are selected from any one or more of MMP2, MMP14, and uPA.

In some embodiments, the activable antibody structure disclosed herein optionally comprises a first linker (linker 1) between BM and CM, and/or optionally comprises a second linker (linker 2) between CM and AB. In some embodiments, the activable antibody disclosed herein in the non-cleaved state has the structure arrangement from N-terminus to C-terminus as follows, BM-linker 1-CM-linker 2-AB or AB-linker 2-CM-linker 1-BM. In some embodiments, the linker 1 or linker 2 disclosed herein independently comprises an amino acid sequence selected from the group consisting of (GS) n₁, (GGS) n₂, (GGGS) n₃, (GGGGS) n₄, (GGSG) n₅, (GGSGG) n₆, (GSGSG) n₇, (GSGGG) n₈, (GGGSG) n₉, (GSSSG) n₁₀, (GGGGSGGGS) n₁₁, or the combination thereof, wherein n₁-n₁₁ are independently any integer equal to or larger than 0. In some embodiments, the linker 1 disclosed herein comprises (GGGGSGGGS) n₁₁, and the linker 2 disclosed herein comprises (GGGGS) n₄, wherein n₁₁ and n₄ are independently any integer equal to or larger than 0. In some embodiment, the linker 1 disclosed herein comprises GGGGSGGGS, and the linker 2 disclosed herein comprises GGGGS. In some embodiments, the activable antibody structure disclosed herein comprises a CM comprising any one of the sequences of SEQ ID NOs: 45-53.

In some embodiments, the activable antibody structure disclosed herein comprises variable regions comprises SEQ ID NO: 54 and/or 56. In some embodiments, the activable antibody structure disclosed herein comprises variable regions comprises SEQ ID NO: 57 and/or 28. In some embodiments, the activable antibody structure disclosed herein comprises variable regions comprises SEQ ID NO: 60 and/or 28.

In one aspect, the present disclosure provides a multi-specific activable antibody structure that in an activated state specifically binds CD3 and at least a second target. In some embodiments, the multi-specific activable antibody structure disclosed herein comprises at least (a) the activable antibody structure disclosed herein as a first activable antibody that specifically binds CD3 in an activated state, and a second antibody or antigen binding fragment thereof that specifically binds the second target, or (b) the activable antibody structure disclosed herein as a first activable antibody that specifically binds CD3 in an activated state, and the activable antibody structure disclosed herein as a second activable antibody that specifically binds the second target in an activated state. In some embodiments, the second target is TAA. In some embodiments, the second target is GPC, preferably GPC3.

In some embodiments, the second activable antibody disclosed herein comprises the variable regions comprising SEQ ID NO: 55. In some embodiments, the second activable antibody disclosed herein comprises variable regions comprising SEQ ID NO: 58 and/or 59.

In some embodiments, the second antibody or antigen binding fragment disclosed herein comprises the variable regions comprising SEQ ID NO: 55. In some embodiments, the second antibody or antigen binding fragment disclosed herein comprises variable regions comprising SEQ ID NO: 58 and/or 59.

In one aspect, the present disclosure discloses an isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the CM, the BM, the activable antibody structure, or the multi-specific activable antibody structure disclosed herein.

In another aspect, the present disclosure discloses a vector comprising the isolated nucleic acid disclosed herein.

In yet another aspect, the present disclosure discloses a host cell comprising the isolated nucleic acid disclosed herein, or the vector disclosed herein.

In still yet another aspect, the present disclosure discloses a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, the isolated nucleic acid, the vector, or the host cell disclosed herein, and a pharmaceutically acceptable carrier or excipient.

In one aspect, the present disclosure provides a method of treating or preventing a condition ameliorated by the stimulation of the immune cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition disclosed herein. In some embodiments of the method of treating or preventing a condition disclosed herein, the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition disclosed herein is administered in combination with other therapeutics. In some embodiments, the other therapeutics is selected from an antibody, a chemotherapeutic agent, and a small molecular drug. In some embodiments, the other therapeutic agent is selected from, for example, a Bruton’s tyrosine kinase (BTK) inhibitor, a tyrosine kinase inhibitor (TKI) , a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a EGFR inhibitor, a VEGF inhibitor, PARP inhibitor Her2, LAG-3, TNFR2 inhibitors.

In some embodiments of the method of treating or preventing a condition disclosed herein, the condition is selected from tumors. In some embodiments, the condition is selected from solid tumors, such as hepatocellular carcinoma (HCC) , gastric cancer (GC) , cholangiocarcinoma, esophageal squamous cell carcinoma (ESCC) , colorectal cancer (CRC) , thyroid cancer, pancreatic cancer, non-small cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , renal cell carcinoma (RCC) , ovarian cancer, breast cancer, uterine cancer, endometrial cancer, testicular cancer, bladder cancer, skin cancer, neuroblastoma, rhabdomyosarcoma, melanoma, osteosarcoma.

In one aspect, the present disclosure provides a method of diagnosing a condition associated with CD3, comprising contacting a sample of subject with the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the activable antibody structure, or the multi-specific activable antibody structure disclosed herein.

In another aspect, the present disclosure provides use of the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition disclosed herein in the preparation of a medicament in the treatment or prevention of a condition ameliorated by the stimulation of the immune cells.

In one aspect, the present disclosure provides use of antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the activable antibody structure, or the multi-specific activable antibody structure disclosed herein in the preparation of a product, such as a kit, for diagnosing a condition associated with CD3.

In yet another aspect, the present disclosure provides a method of inhibiting or weakening the binding of the antibody or antigen binding fragment thereof disclosed herein to CD3, comprising using a BM to mask the binding site of the antibody or antigen binding fragment thereof. In some embodiments, the method comprises linking the antibody or antigen binding fragment thereof disclosed herein to the BM through a CM that is cleavable by proteases. In some embodiments, the BM is any one disclosed herein. In some embodiments, the BM comprises Y₁DGY₂Y₃Y₄Y₅GY₆ITQTPYKVSIS, wherein Y1-Y6 independently are any one of the amino acid residues. In some embodiments, Y1 is Q or E; Y₂ is selected from Y, S or Q; Y₃ is E or D; Y₄ is E or D; Y₅ is M or I; Y₆ is G or S. In some embodiments, the BM comprises any one of the following (a) - (c) : (a) SEQ ID NO: 32 or SEQ ID NO: 32 with 1, 2, or 3 conservative substitutions; (b) SEQ ID NO: 33 or SEQ ID NO: 33 with 1, 2, or 3 conservative substitutions; (c) SEQ ID NO: 34 or SEQ ID NO: 34 with 1, 2, or 3 conservative substitutions. In some embodiments, the BM comprises any one of SEQ ID NOs: 32-34.

In some embodiments, the method comprises linking the antibody or antigen binding fragment thereof disclosed herein to the BM through a CM that is cleavable by proteases. In some embodiments, the BM can be any one disclosed herein, preferably selected from Table 6, such as BM-2, BM-3, BM-4. In some embodiments, the CM can be any one disclosed herein, preferably the one comprises any one or more of the cleavage site (s) selected from Table 7.

In still another aspect, the present disclosure provides a method of linking the antibody or antigen binding fragment thereof disclosed herein to a CM, comprising linking the antibody or antigen binding fragment thereof disclosed herein to a protease cleavage site optionally through a linker or linkers. In some embodiments, the CM can be any one disclosed herein. In some embodiments, the CM comprises cleavage site (s) and linker (s) , and exemplary protease cleavage sites and linkers could be found in Table 7.

Brief Description of Drawings

The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

Figure 1 shows ELISA binding test of antibodies to human CD3ε.

Figure 2 shows ELISA binding test of antibodies to cynomolgus CD3ε.

Figure 3 shows agonism effect of anti-CD3 antibodies in THP-1/Jurkat-NFAT system.

Figure 4 shows the structures of variable regions of the CD3 mAbs.

Figure 5 shows FACS analysis of humanized antibodies binding to Jurkat cells.

Figure 6 shows ability of CD3 blocking moiety in blocking the binding between anti-CD3 mAb, BPCD3-2, and Jurkat cells.

Figure 7A-C show ability of BM-2, BM-3, BM-4 in blocking the binding between anti-CD3 mAbs and Jurkat cells.

Figure 8 shows ability of BM-3 in blocking the binding between anti-CD3 mAbs and Jurkat cells.

Figure 9 shows schematic structure of an activable antibody structure.

Figure 10 shows the T-cell activation upon co-culture with antibody prodrug PRD-1, and GPC3-positive HepG2 in a reporter assay.

Figure 11A shows the binding ability of PRD antibodies to Jurkat cells through FACS.

Figure 11B shows the binding ability of PRD antibodies to HEPG2 cells through FACS.

Figure 12 shows the T-cell activation upon co-culture with antibody prodrugs PRD-4, PRD-5 , and GPC3-positive HepG2 in a reporter assay.

Figure 13 shows the ability of anti-CD3 antibody prodrugs to induce cytokine production in a PBMC assay.

Figure 14 shows the binding efficiency of Jurkat cells to antibody prodrugs. NC consists of Jurkat cell with fresh human serum and second antibody (PE anti-human IgG Fc, Cat. #: 109-115-098, Jackson ImmunoResearch) without treatment of prodrugs.

Figure 15A-B show the anti-tumor activities of the antibody prodrugs in vivo.

Detailed Description of Invention

The aforementioned features and advantages of the disclosure as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken in conjunction with the drawings. The embodiments described herein with reference to drawings are explanatory, illustrative, and shall not be construed to limit the scope of the present disclosure.

The description is not intended to be a detailed catalog of all the different ways in which the disclosure may be implemented or all the features that may be added to the instant disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant disclosure. Hence, the following description is intended to illustrate some particular embodiments of the disclosure, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

Definitions

Unless clearly indicated otherwise, the term “comprise” , “include” , “contain” , and their variations such as “comprising” , “comprises” as used herein should be understood to imply the inclusion of a stated element or step or a group of elements or steps, but not the exclusion of any other element or step or a group of elements or steps.

Unless clearly contraindicated in the context herein or indicated otherwise, the expression of “A and/or B” includes three situations: (1) A, (2) B, and (3) A and B; the expression of “A, B and/or C” includes seven situations: (1) A, (2) B, (3) C, (4) A and B, (5) A and C, (6) B and C, and (7) A, B and C. The meaning of similar expressions can be inferred in this manner.

As used herein, the term "independently" should be interpreted as that multiple events/elements have no effect on each other. For example, "X and Y are independently any of a, b, c" means that X can be any of a, b, c, and Y can be any of a, b, c, and the choice of X and the choice of Y can be the same or different, without the interference of each other.

Unless indicated otherwise, a singular form of a referent shall include its plural counterparts, and plural terms shall include the singular counterpart. For example, “an antibody or antigen binding fragment thereof” includes a plurality of antibodies or antigen binding fragments thereof.

As used herein, the term “subject” includes animals such as vertebrate, preferably mammals, such as dogs, cats, pigs, sheep, horses, goats, rodents (e.g., mice, rates, guinea pigs) or primates (e.g., gorillas, chimpanzees, monkeys, and humans) .

As used herein, the term “antibody” refers to an immunoglobulin (Ig) molecule that specifically binds an antigen. A basic antibody unit is in a tetramer form consisting of two identical light chains and two identical heavy chains. The N-terminal of each chain contains a variable region with highly diverse amino acid sequence, and the rest of each chain contains the constant region. The heavy chain variable region (VH) and light chain variable region (VL) are responsible for antigen binding, while the constant regions may participate in the binding of an immunoglobulin to host cells or factors, including immune cells and components of the complement system.

The light chain variable region (VL) or heavy chain variable region (VH) from N-terminal to C-terminal is in an arrangement of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR represents framework region, and CDR represents complementarity determining region. CDRs 1, 2, and 3 of VL can also be denoted as LCDR1, LCDR2, and LCDR3 respectively; CDRs 1, 2, and 3 of VH can also be denoted as LCDR1, LCDR2, and LCDR3 respectively. The FRs curl to bring the CDRs close to each other, and the CDRs are in a 3D conformation providing an antigen binding surface complementary to the antigen target.

The assignment of amino acids to each domain is in accordance with any conventional protocols, such as the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) and the Chothia definition (Chothia &Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989) . In some preferred embodiments, the present disclosure follows the Kabat definition.

The terms “complementarity determining region” or “CDR, ” as used herein, refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In some embodiments, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3) .

A number of hypervariable region or CDR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (Kabat CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991) ) . Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901 -917 (1987) ) . The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, (Chothia "CDRs" ) and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information (Lafranc et al., Dev. Comp. Immunol. 27 (1) : 55-77 (2003) ) . IMGT is an integrated information system specializing in immunoglobulins (IG) , T-cell receptors (TCR) , and major histocompatibility complex (MHC) of human and other vertebrates.

Unless otherwise stated, in the present invention, the term "CDRs" or "CDR sequences" encompasses CDR sequences determined in any of the ways described above.

As used herein, the term “antibody” should be understood in its broadest interpretation, including, but not limited to monoclonal antibodies (mAbs) , polyclonal antibodies, fusion antibodies, multi-specific antibodies, diabodies, nanobodies, tri-body, chimeric antibodies, humanized antibodies, fully human antibodies, single chain antibodies, antibody fragments that preserve the antigen binding specificity, such as antigen binding fragment. The antibody may contain additional modifications, such as mutations in non-CDR regions, constant regions, glycosylation sites, post-translational modifications and others under the condition that the resulting antibody has the preserved binding specificity to a target antigen.

An immunoglobulin can be categorized into five classes based on heavy chain constant region, that are IgA, IgD, IgE, IgG, and IgM. Further, an immunoglobulin can be further divided into subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2. Whereas, the antibody light chain may be a lambda (λ) chain or a kappa (κ) chain.

The term “antigen binding fragment” as used herein refers to one or more portions of an antibody that preserve the binding specificity to a target antigen. The antigen binding fragment includes, but not limited to VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) ₂, Fd, Fv, scFab, scFv, (scFv) ₂, taFv, DART, (bispecific T-cell engager) , CDR-containing fragments, and others.

As used herein, the term “specifically bind” or “bind” refers to the non-covalent interactions between an immunoglobulin and an antigen for which the immunoglobulin is specific. The strength or affinity of the interaction can be expressed by the equilibrium dissociation constant (K_D or Kd) for an antigen and the corresponding antibody: the lesser the value of the KD, the stronger the binding strength between an epitope and the antibody. Equilibrium dissociation constant (K_D) is calculated as the ratio of k_off/k_on, where the “on rate constant (K_on) ” and the “off rate constant (K_off) ” can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See, Nature 361: 186-87 (1993) ) .

The term “epitope” refers to a site on an antigen to which an antibody binds, and it is an antigenic determinant that interacts with a specific antigen-binding site called the paratope in the variable region of an antibody molecule. A single antigen may have more than one epitope. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the smallest binding site of an antibody and therefore is the specific target of the antibody or antigen binding fragment thereof.

In some embodiment of the present disclosure, an antibody is said to specifically bind to an antigen when a dissociation constant (KD) is less than or equal to 1X10^-6M, in some embodiments less than or equal to 1x10^-7M, in some embodiments less than or equal to 1x10^-8M, and in some embodiments less than or equal to 1x10^-9M, and in some embodiments is in a range between 1x10^-8 to 1x10^-12M.

As used herein, the term “multi-specific” is to be understood as having more than one target binding sites, such as antigen binding sites. The term “multi-specific antibody” , “multi-specific activable antibody” and the like should be interpreted as an entity, an antibody, or an activable antibody having more than one antigen binding sites directed to different antigens or different epitopes. The term “antibody” used within “multi-specific antibody” , “multi-specific activable antibody” and the like should be interpreted in its broadest interpretation as described above.

The term "monoclonal antibody (mAb) ” as used herein refers to a type of immunoglobulin that has identical CDRs, which specifically binds to an antigen or epitope.

As used herein, the term "humanized antibody" generally refers to an antibody containing a heavy and a light chain variable region sequence from a non-human species (e.g., rabbit, mouse, etc. ) , where at least part of the VH and/or VL sequence has been altered to be more "human-like" , i.e. more similar to a human variable sequence. For a type of humanized antibody, at least one human CDR sequence is introduced into the non-human VH and/or VL sequences to replace the non-human CDR counterpart.

As used herein, the term “activable” within “activable antibody” refers to that the binding activity of an antibody could be switched on and off. The “activable” within “multi-specific activable antibody” refers to that the binding activity of a multi-specific antibody for at least a specific target antigen could be switched on and off. In the “on” situation or the activated/cleaved state (for example, in the present of proteases, such as in the tumor-microenvironment) the antibody or multi-specific antibody specifically binds to the specific target antigen/epitope. In the “off” situation or the inactivated/non-cleaved state (for example, without the presence of proteases) the antibody or multi-specific antibody does not have specific binding activity or have weaker binding activity compared to “on” situation to the specific target antigen/epitope. For example, a bispecific activable antibody could consist of two activable antigen binding sites; or one activable antigen binding site and one conventional antigen binding site. In the context of the present disclosure, “antibody prodrug” refers to an antibody drug that does not function in its initial state and becomes an active drug only after being transformed by chemical reaction or enzymatic action within the organism. In a particular case, it refers to an antibody with blocked antigen binding activity, when under certain conditions, such as in the presence of proteases, the antibody prodrug is activated/cleaved to its pertinent antibody that binds specifically with the target antigen/epitope. As used herein, the term “prodrug” , “antibody prodrug” and “activable antibody” can be used interchangeably.

In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal/N-terminal, and the right-hand end corresponds to the carboxy-terminal/C-terminal, in accordance with convention. Likewise, the left-hand end of single-stranded polynucleotide sequence is the 5’ end, the left-hand direction of double-stranded polynucleotide sequences is the 5’ direction, the right-hand end of single-stranded polynucleotide sequence is the 3’ end, and the right-hand direction of double-stranded polynucleotide sequences is the 3’ direction.

As used herein, the term “sequence identity” is usually determined using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit that can be used to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or wild-type proteins and their mutant protein. See, eg, GCG version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, and programs in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provide alignments and percent sequence identity (Pearson) of the region of optimal overlap between the query and search sequences. (2000) , op. cit. ) . Another preferred algorithm is the computer program BLAST, especially BLASTP, BLASTN, using default parameters when comparing the sequences of the present disclosure with databases containing a large number of sequences from different organisms. See, eg, Altschul et al. (1990) J. Mol. Biol. 215: 403410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389402.

In some embodiments, the "sequence identity" is determined by comparing two optimally aligned sequences, wherein the portion of the polynucleotide or polypeptide sequence may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence and multiplying the results by 100 to yield the percentage of sequence identity.

As used herein, the term "conservative substitution" means the substitution of an amino acid that is not essential for the activity of the peptide, or the substitution of an amino acid with another amino acid that has similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc. ) , such that even the substitution of an essential amino acid does not reduce the activity of the peptide. Conservative substitution that provides functionally similar amino acids is well known in the field.

In some embodiments, the conservative substitution could be the one occurs within the same group listed below, (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp. For example, Asp is substituted with Glu, Ala is substituted with Gly or Ser.

Antibody or antigen binding fragment

The present disclosure provides an antibody or antigen binding fragment thereof that specifically binds to CD3. In some embodiments, the antibody or antigen binding fragment thereof disclosed herein comprises a HCDR1 comprising SYWMH (SEQ ID NO: 9) with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising NIYPX₁SX₂X₃TNYDEX₄FKS (SEQ ID NO: 24) with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising DX₅X₆GNYYFDY (SEQ ID NO: 25) with 0, 1, 2, or 3 conservative substitutions, a LCDR1 comprising KSSQSLLNX₇RTRKX₈YLA (SEQ ID NO: 23) with 0, 1, 2, or 3 conservative substitutions, a LCDR2 comprising WASTRES (SEQ ID NO: 12) with 0, 1, 2, or 3 conservative substitutions, and a LCDR3 comprising TQSX₉ X₁₀LRT (SEQ ID NO: 26) with 0, 1, 2, or 3 conservative substitutions, where X_1-10 are independently any amino acid residue. In some embodiments, X₁ is D or G. In some embodiments, X₂ is G or D. In some embodiments, X₃ is S or I. In some embodiments, X₄ is K or R. In some embodiments, X₅ is H, Q or R. In some embodiments, X₆ is Y or S. In some embodiments, X₇ is S or I. In some embodiments, X₈ is S or N. In some embodiments, X₉ is F or Y. In some embodiments, X₁₀ is I or S.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR1 comprising SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 16 or SEQ ID NO: 20 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21 with 0, 1, 2, or 3 conservative substitutions, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 19 with 0, 1, 2, or 3 conservative substitutions, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22 with 0, 1, 2, or 3 conservative substitutions.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comp comprising the amino acid sequence of SEQ ID NO: 13 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 10 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14 with 0, 1, 2, or 3 conservative substitutions.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 21 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 15 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 22 with 0, 1, 2, or 3 conservative substitutions. In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprises the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprises the amino acid sequence of SEQ ID NO: 30 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprises the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprises the amino acid sequence of SEQ ID NO: 15 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprises the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18 with 0, 1, 2, or 3 conservative substitutions. In some embodiments, the above antibody or antigen binding fragment thereof is humanized.

In some embodiment, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a HCDR 1 comprises the amino acid sequence of SEQ ID NO: 9 with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprises the amino acid sequence of SEQ ID NO: 16 with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprises the amino acid sequence of SEQ ID NO: 17 with 0, 1, 2, or 3 conservative substitutions, a LCDR 1 comprises the amino acid sequence of SEQ ID NO: 15 with 0, 1, 2, or 3 conservative substitutions, a LCDR 2 comprises the amino acid sequence of SEQ ID NO: 12 with 0, 1, 2, or 3 conservative substitutions, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18 with 0, 1, 2, or 3 conservative substitutions. In some embodiments, the above antibody or antigen binding fragment thereof is humanized.

In some embodiments, CDR sequences are defined according to Kabat system.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3, comprising any one of the following (a) - (f) with 0, 1, 2, or 3 conservative substitutions: (a) three CDRs of a VH as set forth in SEQ ID NO: 1 and three CDRs of a VL as set forth in SEQ ID NO: 2; (b) three CDRs of a VH as set forth in SEQ ID NO: 3 and three CDRs of a VL as set forth in SEQ ID NO: 4; (c) three CDRs of a VH as set forth in SEQ ID NO: 5 and three CDRs of a VL as set forth in SEQ ID NO: 6; (d) three CDRs of a VH as set forth in SEQ ID NO: 7 and three CDRs of a VL as set forth in SEQ ID NO: 8; (e) three CDRs of a VH as set forth in SEQ ID NO: 27 and three CDRs of a VL as set forth in SEQ ID NO: 28; (f) three CDRs of a VH as set forth in SEQ ID NO: 29 and three CDRs of a VL as set forth in SEQ ID NO: 28. The skilled artisan in the field is able to readily identify CDRs based on the VH and VL provided.

In some embodiments, the VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 1 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, and/or the VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 2 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variants retain the ability of binding to CD3.

In some embodiments, the VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 3 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, and/or the VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 4 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variants retain the ability of binding to CD3..

In some embodiments, the VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 5 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, and/or the VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 6 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variants retain the ability of binding to CD3.

In some embodiments, the VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 7 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, and/or the VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 8 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variants retain the ability of binding to CD3.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 27 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 28. The antibody or antigen binding fragment thereof disclosed herein could be humanized antibody or antigen binding fragment thereof.

In some embodiments, the VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 27 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, and/or the VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 28 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variants retain the ability of binding to CD3.

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 28. The antibody or antigen binding fragment thereof disclosed herein could be humanized antibody or antigen binding fragment thereof.

In some embodiments, the VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 29 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, and/or the VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 28 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variants retain the ability of binding to CD3.

The functional variant comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9%sequence identity to the amino acid sequence of the parent polypeptide. In some preferred embodiments, the insertion, deletion and/or substitution occurs at the FR regions. In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) .

In some embodiments, the antibody or antigen binding fragment thereof disclosed herein that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 27 and/or the VL comprises SEQ ID NO: 28. The antibody or antigen binding fragment thereof disclosed herein could be humanized antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to CD3 comprises a VH and/or a VL, wherein the VH comprises SEQ ID NO: 29 and/or the VL comprises SEQ ID NO: 28. The antibody or antigen binding fragment thereof disclosed herein could be humanized antibody or antigen binding fragment thereof.

The antibody disclosed herein may be any isotype or subtype. In some embodiments, the antibody is IgG isotype. In some embodiments, the antibody is IgG1 subtype, preferably, human IgG1 (hIgG1) subtype.

In some embodiments, the antibody disclosed herein specifically binds to CD3, in particular to CD3 epsilon (CD3ε or CD3e) . In some embodiments, the antibody disclosed herein specifically binds to mammalian CD3ε, such as human CD3ε and cynomolgus CD3ε.

Multi-specific antibody

The present disclosure provides a multi-specific antibody or antigen binding fragment thereof that specifically binds to antigen CD3 (for example CD3ε, such as human CD3ε and cynomolgus CD3ε) and at least another antigen. The multi-specific antibody or antigen binding fragment thereof disclosed herein comprises a first antibody or antigen binding fragment thereof (AB1) that specifically binds to CD3 and at least a second antibody or antigen binding fragment thereof (AB2) that specifically binds a second target, wherein AB1 comprises any one of the antibody or antigen binding fragment thereof disclosed herein. In some embodiments, the second target is a tumor associated antigen (TAA) .

Exemplary tumor associated antigens could be selected from the following: GPC, CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, EGFR, Ep-CAM, EphA3, Her2, Her3, ROR2, PSMA, STEAP1, FGFR2, TROP2, B7-H3, B7-H4, B7-H6, FOLR1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, SSX-2, Fibronectin, MART-2, PDL-1, VEGFR, CLAUDIN, and others.

In some embodiments, the second target is GPC, preferably GPC3.

Fc Variants and modifications

In some embodiments, the antibodies comprise a Fc domain of an isotype selected from the group consisting of IgA, IgD, IgE or IgM Fc domain. In some embodiments, the Fc domain is IgG Fc domain. In some embodiments, the Fc domain is IgG1, IgG2, IgG3 or IgG4 Fc domain. In some embodiments, the Fc domain is IgG1 Fc domain. In some embodiments, the sequence of IgG1 Fc domain is EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62) .

In some embodiments, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. In some embodiments, the modification is “KIH” modification (WO1996027011A1, Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol. 1997 Jul 4; 270 (1) : 26-35. doi: 10.1006/jmbi. 1997.1116. PMID: 9231898. ) .

In some embodiments, the Fc domain comprises one or more Fc modifications increasing stability, increasing half-life, and/or altering effector functions.

In some embodiments, the substitution at a position selected from the group of S239D and I332E ( “DE” ) ; S239D, I332E, and A330L ( “DLE” ) ; S239D; I332E; S298A, E333A, and K334A; G236A, A330L, and I332E ( “GAALIE” ) ; G236A, S239D, A330L, and I332E ( “GASDALIE” ) ; F243L, R292P, Y300L, V305I, and P396L ( “LPLIL” ) ; M252Y, S254T, and T256E ( “YTE” ) ; and T307A, E380A, and N434A ( “AAA” ) (U.S. Pat. No. 10,184,000; Liu et al. Antibodies (Basel) . 2020.9 (4) : 64; Lazar et al. PNAS. 2006.103 (11) : 4005-4010; each of which is herein incorporated by reference in its entirety) .

In some embodiments, the Fc domain can prolong half-life in circulation through its interaction with FcRn. In some embodiments, the substitution at a position selected from the group of M428L and N434S ( “LS” ) ; M252Y and T256D ( “YD” ) ; T256D and T307Q ( “DQ” ) ; and T256D and T307W ( “DW” ) .

Antibody drug conjugate

The present disclosure provides an antibody drug conjugate (ADC) comprising the antibody or antigen binding fragment thereof disclosed herein, or the multi-specific antibody or antigen binding fragment thereof disclosed herein.

The present disclosure provides an ADC comprising the antibody or antigen binding fragment thereof disclosed herein, or the multi-specific antibody or antigen binding fragment thereof disclosed herein, and cytotoxic agent. In some embodiments, the antibody or antigen binding fragment thereof is covalently attached, directly or via a cleavable or non-cleavable linker to the cytotoxic agent.

In some embodiments, the cytotoxic agent may be any compound that results in the death of a cell, or induces cell death, or in some manner decreases cell viability. Preferred cytotoxic agents include, for example, maytansinoids and maytansinoid analogs, taxoids, taxanes such as paclitaxel and docetaxel, CC-1065 and CC-1065 analogs, dolastatin and dolastatin analogs, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs, taxanes such as paclitaxel and docetaxel.

Blocking moiety, cleavable moiety

The present disclosure provides a blocking moiety (BM) that inhibits or weakens the binding of an antibody or antigen binding fragment disclosed herein to CD3. In some embodiments, the BM comprises the sequence of Y₁DGY₂Y₃Y₄Y₅GY₆ITQTPYKVSIS, where Y₁-Y₆ independently are any one of the amino acid residues. In some embodiments, Y1 is Q or E. In some embodiments, Y2 is selected from N, Y, S or Q. In some embodiments, Y3 is E or D. In some embodiments, Y4 is E or D. In some embodiments, Y5 is M or I. In some embodiments, Y6 is G or S. In some embodiments, the BM disclosed herein comprises any one of the SEQ ID NOs: 32-34.

The present disclosure provides a cleavable moiety (CM) subject to cleavage by proteases, comprising any one of the sequences of SEQ ID NOs: 36-44. In one aspect, the present disclosure provides a cleavable moiety (CM) subject to cleavage by proteases, comprising any one or more of the sequences of SEQ ID NOs: 45-53.

The CM may comprise cleavage site (s) , or comprise cleavage site (s) with linker (s) .

Exemplary proteases could be selected from the following: ADAMS, ADAMTS, cysteine proteinase, aspartate proteases, KLKs, serine proteases, elastase, granzyme B, lactoferrin, aspartic cathepsins, MMPs, marapsin, caspase, cysteine cathepsins, plasmin, PSA, tPA, thrombin, tryptase, uPA, and others. Exemplary proteases could also be selected from the following: ADAM8, ADAM9, ADAM10, ADAM I 2, ADAM I 5, ADAM17/TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, Renin, Cathepsin D, Cathepsin E, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Chymase, Cruzipain, DESCI, DPP-4, FAP, Legumain, Otubain-2, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoatase, Hepsin, HtrAl, Human Neutrophil Elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Lactoferrin, Marapsin, Matriptase-2, Meprin, MT-SPl/Matriptase, Neprilysin, NS3/4A, PACE4, Plasmin, PSMA, PSA, BMP-1, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, TMPRSS2, TMPRSS3, TMPRSS4, tPA, Thrombin, Tryptase, and uPA.

Activable antibody structure, multi-specific antibody structure

The present disclosure provides an activable antibody structure, comprising an antibody or antigen binding fragment thereof (AB) ; a blocking moiety (BM) to inhibit or weaken the binding of the antibody or antigen binding fragment thereof to a target antigen when the activable antibody is in a non-cleaved state; and a cleavable moiety (CM) subject to cleavage by proteases. In some embodiments, the BM does not interfere or compete with the AB for binding to the target antigen when the activable antibody is in a cleaved state.

In some embodiments, the activable antibody structure disclosed herein comprises the AB disclosed herein, and the target antigen is CD3. In some embodiments, the target antigen is CD3ε.

In some embodiments, the proteases are selected from any one or more of ADAM8, ADAM9, ADAM10, ADAM I 2, ADAM I 5, ADAM17/TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, Renin, Cathepsin D, Cathepsin E, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V/L2, Cathepsin X/Z/P, Chymase, Cruzipain, DESCI, DPP-4, FAP, Legumain, Otubain-2, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoatase, Hepsin, HtrAl, Human Neutrophil Elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Lactoferrin, Marapsin, Matriptase-2, Meprin, MT-SPl/Matriptase, Neprilysin, NS3/4A, PACE4, Plasmin, PSMA, PSA, BMP-1, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, TMPRSS2, TMPRSS3, TMPRSS4, tPA, Thrombin, Tryptase, and uPA, preferably MMP2, MMP14, and uPA. In some embodiments, the proteases disclosed herein are selected from any one or more of Matrix Metalloproteinases (MMPs) and Urokinase-type plasminogen activator (uPA) .

In some embodiments, the activable antibody structure disclosed herein optionally comprises a first linker (linker 1) between BM and CM, and/or optionally comprises a second linker (linker 2) between CM and AB. In some embodiments, the activable antibody structure disclosed herein in the non-cleaved state has the structure arrangement from N-terminus to C- terminus as follows, BM-linker 1-CM-linker 2-AB or AB-linker 2-CM-linker 1-BM. In some embodiments, the linker 1 or linker 2 disclosed herein independently comprises an amino acid sequence selected from the group consisting of (GS) n₁, (GGS) n₂, (GGGS) n₃, (GGGGS) n₄, (GGSG) n₅, (GGSGG) n₆, (GSGSG) n₇, (GSGGG) n₈, (GGGSG) n₉, (GSSSG) n₁₀, (GGGGSGGGS) n₁₁, or the combination thereof, wherein n₁-n₁₁ are independently any integer equal to or larger than 0. In some embodiments, the linker 1 disclosed herein comprises (GGGGSGGGS) n₁₁, and the linker 2 disclosed herein comprises (GGGGS) n₄, wherein n₁₁ and n₄ are independently any integer equal to or larger than 0. In some embodiment, the linker 1 disclosed herein comprises GGGGSGGGS, and the linker 2 disclosed herein comprises GGGGS. In some embodiments, the activable antibody structure disclosed herein comprises a CM comprising any one of the sequences of SEQ ID NOs: 57-65.

The present disclosure provides a multi-specific activable antibody structure that in an activated state specifically binds CD3 and at least a second target. In some embodiments, the multi-specific activable antibody structure disclosed herein comprises at least the following (a) or (b) , (a) the activable antibody structure disclosed herein as a first activable antibody that specifically binds CD3 in an activated state, and a second antibody or antigen binding fragment thereof that specifically binds the second target; (b) the activable antibody structure of disclosed herein as a first activable antibody that specifically binds CD3 in an activated state, and the activable antibody structure disclosed herein as a second the activable antibody that specifically binds the second target in an activated state. In some embodiments, the second target is a tumor associated antigen (TAA) . In some embodiments, the second target is GPC, preferably GPC3.

In some embodiments, the second activable antibody disclosed herein comprises variable regions comprises SEQ ID NO: 55. In some embodiments, the second activable antibody disclosed herein comprises variable regions comprises SEQ ID NO: 58 and/or 59.

In some embodiments, the second antibody or antigen binding fragment thereof disclosed herein comprises the variable regions comprises SEQ ID NO: 55. In some embodiments, the second antibody or antigen binding fragment thereof disclosed herein comprises variable regions comprises SEQ ID NO: 58 and/or 59.

In some embodiments, the activable antibody structure or multi-specific activable antibody structure disclosed herein will be cleaved/activated at tumor microenvironment, and therefore provides a reduced off-target toxicity that could result from the binding of the antibody to a random location.

Isolated nucleic acid, vector, host cell

The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen binding fragment thereof disclosed herein, or the multi-specific antibody or antigen binding fragment thereof disclosed herein.

The present disclosure provides an isolated nucleic acid comprising a polynucleotide sequence encoding the BM disclosed herein, or the CM disclosed herein.

The present disclosure discloses an isolated nucleic acid comprising a polynucleotide sequence encoding the activable antibody structure disclosed herein, or the multi-specific activable antibody structure disclosed herein.

As used herein, the term "nucleic acid" includes both single-stranded and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

An “isolated” nucleic acid comprising a nucleotide sequence encoding a protein, or portion or fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid encoding the protein, or portion or fragment thereof described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid, therefore, are distinguished from nucleic acid encoding the protein, or portion or fragment thereof described herein existing naturally in cells.

As used herein, the term "polynucleotide" refers to a polymer of nucleotides (nucleotides or deoxynucleotides) of any length. The term refers to the primary structure of a molecule. It is directed to both double-stranded and single-stranded RNA, as well as double-stranded and single-stranded DNA. It also includes polynucleotides modified by methylation and/or terminal modification, as well as unmodified forms. The polynucleotides described herein are not necessarily obtained by physical means, but can be produced by any methods, including, for example, chemical synthesis or DNA replication, reverse transcription or transcription.

The present disclosure provides a vector comprising the isolated nucleic acid disclosed herein.

As used herein, the term “vector” refers to a nucleic acid molecule used as a vehicle to carry genetic material into another cell, where it can be replicated and/or expressed. In some embodiments, the vector is expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV) , a lentiviral vector, or any combination thereof.

The present disclosure provides a host cell comprising the isolated nucleic acid disclosed herein, or the vector disclosed herein.

In some embodiments, the cell can be a prokaryotic cell, fungal cell, yeast cell, or eukaryotic cells such as a mammalian cell. Suitable prokaryotic cells include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonas such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.

Pharmaceutical composition

The present disclosure provides a pharmaceutical composition comprising the antibody or antigen binding fragment thereof disclosed herein, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, the isolated nucleic acid, the vector, or the host cell disclosed herein, and a pharmaceutically acceptable carrier or excipient.

As used herein, the term “pharmaceutical composition” means a mixture of the elements disclosed herein with other chemical components (such as carriers or excipients) that are compatible with pharmaceutical administration. The pharmaceutical composition facilitates the administration of the disclosed elements (such as the antibody, activable antibody disclosed herein) to the subject in need. Various administration methods are known in the art, including but not limited to subcutaneous, intramuscular, oral, transdermal, parenteral, intravenous, intraperitoneal, intrathecal, transpulmonary, transnasal, ocular, systemic, and topical administration.

The pharmaceutical composition disclosed herein may be configured in a dosage form suitable for administration to a subject by the desired route of administration. Said dosage forms include, but are not limited to, tablets, capsules, caplet, pills, soft gel, troche, powders, syrups, elixir, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalations, creams, pastes, lotions, ointment, sprays, lyophilized preparation, injectables, and gels.

As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to the carrier or excipient compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof and/or that such carrier or excipient is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to subject. The term includes all pharmaceutically acceptable materials, solvents, carriers, excipients, stabilizers, diluents, dispersants, suspending agents, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, thickeners and/or excipients. Examples of such carriers or excipients include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5%human serum albumin, liposomes and non-aqueous vehicles such as fixed oils.

Method of treatment, pharmaceutical use, and others

The present disclosure provides a method of treating or preventing a condition ameliorated by the stimulation of the immune cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition disclosed herein.

In some embodiments of the method of treating or preventing a condition disclosed herein, the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition disclosed herein is administered in combination with other therapeutics. In some embodiments, the other therapeutics is selected from an antibody, a chemotherapeutic agent, and a small molecular drug. In some embodiments, the other therapeutic agent is selected from, for example, a Bruton’s tyrosine kinase (BTK) inhibitor, a tyrosine kinase inhibitor (TKI) , a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a EGFR inhibitor, a VEGF inhibitor, PARP inhibitor Her2, LAG-3, TNFR2 inhibitors.

The present disclosure provides a method of diagnosing a condition associated with CD3, comprising contacting a sample of subject with the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the activable antibody structure, or the multi-specific activable antibody structure disclosed herein.

The present disclosure provides use of the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition disclosed herein in the preparation of a medicament in the treatment or prevention of a condition ameliorated by the stimulation of the immune cells.

The present disclosure provides use of the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the activable antibody structure, or the multi-specific activable antibody structure disclosed herein in the preparation of a product, such as a kit, for diagnosing a condition associated with CD3.

As used herein, the term “treat” , “treatment” , or “treating” refers to the alleviation or amelioration of a disease or disorder (i.e., slowing or stopping the progression of a disease or at least one clinical symptom) ; or the alleviation or amelioration of at least one physical parameter or biomarker associated with the disease or disorder.

As used herein, the term “prevent” or “preventing” includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.

As used herein, the term “condition” refers to any alteration in the state of the body or some organs that interrupts or interferes with the performance of a function and/or causes symptoms (e.g., discomfort, dysfunction, distress, or even death) in the subject who is ill or in contact with the patient.

As used herein, the term “therapeutically effective amount” indicates that when comparing to a subject without receiving any treatment, the subject administered with such amount of the therapeutics shows a beneficial effect or therapeutic effect towards a disease, but the amount is sufficiently low in the range of sound medical judgment to avoid serious side effects. The therapeutically effective amount of the antibody, activable antibody and others disclosed herein will vary at least based on the following factors, the route of administration; the severity of the disease being treated; the age, height, weight and physical condition of the patient; the medical history of the patient; the duration of the treatment; the desired therapeutic effect. However, the therapeutically effective amount can still be determined in a conventional manner by those skilled in the art.

The present disclosure provides a method of inhibiting or weakening the binding of the antibody or antigen binding fragment thereof disclosed herein to CD3, comprising using a BM to mask the binding site of the antibody or antigen binding fragment thereof. In some embodiments, the method comprises linking the antibody or antigen binding fragment thereof disclosed herein to the BM through a CM that is cleavable by proteases. In some embodiments, the BM is any one disclosed herein. In some embodiments, the BM comprises Y₁DGY₂Y₃Y₄Y₅GY₆ITQTPYKVSIS, wherein Y₁-Y₆ independently are any one of the amino acid residues. In some embodiments, Y₁ is Q or E; Y₂ is selected from Y, S or Q; Y₃ is E or D; Y₄ is E or D; Y₅ is M or I; Y₆ is G or S. In some embodiments, the BM comprises any one of the following (a) - (c) : (a) SEQ ID NO: 32 or SEQ ID NO: 32 with 1, 2, or 3 conservative substitutions; (b) SEQ ID NO: 33 or SEQ ID NO: 33 with 1, 2, or 3 conservative substitutions; (c) SEQ ID NO: 34 or SEQ ID NO: 34 with 1, 2, or 3 conservative substitutions. In some embodiments, the BM comprises any one of SEQ ID NOs: 32-34.

In some embodiments, the CM can be any one disclosed herein, preferably the one comprises any one or more of the cleavage site (s) selected from Table 7. In particular, the CM could comprise any one set forth in SEQ ID NOs: 45-53.

The present disclosure provides a method of linking the antibody or antigen binding fragment thereof disclosed herein to a CM, comprising linking the antibody or antigen binding fragment thereof disclosed herein to a protease cleavage site optionally through a linker or linkers. In some embodiments, the CM can be any one disclosed herein. In some embodiments, the CM comprises cleavage site (s) and linker (s) . In some embodiments, the cleavage site could be any one or more disclosed herein. In some embodiments, the linkers could be any one disclosed herein. Exemplary cleavage sites and linkers could be found in Table 7.

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the present application described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein.

Examples

The following examples, both actual and prophetic, are provided in conjunction with the accompanying drawings for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope.

The skilled in the art should recognize that various alterations and modifications can be made to the embodiments described herein without departing from the spirit and scope of the present disclosure. Description of well-known functions, structures and the like in the art are omitted from the following description for conciseness.

The experimental methods in the following embodiments, if not otherwise specified, are conventional methods, performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the product protocols. The materials, reagents, and the like used in the following embodiments are commercially available, if not otherwise specified.

Example 1. Generation of anti-CD3 antibodies

Anti-CD3 antibodies were generated by immunizing Balb/c mice (8-10 weeks) with human CD3ε/δ-Fc (Acrobiosystems, Cat No: CDD-H5255) . The titers of anti-CD3 antibody were monitored weekly starting from 3 weeks after the 1^st immunization by ELISA and flow cytometry. After a total of 4 immunizations, mice with high titers were sacrificed. bone marrows were pooled and CD138⁺ plasma cells were enriched using a kit (Miltenyi) . Single cell suspension was loaded on Beacon 14K chips (Berkeley lights) , and was subjected to single B cell screening using CD3ε-coated beads (Acrobiosystems, Cat No: CDE-H5223) . Positive single cells were exported into 96 well plates with lysis buffer, and plates were frozen at -80℃ prior to single cell sequencing. DNA sequences encoding antibodies were determined by Sanger’s sequencing (Sanger, 1977) . The amino acid sequences of representative top clones BPCD3-1, BPCD3-2, BPCD3-14, and BPCD3-17 (VH and VL) are listed in Table 1. The CDR sequences of them are listed in Table 2.

Table 1 Amino acid sequences of representative clones

Table 2 The CDR sequences of representative clones

Example 2. ELISA binding assay

CD3 antigens were immobilized on the high binding 96-well ELISA plate by incubating the wells with 100μl of 1mg/ml human CD3ε (Acro Biosystems, Cat No: CDE-H5223) or cynomolgus CD3ε (Acro Biosystems, Cat No: CDE-C5226) in PBS (pH 7.4) overnight at 4 ℃. The wells were then blocked with blocking buffer (0.05%+ 2%BSA in PBS) for 1h at room temperature. After washing with 300μl of washing buffer (0.05%in PBS) three times, the antibodies (BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17) and the positive control antibody SP34 (Pessano, S., Oettgen, H., Blan, A. K. &Terhorst, C. (1985) EMBO J. 4, 337-344; US2015/0166661A) were diluted with a series of concentrations in dilution buffer (0.05%+ 2%BSA in PBS) and incubated the wells for 1h at room temperature. The wells were then incubated with 100μl of HRP-conjugated anti-human antibody (Jackson ImmunoResearch) in blocking buffer for 1h at room temperature. The wells were then washed three times with washing buffer and treated with 100μl of TMB substrate (catalog TMB-S-001, Innoreagents) for 10 min. The reaction was then stopped by adding 50μl of ELISA stop solution (catalog C1058, Solarbio) , and the absorbance of each well at 450 nm was read immediately using a multimode plate reader (ENVISION, PerkinElmer) .

As shown in Figure 1, antibodies BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17 bind to human CD3ε with a property similar to that of SP34.

As shown in Figure 2, antibodies BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17 bind to cynomolgus CD3ε with a property similar to that of SP34.

Example 3. Activation of T cell induced by CD3 mAb

To verify the agonism effect of monoclonal antibodies (mAbs) against CD3, THP-1/Jurkat-NFAT reporter system (purchased from Vazyme, DD1302-1) was used to determine the antibody function. When the Jurkat-NFAT cells are co-cultured with THP-1 cells and CD3 mAbs, TCR signaling is activated, leading to NFAT transcription and induced luciferase expression.

In brief, THP-1 cells (2×10⁴/well) were co-cultured with Jurkat-NFAT cells (4×10⁴/well) in 96-well flat bottom plates containing serial diluted mAbs of BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17 (atotal volume of 150 μl/well) of at 37℃. After 4 hours incubation, bright-glo (Beyotime, Cat No: RG051M) were added into the corresponding wells and luminescence was read by Envision (PerkinElmer) . SP34 was used as a positive control, and human IgG1 (hIgG1) (Beyotime, Cat No: B117901) was used as a negative control. The luminescence readings are expressed as RLU (relative light unit) and EC50 was calculated (shown in Figure 3) . As shown in Figure 3, Jurkat-NFAT cells are activated by CD3 mAbs.

Example 4. The structure of the CD3 mAbs

As the CD3 mAbs share similar binding affinities, the structures of their CDRs were analyzed. The structures of variable regions (Fv) of the four CD3 mAbs (BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17) were simulated using MOE software (Molecular Operating Environment, version 2022, Chemical Computing Group) . As shown in Figure. 4, all these four mAbs shared a highly similar structure.

Example 5. Humanization of the murine anti-human CD3 mAb

For humanization of the BPCD3-2, human germline IgG genes were searched for sequences that share high degrees of homology to the amino acid sequences of BPCD3-2 variable regions by blasting the human immunoglobulin gene database in NCBI (http: //www. ncbi. nlm. nih. gov/igblast/) website. The human IGVH and IGVL genes that are present in human antibody repertoires with high frequencies (Glanville 2009 PNAS 106: 20216-20221) and are highly homologous to BPCD3-2 were selected as the templates for humanization.

Humanization was carried out by CDR-grafting (Methods in Molecular Biology, Vol 248: Antibody Engineering, Methods and Protocols, Humana Press) and the humanized antibodies (huBPCD3-1, huBPCD3-2) were engineered in human IgG1 (hIgG1) format by pCDNA3.4 vector (Thermo fisher, Cat number: A14697) containing a cDNA encoding a human kappa constant light chain domain or human IgG1 heavy chain (GeneWiz) .

The amino acid sequences and the CDR sequences of humanized clones are listed in Table 3 and 4.

Table 3 Amino acid sequences of humanized clones VH and VL regions

Table 4 Amino acid sequences of humanized clones CDR regions

Example 6. CD3 binding of CD3 mAbs by flow cytometry

The pre-amplified CD3-positive Jurkat cells were adjusted to a density of 2x10⁶/ml, and 100ul/well of cells were taken into 96-well plates. After 300 x g centrifugation for 5 min, the growth medium was discarded. Anti-CD3 antibodies were 5-fold serially diluted from 100 nM with FACS buffer (DPBS+2%FBS) , with different concentration points. 100ul of prepared dilution of BPCD3-2, huBPCD3-1, huBPCD3-2 and SP34 were added to each well. After incubation for 1 hour at 4℃, the cells were washed. 100ul of anti-human IgG Fc secondary antibody AF647 (1: 1000, Jackson, 109-605-098) was added, with 30min incubation. Each cell pellet was resuspended with 200uL of FACS buffer. Cell suspension was analyzed by Flow cytometry (Thermo, Attune Nxt) . The EC50 values were calculated from MFI (mean fluorescence intensity) .

As shown in Figure 5, the BPCD3-2, huBPCD3-1, huBPCD3-2 and SP34 antibodies show binding activity with Jurkat cells. It is shown that the humanized huBPCD3-2 antibody (EC50=0.027) and huBPCD3-1 antibody (EC50=0.032) are superior to SP34 (EC50=0.05) .

Example 7. Affinity determination by BLI

Bio-layer interferometry (BLI) analysis was performed at room temperature using an Octet RH96 Device available from Sartorius. For all measurements, the PBST buffer consisting of 10 mM PBS, PH7.4, 0.05%and 0.1%BSA was used as running buffer. The BPCD3-2 and huBPCD3-2 were directly captured on the AHC (Anti-Human IgG Fc Capture) sensor at a concentration of 10 ug/mL. Gradient concentrations of His-tagged human CD3ε (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25nM indicated as different colors of association/dissociation curves) were then associated with the Fc-tagged antibodies and dissociated with the running buffer subsequently. After each cycle, the sensor surface was regenerated with 0.1 M glycine, pH 1.5. The binding kinetics were all analyzed with the software of Octet Analysis Studio version 12.2.2.26 using a 1: 1 steady-state affinity model. Response was measured as a nm shift in the interference pattern and was recorded and displayed on a sensogram in real time. Equilibrium dissociation constant (K_D) was calculated as the ratio of k_off/k_on, and the results are shown in Table 5.

Table 5. Comparison of antibody binding affinities by BLI

Example 8. Blocking assay of anti-CD3 antibodies with blocking moieties.

Amino acid (AA) residues 1-13 of the blocking moiety of the present disclosure are based on human CD3ε sequence (Table 6) . The blocking moiety functions as a “shield” , blocking the binding of a CD3 mAb to CD3ε. The blocking activity of the CD3 blocking moiety was determined by the binding of the anti-CD3 mAb with the blocking moiety under the incubation with CD3-positive Jurkat cells (ATCC) . Briefly, serially diluted peptides, BM-1, BM-2, BM-3, BM-4, BM-5 (synthesized by Chinese Peptide Company) , were mixed with 30 ng of anti-CD3 mAbs (BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17, or SP34) at 4℃ for 30 minutes, followed by the incubation with CD3-positive Jurkat cells, which were stained with a secondary antibody, AlexaFluor 647-conjugated goat anti-human IgG (Jackson Immunoresearch, 109-605-098) . Immunofluorescence was detected by a flow cytometer (Attune Nxt, Thermo) . The x-axis represents the concentration of peptides, and the y-axis represents MFI (mean fluorescence intensity) . The IC50 values were calculated from MFI.

Figure 6 depicts the result of the blocking assay of anti-CD3 antibody BPCD3-2 with blocking moieties. As shown in Figure 6, all selected blocking moieties block the binding of the BPCD3-2 to Jurkat cells. BM-3 shows the most potent blocking activity (IC50 is 0.105 μM) , which is 2.75-fold more potent than that of the WT peptide (BM-1) .

Figure 7 (A-C) depicts the blocking assay of different anti-CD3 antibodies with blocking moieties (BM-2, BM-3, and BM-4) . As shown in Fig. 7 A-C, BM-2, BM-3 and BM-4 peptides block the binding of different anti-CD3 mAb to CD3+ Jurkat cells.

Table 6. anti-CD3 blocking moieties (BM)

Example 9. Blocking assay of anti-CD3 mAb huBPCD3-2with blocking moieties.

In this assay, serially diluted blocking moiety BM-3 (synthesized by Chinese Peptide Co. ) , starting from 50 μM, were mixed with anti-CD3 mAb huBPCD3-2 (final concentration 0.2 μg/mL) . The antibody-peptide mixtures were then added to Jurkat cells (10⁵/well) in u-bottom 96-well plates on ice for 30 mins, followed by repeated washes for a total of 3 times. Bound primary antibody was captured by anti-human IgG Fc secondary antibody AF647 (1: 1000) for 30 mins, followed by 3 washes. The cell samples were then subjected to a flow cytometry analysis. The x-axis represents the concentration of peptides and the y-axis represents MFI (mean fluorescence intensity) . The IC50 values were calculated from MFI.

As shown in Figure 8, BM-3 blocks the binding of huBPCD3-2 to Jurkat cells.

Example 10. SCREENING OF PROTEASE SENSTIVE CLEAVAGE SITE

Antibody prodrug/activable antibody is a construct consisting of an antibody, a blocking moiety, and a cleavable moiety, for example, as shown in Fig. 9. In protease-rich sites (such as tumor-microenvironment) , the antibody prodrugs/activable antibodies become susceptible to protease cleavage and then turn into active antibodies or cleaved antibodies. The cleavable moiety comprises protease sensitive cleavage sites shown below in Table 7.

Table 7 Cleavable moiety (CM) with protease sensitive cleavage site

The binding ability of prodrugs after MMP digestion was tested. BM-1, SP34 antibody and different cleavage moieties from Table 7 were used to construct prodrugs. Non-cleavable linker (NCL) is a mimic containing only BM-1 and SP34 without a cleavage moiety. Prodrugs were expressed in host cell CHO. Prodrugs (100nM) were digested with 0.25ug/ml MMPs in digestion buffer 37℃ overnight, 20h. 1 x 10⁵/well Jurkat cells in PBS plus 2%FBS. Jurkat cells were incubated with prodrugs (100nM) with or without digestion. 1: 1000 dilution was carried out to anti-human IgG Fc, AF647 (jackson) . Immunofluorescence was detected by a flow cytometer (Attune Nxt, Thermo) . The binding ability was calculated from MFI: MFI of samples /MFI of SP34.

Table 8 shows that during the expression process, the binding ability of prodrug with CM-8 is at an extremely low level, indicating that CM-8 has better stability and is not digested by protease from the host cell. After treating with MMP14, the binding ability of prodrug with CM-8 increased obviously. It is suggested that prodrug with CM-8 could be cleaved by MMP14 and exhibit binding activity.

Table 8. Binding ability of the prodrugs.

Example 11. Construct of prodrugs.

In the present disclosure, Prodrugs (PRD) is bispecific antibody containing Fab 1 and Fab 2. Prodrugs (PRD) are bispecific antibodies comprising a first antigen-binding arm that specifically binds CD3 (Genentech or huBPCD3-2) and a second antigen-binding arm that specifically binds GPC3. The prodrugs harbor a cleavage moiety (CM-8 or CM-12) and a CD3 blocking moiety (BM-1 or BM-3) , which maintain drugs largely non-active without protease cleavage. In protease-rich sites (such as tumor-microenvironment) , prodrugs are susceptible to protease cleavage and turn into active drugs. The components of the Prodrugs are listed in table 9. The sequence of variable regions (Fv) of Prodrugs (CD3 × GPC3) are shown in table 10.

Table 9. Prodrug components

Table 10. Amino acid sequences of antibody prodrugs

Example 12. Prodrug shows great “responsive window” with or without MMP treatment in a reporter assay

A Jurkat-based reporter cell line, Jurkat-NFAT-luciferase (purchased from Vazyme, DD1302-1) , harboring NFAT-driven luciferase expression, was used to determine T-cell activation upon co-culture with prodrugs (pre-treated with proteases versus without protease) and GPC3-positive HepG2 cells (Meisen Cell) . In the protease pre-treatment, prodrugs were resuspended in reaction buffers (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 6.6) , and 5 units of MMP14 (Abcam, Cat#: 8009) at 37℃ overnight for 20 hours. Prior to co-culture of prodrugs with the reporter cell line, HepG2 cells (5x10⁴) were plated onto 96-well plates and cultured overnight. In the co-culture assay, Jurkat-NFAT-luciferase cells (2.5x10⁵) were added to pre-seeded HepG2 culture in the presence of serially diluted prodrugs (protease treatment versus without treatment) and co-cultured at 37℃ for 6 hours. Luciferase activity (luminescence) was determined by adding substrate Bio-glo (Beyotime, RG051M) and measurement with a plate reader (EnVision 2105, Perkin Elmer) .

As shown in Figure 10, PRD-1 prodrug shows great “responsive window” with or without MMP treatment (indicated as the double-arrowed line in Figure. 10) in a reporter assay. cleavage moiety CM-8 works on the prodrug.

Example 13. Binding ability of Prodrugs to Jurkat cells

In the protease treatment, prodrugs were resuspended in reaction buffers (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 6.6 ) , and 5 units of MMP2 (R&D Systems, Cat#: 902-MP-010) plus MMP14 (Abcam, Cat#: 8009) or MMP2 plus MMP14 plus uPA (Acrobiosystem, Cat#: PLU-H5228) at 37℃ for 20 hours. Protease-treated prodrugs were then diluted in FACS buffer (DPBS+2%FBS) and were used to stain CD3⁺ Jurkat cells (2x10⁵) in a similar procedure as described in Example 12. As shown in Figure 11A, prodrugs (PRD-4 and PRD-5) show significant binding to Jurkat cells after protease treatment. In contrast, without protease treatment, PRD-4 and PRD-5 show minimal background staining.

Example 14. Antibody prodrugs bind to HEPG2 cells

To verify whether the binding ability of another Fab will be affected or not, the binding to HEPG2 cells was tested by flow cytometry. The pre-amplified HEPG2 cells were adjusted to a density of 2×10⁴/ml. and 100μl/well cells was taken into 96-well plates. Anti-CD3 antibodies were 5-fold serially diluted from 100 nM with FACS buffer (DPBS+2%FBS) . After incubation for 1 hour at 4℃, the cells were washed at least twice with 200 μL cold (4℃) FACS buffer and the supernatant was discarded. After 1 hour, 100ul anti-human IgG Fc secondary antibody AF647 (1: 1000, Jackson, 109-605-098) was added, with 30min incubation. Afterwards, the cell pellets were resuspended via gently agitation. After repeating 3 times, each cell pellet was resuspended with 200uL of FACS buffer. Flow cytometry (Thermo, Attune Nxt) was used to evaluate the cell suspension. The x-axis represents the concentration of antibodies and the y-axis represents MFI (mean fluorescence intensity) . The EC50 values were calculated from MFI. Both PRD-4 and PRD-5 show comparable binding to HepG2 cells (GPC3-positive) (Fig. 11B) .

Example 15. CD3 Prodrug activity in a reporter assay

A Jurkat-based reporter cell line, Jurkat-NFAT-luciferase (purchased from Vazyme, DD1302-1) , harboring NFAT-driven luciferase expression, was used to determine T-cell activation upon co-culture with prodrugs PRD-4 or PRD-5 (pre-treated with proteases versus without protease treatment) and GPC3-positive HepG2 cells (Meisen Cell) . In the protease pre- treatment, prodrugs were resuspended in reaction buffers (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 6.6 ) , and 5 units of MMP2 (R&D Systems, Cat#: 902-MP-010) plus MMP14 (Abcam, Cat#: 8009) or MMP2plus MMP14 plus uPA (Acrobiosystem, Cat#: PLU-H5228) at 37℃ for 20 hours. Prior to the co-culture of prodrugs with the reporter cell line, HepG2 cells (5x10⁴) were plated onto 96-well plates and cultured overnight. In the co-culture assay, Jurkat-NFAT-luciferase cells (2.5x10⁵) were added to pre-seeded HepG2 culture in the presence of serially diluted prodrugs PRD-4 or PRD-5 (protease treatment vs without treatment) and co-cultured at 37℃ for 6 hours. Luciferase activity (luminescence) was determined by adding substrate Bio-glo (Beyotime, RG051M) , and measurement was taken with a plate reader (EnVision 2105, Perkin Elmer) .

As shown in the reporter assay (Fig. 12) , upon the pre-treatment with proteases, both PRD-4 and PRD-5 show potent T-cell engager activity in the co-culture of HepG2 with the reporter cells, with EC₅₀ values of 0.43 nM and 0.46 nM, respectively.

Example 16. CD3 Prodrug activity in a PBMC assay

To further determine the efficacy of CD3 prodrug using human peripheral blood mononuclear cells (PMBCs) , cryo-preserved PMBCs (10⁵/well, from healthy donors) were co-cultured with HepG2 (10⁴) in the presence of the serially diluted un-treated or protease-treated (as described in Example 6) prodrugs for 24 hours. Cytokines (IFN-γ, IL-2) in the culture supernatants were assayed by ELISA (Thermo, Cat. 88-7025-88; Thermo, Cat. 88-7316-86) . As shown in Figure 13, non-treated prodrugs induce lower background cytokine production, especially IFN-γ. In contrast, protease-treated prodrugs exhibit strong cytokine production with high efficiency (EC₅₀ in IL-2 production: ～0.02 nM; EC₅₀ in IFN-γ production: ～0.25 nM) . The windows are more than 100-fold when comparing non-treated prodrugs with protease-treated prodrugs. Secretion of cytokines indicates non-treated prodrugs were blocked and PBMC were activated by protease-treated prodrugs.

Example 17. Serum stability test of prodrugs.

The environment in human blood is complex and there are many types of enzymes. To ensure that prodrugs can play a better role in the tumor microenvironment, their stability in the serum should be considered.

Fresh human serum was isolated from a healthy donor. Serum was then diluted with DPBS to different concentrations (5%, 10%) . Serum without dilution was set as 100%and DPBS alone was used as a blank control (0%) . Prodrugs PRD-4 and PRD-5 (1000nM) were then mixed with different concentrations of serum as shown in Figure 14, at 37℃ for 20 hours overnight. After incubation, the mixtures were then diluted 10 times with FACS buffer (DPBS+2%FBS) to the working concentration of 100nM. Jurkat cells (10⁵ cells/well) were stained with serum-treated prodrugs, as mentioned above, in 96-well V-bottom plates at 4℃ for 1 hour, followed by 3x repeated washes, and stain with the secondary antibody (PE anti-human IgG Fc, Cat. #: 109-115-098, Jackson ImmunoResearch) . Samples shown in Fig. 14 were analyzed with a flow cytometer. SP34 was used as a positive control. hIgG1 (Beyotime, Cat No: B117901) represents human IgG1 was used as a negative control. NC consists of Jurkat cell with fresh human serum and the second antibody without treatment of prodrugs. The binding efficiency of Jurkat cells by prodrugs was calculated as follows,

Jurkat cell binding efficiency = (MFI of sample-MFI of negative control) / (MFI of positive control-MFI of negative control)

The results in Figure 14 demonstrate that PRD-4 and PRD-5 have a good serum stability.

Example 18. Anti-tumor activities of the prodrugs in vivo

We next determined the in vivo activities of Prodrugs (PRD-4 and PRD-5) . The Prodrugs were further tested in a PBL (peripheral blood lymphocyte) humanized immune system model (PBL-HIS model) . First, the benchmark antibody CX-PC4 (anti-CD3 × GPC3) was constructed. CX-PC4 comprises a first antigen-binding arm that specifically binds CD3 and a second antigen-binding arm that specifically binds GPC3. The first antigen-binding arm is a scFv linking to BM (blocking moiety) and CM (cleavable moiety) , the sequence of which was published in the prior art (WO2019213444A1) . The variable regions of CX-PC4 were listed separately in Table 12. The PRD-AC is PRD-4 without BM and CM which remains constantly active.

Briefly, the human hepatoma-derived huh7 tumor cell line (Procell, cl-0120) was inoculated subcutaneously with 5x10⁶ cells per mouse on the shoulder of NOG mice (female, 7-8w, Beijing Vital River Laboratory Animal Technology Co., Ltd) with 50%Matrigel (Absin) on day 0 (the day of tumor inoculation was defined as D0) . On Day 3, mice were randomized into six groups with 7 mice per group when the volume of tumor was palpable (～50mm3-100mm3) . Treatment groups and dosages were shown as Table 11. Drug administration was started on day 5 after 1x10⁷ cells /per mouse of human PBMC (Milestone Biotechnologies) were intravenously injected on day 4. Animals were dosed intraperitoneally weekly for 3 weeks with drugs (dosage were shown above) , and tumor volumes and body weights were recorded 2-3 times per week. Tumor volume (TV) was calculated according to the formula: TV (mm³) =LxW2/2, where L is the length of the tumor and W is the width of the tumor.

The data of the in vivo test shows that prodrugs have great anti-tumor capacity. As shown in Fig. 15A-15B, treatment with the PRD-AC leads to tumor regressions in a dose-dependent manner in established xenograft tumors in PBMC-engrafted NOG mice. Similarly, the masked PRD-4 and PRD-5 prodrugs also result in tumor regression significantly.

Table 11. Treatment and dosages for various groups

Table 12. Sequences for chains of CX-PC4

Claims

An antibody or antigen binding fragment thereof that specifically binds to CD3, comprising a HCDR1 comprising SYWMH (SEQ ID NO: 9) with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising NIYPX₁SX₂X₃TNYDEX₄FKS (SEQ ID NO: 24) with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising DX₅X₆GNYYFDY (SEQ ID NO: 25) with 0, 1, 2, or 3 conservative substitutions, a LCDR1 comprising KSSQSLLNX₇RTRKX₈YLA (SEQ ID NO: 23) with 0, 1, 2, or 3 conservative substitutions, a LCDR2 comprising WASTRES (SEQ ID NO: 12) with 0, 1, 2, or 3 conservative substitutions, and a LCDR3 comprising TQSX₉X₁₀LRT (SEQ ID NO: 26) with 0, 1, 2, or 3 conservative substitutions, wherein X_1-10 are independently any amino acid residue.
The antibody or antigen binding fragment thereof of claim 1, wherein X₁ is D or G, X₂ is G or D, X₃ is S or I, X₄ is K or R, X₅ is H, Q or R, X₆ is Y or S, X₇ is S or I, X₈ is S or N, X₉ is F or Y, X₁₀ is I or S.
The antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 16 or SEQ ID NO: 20, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 19 and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22.
The antibody or antigen binding fragment thereof of any one of claims 1-2, wherein the HCDRs 1-3 and LCDRs1-3 are selected from any one of the following (a) - (d) with 0, 1, 2, or 3 conservative substitutions:

(a) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 11 and 13 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 10, 12 and 14 respectively;

(b) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 16 and 17 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 15, 12 and 18 respectively;

(c) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 11 and 13 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 19, 12 and 14 respectively; and

(d) the HCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 9, 20 and 21 respectively, and the LCDRs 1-3 comprise the amino acid sequences of SEQ ID NO: 15, 12 and 22 respectively.
An antibody or antigen binding fragment thereof that specifically binds to CD3, comprising a VH and/or a VL, wherein the VH and/or VL are selected from any one of the following (a) - (d) :

(a) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 2;

(b) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 3 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 4;

(c) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6; and

(d) the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 7 and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 8.
The antibody or antigen binding fragment thereof of claim 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and/or the VL comprises the amino acid sequence of SEQ ID NO: 2.
The antibody or antigen binding fragment thereof of claim 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 3 and/or the VL comprises the amino acid sequence of SEQ ID NO: 4.
The antibody or antigen binding fragment thereof of claim 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 5 and/or the VL comprises the amino acid sequence of SEQ ID NO: 6.
The antibody or antigen binding fragment thereof of claim 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and/or the VL comprises the amino acid sequence of SEQ ID NO: 8.
An antibody or antigen binding fragment thereof that specifically binds to CD3, comprising any one of the following (a) - (f) with 0, 1, 2, or 3 conservative substitutions:

(a) three CDRs of a VH as set forth in SEQ ID NO: 1 and three CDRs of a VL as set forth in SEQ ID NO: 2;

(b) three CDRs of a VH as set forth in SEQ ID NO: 3 and three CDRs of a VL as set forth in SEQ ID NO: 4;

(c) three CDRs of a VH as set forth in SEQ ID NO: 5 and three CDRs of a VL as set forth in SEQ ID NO: 6;

(d) three CDRs of a VH as set forth in SEQ ID NO: 7 and three CDRs of a VL as set forth in SEQ ID NO: 8;

(e) three CDRs of a VH as set forth in SEQ ID NO: 27 and three CDRs of a VL as set forth in SEQ ID NO: 28;

(f) three CDRs of a VH as set forth in SEQ ID NO: 29 and three CDRs of a VL as set forth in SEQ ID NO: 28.
An antibody or antigen binding fragment thereof that specifically binds to CD3, comprising a HCDR1 comprising SYWMH (SEQ ID NO: 9) with 0, 1, 2, or 3 conservative substitutions, a HCDR2 comprising NIYPGSGSTNYDEKFKS (SEQ ID NO: 16) with 0, 1, 2, or 3 conservative substitutions or NIYPGSGSTNYAQKFQG (SEQ ID NO: 30) with 0, 1, 2, or 3 conservative substitutions, a HCDR3 comprising DQYGNYYFDY (SEQ ID NO: 17) with 0, 1, 2, or 3 conservative substitutions, a LCDR1 comprising KSSQSLLNSRTRKNYLA (SEQ ID NO: 15) with 0, 1, 2, or 3 conservative substitutions, a LCDR2 comprising WASTRES (SEQ ID NO: 12) with 0, 1, 2, or 3 conservative substitutions, and a LCDR3 comprising TQSYILRT (SEQ ID NO: 18) with 0, 1, 2, or 3 conservative substitutions.
An antibody or antigen binding fragment thereof that specifically binds to CD3, comprising a VH and/or a VL, wherein the VH comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 27 or SEQ ID NO: 29, and/or the VL comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 28.
The antibody or antigen binding fragment thereof of claim 12, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 or 29, and/or the VL comprises the amino acid sequence of SEQ ID NO: 28.
A multi-specific antibody or antigen binding fragment thereof comprises a first antibody or antigen binding fragment thereof (AB1) that specifically binds CD3 and at least a second antibody or antigen binding fragment thereof (AB2) that specifically binds a second target, wherein AB1 comprises any one of the antibody or antigen binding fragment thereof of claims 1-13.
The multi-specific antibody or antigen binding fragment thereof of claim 14, wherein the second target is a tumor associated antigen.
The multi-specific antibody or antigen binding fragment thereof of claim 15, wherein the tumor associated antigen is selected from GPC, CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, EGFR, Ep-CAM, EphA3, Her2, Her3, ROR2, PSMA, STEAP1, FGFR2, TROP2, B7-H3, B7-H4, B7-H6, FOLR1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, SSX-2, Fibronectin, MART-2, PDL-1, VEGFR, CLAUDIN, and others.
The multi-specific antibody or antigen binding fragment thereof of claim 14 or 15, wherein the second target is GPC, preferably GPC3.
An antibody conjugate (ADC) comprising the antibody or antigen binding fragment thereof any one of the claims 1-13, or the multi-specific antibody or antigen binding fragment thereof of any one of claims 14-17.
A blocking moiety (BM) , comprising the sequence of Y₁DGY₂Y₃Y₄Y₅GY₆ITQTPYKVSIS, wherein Y₁-Y₆ independently are any one of the amino acid residues.
The blocking moiety of claim 19, wherein Y₁ is Q or E; Y₂ is selected from Y, S or Q; Y₃ is E or D; Y₄ is E or D; Y₅ is M or I; Y₆ is G or S.
The blocking moiety of claim 20, comprising any one of SEQ ID NOs: 32-34.
A cleavable moiety (CM) subject to cleavage by proteases, comprising any one or more of the sequence (s) selected from SEQ ID NOs: 36-44.
The CM of claim 22, comprising any one or more of the sequence (s) selected from SEQ ID NOs: 45-53.
An activable antibody structure, comprising an antibody or antigen binding fragment thereof (AB) ; a blocking moiety (BM) to inhibit or weaken the binding of the antibody or antigen binding fragment thereof to a target antigen when the activable antibody is in a non-cleaved state; and a cleavable moiety (CM) subject to cleavage by proteases.
The activable antibody structure of claim 24, wherein the activable antibody structure in the non-cleaved state has a structural arrangement from N-terminus to C-terminus as follows, BM-CM-AB or AB-CM-BM.
The activable antibody structure of claim 24 or 25, wherein the antibody or antigen binding fragment thereof is that of any one of claims 1-13, and the target antigen is CD3.
The activable antibody structure of any one of claims 24-26, wherein the BM is that of any one of claims 19-21.
The activable antibody structure of any one of claims 24-27, wherein the CM is that of claim 22 or 23.
The activable antibody structure of any one of claims 24-28, wherein the proteases are selected from any one or more of Matrix Metalloproteinases (MMPs) and Urokinase-type plasminogen activator (uPA) .
The activable antibody structure of claim 29, wherein the proteases are selected from any one or more of MMP2, MMP14, and uPA.
The activable antibody structure of any one of claims 24-30 optionally comprises a first linker (linker 1) between BM and CM, and/or optionally comprises a second linker (linker 2) between CM and AB.
The activable antibody structure of claim 31, wherein the activable antibody in the non-cleaved state has the structure arrangement from N-terminus to C-terminus as follows, BM-linker 1-CM-linker 2-AB or AB-linker 2-CM-linker 1-BM.
The activable antibody structure of any one of claims 24-32, comprising any one of the following (a) - (c) :

(a) SEQ ID NO: 54 and/or 56;

(b) SEQ ID NO: 57 and/or 28;

(c) SEQ ID NO: 60 and/or 28.
A multi-specific activable antibody structure that in an activated state specifically binds CD3 and at least a second target, wherein the multi-specific activable antibody structure comprises at least the following (a) or (b) :

(a) the activable antibody structure of any one of claims 24-33 as a first activable antibody that specifically binds CD3 in an activated state, and a second antibody or antigen binding fragment thereof that specifically binds the second target;

(b) the activable antibody structure of any one of claims 24-33 as a first activable antibody that specifically binds CD3 in an activated state, and the activable antibody structure of claim 24 or 25 as a second activable antibody that specifically binds the second target in an activated state.
The multi-specific activable antibody structure of claim 34, wherein the second target is a tumor associated antigen (TAA) .
The multi-specific activable antibody structure of claim 34, wherein the second target is GPC, preferably GPC3.
The multi-specific activable antibody structure of claim 36, wherein the second antibody or antigen binding fragment thereof, or the second activable antibody comprises any one of the following (a) - (b) :

(a) SEQ ID NO: 55;

(b) SEQ ID NO: 58 and/or 59.
An isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen binding fragment thereof of any one of claims 1-13, the multi-specific antibody or antigen binding fragment thereof of any one of claims 14-17, the BM of any one of claims 19-21, the CM of claim 22 or 23, the activable antibody structure of any one of claims 24-33, or the multi-specific activable antibody structure of any one of claims 34-37.
A vector comprising the isolated nucleic acid of claim 38.
A host cell comprising the isolated nucleic acid of claim 38, or the vector of claim 39.
A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of claims 1-13, the multi-specific antibody or antigen binding fragment thereof of any one of claims 14-17, the ADC of claim 18, the activable antibody structure of any one of claims 24-33, the multi-specific activable antibody structure of any one of claims 34-37, the isolated nucleic acid of claim 38, the vector of claim 39, or the host cell of claim 40, and a pharmaceutically acceptable carrier or excipient.
A method of treating or preventing a condition ameliorated by the stimulation of the immune cells in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen binding fragment thereof of any one of claims 1-13, the multi-specific antibody or antigen binding fragment thereof of any one of claims 14-17, the ADC of claim 18, the activable antibody structure of any one of claims 24-33, the multi-specific activable antibody structure of any one of claims 34-37, or the pharmaceutical composition of claim 41.
The method of claim 42, wherein the antibody or antigen binding fragment thereof, the multi-specific antibody or antigen binding fragment thereof, the ADC, the activable antibody structure, the multi-specific activable antibody structure, or the pharmaceutical composition is administered in combination with other therapeutics.
The method of claim 42 or 43, wherein the condition is selected from tumor.
The method of claim 44, wherein the condition is selected from solid tumors, such as hepatocellular carcinoma (HCC) , gastric cancer (GC) , cholangiocarcinoma, esophageal squamous cell carcinoma (ESCC) , colorectal cancer (CRC) , thyroid cancer, pancreatic cancer non-small cell lung cancer (NSCLC) , small cell lung cancer (SCLC) , renal cell carcinoma (RCC) , ovarian cancer, breast cancer, uterine cancer, endometrial cancer, testicular cancer, bladder cancer, skin cancer, neuroblastoma, rhabdomyosarcoma, melanoma, osteosarcoma.
A method of diagnosing a condition associated with CD3, comprising contacting a sample of subject with the antibody or antigen binding fragment thereof of any one of claims 1-13, the multi-specific antibody or antigen binding fragment thereof of any one of claims 14-17, the activable antibody structure of any one of claims 24-33, or the multi-specific activable antibody structure of any one of claims 34-37.
A method of inhibiting or weakening the binding of the antibody or antigen binding fragment thereof of any one of claims 1-13 to CD3, comprising using a BM to mask the binding site of the antibody or antigen binding fragment thereof.
The method of claim 47, wherein the BM is that of any one of claims 19-21.
The method of claim 47 or 48, comprising linking the antibody or antigen binding fragment thereof to the BM through a CM cleavable by proteases.
The method of claim 49, wherein the CM is that of claim 22 or 23.
A method of linking the antibody or antigen binding fragment thereof of any one of claims 1-13 to a CM, comprising linking the antibody or antigen binding fragment thereof to a protease cleavage site optionally through a linker.
The method of claim 51, wherein the CM is that of claim 22 or 23.