Anti-CD3 activatable antibodies and uses thereof

Abstract

The present invention relates to anti-CD3 antibodies, activatable antibody structures, and multispecific activatable antibody structures comprising anti-CD3 antibodies. The invention also relates to a therapeutic application of the invention, in particular to an application in tumor treatment.

Description

Cross-reference to related applications

[0001] This application claims priority to PCT application No. PCT / CN2023 / 124115, filed on October 11, 2023. The contents of the earlier PCT application are considered part of this disclosure and are incorporated herein by reference in their entirety.

[0002] References to sequence lists The sequence list titled DCF230804WO-SequenceListing.xml, created on October 9, 2023, and measuring 57,369 bytes, is incorporated into this article by reference in its entirety. Technical Field

[0003] This disclosure relates to cancer immunotherapy. It targets anti-CD3 antibodies, activatable antibodies containing such antibodies, and the therapeutic use of the aforementioned antibodies, particularly in the treatment, prevention, and diagnosis of conditions such as cancer that are improved by stimulating immune cells. Background Technology

[0004] The statements in this section are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0005] The nonspecificity of novel, potent biotherapies that improve the treatment of conditions such as cancer by stimulating immune cells, coupled with off-target toxicity due to the broad expression of targets in patients, limits the use of such therapies. By enhancing target specificity, antibody prodrugs / activatable antibodies can broaden or even create therapeutic windows for biotherapies that are difficult or impossible to use due to poor patient tolerance. However, significant challenges remain due to limited experience with antibody prodrugs / activatable antibodies, including designing molecules to adequately treat conditions while minimizing off-target toxicity.

[0006] CD3 (Cluster 3 of Differentiation) is a protein complex and T-cell co-receptor involved in activating cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). It consists of four distinct chains: one CD3γ chain, one CD3δ chain, and two CD3ε chains in mammals. The CD3 complex acts as a signaling module for the T-cell receptor (TCR), non-covalently binding to the antigen-binding a / b chains of the TCR. Because CD3 directly contributes to T-cell activation, it is an ideal target for various therapeutic and / or diagnostic applications. For example, anti-CD3 antibodies can stimulate T cells and can be used in medical treatments such as anticancer therapies. Therefore, there is a need to develop antibodies and therapeutics targeting the CD3 / TCR pathway with improved off-target toxicity and / or enhanced efficacy. Summary of the Invention

[0007] For the purposes described above, this disclosure provides, on the one hand, antibodies that specifically bind to CD3 or antigen-binding fragments thereof. In some embodiments, the antibody or antigen-binding fragment thereof described in this disclosure comprises: HCDR1, wherein HCDR1 comprises SYWMH (SEQ ID NO: 9) having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises NIYPX1SX2X3TNYDEX4FKS (SEQ ID NO: 24) having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises DX5X6GNYYFDY (SEQ ID NO: 25) having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises KSSQSLLNX7RTRKX8YLA (SEQ ID NO: 23) having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises WASTRES (SEQ ID NO: 12) having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises TQSX9X having 0, 1, 2 or 3 conserved substitutions. 10 LRT (SEQ ID NO: 26), where X 1-10 Independently, it is 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, X... 10 It is either I or S.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 16 or SEQ ID NO: 20; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 19 and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises HCDRs 1-3 and LCDRs 1-3 selected from any one of the following (a)-(d) having 0, 1, 2 or 3 conserved substitutions: (a) HCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, 11 and 13, respectively, and LCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 10, 12 and 14, respectively; (b) HCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, 16 and 17, respectively, and LCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 15, 12 and 18, respectively; (c) HCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, 11 and 13, respectively, and LCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 19, 12 and 14, respectively; and (d) HCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, 11 and 13, respectively, and LCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 19, 12 and 14, respectively; and (d) HCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 9, 11 and 13, respectively, and LCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 19, 12 and 14, respectively; and (d) HCDRs 1-3 comprising the amino acid sequences of SEQ ID NO: 10, 12 and 14, respectively. The amino acid sequences of NO: 9, 20 and 21, and LCDRs 1-3 contain the amino acid sequences of SEQ ID NO: 15, 12 and 22, respectively.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 10 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2 or 3 conserved substitutions.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:16 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:17 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:15 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO:12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO:18 having 0, 1, 2 or 3 conserved substitutions.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 19 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2 or 3 conserved substitutions.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 20 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 21 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 22.

[0014] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises: HCDR1, wherein HCDR1 comprises SYWMH (SEQ ID NO: 9) having 0, 1, 2, or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises NIYPGSGSTNYDEKFKS (SEQ ID NO: 16) or NIYPGSGSTNYAQKFQG (SEQ ID NO: 30) having 0, 1, 2, or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises DQYGNYYFDY (SEQ ID NO: 17) having 0, 1, 2, or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises KSSQSLLNSRTRKNYLA (SEQ ID NO: 15) having 0, 1, 2, or 3 conserved substitutions; and LCDR2, wherein LCDR2 comprises WASTRES (SEQ ID NO: 9) having 0, 1, 2, or 3 conserved substitutions. (SEQ ID NO: 12); and LCDR3, wherein LCDR3 comprises TQSYILRT (SEQ ID NO: 18) having 0, 1, 2 or 3 conserved substitutions. In some embodiments, the antibody or antigen-binding fragment thereof described herein is humanized.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in this disclosure comprises any one of the following (a)-(f) having 0, 1, 2 or 3 conserved substitutions: (a) three CDRs of VH as shown in SEQ ID NO: 1 and three CDRs of VL as shown in SEQ ID NO: 2; (b) three CDRs of VH as shown in SEQ ID NO: 3 and three CDRs of VL as shown in SEQ ID NO: 4; (c) three CDRs of VH as shown in SEQ ID NO: 5 and three CDRs of VL as shown in SEQ ID NO: 6; (d) three CDRs of VH as shown in SEQ ID NO: 7 and three CDRs of VL as shown in SEQ ID NO: 8; (e) three CDRs of VH as shown in SEQ ID NO: 27 and three CDRs of VL as shown in SEQ ID NO: 28; (f) three CDRs of VH as shown in SEQ ID NO: 29 and three CDRs of VL as shown in SEQ ID NO: 28.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 2.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 4.

[0018] In some embodiments, the CD3-specific antibody or its antigen-binding fragment described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 6.

[0019] In some embodiments, the CD3-specific antibody or its antigen-binding fragment described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 8.

[0020] In some embodiments, the CD3-specific antibody or its antigen-binding fragment described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 28.

[0021] In some embodiments, the CD3-specific antibody or its antigen-binding fragment described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 28.

[0022] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 1 and / or the VL comprises SEQ ID NO: 2. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 3 and / or the VL comprises SEQ ID NO: 4. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 5 and / or the VL comprises SEQ ID NO: 6. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 7 and / or the VL comprises SEQ ID NO: 8.

[0023] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 27 and / or the VL comprises SEQ ID NO: 28. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or 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 described in this disclosure is humanized.

[0024] On one hand, this disclosure provides a multispecific antibody or antigen-binding fragment thereof comprising a first antibody or antigen-binding fragment thereof specifically binding to CD3 (AB1) and at least one second antibody or antigen-binding fragment thereof specifically binding to a second target (AB2), wherein AB1 comprises any of the antibodies or antigen-binding fragments thereof described in this disclosure. In some embodiments, the second target is a tumor-associated antigen (TAA). In some embodiments, the second target is a GPC, preferably GPC3.

[0025] In some implementations, tumor-associated antigens may 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.

[0026] In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 55. In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 58. In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 59. In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 58 and 59.

[0027] On the other hand, this disclosure provides an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof described in this disclosure, or the multispecific antibody or antigen-binding fragment thereof described in this disclosure.

[0028] On the one hand, this disclosure provides an isolated nucleic acid comprising a polynucleotide sequence, said polynucleotide sequence encoding the antibody or antigen-binding fragment thereof described in this disclosure or the multispecific antibody described in this disclosure.

[0029] On the other hand, this disclosure provides a carrier containing the isolated nucleic acid described in this disclosure.

[0030] In another aspect, this disclosure provides a host cell comprising the isolated nucleic acid or the vector described in this disclosure.

[0031] On the one hand, this disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in this disclosure, a multispecific antibody or antigen-binding fragment thereof as described in this disclosure, an ADC as described in this disclosure, an isolated nucleic acid as described in this disclosure, a carrier as described in this disclosure, or a host cell as described in this disclosure, and a pharmaceutically acceptable carrier or excipient.

[0032] On the one hand, this disclosure provides a method for treating or preventing disease by stimulating immune cells in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in this disclosure, a multispecific antibody or antigen-binding fragment thereof described in this disclosure, an ADC described in this disclosure, or a pharmaceutical composition described in this disclosure.

[0033] In some embodiments of the methods disclosed herein, the antibodies or antigen-binding fragments thereof, multispecific antibodies or antigen-binding fragments thereof, ADCs, or pharmaceutical compositions described herein are administered in combination with other therapeutic agents. In some embodiments, the other therapeutic agents are selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the other therapeutic agents are selected from, for example, Bruton's tyrosine kinase (BTK) inhibitors, tyrosine kinase inhibitors (TKIs), PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, EGFR inhibitors, VEGF inhibitors, PARP inhibitors, Her2, LAG-3, and TNFR2 inhibitors.

[0034] In some embodiments of the methods disclosed herein, the disease is selected from tumors, and in some embodiments, the disease 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, and osteosarcoma.

[0035] In another aspect, this disclosure provides the use of the antibodies or antigen-binding fragments thereof described herein, the multispecific antibodies or antigen-binding fragments thereof described herein, the ADCs or pharmaceutical compositions described herein in the preparation of medicaments for treating or preventing conditions improved by stimulating immune cells.

[0036] On one hand, this disclosure provides a blocking portion (BM) that inhibits or attenuates the binding of the antibody or antigen-binding fragment of this disclosure to CD3. In some embodiments, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment of this disclosure that specifically binds to CD3. In some embodiments, the BM comprises the sequence Y1DGY2Y3Y4Y5GY6ITQTPYKVSIS, wherein Y1-Y6 are independently 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 described in this disclosure comprises any one of the following (a)-(c): (a) SEQ ID NO: 32 or SEQ ID NO: 32 having 1, 2, or 3 conservative substitutions; (b) SEQ ID NO: 33 or SEQ ID NO: 33 having 1, 2, or 3 conservative substitutions; (c) SEQ ID NO: 34 or SEQ ID NO: 34 having 1, 2, or 3 conservative substitutions. In some embodiments, the BM described in this disclosure comprises any one of SEQ ID NOs: 32-34.

[0037] On one hand, this disclosure provides a cleavable portion (CM) that can be cleaved by a protease, comprising any one of the sequences of SEQ ID NOs: 36-44. On the other hand, this disclosure provides a cleavable portion (CM) that can be cleaved by a protease, comprising any one or more of the sequences of SEQ ID NOs: 45-53.

[0038] On the one hand, this disclosure provides an isolated nucleic acid comprising a polynucleotide sequence, said polynucleotide sequence encoding the BM or CM described in this disclosure.

[0039] On the other hand, this disclosure provides a carrier containing the isolated nucleic acid described in this disclosure.

[0040] In another aspect, this disclosure provides a host cell comprising the isolated nucleic acid or the vector described in this disclosure.

[0041] On one hand, this disclosure provides an activatable antibody structure comprising an antibody or an antigen-binding fragment thereof (AB); a blocking portion (BM) that, when the activatable antibody is in an uncleaved state, inhibits or weakens the binding of the antibody or the antigen-binding fragment thereof to a target antigen; and a cleavable portion (CM) that can be cleaved by a protease.

[0042] On one hand, this disclosure provides an activatable antibody structure comprising an antibody or an antigen-binding fragment thereof (AB); a blocking portion (BM) that, when the activatable antibody is in an uncleaved state, inhibits or weakens the binding of the antibody or its antigen-binding fragment to a target antigen; and a cleavable portion (CM) that can be cleaved by a protease. In some embodiments, when the activatable antibody is in a cleaved state, the BM does not interfere with or compete for the binding of AB to the target antigen.

[0043] In some embodiments, the activatable antibody structure in the uncut state described herein has the following structural arrangement from N-terminus to C-terminus: BM-CM-AB or AB-CM-BM.

[0044] In some embodiments, the activatable antibody structure described herein comprises the AB described herein, and the target antigen is CD3.

[0045] In some embodiments, the activatable antibody structure described herein comprises the BM described herein.

[0046] In some embodiments, the activatable antibody structure described herein comprises the CM described herein.

[0047] In some embodiments, the protease is selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, and caspase 1. 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, Chymotrypsin, Cruzipain, DESCI, DPP-4, FAP, Legumin, Otubain-2, Elastase, FVIIa, FIXA, FXa, FXIa, FXIIa, Granzyme B, Guanidylbenzoate esterase (G Human anidinobenzoatase, heepsin, HtrA1, human neutrophil elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, lactoferrin, Marapsin, Matriptase-2, meprin, MT-SP1 / Matriptase, enkephalin, 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, trypsin, and uPA. In some embodiments, the protease is selected from any one or more of MMP2, MMP14, and uPA.

[0048] In some embodiments, the activatable antibody structure of this disclosure optionally includes a first linker (linker 1) between BM and CM, and / or optionally includes a second linker (linker 2) between CM and AB. In some embodiments, the activatable antibody in its uncleaved state of this disclosure has the following structural arrangement from N-terminus to C-terminus: BM-linker 1-CM-linker 2-AB or AB-linker 2-CM-linker 1-BM. In some embodiments, linker 1 or linker 2 of this disclosure independently includes (GS)n1, (GGS)n2, (GGGS)n3, (GGGGS)n4, (GGSG)n5, (GGSGG)n6, (GSGSG)n7, (GSGGG)n8, (GGGSG)n9, (GSSSG)n 10 (GGGGSGGGS)n 11 The amino acid sequence of a group composed of or a combination thereof, wherein n1-n 11 Independently, it is any integer equal to or greater than 0. In some embodiments, the connector 1 described in this disclosure contains (GGGGSGGGS)n 11 Furthermore, the connector 2 described in this disclosure comprises (GGGGS)n4, wherein n 11 n4 is independently any integer equal to or greater than 0. In some embodiments, adapter 1 of this disclosure comprises GGGGSGGGS, and adapter 2 of this disclosure comprises GGGGS. In some embodiments, the activatable antibody structure of this disclosure comprises CM, said CM comprising any one of the sequences of SEQ ID NOs: 45-53.

[0049] In some embodiments, the activatable antibody structure of this disclosure includes a variable region comprising SEQ ID NO: 54 and / or 56. In some embodiments, the activatable antibody structure of this disclosure includes a variable region comprising SEQ ID NO: 57 and / or 28. In some embodiments, the activatable antibody structure of this disclosure includes a variable region comprising SEQ ID NO: 60 and / or 28.

[0050] On one hand, this disclosure provides a multispecific activatable antibody structure that, in an activated state, specifically binds to CD3 and at least a second target. In some embodiments, the multispecific activatable antibody structure of this disclosure comprises at least (a) the activatable antibody structure of this disclosure as a first activatable antibody that specifically binds to CD3 in an activated state, and a second antibody or its antigen-binding fragment that specifically binds to a second target; or (b) the activatable antibody structure of this disclosure as a first activatable antibody that specifically binds to CD3 in an activated state, and the activatable antibody structure of this disclosure as a second activatable antibody that specifically binds to a second target in an activated state. In some embodiments, the second target is TAA. In some embodiments, the second target is GPC, preferably GPC3.

[0051] In some implementations, tumor-associated antigens may 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.

[0052] In some embodiments, the second activatable antibody of this disclosure includes a variable region comprising SEQ ID NO: 55. In some embodiments, the second activatable antibody of this disclosure includes a variable region comprising SEQ ID NO: 58 and / or 59.

[0053] In some embodiments, the second antibody or antigen-binding fragment of this disclosure includes a variable region comprising SEQ ID NO: 55. In some embodiments, the second antibody or antigen-binding fragment of this disclosure includes a variable region comprising SEQ ID NO: 58 and / or 59.

[0054] On one hand, this disclosure discloses an isolated nucleic acid comprising a polynucleotide sequence, the polynucleotide sequence encoding the antibody or antigen-binding fragment thereof described in this disclosure, the multispecific antibody or antigen-binding fragment thereof, the CM, the BM, the activatable antibody structure, or the polynucleotide sequence of the multispecific activatable antibody structure.

[0055] On the other hand, this disclosure discloses a carrier containing the isolated nucleic acid described in this disclosure.

[0056] In another aspect, this disclosure discloses a host cell comprising the isolated nucleic acid or the vector described in this disclosure.

[0057] In another aspect, this disclosure discloses a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the multispecific antibody or antigen-binding fragment thereof, the ADC, the activatable antibody structure, the multispecific activatable antibody, the isolated nucleic acid, the carrier or the host cell, and a pharmaceutically acceptable carrier or excipient.

[0058] On one hand, this disclosure provides a method for treating or preventing disease by stimulating immune cells in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in this disclosure, the multispecific antibody or antigen-binding fragment thereof, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, or the pharmaceutical composition thereof. In some embodiments of the method for treating or preventing disease according to this disclosure, the antibody or antigen-binding fragment thereof described in this disclosure, the multispecific antibody or antigen-binding fragment thereof, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, or the pharmaceutical composition thereof are administered in combination with other therapeutic agents. In some embodiments, the other therapeutic agents are selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the other therapeutic agents are selected from, for example, Bruton's tyrosine kinase (BTK) inhibitors, tyrosine kinase inhibitors (TKIs), PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, EGFR inhibitors, VEGF inhibitors, PARP inhibitors, Her2, LAG-3, and TNFR2 inhibitors.

[0059] In some embodiments of the methods for treating or preventing diseases described in this disclosure, the disease is selected from tumors. In some embodiments, the disease 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, and osteosarcoma.

[0060] On the one hand, this disclosure provides a method for diagnosing CD3-related conditions, including exposing a subject's sample to the antibody or antigen-binding fragment thereof described in this disclosure, the multispecific antibody or antigen-binding fragment thereof, the activatable antibody structure, or the multispecific activatable antibody structure.

[0061] On the other hand, this disclosure provides the use of the antibody or antigen-binding fragment thereof described herein, the multispecific antibody or antigen-binding fragment thereof, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, or the pharmaceutical composition thereof in the preparation of a medicament for treating or preventing a condition that is improved by stimulating immune cells.

[0062] On the one hand, this disclosure provides the use of the antibodies or antigen-binding fragments thereof described herein, the multispecific antibodies or antigen-binding fragments thereof, the activatable antibody structures, or the multispecific activatable antibody structures in the preparation of products (such as kits) for diagnosing CD3-related conditions.

[0063] In another aspect, this disclosure provides a method for inhibiting or weakening the binding of the antibody or its antigen-binding fragment described herein to CD3, including using a masking membrane (BM) to mask the binding site of the antibody or its antigen-binding fragment. In some embodiments, the method includes linking the antibody or its antigen-binding fragment described herein to the BM via a protease-cleavable membrane (CM). In some embodiments, the BM is any of those disclosed herein. In some embodiments, the BM comprises Y1DGY2Y3Y4Y5GY6ITQTPYKVSIS, wherein Y1-Y6 are independently 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; and 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 having 1, 2, or 3 conservative substitutions; (b) SEQ ID NO: 33 or SEQ ID NO: 33 having 1, 2, or 3 conservative substitutions; (c) SEQ ID NO: 34 or SEQ ID NO: 34 having 1, 2, or 3 conservative substitutions. In some embodiments, the BM comprises any one of SEQ ID NOs: 32-34.

[0064] In some embodiments, the method includes linking the antibody or its antigen-binding fragment described herein to the BM via a CM that can be cleaved by a protease. In some embodiments, the BM can be any of those disclosed herein, preferably selected from Table 6, such as BM-2, BM-3, BM-4. In some embodiments, the CM can be any of those disclosed herein, preferably, the CM includes any one or more cleavage sites selected from Table 7.

[0065] In another aspect, this disclosure provides a method for linking an antibody or antigen-binding fragment thereof described herein to a CM, comprising optionally linking the antibody or antigen-binding fragment thereof described herein to a protease cleavage site via one or more adapters. In some embodiments, the CM may be any of those disclosed herein. In some embodiments, the CM includes a cleavage site and an adapter, and exemplary protease cleavage sites and adapters are shown in Table 7. Attached Figure Description

[0066] The following is a brief description of the accompanying drawings, which are presented for the purpose of illustrating exemplary embodiments of this disclosure and not for limiting purposes.

[0067] Figure 1 The ELISA binding assay for antibody-mediated binding to human CD3ε is shown.

[0068] Figure 2 The ELISA binding assay of the antibody to CD3ε in cynomolgus monkeys was demonstrated.

[0069] Figure 3 This study demonstrated the agonism effect of the anti-CD3 antibody in the THP-1 / Jurkat-NFAT system.

[0070] Figure 4 The structure of the variable region of CD3 mAb is shown.

[0071] Figure 5 FACS analysis of the humanized antibody bound to Jurkat cells is shown.

[0072] Figure 6 The ability of the CD3 blocking moiety to block the binding between anti-CD3 mAb, BPCD3-2, and Jurkat cells was demonstrated.

[0073] Figure 7A -C indicates the ability of BM-2, BM-3, and BM-4 to block the binding between anti-CD3 mAb and Jurkat cells.

[0074] Figure 8This demonstrated the ability of BM-3 to block the binding between anti-CD3 mAb and Jurkat cells.

[0075] Figure 9 A schematic structure of an activatable antibody structure is shown.

[0076] Figure 10 This study demonstrates T cell activation during co-culture with antibody prodrug PRD-1 and GPC3-positive HepG2 in a reporter assay.

[0077] Figure 11A The binding ability of PRD antibody to Jurkat cells was demonstrated by FACS detection.

[0078] Figure 11B The binding ability of PRD antibody to HEPG2 cells was demonstrated by FACS detection.

[0079] Figure 12 The study demonstrated T cell activation during co-culture with antibody prodrugs PRD-4, PRD-5, and GPC3-positive HepG2 in a reporter assay.

[0080] Figure 13 This demonstrates the ability of the anti-CD3 antibody prodrug to induce cytokine production in PBMC assays.

[0081] Figure 14 The binding efficiency of Jurkat cells to the antibody prodrug was demonstrated. NC consisted of Jurkat cells with fresh human serum and a second antibody (PE anti-human IgG Fc, Cat.#: 109-115-098, Jackson ImmunoResearch), without prodrug treatment.

[0082] Figure 15A -B indicates the antitumor activity of the antibody prodrug in vivo. Detailed Implementation

[0083] The foregoing features and advantages, as well as their additional features and advantages, of this disclosure will become clearer in the following detailed description of the embodiments taken in conjunction with the accompanying drawings. The embodiments described herein with reference to the accompanying drawings are illustrative and explanatory, and should not be construed as limiting the scope of this disclosure.

[0084] This description is not intended to be a detailed list of all different ways in which this disclosure can be implemented or all features that can be added to this disclosure. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be removed from that embodiment. Furthermore, given that this disclosure is not departing from it, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art. Therefore, the following description is intended to illustrate some specific embodiments of this disclosure, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of experiments conducted under this disclosure, preferred materials and methods are described herein. The following terminology will be used in describing and claiming protection for this disclosure.

[0086] definition Unless otherwise expressly stated, the terms “comprise,” “include,” “contain,” and variations thereof (such as “comprise” as used herein) should be understood to imply the inclusion of the said element or step or group of elements or steps, but not to exclude any other element or step or group of elements or steps.

[0087] Unless explicitly prohibited or otherwise indicated in the context of this article, the expression “A and / or B” includes three cases: (1) A, (2) B, and (3) A and B; the expression “A, B, and / or C” includes seven cases: (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 way.

[0088] As used herein, the term "independently" should be interpreted as multiple events / elements having no effect on each other. For example, "X and Y are independently any one of a, b, c" means that X can be any one of a, b, c, and Y can be any one of a, b, c, and the choice of X and the choice of Y can be the same or different without interfering with each other.

[0089] Unless otherwise specified, singular indicators shall include their plural counterparts, and plural terms shall include their singular counterparts. For example, “antibody or antigen-binding fragment thereof” includes a variety of antibodies or antigen-binding fragments thereof.

[0090] As used herein, the term “subject” includes animals such as vertebrates, preferably mammals such as dogs, cats, pigs, sheep, horses, goats, rodents (e.g., mice, rats, guinea pigs) or primates (e.g., gorillas, chimpanzees, monkeys, and humans).

[0091] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule that specifically binds to an antigen. A basic antibody unit is a tetramer consisting of two identical light chains and two identical heavy chains. The N-terminus of each chain contains a variable region with a highly diverse amino acid sequence, and the remainder of each chain contains a constant region. The heavy chain variable region (VH) and light chain variable region (VL) are responsible for antigen binding, while the constant region can participate in the binding of the immunoglobulin to host cells or factors, including components of the immune cell and complement system.

[0092] The light chain variable regions (VL) or heavy chain variable regions (VH) are arranged from the N-terminus to the C-terminus in the pattern FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR represents the framework region and CDR represents the complementarity-determining region. CDRs 1, 2, and 3 of the VL can also be represented as LCDR1, LCDR2, and LCDR3, respectively; similarly, CDRs 1, 2, and 3 of the VH can also be represented as LCDR1, LCDR2, and LCDR3, respectively. The FRs are coiled to bring the CDRs closer together, and the CDRs are in a 3D conformation, thus providing an antigen-binding surface complementary to the antigen target.

[0093] The allocation of amino acids in the various domains is based on any conventional scheme, 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, this disclosure follows the Kabat definition.

[0094] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid sequence within an antibody variable region that confers antigen specificity and binding affinity. In some embodiments, three CDRs (HCDR1, HCDR2, and HCDR3) are present in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, and LCDR3) are present in each light chain variable region.

[0095] Many hypervariable regions or CDR classification criteria are adopted and covered in this paper. 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 to the location of structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops (Chothia “CDRs”) and are used by Oxford Molecular’s ​​AbM antibody modeling software. “Contact” hypervariable regions are based on the analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system specifically for immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complexes (MHC) in humans and other vertebrates.

[0096] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0097] As used herein, the term "antibody" should be understood in its broadest sense, including but not limited to monoclonal antibodies (mAbs), polyclonal antibodies, fusion antibodies, multispecific antibodies, biantibodies, nanobodies, triantibodies, chimeric antibodies, humanized antibodies, fully human antibodies, single-chain antibodies, and antibody fragments that retain antigen-binding specificity, such as antigen-binding fragments. While ensuring that the resulting antibody retains the binding specificity to the target antigen, the antibody may contain additional modifications, such as mutations in non-CDR regions, constant regions, glycosylation sites, and post-translational modifications.

[0098] Immunoglobulins can be classified into five classes based on the constant heavy chain: IgA, IgD, IgE, IgG, and IgM. Furthermore, immunoglobulins can be further subdivided into subtypes such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. However, the antibody light chain can be either a λ chain or a κ chain.

[0099] As used herein, the term "antigen-binding fragment" refers to one or more portions of an antibody that maintains binding specificity to a target antigen. Antigen-binding fragments include, but are not limited to, VH, VL, VHH (sdAb or dAb), Fab, Fab', F(ab')2, Fd, Fv, scFab, scFv, (scFv)2, taFv, DART, BiTE® (bispecific T-cell connector), fragments containing a CDR, and other types.

[0100] As used herein, the terms "specific binding" or "binding" refer to the non-covalent interaction between an immunoglobulin and an antigen to which the immunoglobulin specifically binds. The strength or affinity of this interaction can be determined by the equilibrium dissociation constant (K0) of the antigen and the corresponding antibody. D Or Kd) is used to represent: the smaller the KD value, the stronger the binding strength between the epitope and the antibody. Equilibrium dissociation constant (K D ) is calculated as k off / k on The ratio, where "binding rate constant (K)" on ")" and "dissociation rate constant (K)" off The concentration can be determined by calculations of the actual binding and dissociation rates. (See Nature 361:186-87 (1993)).

[0101] The term "epitope" refers to a site on an antigen where an antibody binds, and it is an antigenic determinant that interacts with a specific antigen-binding site called a complementary site in the variable region of an antibody molecule. A single antigen may have multiple epitopes. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes typically contain at least three, and more often at least five or eight to ten amino acids, in a distinctive spatial conformation. The epitope defines the minimum binding site of an antibody and is therefore a specific target of the antibody or its antigen-binding fragment.

[0102] In some embodiments of this disclosure, when the dissociation constant (KD) is less than or equal to 1 x 10⁻⁶ -6 M, in some implementations, is less than or equal to 1x10 -7 M, in some implementations, is less than or equal to 1x10 -8 M, and in some implementations less than or equal to 1x10 -9 M, and in some implementations in 1x10 -8 Up to 1x10 -12 When the value is within the range of M, it is considered that the antibody specifically binds to the antigen.

[0103] As used herein, the term "multispecific" should be understood as having multiple target binding sites, such as antigen-binding sites. The terms "multispecific antibody," "multispecific activatable antibody," etc., should be interpreted as entities, antibodies, or activatable antibodies having multiple antigen-binding sites against different antigens or different epitopes. The term "antibody" as used in "multispecific antibody," "multispecific activatable antibody," etc., should be understood in its broadest interpretation as described above.

[0104] As used herein, the term “monoclonal antibody (mAb)” refers to a type of immunoglobulin with the same CDR that specifically binds to an antigen or epitope.

[0105] As used herein, the term "humanized antibody" generally refers to an antibody containing heavy and light chain variable region sequences derived from a non-human species (e.g., rabbit, mouse, etc.), wherein at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to human variable sequences. For a class of humanized antibodies, at least one human CDR sequence is introduced into the non-human VH and / or VL sequences to replace the non-human CDR counterpart.

[0106] As used herein, the term "activatable antibody" means that the binding activity of the antibody can be turned on and off. The term "multispecific activatable antibody" means that the binding activity of the multispecific antibody against at least a specific target antigen can be turned on and off. In the "on" state or in the activated / cleaved state (e.g., in the presence of a protease, such as in a tumor microenvironment), the antibody or multispecific antibody specifically binds to the specific target antigen / epitope. In the "off" state or in the inactivated / uncleaved state (e.g., in the absence of a protease), the antibody or multispecific antibody does not have specific binding activity against the specific target antigen / epitope or has weaker binding activity compared to the "on" state. For example, a bispecific activatable antibody may consist of two activatable antigen-binding sites; or it may consist of one activatable antigen-binding site and one conventional antigen-binding site. In the context of this disclosure, an "antibody prodrug" refers to an antibody drug that is inactive in its initial state and only becomes an active drug after conversion through chemical reactions or enzymatic processes in vivo. In specific contexts, it refers to an antibody with blocking antigen-binding activity, where, under certain conditions, such as in the presence of a protease, the antibody prodrug is activated / cleaved into an associated antibody that specifically binds to the target antigen / epitope. As used herein, the terms "prodrug," "antibody prodrug," and "activatable antibody" are used interchangeably.

[0107] In the polypeptide notation used in this article, by convention, the left-hand end corresponds to the amino terminus / N-terminus, and the right-hand end corresponds to the carboxyl terminus / C-terminus. Similarly, the left-hand end of a single-stranded polynucleotide sequence is the 5' end, the left-hand direction of a double-stranded polynucleotide sequence is the 5' direction, the right-hand end of a single-stranded polynucleotide sequence is the 3' end, and the right-hand direction of a double-stranded polynucleotide sequence is the 3' direction.

[0108] As used herein, the term "sequence identity" is typically determined using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. For example, GCG software includes programs such as Gap and Bestfit, which can be used to determine sequence homology or sequence identity between closely related peptides (such as homologous peptides from organisms of different species) or wild-type proteins and their mutant counterparts. See, for example, GCG version 6.1. Peptide sequences can also be compared using programs in FASTA and GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments of the best overlapping regions between query and search sequences and a percentage of sequence identity (Pearson. (2000), op. cit.). Another preferred algorithm is the computer program BLAST, particularly BLASTP and BLASTN, which uses default parameters when comparing sequences disclosed herein with databases containing large numbers of sequences from different organisms. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389402.

[0109] In some implementations, "sequence identity" is determined by comparing two best-aligned sequences, where a portion of the polynucleotide or polypeptide sequence may contain 20% or less of additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0110] As used herein, the term "conservative substitution" refers to the substitution of an amino acid that is not essential for the activity of a peptide, or the substitution of an amino acid with another amino acid having similar properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.) such that the substitution of an essential amino acid does not reduce the activity of the peptide. Conservative substitutions of functionally similar amino acids are well known in the art.

[0111] In some embodiments, conservative substitutions may occur 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 may be substituted with Glu, and Ala may be substituted with Gly or Ser. Antibodies or antigen binding fragments This disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to CD3. In some embodiments, the antibodies or antigen-binding fragments thereof described in this disclosure comprise: HCDR1, wherein HCDR1 comprises SYWMH (SEQ ID NO: 9) having 0, 1, 2, or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises NIYPX1SX2X3TNYDEX4FKS (SEQ ID NO: 24) having 0, 1, 2, or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises DX5X6GNYYFDY (SEQ ID NO: 25) having 0, 1, 2, or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises KSSQSLLNX7RTRKX8YLA (SEQ ID NO: 23) having 0, 1, 2, or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises WASTRES (SEQ ID NO: 12) having 0, 1, 2, or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises TQSX9X having 0, 1, 2, or 3 conserved substitutions. 10 LRT (SEQ ID NO: 26), where X 1-10Independently, it is 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, X... 10 It is either I or S.

[0112] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof disclosed herein comprises: HCDR1, wherein HCDR1 comprises SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises an amino acid sequence having 0, 1, 2 or 3 conserved substitutions of SEQ ID NO: 11, SEQ ID NO: 16 or SEQ ID NO: 20; HCDR3, wherein HCDR3 comprises an amino acid sequence having 0, 1, 2 or 3 conserved substitutions of SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 21; LCDR1, wherein LCDR1 comprises an amino acid sequence having 0, 1, 2 or 3 conserved substitutions of SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 19; and LCDR3, wherein LCDR3 comprises an amino acid sequence having 0, 1, 2 or 3 conserved substitutions of SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 22.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 10 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2 or 3 conserved substitutions.

[0114] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 16 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 18 having 0, 1, 2 or 3 conserved substitutions.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 11 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 13 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 19 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 14 having 0, 1, 2 or 3 conserved substitutions.

[0116] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2 or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 20 having 0, 1, 2 or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 21 having 0, 1, 2 or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2 or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2 or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 22 having 0, 1, 2 or 3 conserved substitutions.

[0117] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2, or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 30 having 0, 1, 2, or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2, or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2, or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2, or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 18 having 0, 1, 2, or 3 conserved substitutions. In some embodiments, the above-described antibody or antigen-binding fragment thereof is humanized.

[0118] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises: HCDR1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 9 having 0, 1, 2, or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO: 16 having 0, 1, 2, or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO: 17 having 0, 1, 2, or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 15 having 0, 1, 2, or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises the amino acid sequence of SEQ ID NO: 12 having 0, 1, 2, or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises the amino acid sequence of SEQ ID NO: 18 having 0, 1, 2, or 3 conserved substitutions. In some embodiments, the above-described antibody or antigen-binding fragment thereof is humanized.

[0119] In some implementations, the CDR sequence is defined according to the Kabat system.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in this disclosure comprises any one of the following (a)-(f) having 0, 1, 2 or 3 conserved substitutions: (a) three CDRs of VH as shown in SEQ ID NO: 1 and three CDRs of VL as shown in SEQ ID NO: 2; (b) three CDRs of VH as shown in SEQ ID NO: 3 and three CDRs of VL as shown in SEQ ID NO: 4; (c) three CDRs of VH as shown in SEQ ID NO: 5 and three CDRs of VL as shown in SEQ ID NO: 6; (d) three CDRs of VH as shown in SEQ ID NO: 7 and three CDRs of VL as shown in SEQ ID NO: 8; (e) three CDRs of VH as shown in SEQ ID NO: 27 and three CDRs of VL as shown in SEQ ID NO: 28; (f) three CDRs of VH as shown in SEQ ID NO: 29 and three CDRs of VL as shown in SEQ ID NO: 28. Those skilled in the art can easily identify CDRs based on the provided VH and VL.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 according to this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 2.

[0122] In some embodiments, VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 1 formed by inserting, deleting and / or substituting one or more amino acids therein, and / or VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 2 formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to CD3.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD3 as described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 4.

[0124] In some embodiments, VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 3 formed by inserting, deleting and / or substituting one or more amino acids therein, and / or VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 4 formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to CD3.

[0125] In some embodiments, the CD3-specific antibody or its antigen-binding fragment described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 6.

[0126] In some embodiments, VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 5 formed by inserting, deleting and / or substituting one or more amino acids therein, and / or VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 6 formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains its ability to bind to CD3.

[0127] In some embodiments, the CD3-specific antibody or its antigen-binding fragment described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 8.

[0128] In some embodiments, VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 7 formed by inserting, deleting and / or substituting one or more amino acids therein, and / or VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 8 formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to CD3.

[0129] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 28. The antibody or antigen-binding fragment thereof described in this disclosure may be a humanized antibody or antigen-binding fragment thereof.

[0130] In some embodiments, VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 27 formed by inserting, deleting and / or substituting one or more amino acids therein, and / or VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 28 formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains its ability to bind to CD3.

[0131] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises VH and / or 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 with 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 with SEQ ID NO: 28. The antibody or antigen-binding fragment thereof described in this disclosure may be a humanized antibody or antigen-binding fragment thereof.

[0132] In some embodiments, VH comprises a functional variant of the amino acid sequence of SEQ ID NO: 29 formed by inserting, deleting and / or substituting one or more amino acids therein, and / or VL comprises a functional variant of the amino acid sequence of SEQ ID NO: 28 formed by inserting, deleting and / or substituting one or more amino acids therein, provided that the functional variant retains the ability to bind to CD3.

[0133] The functional variant comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity with the parent polypeptide. In some preferred embodiments, insertions, deletions, and / or substitutions occur in the FR region. In some embodiments, the substitution of one or more amino acids may be a conserved substitution of one or more amino acids.

[0134] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 1 and / or the VL comprises SEQ ID NO: 2. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 3 and / or the VL comprises SEQ ID NO: 4. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 5 and / or the VL comprises SEQ ID NO: 6. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or VL, wherein the VH comprises SEQ ID NO: 7 and / or the VL comprises SEQ ID NO: 8.

[0135] In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof described in this disclosure comprises VH and / or 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 described in this disclosure may be a humanized antibody or antigen-binding fragment thereof. In some embodiments, the CD3-specific antibody or antigen-binding fragment thereof comprises VH and / or 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 described in this disclosure may be a humanized antibody or antigen-binding fragment thereof.

[0136] The antibodies described in this disclosure can be any isotype or subtype. In some embodiments, the antibody is an IgG isotype. In some embodiments, the antibody is an IgG1 subtype, preferably a human IgG1 (hIgG1) subtype.

[0137] In some embodiments, the antibodies described in this disclosure specifically bind to CD3, particularly CD3ε (CD3ε or CD3e). In some embodiments, the antibodies described in this disclosure specifically bind to mammalian CD3ε, such as human CD3ε and cynomolgus monkey CD3ε.

[0138] Multispecific antibodies This disclosure provides multispecific antibodies or antigen-binding fragments thereof that specifically bind to antigen CD3 (e.g., CD3ε, such as human CD3ε and cynomolgus monkey CD3ε) and at least one other antigen. The multispecific antibodies or antigen-binding fragments thereof described in this disclosure comprise a first antibody or antigen-binding fragment thereof that specifically binds to CD3 (AB1) and at least one second antibody or antigen-binding fragment thereof that specifically binds to a second target, wherein AB1 comprises any of the antibodies or antigen-binding fragments thereof described in this disclosure. In some embodiments, the second target is a tumor-associated antigen (TAA).

[0139] Exemplary tumor-associated antigens may be selected from the following: GPC, CEA, immature laminin receptor, TAG-72, HPVE6, 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.

[0140] In some implementations, the second target is GPC, preferably GPC3.

[0141] In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 55. In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 58. In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 59. In some embodiments, AB2 of this disclosure includes a variable region including SEQ ID NO: 58 and 59.

[0142] Fc variants and modifications In some embodiments, the antibody comprises a homotype Fc domain selected from the group consisting of IgA, IgD, IgE, or IgM Fc domains. In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the sequence of the IgG1 Fc domain is EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62).

[0143] In some embodiments, the Fc domain includes modifications that promote association between the first and second subunits of the Fc domain. In some embodiments, the modification is a “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.).

[0144] In some implementations, the Fc domain includes one or more Fc modifications that can increase stability, increase half-life, and / or alter the effect function.

[0145] In some embodiments, the substitution is selected from sites in the group consisting 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”) (US 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 these citations is incorporated herein by reference in its entirety.

[0146] In some embodiments, the Fc domain extends the cyclic half-life by interacting with FcRn. In some embodiments, the substitution is selected from sites in the group consisting of: M428L and N434S (“LS”); M252Y and T256D (“YD”); T256D and T307Q (“DQ”); and T256D and T307W (“DW”).

[0147] Antibody conjugates This disclosure provides an antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof described in this disclosure, or the multispecific antibody or antigen-binding fragment thereof described in this disclosure.

[0148] This disclosure provides an ADC comprising an antibody or antigen-binding fragment thereof as described in this disclosure, or a multispecific antibody or antigen-binding fragment thereof as described in this disclosure, and a cytotoxic agent. In some embodiments, the antibody or antigen-binding fragment thereof is covalently linked to the cytotoxic agent directly or through a cleavable or incleavable linker.

[0149] In some embodiments, the cytotoxic agent can be any compound that causes or induces cell death or reduces cell viability in some way. Preferred cytotoxic agents include, for example, maytansine compounds and maytansine analogues, taxoids, taxanes (such as paclitaxel and docetaxel), CC-1065 and CC-1065 analogues, doralastatin and doralastatin analogues, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, chachiin, tubulolysin and tubulolysin analogues, ducamycin and ducamycin analogues, and taxanes (such as paclitaxel and docetaxel).

[0150] Blocking moieties, cleavable moieties This disclosure provides a blocking portion (BM) that inhibits or attenuates the binding of the antibody or antigen-binding fragment disclosed herein to CD3. In some embodiments, the BM comprises the sequence Y1DGY2Y3Y4Y5GY6ITQTPYKVSIS, wherein Y1-Y6 are each independently any amino acid residue. 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 of this disclosure comprises any one of SEQ ID NOs: 32-34.

[0151] This disclosure provides a cleavable portion (CM) that can be cleaved by a protease, comprising any one of the sequences in SEQ ID NOs: 36-44. In another aspect, this disclosure provides a cleavable portion (CM) that can be cleaved by a protease, comprising any one or more sequences in SEQ ID NOs: 45-53.

[0152] The CM may include a cutting point, or a cutting point with a connector.

[0153] Exemplary proteases may be selected from the following: ADAMS, ADAMTS, cysteine ​​protease, aspartic protease, KLK, serine protease, elastase, granzyme B, lactoferrin, aspartic cathepsin, MMP, marapsin, caspase, caspase, plasmin, PSA, tPA, thrombin, trypsin, uPA, and others. Exemplary proteases may also be selected from the following: ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1 (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, Chymotrypsin, Cruzipain, DESCI, DPP-4, FAP, Legumin, Otubain-2, Elastase, FVIIa, FIXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoatase, Hepsin, HtrA1, Human Neutrophil Elastase, KLK4 KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, lactoferrin, Marapsin, Matriptase-2, Meprin, MT-SP1 / Matriptase, enkephalin, 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, trypsin, and uPA.

[0154] Activatable antibody structures, multispecific antibody structures This disclosure provides an activatable antibody structure comprising an antibody or an antigen-binding fragment thereof (AB); a blocking portion (BM) that, when the activatable antibody is in an uncleaved state, inhibits or weakens the binding of the antibody or its antigen-binding fragment to a target antigen; and a cleavable portion (CM) that can be cleaved by a protease. In some embodiments, when the activatable antibody is in a cleaved state, the BM does not interfere with or compete for the binding of AB to the target antigen.

[0155] In some embodiments, the uncut, activatable antibody structure described in this disclosure is arranged from the N-terminus to the C-terminus as follows: BM-CM-AB or AB-CM-BM.

[0156] In some embodiments, the activatable antibody structure described herein comprises AB as described herein, and the target antigen is CD3. In some embodiments, the target antigen is CD3ε.

[0157] In some embodiments, the activatable antibody structure described herein comprises the BM described herein.

[0158] In some embodiments, the activatable antibody structure described herein comprises the CM described herein.

[0159] In some embodiments, the protease is selected from any one or more of the following: ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1. 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, Chymotrypsin, Cruzipain, DESCI, DPP-4, FAP, Legumin, Otubain-2, Elastase, FVIIa, FIXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoatase, Hepsin, HtrA1, Human Neutrophil Elastase, KLK4, KLK5, KLK 6. KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, lactoferrin, Marapsin, Matriptase-2, Meprin, MT-SP1 / Matriptase, enkephalin, 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, trypsin, and uPA, preferably MMP2, MMP14, and uPA. In some embodiments, the protease described in this disclosure is selected from any one or more of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA).

[0160] In some embodiments, the activatable antibody structure of this disclosure optionally includes a first linker (linker 1) between BM and CM, and / or optionally includes a second linker (linker 2) between CM and AB. In some embodiments, the activatable antibody structure in its uncut state of this disclosure is arranged from the N-terminus to the C-terminus as follows: BM-linker 1-CM-linker 2-AB or AB-linker 2-CM-linker 1-BM. In some embodiments, linker 1 or linker 2 of this disclosure is independent and 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)n 10 (GGGGSGGGS)n 11 Or a combination thereof, where n1-n 11 Each is independently any integer equal to or greater than 0. In some embodiments, the connector 1 described in this disclosure contains (GGGGSGGGS)n 11 Furthermore, the connector 2 described in this disclosure comprises (GGGGS)n4, wherein n 11 n4 is independent and is any integer equal to or greater than 0. In some embodiments, adapter 1 of this disclosure comprises GGGGSGGGS, and adapter 2 of this disclosure comprises GGGGS. In some embodiments, the activatable antibody structure of this disclosure comprises CM, said CM comprising any one of the sequences in SEQ ID NO: 57-65.

[0161] In some embodiments, the activatable antibody structure of this disclosure includes a variable region comprising SEQ ID NO: 54 and / or 56. In some embodiments, the activatable antibody structure of this disclosure includes a variable region comprising SEQ ID NO: 57 and / or 28. In some embodiments, the activatable antibody structure of this disclosure includes a variable region comprising SEQ ID NO: 60 and / or 28.

[0162] This disclosure provides a multispecific activatable antibody structure that, in an activated state, specifically binds to CD3 and at least one second target. In some embodiments, the multispecific activatable antibody structure of this disclosure comprises at least one of the following (a) or (b): (a) the activatable antibody structure of this disclosure as a first activatable antibody that specifically binds to CD3 in an activated state, and a second antibody or its antigen-binding fragment that specifically binds to the second target; (b) the activatable antibody structure of this disclosure as a first activatable antibody that specifically binds to CD3 in an activated state, and the activatable antibody structure of this disclosure as a second activatable antibody that specifically binds to 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 a GPC, preferably GPC3.

[0163] In some embodiments, the second activatable antibody of this disclosure includes a variable region comprising SEQ ID NO: 55. In some embodiments, the second activatable antibody of this disclosure includes a variable region comprising SEQ ID NO: 58 and / or 59.

[0164] In some embodiments, the second antibody or antigen-binding fragment thereof described in this disclosure includes a variable region comprising SEQ ID NO: 55. In some embodiments, the second antibody or antigen-binding fragment thereof described in this disclosure includes a variable region comprising SEQ ID NO: 58 and / or 59.

[0165] In some embodiments, the activatable antibody structure or multispecific activatable antibody structure described in this disclosure will be cleaved / activated in the tumor microenvironment, and thus provide the ability to reduce off-target toxicity that may result from antibody binding at random sites.

[0166] Isolated nucleic acids, vectors, host cells This disclosure provides an isolated nucleic acid comprising a polynucleotide sequence, said polynucleotide sequence encoding an antibody or antigen-binding fragment thereof as described in this disclosure, or a multispecific antibody or antigen-binding fragment thereof as described in this disclosure.

[0167] This disclosure provides an isolated nucleic acid comprising a polynucleotide sequence, said polynucleotide sequence comprising encoding the BM or CM described in this disclosure.

[0168] This disclosure discloses an isolated nucleic acid comprising a polynucleotide sequence, said polynucleotide sequence comprising encoding the activatable antibody structure or the multispecific activatable antibody described in this disclosure.

[0169] As used herein, the term "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. The nucleic acid can be a ribonucleotide or deoxyribonucleotide, or a modified form of any type of nucleotide. Modifications include base modifications, such as bromouridine and inosine derivatives; ribose modifications, such as 2',3'-dideoxyribose; and internucleotide bonding modifications, such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioate, phosphoraniladate, and aminophosphates.

[0170] The “isolated” nucleic acid described herein comprises a nucleotide sequence encoding the protein or a portion or fragment thereof described herein, and is a nucleic acid molecule identified and isolated from at least one contaminating nucleic acid molecule associated with its production environment. Preferably, the isolated nucleic acid is not associated with any component associated with the production environment. The isolated nucleic acid encoding the protein or a portion or fragment thereof described herein is in a form different from the form or state that exists in nature. Therefore, the isolated nucleic acid is different from the nucleic acid encoding the protein or a portion or fragment thereof that naturally exists in cells as described herein.

[0171] 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 refers to both double-stranded and single-stranded RNA and double-stranded and single-stranded DNA. It also includes polynucleotides modified by methylation and / or terminal modifications, as well as their unmodified forms. The polynucleotides described herein are not necessarily obtained by physical means, but can be produced by any method, including, for example, chemical synthesis or DNA replication, reverse transcription, or transcription.

[0172] This disclosure provides a vector containing the isolated nucleic acid described herein.

[0173] As used herein, the term "vector" refers to a nucleic acid molecule used as a medium to carry genetic material into another cell, where it can be replicated and / or expressed. In some embodiments, the vector is an 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 adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0174] This disclosure provides a host cell comprising the isolated nucleic acid or the vector described in this disclosure.

[0175] In some embodiments, the cells may be prokaryotic cells, fungal cells, yeast cells, or eukaryotic cells such as mammalian cells. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive microorganisms, for example: Enterobacteriaceae, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens and Shigella), Bacillus (e.g., Bacillus subtilis and Bacillus licheniformis), Pseudomonas (e.g., Pseudomonas aeruginosa), and Streptomyces. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0176] Pharmaceutical compositions This disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the multispecific antibody or antigen-binding fragment thereof described herein, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, the isolated nucleic acid, the vector or the host cell, and a pharmaceutically acceptable carrier or excipient.

[0177] As used herein, the term "pharmaceutical composition" refers to a mixture of the ingredients disclosed herein with other chemical components (such as carriers or excipients) compatible with pharmaceutical administration. The pharmaceutical composition facilitates the administration of the disclosed ingredients (such as antibodies or activatable antibodies disclosed herein) to a subject in need. Various methods of administration are known in the art, including but not limited to subcutaneous, intramuscular, oral, transdermal, parenteral, intravenous, intraperitoneal, intrathecal, pulmonary, nasal, ocular, systemic, and local administration.

[0178] The pharmaceutical compositions disclosed herein can be formulated into dosage forms suitable for administration to a subject via a desired route of administration. These dosage forms include, but are not limited to, tablets, capsules, pouches, pills, soft gels, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalers, creams, pastes, lotions, ointments, sprays, lyophilized formulations, injections, and gels.

[0179] As used herein, the term "pharmaceutically acceptable carrier or excipient" means a carrier or excipient that is compatible with the other components of the composition and substantially harmless to the recipient, and / or a carrier or excipient that has been approved or may be approved for inclusion in a pharmaceutical composition for parenteral administration to a subject. This 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 solution, glucose solution and 5% human serum albumin, liposomes, and non-aqueous media such as fixed oils.

[0180] Methods of treatment, pharmaceutical uses and others This disclosure provides a method for treating or preventing a condition by stimulating immune cells in a subject in need, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in this disclosure, the multispecific antibody or antigen-binding fragment thereof, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, or the pharmaceutical composition.

[0181] In some embodiments of the methods for treating or preventing diseases disclosed herein, the antibodies or antigen-binding fragments thereof, multispecific antibodies or antigen-binding fragments thereof, ADCs, activatable antibody structures, multispecific activatable antibody structures, or pharmaceutical compositions described herein are administered in combination with other therapeutic agents. In some embodiments, the other therapeutic agents are selected from antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the other therapeutic agents are selected from, for example, Bruton's tyrosine kinase (BTK) inhibitors, tyrosine kinase inhibitors (TKIs), PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, EGFR inhibitors, VEGF inhibitors, PARP inhibitors, Her2, LAG-3, and TNFR2 inhibitors.

[0182] In some embodiments of the methods for treating or preventing diseases described in this disclosure, the disease is selected from tumors. In some embodiments, the disease 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, and osteosarcoma.

[0183] This disclosure provides a method for diagnosing CD3-related conditions, comprising exposing a subject's sample to the antibody or antigen-binding fragment thereof of this disclosure, the multispecific antibody or antigen-binding fragment thereof, the activatable antibody structure, or the multispecific activatable antibody structure.

[0184] This disclosure provides the use of the antibody or antigen-binding fragment thereof, the multispecific antibody or antigen-binding fragment thereof, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, or the pharmaceutical composition thereof in the preparation of a medicament for treating or preventing a condition that is improved by stimulating immune cells.

[0185] This disclosure provides the use of the antibodies or antigen-binding fragments thereof described herein, the multispecific antibodies or antigen-binding fragments thereof, the activatable antibody structures, or the multispecific activatable antibody structures in the preparation of products (such as kits) for diagnosing CD3-related conditions.

[0186] As used herein, the term “treatment” means to reduce or improve a disease or disorder (i.e., to slow or stop the progression of the disease or at least one clinical symptom); or to reduce or improve at least one physical parameter or biomarker associated with a disease or disorder.

[0187] As used in this article, the term "prevention" includes providing preventative measures for the onset or recurrence of a disease in individuals who may be susceptible to the disease but have not yet been diagnosed with it.

[0188] As used herein, the term “symptom” refers to any alteration in the state of the body or organs that interrupts or interferes with the performance of function and / or causes symptoms (e.g., discomfort, dysfunction, pain, or even death) in a subject who is ill or in contact with the patient.

[0189] As used herein, the term "therapeutic effective amount" means that, when administered to a subject who has not received any treatment, such an amount of therapeutic agent shows a beneficial or therapeutic effect on the disease, and that the amount is sufficiently low within a reasonable medical judgment to avoid serious side effects. The therapeutically effective amounts of antibodies, activatable antibodies, and other therapeutically effective amounts described in this disclosure will vary at least due to the following factors: route of administration; severity of the disease being treated; patient's age, height, weight, and physical condition; patient's medical history; duration of treatment; and desired therapeutic effect. However, those skilled in the art can determine the therapeutically effective amount using conventional methods.

[0190] This disclosure provides methods for inhibiting or attenuating the binding of the antibody or antigen-binding fragment thereof described herein to CD3, including using a masking membrane (BM) to mask the binding site of the antibody or antigen-binding fragment thereof. In some embodiments, the method includes linking the antibody or antigen-binding fragment thereof described herein to the BM via a protease-cleavable cleavable membrane (CM). In some embodiments, the BM is any of those disclosed herein. In some embodiments, the BM comprises Y1DGY2Y3Y4Y5GY6ITQTPYKVSIS, wherein Y1-Y6 are independently 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; and 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 having 1, 2, or 3 conservative substitutions; (b) SEQ ID NO: 33 or SEQ ID NO: 33 having 1, 2, or 3 conservative substitutions; (c) SEQ ID NO: 34 or SEQ ID NO: 34 having 1, 2, or 3 conservative substitutions. In some embodiments, the BM comprises any one of SEQ ID NOs: 32-34.

[0191] In some embodiments, the CM can be any of those described in this disclosure, preferably comprising one or more cleavage sites selected from Table 7. In particular, the CM can comprise any of the sequences shown in SEQ ID NOs: 45-53.

[0192] This disclosure provides a method for ligating an antibody or antigen-binding fragment thereof described herein to a CM, comprising optionally ligating the antibody or antigen-binding fragment thereof to a protease cleavage site via one or more adapters. In some embodiments, the CM may be any of those disclosed herein. In some embodiments, the CM includes a cleavage site and an adapter. In some embodiments, the cleavage site may be any one or more of those disclosed herein. In some embodiments, the adapter may be any of those disclosed herein. Exemplary cleavage sites and adapters are shown in Table 7.

[0193] For those skilled in the art, other suitable modifications and adjustments to this application described herein will be obvious without departing from the scope of this disclosure or its embodiments, and can be implemented using suitable equivalents, all of which fall within the scope of obviousness.

[0194] Example The following embodiments (including actual embodiments and predictive embodiments) and the accompanying drawings are intended to illustrate specific implementations or features of the present invention, and are not intended to limit the scope of the present invention.

[0195] Those skilled in the art will understand that various modifications and changes can be made to the embodiments described herein without departing from the spirit and scope of this disclosure. For the sake of brevity, descriptions of functions and structures that are common knowledge in the art are omitted below.

[0196] Unless otherwise stated, the experimental methods used in the following embodiments are all conventional methods and can be carried out in accordance with the technical conditions or product instructions described in the literature in this field; unless otherwise stated, the materials and reagents used in the following embodiments are all commercially available.

[0197] Example 1. Generation of anti-CD3 antibody Balb / c mice were immunized with human CD3ε / δ-Fc (Acrobiosystems, Cat No: CDD-H5255) for 8-10 weeks to induce anti-CD3 antibodies. Starting from week 3 after the initial immunization, anti-CD3 antibody titers were monitored weekly by ELISA and flow cytometry. After a total of four immunizations, mice with high titers were sacrificed, bone marrow samples were collected and mixed, and CD138 was enriched using a kit (Miltenyi). + Plasma cells. Single-cell suspensions were loaded onto a Beacon 14K chip (Berkeleylights) and single B-cell screening was performed using CD3ε-coated beads (Acrobiosystems, Cat No: CDE-H5223). Positive single cells were transferred to 96-well plates via lysis buffer and... Frozen at 80°C for subsequent single-cell sequencing. The DNA sequence encoding the antibody was determined by Sanger sequencing (Sanger, 1977). The amino acid sequences of representative dominant clones BPCD3-1, BPCD3-2, BPCD3-14, and BPCD3-17 (VH and VL) are listed in Table 1. Their CDR sequences are listed in Table 2.

[0198] Table 1. Amino acid sequences of representative clones

[0199] Table 2. CDR sequences of representative clones

[0200] Example 2. ELISA Binding Assay CD3 antigen was immobilized onto a 96-well high-binding ELISA plate by adding 100 μl of 1 mg / ml human CD3ε (Acro Biosystems, Cat No: CDE-H5223) or cynomolgus monkey CD3ε (Acro Biosystems, Cat No: CDE-C5226) to the wells in PBS (pH 7.4) and incubating overnight at 4°C. The wells were then blocked for 1 h at room temperature with blocking buffer (PBS containing 0.05% Tween® 20 + 2% BSA). After washing three times with 300 μl of wash buffer (PBS containing 0.05% Tween® 20), the antibodies (BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17) and the positive control antibody SP34 (Pessano, S., Oettgen, H., Blan, AK & Terhorst, C. (1985) EMBO J.4, 337-344; US2015 / 0166661A) were serially diluted with dilution buffer (PBS containing 0.05% Tween® 20 + 2% BSA) and incubated at room temperature for 1 h. Then, 100 μl of HRP-conjugated anti-human antibody (Jackson Immuno Research) was added to the plate and incubated in blocking buffer at room temperature for 1 h. The wells were then washed three times with washing buffer and treated with 100 μl of TMB substrate (Innoreagent, Cat No: TMB-S-001) for 10 minutes. The reaction was then terminated by adding 50 μl of ELISA stop solution (Solarbio, Cat No: C1058), and the absorbance of each well at 450 nm was immediately read using a multimode microplate reader (ENVISION, PerkinElmer).

[0201] like Figure 1 As shown, the binding properties of antibodies BPCD3-1, BPCD3-2, BPCD3-14, and BPCD3-17 to human CD3ε are similar to those of SP34.

[0202] like Figure 2 As shown, the binding properties of antibodies BPCD3-1, BPCD3-2, BPCD3-14, and BPCD3-17 to CD3ε in cynomolgus monkeys are similar to those of SP34.

[0203] Example 3. CD3 mAb-induced T cell activation To verify the agonistic effect of monoclonal antibodies (mAbs) on CD3, the THP-1 / Jurkat-NFAT reporter system (purchased from Vazyme, DD1302-1) was used to determine antibody function. When Jurkat-NFAT cells were co-cultured with THP-1 cells and CD3 mAb, the T cell receptor (TCR) signaling pathway was activated, leading to NFAT transcription and mediating luciferase expression.

[0204] In short, THP-1 cells (2 × 10⁻⁶) 4 / well) and Jurkat-NFAT cells (4×10⁶) 4 / well) were co-cultured at 37°C in 96-well plates containing serially diluted mAbs of BPCD3-1, BPCD3-2, BPCD3-14, and BPCD3-17 (total volume 150 μl / well). After 4 hours of incubation, bright-glo (Beyotime, Cat No: RG051M) was added to the corresponding wells, and the luminescence values ​​were read using Envision (PerkinElmer). SP34 was used as a positive control, and human IgG1 (hIgG1) (Beyotime, Cat No: B117901) was used as a negative control. Luminescence readings are expressed as RLU (relative light units), and EC50 was calculated (see results in [link to results]). Figure 3 ).like Figure 3 As shown, Jurkat-NFAT cells can be activated by CD3 mAb.

[0205] Example 4. Structure of CD3 mAb Because the aforementioned CD3 mAbs possess similar affinities, the structures of their CDRs were analyzed. Structural simulations of the variable regions (Fv) of four CD3 mAbs (BPCD3-1, BPCD3-2, BPCD3-14, and BPCD3-17) were performed using MOE software (Molecular Operation Environment, 2022, Chemical Computing Group). Figure 4 As shown, all four mAbs have highly similar structures.

[0206] Example 5. Humanization of mouse anti-human CD3 mAb To humanize BPCD3-2, through NCBI ( http: / / www.ncbi.nlm.nih.gov / igblast / The human immunoglobulin gene database was compared on the website to search for human germline IgG gene sequences that were highly homologous to the amino acid sequence of the variable region of BPCD3-2. Human IGVH and IGVL genes, which frequently appear in human antibody libraries (Glanville 2009 PNAS 106:20216-20221) and are highly homologous to BPCD3-2, were selected as humanization templates.

[0207] Humanization was achieved through complementarity-determining region (CDR) transplantation (Methods in Molecular Biology, Vol248: Antibody Engineering, Methods and Protocols, Humana Press), and humanized antibodies (huBPCD3-1, huBPCD3-2) were constructed in the form of human IgG1 (hIgG1) using the pCDNA3.4 vector (Thermo Fisher, Cat No: A14697). The vector contains cDNA (GeneWiz) encoding the human κ constant light chain domain or the human IgG1 heavy chain.

[0208] The amino acid sequences and CDR sequences of the humanized clones are listed in Tables 3 and 4, respectively.

[0209] Table 3. Humanized Cloning VH and VL amino acid sequence of the region

[0210] Table 4. Humanized Cloning CDR amino acid sequence of the region

[0211] Example 6. CD3 binding of CD3 mAb by flow cytometry Pre-expanded CD3-positive Jurkat cells were adjusted to 2 × 10⁻⁶. 6At a density of 1 / ml, 100 μl of cells were added to each well of a 96-well plate. After centrifugation at 300×g for 5 minutes, the growth medium was discarded. Anti-CD3 antibody was serially diluted 5-fold from 100 nM to different concentrations using FACS buffer (DPBS + 2% FBS). 100 μl of the prepared BPCD3-2, huBPCD3-1, huBPCD3-2, and SP34 dilutions were added to each well. After incubation at 4°C for 1 hour, the cells were washed. 100 μl of AF647-labeled anti-human IgG Fc secondary antibody (Jackson, Cat No: 109-605-098, dilution 1:1000) was added, and the cells were incubated for 30 min. Each cell pellet was resuspended in 200 μL of FACS buffer. The cell suspension was analyzed by flow cytometry (Thermo, Attune Nxt). The EC50 value was calculated based on the mean fluorescence intensity (MFI).

[0212] like Figure 5 As shown, antibodies against BPCD3-2, huBPCD3-1, huBPCD3-2, and SP34 all exhibited binding activity to Jurkat cells. Among them, the humanized antibodies huBPCD3-2 (EC50=0.027) and huBPCD3-1 (EC50=0.032) were superior to SP34 (EC50=0.05).

[0213] Example 7. Affinity determination by BLI Biolayer interferometry (BLI) analysis was performed at room temperature using an Octet RH96 device purchased from Sartorius. For all assays, a PBST buffer consisting of 10 mM PBS, pH 7.4, 0.05% Tween® 20, and 0.1% BSA was used as the run buffer. BPCD3-2 and huBPCD3-2 were directly captured onto the AHC (anti-human IgG Fc capture) sensor at a concentration of 10 μg / mL. His-tagged human CD3ε was then bound to the Fc-tagged antibody at gradient concentrations (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 nM, represented by binding / dissociation curves of different colors), followed by dissociation with the run buffer. After each cycle, the sensor surface was regenerated with 0.1 M glycine (pH 1.5). All binding kinetic data were analyzed using a 1:1 steady-state affinity model in OctetAnalysis Studio software (version 12.2.2.26). The response signal is measured as nanometer (nm) level optical interference offset values ​​and is recorded in real time and displayed on the sensor map. The equilibrium dissociation constant (K0) D By calculating the dissociation rate constant (k) off ) and binding rate constant (kon The ratio of ) is obtained, and the results are listed in Table 5.

[0214] Table 5. Comparison of antibody binding affinity by BLI

[0215] Example 8. Blocking assay of anti-CD3 antibody with blocking motif The blocking moiety described in this disclosure comprises amino acid residues 1-13 (AA) based on the human CD3ε sequence. The blocking moiety acts as a “shield” to block the binding of CD3 mAb to CD3ε. The blocking activity of the CD3 blocking moiety is determined by the binding of anti-CD3 mAb to the blocking moiety during incubation with CD3-positive Jurkat cells (ATCC). In short, serially diluted peptides BM-1, BM-2, BM-3, BM-4, and BM-5 (synthesized from a peptide biochemical agent) are mixed with 30 ng of anti-CD3 mAb (BPCD3-1, BPCD3-2, BPCD3-14, BPCD3-17, or SP34) at 4°C for 30 min, followed by co-incubation with CD3-positive Jurkat cells stained with goat anti-human IgG conjugated to the secondary antibody Alexa Fluor 647 (Jackson Immunoresearch, 109-605-098). Immunofluorescence is detected by flow cytometry (Attune Nxt, Thermo). The x-axis represents the peptide concentration, and the y-axis represents the mean fluorescence intensity (MFI). The half-maximal inhibitory concentration (IC50) is calculated based on the MFI.

[0216] Figure 6 The results of the blocking assay for the blocking component and the anti-CD3 antibody BPCD3-2 were described. For example... Figure 6 As shown, all selected blocking components blocked the binding of BPCD3-2 to Jurkat cells. BM-3 showed the most potent blocking activity (IC50 of 0.105 μM), which was 2.75 times more potent than the WT peptide (BM-1).

[0217] Figure 7 (AC) shows the blocking assays of the blocking components (BM-2, BM-3, and BM-4) with different anti-CD3 antibodies. Figure 7A As shown in -C, BM-2, BM-3, and BM-4 peptides can all block the binding of different anti-CD3 mAb to CD3+Jurkat cells.

[0218] Table 6. Anti-CD3 blocking component (BM)

[0219] Example 9. Determination of the blocking component and the blocking effect of anti-CD3 mAb huBPCD3-2 In this assay, serially diluted blocking fraction BM-3 (biochemically synthesized from a medium peptide) starting at 50 μM was mixed with anti-CD3 mAb huBPCD3-2 (final concentration 0.2 μg / mL). The antibody-peptide mixture was then added to Jurkat cells (103) placed in U-bottom 96-well plates on ice. 5 Cells were washed for 30 minutes in each well (1:1000) and then washed a total of 3 times. The bound primary antibody was captured by anti-human IgG Fc secondary antibody AF647 (1:1000) for 30 minutes, followed by 3 washes. Cell samples were then analyzed by flow cytometry. The x-axis represents peptide concentration, and the y-axis represents MFI (mean fluorescence intensity). IC50 values ​​were calculated from the MFI.

[0220] like Figure 8 As shown, BM-3 blocks the binding of huBPCD3-2 to Jurkat cells.

[0221] Example 10. Screening for protease-sensitive cleavage sites Antibody prodrugs / activatable antibodies are constructs composed of an antibody, a blocking moiety, and a cleavable moiety, for example, such as... Figure 9 As shown in Table 7, in protease-rich sites (such as the tumor microenvironment), antibody prodrugs / activatable antibodies become sensitive to protease cleavage and then become active antibodies or post-cleavage antibodies. The cleavable portions include protease-sensitive cleavage sites as shown in Table 7 below.

[0222] Table 7. Cleavable moieties (CM) with protease-sensitive cleavage sites

[0223] The binding affinity of the prodrugs after MMP digestion was tested. BM-1 and SP34 antibodies and different cleavage fragments listed in Table 7 were used to construct the prodrugs. Uncleavable linkers (NCLs) were mimics containing only BM-1 and SP34 without cleavage fragments. All prodrugs were expressed in host cell CHO. The prodrugs (100 nM) were digested with 0.25 μg / ml MMPs in digestion buffer at 37°C for 20 h (overnight). Jurkat cells (1 × 10⁻⁶) were then digested. 5 Jurkat cells (cells / well) were placed in PBS with 2% FBS added. The cells were incubated with pre-digested / undigested prodrugs (100 nM). AF647-labeled anti-human IgG Fc (Jackson Immuno Research, 1:1000 dilution) was added. Immunofluorescence was detected by flow cytometry (Attune Nxt, Thermo). Binding capacity was calculated based on MFI: MFI of sample / MFI of SP34.

[0224] As shown in Table 8, the binding affinity of the prodrug to CM-8 was extremely low, indicating that CM-8 exhibits better stability during expression and is not digested by host cell proteases. Treatment with MMP14 significantly increased the binding affinity of the prodrug to CM-8, suggesting that the prodrug containing CM-8 can be cleaved by MMP14 and exhibit binding activity.

[0225] Table 8. Binding capacity of prodrugs

[0226] Example 11. Construction of the prodrug In this disclosure, the prodrug (PRD) is a bispecific antibody comprising Fab 1 and Fab 2. The prodrug (PRD) is a bispecific antibody comprising a first antigen-binding arm (Genentech or huBPCD3-2) that specifically binds to CD3 and a second antigen-binding arm that specifically binds to GPC3. The prodrug further integrates a cleavage portion (CM-8 or CM-12) and a CD3 blocking portion (BM-1 or BM-3), which allows the prodrug to remain substantially inactive in the absence of protease cleavage; at protease-rich sites (such as the tumor microenvironment), the prodrug is cleaved into the active drug by protease-sensitive reactions. The prodrug components are listed in Table 9. The sequence of the variable region (Fv) of the prodrug (CD3×GPC3) is shown in Table 10.

[0227] Table 9. Prodrug Composition

[0228] Table 10. Amino acid sequences of antibody prodrugs

[0229] Example 12. In the reporter assay, the prodrug exhibited a significant response window with and without MMP treatment. The Jurkat-NFAT-luciferase reporter cell line (Vazyme DD1302-1), with NFAT-driven luciferase expression, was used to determine T cell activation when co-cultured with the prodrug (pretreated with protease, versus without protease pretreatment) and GPC3-positive HepG2 cells (Meisen cells). Under protease pretreatment conditions, the prodrug was resuspended overnight for 20 hours at 37°C in reaction buffer (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 6.6) and 5 units of MMP14 (Abcam, Cat#:8009). Before co-culturing the prodrug with the reporter cell line, HepG2 cells (5 × 10⁶ cells) were... 4Jurkat-NFAT-luciferase cells (2.5 × 10⁶ cells) were seeded in 96-well plates and cultured overnight. In co-culture assays, Jurkat-NFAT-luciferase cells (2.5 × 10⁶ cells) were seeded in 96-well plates and cultured overnight. 5 Pre-inoculated HepG2 cultures were added, along with serially diluted prodrugs (protease-treated and unprotease-treated), and co-cultured at 37°C for 6 hours. Luciferase activity (luminescence) was determined by adding the substrate Bio-glo (Beyotime, Cat No: RG051M) and detecting it using an ELISA reader (EnVision 2105, Perkin Elmer).

[0230] like Figure 10 As shown, the PRD-1 prodrug exhibited a significant "response window" with and without MMP treatment. Figure 10 (Illustrated by double arrows in the middle), the cut portion of CM-8 can exert its effect on the prodrug.

[0231] Example 13. Binding ability of prodrug to Jurkat cells In protease treatment, the prodrug was resuspended in reaction buffer (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 6.6), and 5 units of MMP2 (R&D Systems, Cat No: 902-MP-010) and MMP14 (Abcam, Cat#: 8009), or MMP2, MMP14, and uPA (Acrobiosystem, Cat#: PLU-H5228) were added and incubated at 37°C for 20 hours. Subsequently, the prodrug treated with protease was diluted with FACS buffer (DPBS + 2% FBS) and CD3 was treated in a similar manner to that in Example 12. + Jurkat cells (2 × 10) 5 Staining is performed. For example... Figure 11A As shown, the prodrugs (PRD-4 and PRD-5) treated with the protease exhibited significant binding to Jurkat cells. Conversely, without protease treatment, PRD-4 and PRD-5 showed extremely low levels of background staining.

[0232] Example 14. Binding of antibody prodrug to HEPG2 cells To verify whether the binding ability of another Fab was affected, flow cytometry was used to bind it to HEPG2 cells. The pre-expanded HEPG2 cells were adjusted to 2 × 10⁶ cells / cells. 4Cells were collected at a density of 100 μl / well and added to 96-well plates. Anti-CD3 antibody was serially diluted 5-fold from 100 nM using FACS buffer (DPBS + 2% FBS). Cells were incubated at 4°C for 1 hour, followed by washing at least twice with 200 μL of pre-chilled (4°C) FACS buffer, discarding the supernatant. After 1 hour, 100 μl of AF647-labeled anti-human IgG Fc secondary antibody (Jackson, Cat No: 109-605-098, 1:1000 dilution) was added, and incubation was continued for 30 min. The cell pellet was then resuspended with gentle agitation. After three repetitions, each cell pellet was resuspended with 200 μL of FACS buffer. Flow cytometry (Thermo, Attune Nxt) was used to analyze the cell suspensions. The x-axis represents antibody concentration, and the y-axis represents mean fluorescence intensity (MFI). EC was calculated based on the MFI values. 50 Values. Both PRD-4 and PRD-5 showed binding comparable to HepG2 cells (GPC3 positive). Figure 11B ).

[0233] Example 15. Activity of CD3 prodrug in reporter assay The JurkatNFAT-luciferase reporter cell line (Vazyme, Cat No: DD1302-1), based on Jurkat and exhibiting NFAT-driven luciferase expression, was used to determine T cell activation when the prodrug PRD-4 or PRD-5 (pretreated with protease, versus untreated) was co-cultured with GPC3-positive HepG2 cells (Meisen cells). Under protease pretreatment conditions, the prodrug was resuspended for 20 hours at 37°C in reaction buffer (50 mM Tris, 3 mM CaCl2, 1 μM ZnCl2, pH 6.6) and 5 units of MMP2 (R&D Systems, Cat No: 902-MP-010) with MMP14 (Abcam, Cat No: 8009); or MMP2, MMP14, and uPA (Acrobiosystem, Cat No: PLU-H5228). Before co-culturing the prodrug with the reporter cell line, HepG2 cells (5 × 10⁶) were... 4 Jurkat-NFAT-luciferase cells (2.5 × 10⁶ cells) were seeded in 96-well plates and cultured overnight. In co-culture assays, Jurkat-NFAT-luciferase cells (2.5 × 10⁶ cells) were seeded in 96-well plates and cultured overnight. 5Pre-inoculated HepG2 cultures were added, along with serially diluted prodrugs PRD-4 or PRD-5 (pre-treated and untreated proteins), and co-cultured at 37°C for 6 hours. Luciferase activity (luminescence) was determined by adding the substrate Bio-glo (Beyotime, Cat No: RG051M) and detecting it using an ELISA reader (EnVision 2105, Perkin Elmer).

[0234] As shown in the report ( Figure 12 After pretreatment with proteases (MMP2 / MMP14 / uPA), both PRD-4 and PRD-5 exhibited significant T cell activation activity in HepG2 co-cultures with reporter cells, with their EC50... 50 The values ​​are 0.43 nM and 0.46 nM, respectively.

[0235] Example 16. Activity of CD3 prodrug in PBMC assay To further determine the efficacy of CD3 prodrugs using human peripheral blood mononuclear cells (PMBCs), frozen PMBCs (10 5 / hole, from a healthy donor) and HepG2 (10 4 The cultures were co-cultured for 24 hours in the presence of either serially diluted protease pretreatment or no prodrug (as described in Example 6). Cytokines (IFN-γ, IL-2) in the culture supernatant were measured by ELISA (Thermo, Cat No: 88-7025-88; Thermo, Cat No: 88-7316-86). Figure 13 As shown, the untreated prodrug induced only very low production of background cytokines, especially IFN-γ. In contrast, the prodrug treated with the protease exhibited highly efficient and potent cytokine production (ECG). 50 In IL-2 production: ~0.02 nM; EC 50 In IFN-γ production: ~0.25 nM). The activity window of protease treatment is more than 100-fold compared to the untreated prodrug. Cytokine secretion indicates that the untreated prodrug is blocked, and PBMCs are activated by the protease-treated prodrug.

[0236] Example 17. Serum stability test of the prodrug.

[0237] Human blood has a complex environment and contains a variety of enzymes. To ensure that prodrugs can play a better role in the tumor microenvironment, it is necessary to evaluate their stability in serum.

[0238] Fresh human serum was isolated from healthy donors and then diluted to different concentrations (5%, 10%) with DPBS. Undiluted serum was set as 100%, and DPBS alone served as a blank control (0%). The prodrugs PRD-4 and PRD-5 (1000 nM) were then mixed with the serum at different concentrations and incubated at 37°C for 20 hours (overnight). Figure 14 As shown. After incubation, the mixture was diluted 10-fold with FACS buffer (DPBS + 2% FBS) to reduce the working concentration to 100 nM. As described above, Jurkat cells (10 5 Cells / well were stained with serum-treated prodrugs in 96-well V plates at 4°C for 1 hour, followed by three washes, and then stained with PE-labeled anti-human IgG Fc secondary antibody (Jackson ImmunoResearch, Cat No: 109-115-098). Analysis was performed using flow cytometry. Figure 14 The samples shown are labeled SP34 as a positive control. Human IgG1 (Beyotime, Cat No: B117901) represents human IgG1 and is used as a negative control. NC consists of Jurkat cells with fresh human serum and a secondary antibody, without prodrug treatment. The binding efficiency of Jurkat cells was calculated using the prodrug as follows: Jurkat cell binding efficiency = (MFI of sample - MFI of negative control) / (MFI of positive control - MFI of negative control) Figure 14 The results showed that PRD-4 and PRD-5 had good serum stability.

[0239] Example 18. Antitumor activity of the prodrug in vivo We next determined the in vivo activity of the 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 control antibody CX-PC4 (anti-CD3×GPC3) was constructed. CX-PC4 contains a first antigen-binding arm that specifically binds to CD3 and a second antigen-binding arm that specifically binds to GPC3. The first antigen-binding arm is an scFv linked to the BM (blocking moiety) and CM (cleavable moiety), the sequences of which have been disclosed in the prior art (WO2019213444A1). The variable regions of CX-PC4 are listed in Table 12. PRD-AC is PRD-4 without BM and CM, and it maintains its activity.

[0240] In short, on day 0 (i.e., the tumor inoculation day, defined as D0), the human liver cancer-derived cell line huh7 (Procell, Cat No: cl-0120) was inoculated with 50% substrate gel (Absin) at a rate of 5 × 10⁶ cells per mouse.6 A dose of [number] cells was subcutaneously injected into the shoulder of NOG mice (female, 7-8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.). On day 3, the mice were randomly divided into six groups of 7 mice each, at which point the tumor volume was perceptible (~50 mm). 3 -100 mm 3 The treatment groups and dosages are shown in Table 11. On day 4, each mouse was injected intravenously with 1×10⁻⁶ mg / L of the drug. 7 Human PBMCs (Milestone Biotechnologies) were administered the drug starting on day 5. Animals received the drug intraperitoneally once weekly for 3 weeks (dosage as described above), with tumor volume and body weight recorded 2-3 times weekly. Tumor volume (TV) was calculated using the formula TV (mm). 3 =LxW 2 The value is calculated as / 2, where L is the long axis of the tumor and W is the short axis of the tumor.

[0241] In vivo experimental data showed that the prodrug possessed significant antitumor activity. For example... Figure 15A-15B As shown, in a PBMC-reconstructed NOG mouse xenograft tumor model, PRD-AC treatment induced tumor regression in a dose-dependent manner. Similarly, the masked prodrugs PRD-4 and PRD-5 also significantly inhibited tumor growth.

[0242] Table 11. Treatments and dosages for each group

[0243] Table 12. Sequence of the CX-PC4 chain

Claims

1. An antibody that specifically binds to CD3 or an antigen-binding fragment thereof, comprising: HCDR1, wherein HCDR1 comprises SYWMH (SEQ ID NO: 9) having 0, 1, 2, or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises NIYPX1SX2X3TNYDEX4FKS (SEQ ID NO: 24) having 0, 1, 2, or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises DX5X6GNYYFDY (SEQ ID NO: 25) having 0, 1, 2, or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises KSSQSLLNX7RTRKX8YLA (SEQ ID NO: 23) having 0, 1, 2, or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises WASTRES (SEQ ID NO: 12) having 0, 1, 2, or 3 conserved substitutions; and LCDR3, wherein LCDR3 comprises TQSX9X having 0, 1, 2, or 3 conserved substitutions. 10 LRT (SEQ ID NO: 26), where X 1-10 It can be any amino acid residue on its own.

2. The antibody or antigen-binding fragment thereof according to 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, X 10 It is either I or S.

3. The antibody or antigen-binding fragment thereof according to 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.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, wherein the HCDR 1-3 and LCDR 1-3 are selected from any one of the following (a)-(d) having 0, 1, 2 or 3 conserved substitutions: (a) The HCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 9, 11 and 13, and the LCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 10, 12 and 14; (b) The HCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 9, 16 and 17, and the LCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 15, 12 and 18; (c) The HCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 9, 11 and 13, and the LCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 19, 12 and 14; and (d) The HCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 9, 20 and 21, and the LCDR 1-3 respectively contain the amino acid sequences of SEQ ID NO: 15, 12 and 22.

5. An antibody that specifically binds to CD3 or an antigen-binding fragment thereof, comprising VH and / or VL, wherein the VH and / or VL is selected from any one of (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 with 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 with 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 with 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 with 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 with 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 with SEQ ID NO: 6; and (d) The VH contains an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 7, and / or the VL contains an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:

8.

6. The antibody or antigen-binding fragment thereof according to 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.

7. The antibody or antigen-binding fragment thereof according to 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.

8. The antibody or antigen-binding fragment thereof according to 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.

9. The antibody or antigen-binding fragment thereof according to 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.

10. An antibody that specifically binds to CD3 or an antigen-binding fragment thereof, comprising any one of the following (a)-(f) having 0, 1, 2 or 3 conserved substitutions: (a) The three CDRs of VH as shown in SEQ ID NO: 1 and the three CDRs of VL as shown in SEQ ID NO: 2; (b) The three CDRs of VH as shown in SEQ ID NO:3 and the three CDRs of VL as shown in SEQ ID NO:4; (c) The three CDRs of VH as shown in SEQ ID NO: 5 and the three CDRs of VL as shown in SEQ ID NO: 6; (d) The three CDRs of VH as shown in SEQ ID NO: 7 and the three CDRs of VL as shown in SEQ ID NO: 8; (e) The three CDRs of VH as shown in SEQ ID NO: 27 and the three CDRs of VL as shown in SEQ ID NO: 28; (f) The three CDRs of VH as shown in SEQ ID NO: 29 and the three CDRs of VL as shown in SEQ ID NO:

28.

11. An antibody that specifically binds to CD3 or an antigen-binding fragment thereof, comprising: HCDR1, wherein HCDR1 comprises SYWMH (SEQ ID NO: 9) having 0, 1, 2, or 3 conserved substitutions; HCDR2, wherein HCDR2 comprises NIYPGSGSTNYDEKFKS (SEQ ID NO: 16) or NIYPGSGSTNYAQKFQG (SEQ ID NO: 30) having 0, 1, 2, or 3 conserved substitutions; HCDR3, wherein HCDR3 comprises DQYGNYYFDY (SEQ ID NO: 17) having 0, 1, 2, or 3 conserved substitutions; LCDR1, wherein LCDR1 comprises KSSQSLLNSRTRKNYLA (SEQ ID NO: 15) having 0, 1, 2, or 3 conserved substitutions; LCDR2, wherein LCDR2 comprises WASTRES (SEQ ID NO: 9) having 0, 1, 2, or 3 conserved substitutions. NO: 12); and LCDR3, wherein the LCDR3 comprises TQSYILRT (SEQ ID NO: 18) having 0, 1, 2 or 3 conservative substitutions.

12. An antibody that specifically binds to CD3 or an antigen-binding fragment thereof, comprising VH and / or 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 with 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 with SEQ ID NO:

28.

13. The antibody or antigen-binding fragment thereof according to 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.

14. A multispecific antibody or an antigen-binding fragment thereof comprises a first antibody or an antigen-binding fragment thereof (AB1) that specifically binds to CD3 and at least one second antibody or an antigen-binding fragment thereof (AB2) that specifically binds to a second target, wherein AB1 comprises any one of the antibodies or antigen-binding fragments thereof of claims 1-13.

15. The multispecific antibody or its antigen-binding fragment according to claim 14, wherein the second target is a tumor-associated antigen.

16. The multispecific antibody or antigen-binding fragment thereof according to 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.

17. The multispecific antibody or antigen-binding fragment thereof according to claim 14 or 15, wherein the second target is GPC, preferably GPC3.

18. An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof of any one of claims 1-13, or the multispecific antibody or antigen-binding fragment thereof of any one of claims 14-17.

19. The blocking part (BM) contains the sequence Y1DGY2Y3Y4Y5GY6ITQTPYKVSIS, wherein Y1-Y6 are any one of the amino acid residues.

20. The blocking portion according to 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; and Y6 is G or S.

21. The blocking portion of claim 20, comprising any one of SEQ ID NOs: 32-34.

22. A cleavable portion (CM) that can be cleaved by a protease, comprising any one or more sequences selected from SEQ ID NOs: 36-44.

23. The CM of claim 22, comprising any one or more sequences selected from SEQ ID NOs: 45-53.

24. An activatable antibody structure comprising an antibody or an antigen-binding fragment thereof (AB); a blocking portion (BM) that, when the activatable antibody is in an uncleaved state, inhibits or weakens the binding of the antibody or the antigen-binding fragment thereof to a target antigen; and a cleavable portion (CM) that can be cleaved by a protease.

25. The activatable antibody structure of claim 24, wherein the activatable antibody structure in the uncleaved state has the following structural arrangement from the N-terminus to the C-terminus: BM-CM-AB or AB-CM-BM.

26. The activatable antibody structure according to claim 24 or 25, wherein the antibody or its antigen-binding fragment is the antibody or its antigen-binding fragment according to any one of claims 1-13, and the target antigen is CD3.

27. The activatable antibody structure according to any one of claims 24-26, wherein the BM is the BM according to any one of claims 19-21.

28. The activatable antibody structure according to any one of claims 24-27, wherein the CM is the CM according to claim 22 or 23.

29. The activatable antibody structure according to any one of claims 24-28, wherein the protease is selected from any one or more matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA).

30. The activatable antibody structure of claim 29, wherein the protease is selected from any one or more of MMP2, MMP14, and uPA.

31. The activatable antibody structure according to any one of claims 24-30, optionally comprising a first linker (linker 1) between BM and CM, and / or optionally comprising a second linker (linker 2) between CM and AB.

32. The activatable antibody structure according to claim 31, wherein the activatable antibody in the uncut state has the following structural arrangement from the N-terminus to the C-terminus: BM-connector 1-CM-connector 2-AB or AB-connector 2-CM-connector 1-BM.

33. The activatable antibody structure according to 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.

34. A multispecific activatable antibody structure that, in an activated state, specifically binds to CD3 and at least one second target, wherein the multispecific activatable antibody structure comprises at least the following (a) or (b): (a) The activatable antibody structure of any one of claims 24-33: wherein the first activatable antibody specifically binds to CD3 in the activated state, and the second antibody or its antigen-binding fragment specifically binds to the second target; (b) The activatable antibody structure of any one of claims 24-33: wherein the first activatable antibody specifically binds to CD3 in the activated state, and the activatable antibody structure of claim 24 or 25 serves as the second activatable antibody, which specifically binds to the second target in the activated state.

35. The multispecific activatable antibody structure according to claim 34, wherein the second target is a tumor-associated antigen (TAA).

36. The multispecific activatable antibody structure according to claim 34, wherein the second target is GPC, preferably GPC3.

37. The multispecific activatable antibody structure of claim 36, wherein the second antibody or its antigen-binding fragment or the second activatable antibody comprises any one of the following (a)-(b): (a) SEQ ID NO: 55; (b) SEQ ID NO: 58 and / or 59.

38. An isolated nucleic acid comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-13, a multispecific antibody or antigen-binding fragment thereof as claimed in any one of claims 14-17, a BM as claimed in any one of claims 19-21, a CM as claimed in any one of claims 22 or 23, an activatable antibody structure as claimed in any one of claims 24-33, or a multispecific activatable antibody structure as claimed in any one of claims 34-37.

39. A vector comprising the isolated nucleic acid of claim 38.

40. A host cell comprising the isolated nucleic acid of claim 38 or the vector of claim 39.

41. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-13, the multispecific antibody or antigen-binding fragment thereof of any one of claims 14-17, the ADC of claim 18, the activatable antibody structure of any one of claims 24-33, the multispecific activatable antibody structure of any one of claims 34-37, the isolated nucleic acid of claim 38, the carrier of claim 39, or the host cell of claim 40, and a pharmaceutically acceptable carrier or excipient.

42. A method of treatment or prevention for improving a condition in a subject in need by stimulating immune cells, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-13, a multispecific antibody or antigen-binding fragment thereof as described in any one of claims 14-17, an ADC as described in claim 18, an activatable antibody structure as described in any one of claims 24-33, a multispecific activatable antibody structure as described in any one of claims 34-37, or a pharmaceutical composition as described in claim 41.

43. The method of claim 42, wherein the antibody or its antigen-binding fragment, the multispecific antibody or its antigen-binding fragment, the ADC, the activatable antibody structure, the multispecific activatable antibody structure, or the pharmaceutical composition is administered in combination with other therapeutic agents.

44. The method according to claim 42 or 43, wherein the disease is selected from tumors.

45. The method of claim 44, wherein the disease 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, and osteosarcoma.

46. ​​A method for diagnosing a CD3-related condition, comprising exposing a subject’s sample to an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-13, a multispecific antibody or antigen-binding fragment thereof as claimed in any one of claims 14-17, an activatable antibody structure as claimed in any one of claims 24-33, or a multispecific activatable antibody structure as claimed in any one of claims 34-37.

47. A method for inhibiting or weakening the binding of an antibody or antigen-binding fragment thereof according to any one of claims 1-13 to CD3, comprising using BM to mask the binding site of the antibody or antigen-binding fragment thereof.

48. The method of claim 47, wherein the BM is the BM of any one of claims 19-21.

49. The method of claim 47 or 48, comprising linking the antibody or its antigen-binding fragment to the BM via a CM that can be cleaved by a protease.

50. The method of claim 49, wherein the CM is the CM of claim 22 or 23.

51. A method for linking an antibody or antigen-binding fragment thereof according to any one of claims 1-13 to a CM, comprising optionally linking the antibody or antigen-binding fragment thereof to a protease cleavage site via a connector.

52. The method of claim 51, wherein the CM is the CM of claim 22 or 23.

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