CN116120461B

CN116120461B - Novel anti-drug antibodies and uses thereof

Info

Publication number: CN116120461B
Application number: CN202310093475.9A
Authority: CN
Inventors: 宇文辉; 陈朋; 侯冰; 单波; 梅建明
Original assignee: Deqi Hangzhou Biology Co ltd
Current assignee: Deqi Hangzhou Biology Co ltd
Priority date: 2022-04-29
Filing date: 2023-02-02
Publication date: 2023-09-29
Anticipated expiration: 2043-02-02
Also published as: CN116120461A

Abstract

The present disclosure provides novel anti-drug antibodies (in particular, anti-PD-L1/4-1 BB bispecific antibodies), isolated polynucleotides encoding the same, compositions comprising the same, and uses thereof.

Description

Novel anti-drug antibodies and uses thereof

Technical Field

The present disclosure relates generally to novel anti-drug antibodies (in particular, anti-drug antibodies to PD-L1/4-1BB bispecific antibodies), antigen-binding fragments thereof, and uses thereof.

Background

Anti-drug antibodies (ADA) are commonly used to detect and quantify therapeutic antibodies during the drug development process. For example, ADA can be used in pharmacokinetic or immunogenicity assays for therapeutic antibodies. In addition, ADA can also be used as a Neutralizing Antibody (NAB) to neutralize the activity of therapeutic antibodies, thereby reducing side effects (e.g., cytokine storms) caused by therapeutic antibodies.

ATG-101 is a PD-L1/4-1BB bispecific antibody designed to activate 4-1BB positive T cells in a manner dependent on PD-L1 cross-linking and to effectively treat tumors without off-target hepatotoxicity (on-target-off-tumor liver toxicity) (see Hui Yuwen et al, J.cancer immunotherapy (J Immunother Cancer) 2021;9 (Prop 2): A1-A1054). In preclinical and clinical studies of ATG-101, molecules capable of recognizing and binding ATG-101 with high affinity are required to detect ATG-101 and thus study PK/PD characteristics of ATG-101.

There remains a need for novel anti-drug antibodies (in particular, anti-drug antibodies to PD-L1/4-1BB bispecific antibodies).

Disclosure of Invention

Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. For example, "an antibody" means one antibody or more than one antibody.

In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof comprising:

one or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) comprised within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: SEQ ID NOS 6, 13, 20, 27, 34, 41, 48 and 55; and/or

One or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and LCDR 3) comprised within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS.7, 14, 21, 28, 35, 42, 49 and 56.

In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure comprises at least one heavy or light chain Complementarity Determining Region (CDR) comprising an amino acid sequence selected from the group consisting of: 1, 2, 3, 4, NAK, 5, 8, 9, 10, 11, RAN, 12, 15, 16, 17, 18, AAS, 19, 22, 23, 24, 25, AAS, 26, 29, 30, 31, 32, STS, 33, 36, 37, 38, 39, NAK, 40, 43, 44, 45, 46, NAK, 47, 50, 51, 52, 53, QMS and 54.

In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a VH region comprising one or two or three of HCDR1, HCDR2, and HCDR3, the HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of: 1, 2, 3, 8, 9, 10, 15, 16, 17, 22, 23, 24, 29, 30, 31, 36, 37, 38, 43, 44, 45, 50, 51 and 52.

In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure comprises a VL region comprising one or two or three of LCDR1, LCDR2, and LCDR3, the LCDR1, LCDR2, and LCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs 4, NAK, 5, 11, RAN, 12, 18, AAS, 19, 25, AAS, 26, 32, STS, 33, 39, NAK, 40, 46, NAK, 47, 53, QMS and 54.

In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure comprises:

HCDR1, said HCDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs 1, 8, 15, 22, 29, 36, 43 and 50;

HCDR2, said HCDR2 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs 2, 9, 16, 23, 30, 37, 44 and 51; and

HCDR3, said HCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs 3, 10, 17, 24, 31, 38, 45 and 52.

LCDR1, said LCDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOS 4, 11, 18, 25, 32, 39, 46 and 53;

LCDR2, said LCDR2 comprising an amino acid sequence selected from the group consisting of: NAK, RAN, AAS, AAS, STS, NAK, NAK and QMS; and

LCDR3, said LCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOS 5, 12, 19, 26, 33, 40, 47 and 54.

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 1, said HCDR2 comprising an amino acid sequence of SEQ ID No. 2, said HCDR3 comprising an amino acid sequence of SEQ ID No. 3;

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 8, said HCDR2 comprising an amino acid sequence of SEQ ID No. 9, said HCDR3 comprising an amino acid sequence of SEQ ID No. 10;

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 15, said HCDR2 comprising an amino acid sequence of SEQ ID No. 16, said HCDR3 comprising an amino acid sequence of SEQ ID No. 17;

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 22, said HCDR2 comprising an amino acid sequence of SEQ ID No. 23, said HCDR3 comprising an amino acid sequence of SEQ ID No. 24;

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 29, said HCDR2 comprising an amino acid sequence of SEQ ID No. 30, said HCDR3 comprising an amino acid sequence of SEQ ID No. 31;

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 36, said HCDR2 comprising an amino acid sequence of SEQ ID No. 37, said HCDR3 comprising an amino acid sequence of SEQ ID No. 38;

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 43, said HCDR2 comprising an amino acid sequence of SEQ ID No. 44, and said HCDR3 comprising an amino acid sequence of SEQ ID No. 45; or (b)

HCDR1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 50, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 51 and said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 52.

LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 4, LCDR2 comprises an amino acid sequence as shown in NAK, and LCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 5;

LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 11, LCDR2 comprises the amino acid sequence shown in RAN, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 12;

LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown as SEQ ID NO. 18, LCDR2 comprises the amino acid sequence shown as AAS, and LCDR3 comprises the amino acid sequence shown as SEQ ID NO. 19;

LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 25, LCDR2 comprises the amino acid sequence shown in AAS, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 26;

LCDR1, LCDR2 and LCDR3, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 32, said LCDR2 comprising an amino acid sequence shown as STS, said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 33;

LCDR1, LCDR2 and LCDR3, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 39, said LCDR2 comprising an amino acid sequence shown as NAK, and said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 40;

LCDR1, LCDR2 and LCDR3, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:46, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 47; or (b)

LCDR1, LCDR2 and LCDR3, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO:53, said LCDR2 comprising the amino acid sequence shown as QMS and said LCDR3 comprising the amino acid sequence shown as SEQ ID NO: 54.

In some embodiments, the antibodies of the present disclosure, or antigen-binding fragments thereof, comprise HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, comprised within a VH/VL amino acid sequence pair selected from the group consisting of: SEQ ID NOS 6 and 7, 13 and 14, 20 and 21, 27 and 28, 34 and 35, 41 and 42, 48 and 49, and 55 and 56.

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence shown as SEQ ID No. 1, said HCDR2 comprising an amino acid sequence shown as SEQ ID No. 2, said HCDR3 comprising an amino acid sequence shown as SEQ ID No. 3, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 4, said LCDR2 comprising an amino acid sequence shown as NAK, said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 5;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 8, said HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 9, said HCDR3 comprising an amino acid sequence as shown in SEQ ID No. 10, said LCDR1 comprising an amino acid sequence as shown in SEQ ID No. 11, said LCDR2 comprising an amino acid sequence as shown in RAN, said LCDR3 comprising an amino acid sequence as shown in SEQ ID No. 12;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence shown as SEQ ID No. 15, said HCDR2 comprising an amino acid sequence shown as SEQ ID No. 16, said HCDR3 comprising an amino acid sequence shown as SEQ ID No. 17, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 18, said LCDR2 comprising an amino acid sequence shown as AAS, said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 19;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence of SEQ ID No. 22, said HCDR2 comprising an amino acid sequence of SEQ ID No. 23, said HCDR3 comprising an amino acid sequence of SEQ ID No. 24, said LCDR1 comprising an amino acid sequence of SEQ ID No. 25, said LCDR2 comprising an amino acid sequence of AAS, said LCDR3 comprising an amino acid sequence of SEQ ID No. 26;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 29, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO. 30, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 31, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 32, said LCDR2 comprising an amino acid sequence as shown in STS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 33;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence shown as SEQ ID No. 36, said HCDR2 comprising an amino acid sequence shown as SEQ ID No. 37, said HCDR3 comprising an amino acid sequence shown as SEQ ID No. 38, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 39, said LCDR2 comprising an amino acid sequence shown as NAK, said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 40;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 43, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO. 44, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 45, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 46, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 47; or (b)

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 50, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO. 51, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 52, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 53, said LCDR2 comprising an amino acid sequence as shown in QMS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 54.

In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a VH region having an amino acid sequence as set forth in SEQ ID NOs 6, 13, 20, 27, 34, 41, 48, and 55 or a homologous sequence having at least 80% sequence identity to SEQ ID NOs 6, 13, 20, 27, 34, 41, 48, and 55.

In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure comprises a VL region having an amino acid sequence as set forth in SEQ ID NOs 7, 14, 21, 28, 35, 42, 49 and 56 or a homologous sequence having at least 80% sequence identity to SEQ ID NOs 7, 14, 21, 28, 35, 42, 49 and 56.

In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure comprises a VH/VL amino acid sequence pair selected from the group consisting of: SEQ ID NOS 6 and 7, 13 and 14, 20 and 21, 27 and 28, 34 and 35, 41 and 42, 48 and 49, and 55 and 56.

In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, further comprise one or more amino acid residue substitutions or modifications, but still retain specific binding affinity for ATG-101. In some embodiments, at least one of the substitutions or modifications is in one or more CDR sequences of the VH region or the VL region. In some embodiments, at least one of the substitutions or modifications is in one or more non-CDR sequences of the VH region or the VL region. In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, further comprise one or more unnatural amino acid (NNAA) substitutions. In some embodiments, the NNAA is capable of being conjugated.

In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, are capable of specifically binding to the PD-L1 binding portion and/or 4-1BB binding portion of ATG-101, as measured by an ELISA assay. In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure competes with ATG-101 for binding to PD-L1 and/or 4-1BB.

In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment as described above for binding to ATG-101.

In some embodiments, the antibodies of the present disclosure or antigen-binding fragments thereof are chimeric, humanized, or human antibodies or antigen-binding fragments thereof.

In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, are labeled antibodies, bivalent antibodies, anti-idiotype antibodies, or fusion proteins.

In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, are bifunctional antibodies (diabodies), fab ', F (ab') ₂ Fd, fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ₂ Bispecific dsFv (dsFv-dsFv'), disulfide stabilized bifunctional antibodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), camelized single domain antibodies, nanobodies, domain antibodies or bivalent domain antibodies.

In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, further comprise an Fc region. In some embodiments, the Fc region is that of a human immunoglobulin (Ig). In some embodiments, the Fc region is that of a human IgG. In some embodiments, the Fc region is derived from human IgG1, igG2, igG3, or IgG4. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOS 75-78.

In some embodiments, the light chain of an antibody or antigen binding fragment thereof of the disclosure is a lambda light chain or a kappa light chain.

In some embodiments, the antibodies of the present disclosure, or antigen-binding fragments thereof, are bispecific or multispecific antibodies, or antigen-binding fragments thereof. In some embodiments, the antibodies of the present disclosure, or antigen binding fragments thereof, are capable of specifically binding to the PD-L1 binding portion and the 4-1BB binding portion of ATG-101.

i. one or two or three of heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) comprised within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: SEQ ID NOS 6, 13, 20 and 27;

One or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) comprised within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: 34, 41, 48 and 55;

one or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and/or LCDR 3) comprised within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS 7, 14, 21 and 28; and

one or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and/or LCDR 3) comprised within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS.35, 42, 49 and 56.

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 1, said HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 2, said HCDR3 comprising an amino acid sequence as shown in SEQ ID No. 3, said LCDR1 comprising an amino acid sequence as shown in SEQ ID No. 4, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID No. 5, said HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 29, said HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 30, said HCDR3 comprising an amino acid sequence as shown in SEQ ID No. 31, said LCDR1 comprising an amino acid sequence as shown in SEQ ID No. 32, said LCDR2 comprising an amino acid sequence as shown in STS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID No. 33;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:1, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:2, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:3, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:4, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:5, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:36, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:37, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:38, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:39, said LCDR2 comprising an amino acid sequence as shown in NAK, and said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 40;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:1, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:2, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:3, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:4, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:5, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:43, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:44, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:45, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:46, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 47;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:1, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:2, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:3, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:4, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:5, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:50, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:51, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:52, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:53, said LCDR2 comprising an amino acid sequence as shown in QMS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 54;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:8, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:9, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:10, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:11, said LCDR2 comprising an amino acid sequence as shown in RAN, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:12, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:29, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:30, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:31, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:32, said LCDR2 comprising an amino acid sequence as shown in STS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 33;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:8, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:9, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:10, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:11, said LCDR2 comprising an amino acid sequence as shown in RAN, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:12, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:36, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:37, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:38, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:39, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 40;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:8, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:9, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:10, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:11, said LCDR2 comprising an amino acid sequence as shown in RAN, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:12, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:43, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:44, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:45, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:46, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 47;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:8, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:9, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:10, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:11, said LCDR2 comprising an amino acid sequence as shown in RAN, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:12, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:50, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:51, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:52, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:53, said LCDR2 comprising an amino acid sequence as shown in QMS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 54;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:15, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:16, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:17, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:18, said LCDR2 comprising an amino acid sequence as shown in AAS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:19, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:29, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:30, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:31, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:32, said LCDR2 comprising an amino acid sequence as shown in STS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 33;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:15, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:16, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:17, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:18, said LCDR2 comprising an amino acid sequence as shown in AAS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:19, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:36, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:37, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:38, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:39, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 40;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:15, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:16, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:17, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:18, said LCDR2 comprising an amino acid sequence as shown in AAS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:19, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:43, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:44, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:45, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:46, said LCDR2 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 47;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:15, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:16, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:17, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:18, said LCDR2 comprising an amino acid sequence as shown in AAS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO:19, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO:50, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO:51, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO:52, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO:53, said LCDR2 comprising an amino acid sequence as shown in QMS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO: 54;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 22, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 23, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 24, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 25, said LCDR2 comprising the amino acid sequence shown as AAS, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 26, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 29, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 30, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 31, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 32, said LCDR2 comprising the amino acid sequence shown as STS, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 33;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 22, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 23, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 24, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 25, said LCDR2 comprising the amino acid sequence shown as AAS, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 26, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 36, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 37, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 38, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 39, said LCDR2 comprising the amino acid sequence shown as NAK, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 40;

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 22, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 23, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 24, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 25, said LCDR2 comprising the amino acid sequence shown as AAS, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 26, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 43, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 44, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 45, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 46, said LCDR2 comprising the amino acid sequence shown as NAK, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 47; or (b)

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 22, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 23, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 24, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 25, said LCDR2 comprising the amino acid sequence shown as AAS, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 26, said HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 50, said HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 51, said HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 52, said LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 53, said LCDR2 comprising the amino acid sequence shown as QMS, said LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 54.

In some embodiments, an antibody or antigen binding fragment thereof of the present disclosure is linked to one or more conjugate moieties. In some embodiments, the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., luminescent label, fluorescent label, enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification moiety, or other anticancer drug. In some embodiments, the conjugate moiety is covalently linked directly or through a linker.

In another aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof of the present disclosure and one or more carriers.

In another aspect, the present disclosure provides a chimeric antigen receptor comprising an antibody or antigen binding fragment thereof of the present disclosure, a transmembrane region, and an intracellular signaling region. In some embodiments, the transmembrane region comprises a transmembrane region of CD3, CD4, CD8 or CD 28. In some embodiments, the intracellular signaling region is selected from the group consisting of: an intracellular signal region sequence of CD3, fccRI, CD27, CD28, CD137, CD134, myD88, CD40, CD278, TLR, or a combination thereof. In some embodiments, the antigen binding fragment of the chimeric antigen receptor is an scFv.

In another aspect, the present disclosure provides a chimeric antigen receptor, wherein the chimeric antigen receptor is transplanted into an allogeneic, autologous or xenogeneic cell.

In another aspect, the present disclosure provides a chimeric antigen receptor, wherein the chimeric antigen receptor is transplanted into an immune effector cell.

In another aspect, the present disclosure provides a chimeric antigen receptor, wherein the chimeric antigen receptor is transplanted into a T cell, a natural killer cell, a macrophage, or a tumor-infiltrating lymphocyte.

In another aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof or chimeric antigen receptor of the present application, and one or more carriers.

In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen binding fragment thereof of the present disclosure and/or a chimeric antigen receptor of the present disclosure.

In another aspect, the present disclosure provides a vector comprising an isolated polynucleotide of the present disclosure.

In another aspect, the present disclosure provides a host expression system comprising a vector of the present disclosure or a polynucleotide of the present disclosure having integrated into its genome. In some embodiments, the host expression system of the present disclosure is a microorganism, a yeast, or a mammalian cell, wherein the microorganism is selected from the group consisting of escherichia coli (e.coli) and bacillus subtilis (b.subtilis), optionally wherein the yeast is Saccharomyces (Saccharomyces), and optionally wherein the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

In another aspect, the present disclosure provides a virus comprising a vector of the present disclosure.

In another aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof or chimeric antigen receptor of the present disclosure, the method comprising culturing a host expression system of the present disclosure under conditions that express the antibody or antigen-binding fragment thereof or chimeric antigen receptor of the present disclosure.

In another aspect, the present disclosure provides a method of detecting the presence or amount of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof of the present disclosure and/or a composition of the present disclosure and/or a chimeric antigen receptor of the present disclosure and determining the presence or amount of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in the sample. In some embodiments, the method is a non-diagnostic method. In some embodiments, the method is a diagnostic method. In some embodiments, the methods are used in pharmacokinetic, drug resistance or neutralization assays for anti-PD-L1 antibodies, anti-4-1 BB antibodies, or bispecific PD-L1/4-1BB antibodies. In some embodiments, the method is used in a pharmacokinetic, drug resistance or neutralization assay for ATG-101.

In another aspect, the present disclosure provides a method of reducing side effects caused by administration of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody to a subject, the method comprising administering to the subject an antibody of the present disclosure or an antigen-binding fragment thereof and/or a composition of the present disclosure and/or a chimeric antigen receptor of the present disclosure. In some embodiments, the side effect is a cytokine storm. In some embodiments, the anti-PD-L1 antibody, the anti-4-1 BB antibody, or the bispecific PD-L1/4-1BB antibody is administered to the subject to prevent or treat a disease, disorder, or condition associated with PD-L1 and/or 4-1 BB.

In some embodiments, the disease, disorder, or condition associated with PD-L1 and/or 4-1BB is cancer. In some embodiments, the cancer is a solid tumor or a hematological tumor. In some embodiments, the disease, disorder, or condition associated with PD-L1 and/or 4-1BB is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer (NSCLC), small Cell Lung Cancer (SCLC) (lung adenocarcinoma or lung squamous cell carcinoma), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumors, skin cancer, gall bladder cancer, head and neck cancer, squamous cell cancer, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), anal region cancer (e.g., anal squamous cell cancer), gastric cancer (gastric or stomach cancer) (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma/hepatoma or liver cancer), cholangiocarcinoma, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial or uterine cancer, osteosarcoma, vaginal cancer, vulval cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, real world cancer, penile cancer, kidney or urinary tract cancer (e.g., renal rhabdoid tumor), cutaneous T cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, chorioallantoic papilloma, renal cell carcinoma, renal pelvis carcinoma, central Nervous System (CNS) neoplasm, soft tissue sarcoma (e.g., rhabdomyosarcoma, fibrosarcoma, kaposi's sarcoma), spinal cord shaft tumor, glioma (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma), eye cancer (e.g., retinoblastoma), brain stem glioma or mixed glioma such as oligodendroastrocytoma, brain tumor (e.g., glioblastoma/glioblastoma multiforme (GBM), non-glioblastoma brain tumor or meningioma), cutaneous or intraocular melanoma, thrombocythemia, mesothelioma, mycosis fungoides, sezary syndrome (Sezary syndrome), primary myelofibrosis, isolated plasmacytomer, vestibular schwannoma, ewing's sarcoma, chondrosarcoma, MYH-related polyposis, pituitary adenoma, pediatric cancers such as pediatric sarcomas (e.g., neuroblastoma, rhabdomyosarcoma and osteosarcoma), hematologic cancers, hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia (e.g., lymphoblastic/lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), chronic Myelogenous Leukemia (CML), acute Myelogenous Leukemia (AML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy Cell Leukemia (HCL), burkitt's Lymphoma (BL), multiple myeloma (e.g., recurrent or refractory multiple myeloma), T-cell or B-cell lymphoma, mantle Cell Lymphoma (MCL) (e.g., recurrent or refractory mantle cell lymphoma), malignant melanoma, diffuse large B-cell lymphoma (DLBCL), DLBCL caused by follicular lymphoma, advanced B-cell lymphoma, primary mediastinum large B-cell lymphoma, follicular Lymphoma (FL) and primary mediastinum B-cell lymphoma.

In some embodiments, the antibodies of the present disclosure or antigen-binding fragments thereof and/or the compositions of the present disclosure and/or the chimeric antigen receptors of the present disclosure are used to neutralize the anti-PD-L1 antibody, the anti-4-1 BB antibody, or the bispecific PD-L1/4-1BB antibody.

In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof of the present disclosure and/or a composition of the present disclosure and/or a chimeric antigen receptor of the present disclosure.

In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof of the present disclosure, and/or a chimeric antigen receptor of the present disclosure, and/or a composition of the present disclosure, which can be used to detect an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in a sample.

In another aspect, the present disclosure provides a kit comprising an antibody of the present disclosure, or an antigen-binding fragment thereof, and/or a chimeric antigen receptor of the present disclosure, and/or a composition of the present disclosure, for detecting an anti-drug antibody (ADA) that is an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody.

In another aspect, the present disclosure provides for the use of an antibody or antigen binding fragment thereof provided herein, and/or a chimeric antigen receptor of the disclosure, and/or a composition of the disclosure, in the preparation of a kit for use in a method for detecting an anti-drug antibody (ADA) to a PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody.

In some embodiments, the sample is selected from the group consisting of: tissue sections, biopsies, paraffin embedded tissue, body fluids, colonic exudates, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof.

In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure includes a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof.

In some embodiments, the first antibody or antigen binding fragment thereof comprises:

i. one or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) included within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: SEQ ID NOS 6, 13, 20 and 27; and

one or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and LCDR 3) included within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS.7, 14, 21 and 28.

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence shown as SEQ ID No. 15, said HCDR2 comprising an amino acid sequence shown as SEQ ID No. 16, said HCDR3 comprising an amino acid sequence shown as SEQ ID No. 17, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 18, said LCDR2 comprising an amino acid sequence shown as AAS, said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 19; or (b)

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 22, said HCDR2 comprising an amino acid sequence as shown in SEQ ID NO. 23, said HCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 24, said LCDR1 comprising an amino acid sequence as shown in SEQ ID NO. 25, said LCDR2 comprising an amino acid sequence as shown in AAS, said LCDR3 comprising an amino acid sequence as shown in SEQ ID NO. 26.

In some embodiments, the second antibody or antigen binding fragment thereof comprises:

i. one or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) included within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: 34, 41, 48 and 55; and

one or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and LCDR 3) included within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS.35, 42, 49 and 56.

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence shown as SEQ ID No. 43, said HCDR2 comprising an amino acid sequence shown as SEQ ID No. 44, said HCDR3 comprising an amino acid sequence shown as SEQ ID No. 45, said LCDR1 comprising an amino acid sequence shown as SEQ ID No. 46, said LCDR2 comprising an amino acid sequence shown as NAK, said LCDR3 comprising an amino acid sequence shown as SEQ ID No. 47; or (b)

In some embodiments, the bodily fluid is selected from the group consisting of: whole blood, serum, plasma, urine, mucus, saliva, peritoneal fluid, pleural fluid, synovial fluid, cerebrospinal fluid, pleural effusion, peritoneal effusion, and any combination thereof.

In some embodiments, the bispecific PD-L1/4-1BB antibody is ATG-101.

Drawings

FIGS. 1A through 1F show the binding affinities of antibody clones 10F10G7, 9D6E11, 27E8A8, 31B6G8, 50D6E10, 58H6F8, 63C9C12 and 75H9F7 for ATG-101 and IgG, respectively.

FIGS. 2A to 2C show the binding affinity and competition capacity of selected antibody clones (10F 10G7, 9D6E11, 27E8A8, 31B6G8, 50D6E10, 58H6F8, 63C9C12, 75H9F 7) against ATG-101 for human PD-L1 protein or human 4-1BB protein. Mouse IgG1 was used as a non-competitive negative control.

FIG. 3 shows the structure of ATG-101.

Detailed Description

The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. As such, the particular modifications discussed should not be construed as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included herein. All documents, including publications, patents and patent applications cited herein are incorporated by reference in their entirety.

Definition of the definition

The term "antibody" as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a particular antigen. Natural intact antibodies comprise two heavy (H) chains and two light (L) chains. Mammalian heavy chains are divided into α, δ, ε, γ and μ, each heavy chain comprising a variable region (VH) and a first constant region, a second constant region, a third constant region and optionally a fourth constant region (CH 1, CH2, CH3, CH4, respectively); mammalian light chains are classified as either lambda or kappa, and each light chain includes a variable region (VL) and a constant region. The antibody is "Y" shaped, wherein the stem of the Y-shaped structure comprises a second constant region and a third constant region of two heavy chains that are joined together by disulfide bonds. Each arm of Y comprises a variable region and a first constant region of a single heavy chain in combination with a variable region and a constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain typically contains three hypervariable regions, known as Complementarity Determining Regions (CDRs) (light chain CDRs comprising LCDR1, LCDR2, LCDR3, heavy chain CDRs comprising HCDR1, HCDR2, HCDR 3). The three CDRs are separated by flanking regions called Framework Regions (FR) (light chain FR includes LFR1, LFR2, LFR3 and LFR4, and heavy chain FR includes HFR1, HFR2, HFR3 and HFR 4) that are more highly conserved than the CDRs and form scaffolds to support highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions. Antibodies can be classified into several classes based on the amino acid sequence of their heavy chain constant region. The five main classes or isotypes of antibodies are IgA, igD, igE, igG and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), igG4 (gamma 4 heavy chain), igA1 (alpha 1 heavy chain) or IgA2 (alpha 2 heavy chain).

In certain embodiments, the antibodies provided herein encompass any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that includes one or more (e.g., 1, 2, 3, 4, 5, or 6) CDRs, or any other antibody fragment that binds an antigen but does not include the complete native antibody structure. Examples of antigen binding fragments include, but are not limited to, bifunctional antibodies (diabodies), fab ', F (ab') ₂ Fd, fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ₂ Bispecific dsFv (dsFv-dsFv'), disulfide stabilized bifunctional antibodies (ds diabody), single chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelid single domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. The antigen binding fragment is capable of binding the same antigen or epitope as the parent antibody.

"Fab" of an antibody refers to a portion of an antibody consisting of a single light chain (including variable and constant regions) and the variable and first constant regions of a single heavy chain joined by disulfide bonds.

"Fab'" refers to a Fab fragment which comprises a portion of the hinge region.

“F(ab’) ₂ "refers to a dimer of Fab'.

"Fc" of an antibody (e.g., an IgG, igA, or IgD isotype) refers to the portion of the antibody that is composed of the second constant domain and the third constant domain of the first heavy chain that binds to the second constant domain and the third constant domain of the second heavy chain via disulfide bonds. The Fc of IgM and IgE isotype antibodies further comprises a fourth constant domain. The Fc portion of antibodies is responsible for a number of different effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

"Fv" of an antibody refers to the smallest antibody fragment that contains the complete antigen binding site. Fv fragments consist of a single light chain variable region in combination with a single heavy chain variable region.

"Single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region, either directly linked to each other or linked to each other by a linker (e.g., a peptide sequence) (Huston JS et al, proc. Natl. Acad. Sci. USA (Proc Natl Acad Sci USA), 85:5879 (1988)).

"Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of the antibody.

"camelized single domain antibody", "heavy chain antibody" or "HCAb" refers to an antibody containing two VH domains and no light chain (Riechmann L. And Muyldermans S., "J.Immunol.methods (J Immunol Methods)," 12 months for 10 days; 231 (1-2): 25-38 (1999); muyldermans S., "J.Biotechnol.)," 6 months; 74 (4): 277-302 (2001); WO94/04678; WO94/25591; U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (camel, dromedary and llama). The camelized antibody has a confirmed antigen binding full function despite deletion of the light chain (Hamers-Casterman C. Et al, nature) 6 months 3, 363 (6428) 446-8 (1993), nguyen VK. et al, immunogenetics (Immunogenetics) 4 months, 54 (1) 39-47 (2002), nguyen VK. et al, immunology (Immunology) 5 months, 109 (1) 93-101 (2003)). The variable region (VHH domain) of a heavy chain antibody represents the smallest known antigen binding unit generated by an adaptive immune response (Koch-Nolte F. Et al, journal of the American society of laboratory Biotechnology (FASEB J.)) 11 months; 21 (13): 3490-8 electronic version 2007, 6, 15 (2007)).

"nanobody" refers to an antibody fragment consisting of a VHH domain from a chain antibody and two constant domains CH2 and CH 3.

"diabodies", "diabodies" or "dAbs" include small antibody fragments having two antigen binding sites, wherein the fragments comprise a VH domain and a VL domain (VH-VL or VL-VH) linked on the same polypeptide chain (see, e.g., holliger P. Et al, proc. Natl. Acad. Sci. USA, 7, 15 days; 90 (14): 6444-8 (1993); EP404097; WO 93/11161). By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand, thereby creating two antigen binding sites. The antigen binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes).

"domain antibody" refers to an antibody fragment containing only heavy chain variable regions or light chain variable regions. In certain cases, two or more VH domains are covalently joined by a peptide linker to produce a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens (or epitopes).

As used herein, the term "valency" refers to the presence of a specified number of antigen binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen binding fragment having only one antigen binding site; and the term "multivalent" refers to an antibody or antigen binding fragment having multiple antigen binding sites. Thus, the terms "divalent", "tetravalent" and "hexavalent" denote the presence of two antigen binding sites, four antigen binding sites and six antigen binding sites, respectively, in an antigen binding molecule. In some embodiments, the antibody or antigen binding fragment thereof is bivalent.

As used herein, a "bispecific" antibody refers to an artificial antibody having fragments derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

As used herein, a "multispecific" antibody refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes of the same antigen. The multispecific antibodies may bind to, for example, two, three, four, five, or more different antigens, or may bind to two, three, four, five, or more different epitopes of the same antigen.

In certain embodiments, the "scFv dimer" is a bivalent bifunctional antibody (diabody) or a bispecific scFv (BsFv) comprising two VH-VL (linked by a peptide linker) portions that dimerize such that the VH of one portion cooperates with the VL of the other portion to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, a "scFv dimer" is a bispecific bifunctional antibody comprising VH1-VL2 (linked by a peptide linker) and VL1-VH2 (also linked by a peptide linker) that are associated with each other such that VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperating pair has a different antigen specificity.

"dsFv" refers to a disulfide stabilized Fv fragment in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is disulfide. In some embodiments, "(dsFv) ₂ "or" (dsFv-dsFv') "includes three peptide chains: the two VH moieties are linked by a peptide linker (e.g., a long flexible linker) and are bound to the two VL moieties, respectively, by disulfide bonds. In some embodiments, dsFv-dsFv' having dual specificity, wherein each pair of heavy and light chains paired by disulfide bonds has Different antigen specificity.

As used herein, the term "chimeric" refers to an antibody or antigen binding fragment having a portion of the heavy and/or light chain derived from one species and the remainder of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

As used herein, the term "humanized" refers to antibodies or antigen binding fragments that comprise CDRs derived from a non-human animal, FR regions derived from a human, and constant regions (when applicable) derived from a human. The CDRs of the humanized antibodies provided by the present disclosure can contain mutations compared to the CDRs of their parent antibodies.

As used herein, the term "affinity" refers to the strength of a non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or antigen-binding fragment thereof and an antigen.

Antibodies or antigen binding fragments thereof that "specifically bind" (specifically binds/specific binding) to a target (e.g., an epitope) are well known in the art and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates more frequently, more rapidly, longer in duration, and/or with greater affinity than it does with a particular cell or substance. An antibody "specifically binds" to a target if it binds more strongly, more readily, and/or for a longer duration than it does with other substances. For example, an antibody that specifically binds to a PD-L1 epitope is an antibody that binds to this PD-L1 epitope with greater affinity, higher affinity, easier and/or longer duration than binds to other PD-L1 epitopes or non-PD-L1 epitopes. It will also be appreciated by reading this definition that, for example, an antibody (or portion or epitope) that specifically binds to a first target may or may not specifically bind to a second target. As such, "specific binding" (specific binding/specifically bind) does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding refers to specific binding.

As used herein, the term "epitope" refers to a specific set of atoms or amino acids on an antigen to which an antibody binds. If both antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitope within the antigen. Epitopes can be linear or spatially conformational (i.e., include spaced apart amino acid residues). For example, an antibody or antigen-binding portion may be considered to bind the same or closely related epitope as a reference antibody if the antibody or antigen-binding fragment blocks at least 85%, or at least 90%, or at least 95% of the binding of the reference antibody to the antigen.

As used herein, the term "amino acid" refers to a polypeptide containing an amino group (-NH) ₂ ) And a carboxyl (-COOH) functional group, and a side chain unique to each amino acid. Amino acid names are also indicated in the present disclosure in standard single-letter or three-letter codes, summarized below:

"conservative substitution" with respect to an amino acid sequence refers to the replacement of an amino acid residue with a different amino acid residue having a side chain of similar physicochemical properties. For example, conservative substitutions may be made between amino acid residues having a hydrophobic side chain (e.g., met, ala, val, leu and Ile), amino acid residues having a neutral hydrophilic side chain (e.g., cys, ser, thr, asn and gin), amino acid residues having an acidic side chain (e.g., asp, glu), amino acid residues having a basic side chain (e.g., his, lys, and Arg), or amino acid residues having an aromatic side chain (e.g., trp, tyr, and Phe). As is known in the art, conservative substitutions typically do not cause a significant change in the conformational structure of the protein, and thus may preserve the biological activity of the protein.

As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to another sequence when optimally aligned.

"percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or reference sequence (whichever is shorter). Conservative substitutions of amino acid residues may or may not be considered as identical residues. The alignment can be accomplished, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the national institute of biological technology (U.S. national Center for Biotechnology Information, NCBI), see also Altschul S.F. et al, (J. Mol. Biol.), 215:403-410 (1990), stephen F. Et al, (Nucleic Acids Res.), 25:3389-3402 (1997)), clustalW2 (available on the website of the European institute of biological information (European Bioinformatics Institute), see also Higgins D.G. et al, methods of enzymology (Methods In Enzymology), 266:383-402 (1996), larkin M.A. et al, (Bioinformatics) (British oxford.), 23 (21) 2947-8 (2007) or Megalign (DNASTAR) software to determine the identity of amino acid sequences, or Nucleic acid sequences, and the appropriate parameters can be selected by a person in accordance with the appropriate algorithm of the art, for example, by appropriate choice of the appropriate parameters.

As used herein, "effector function" refers to biological activity caused by the binding of an Fc region of an antibody to its effectors (e.g., C1 complex and Fc receptor). Exemplary effector functions include: complement Dependent Cytotoxicity (CDC) mediated by the interaction of the antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by binding of the Fc region of an antibody to an Fc receptor on an effector cell; phagocytosis. Various assays, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays, can be used to assess effector function.

As used herein, "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which effector cells expressing an Fc receptor (FcR) recognize a binding antibody or antigen binding fragment on a target cell and subsequently cause lysis of the target cell. "ADCC activity" or "ADCC effect" refers to the ability of an antibody or antigen binding fragment bound to a target cell to initiate an ADCC reaction as described above.

As used herein, "complement dependent cytotoxicity" or "CDC" refers to a mechanism by which an antibody can mediate specific target cell lysis by activating the complement system of an organism. In CDC, C1q binds to antibodies and this binding triggers the complement cascade, which results in the formation of a Membrane Attack Complex (MAC) at the target cell surface (C5 b to C9) as a result of classical pathway complement activation. "CDC activity" or "CDC effect" refers to the ability of an antibody or antigen binding fragment to bind to a target cell to elicit a CDC reaction as described above.

As used herein, "target cell" refers to a cell that includes an Fc region to which an antibody specifically binds. An "effector cell" is a leukocyte that expresses one or more Fc receptors and performs effector functions. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMC), natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; of these, PBMC and NK cells are preferred. Effector cells may be isolated from their natural sources, for example from blood or PBMCs as known in the art.

"isolated" substances have been altered manually by natural states. If a certain "isolated" composition or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally occurring in a living animal is not "isolated," but may be considered "isolated" if the polynucleotide or polypeptide is sufficiently separated from the material in which it coexists in its natural state and exists in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, as determined by an electrophoretic method (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or a chromatographic method (e.g., ion exchange chromatography or reverse phase HPLC).

As used herein, the term "vector" refers to a vector into which a polynucleotide encoding a protein may be operably inserted so as to cause expression of the protein. Vectors may be used to transform, transduce or transfect host cells such that the genetic elements carried thereby are expressed within the host cells. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV 40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The carrier may also include materials that assist in its entry into the cell, including but not limited to viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) comprising a nucleic acid sequence provided herein encoding an antibody or antigen binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1 a) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40), lambda and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-Gseu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and/or vector may or has been introduced.

The term "subject" includes both human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. The terms "patient," "subject," or "individual" are used interchangeably herein, except where indicated.

The term "anti-tumor activity" means a decrease in proliferation, viability or metastatic activity of tumor cells. For example, anti-tumor activity may be indicated by a decrease in the growth rate of abnormal cells or a decrease in tumor size stability or a decrease in survival due to treatment or due to treatment, as compared to a control without treatment. Such activity may be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, mouse Mammary Tumor Virus (MMTV) models, and other known models known in the art to study anti-tumor activity.

As used herein, "treating" or "treatment" of a disease, disorder or condition includes preventing or alleviating the disease, disorder or condition, slowing the onset or rate of progression of the disease, disorder or condition, reducing the risk of developing the disease, disorder or condition, preventing or slowing the progression of symptoms associated with the disease, disorder or condition, reducing or ending symptoms associated with the disease, disorder or condition, causing complete or partial regression of the disease, disorder or condition, curing the disease, disorder or condition, or some combination thereof.

As used herein, the term "biological sample" or "sample" refers to a biological composition obtained from or derived from a subject of interest that contains cells and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical, and/or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs and/or biological fluids of a subject obtained by any method known to those of skill in the art. In some embodiments, the biological sample is a body fluid sample. In some embodiments, the bodily fluid sample is whole blood, plasma, serum, mucus (including nasal drainage and sputum), peritoneal fluid, pleural fluid, thoracic fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracic fluid, peritoneal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is a tissue or cell obtained from the stomach, heart, liver, spleen, lung, kidney, skin, or blood vessel of the subject.

As used herein, "PD-L1", also referred to as B7-H1 or CD274, refers to the programmed cell death ligand 1 (PD-L1, see, e.g., freeman et al (2000), "journal of experimental medicine (j.exp. Med.))," 192:1027) protein, and includes any variant, conformation, isoform and species homolog of PD-L1 that is expressed by cells naturally or by cells transfected with the PD-L1 gene. For example, PD-L1 described herein may refer to PD-L1 proteins derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). It is expressed in placenta, spleen, lymph node, thymus, heart, fetal liver, and can also be found on many tumor cells or cancer cells. NCBI accession number: NP 054862.1 (representative amino acid sequence) and NCBI accession numbers: exemplary sequences of human PD-L1 are disclosed in NM-014143.3 (representative nucleic acid sequences). Exemplary sequences of non-human PD-L1 include: cynomolgus monkey (Macaca fascicularis) (monkey) PD-L1 protein (NCBI Ref Seq No. xm_ 005581779.3); mice (Mus musculus) (mice) PD-L1 protein (NCBI Ref Seq No. adk 70950.1); brown mice (Rattus norvegicus) (rats) PD-L1 protein (NCBI Ref Seq No. NP-001178883.1). In certain embodiments, PD-L1 as used herein may also be recombinant PD-L1, which recombinant PD-L1 may optionally be expressed in the form of a recombinant PD-L1 complex. The recombinant PD-L1 complex may be expressed on the cell surface or may be expressed in a soluble form that does not bind to the cell surface. In certain embodiments, PD-L1 is human PD-L1. The terms "PD-L1", "PDL1", "B7H1", "B7-H1" and "CD274" are used interchangeably in this disclosure.

As used herein, the term "4-1BB", also known as CD137 or tumor necrosis factor receptor superfamily member 9 (TNFRSF 9), refers to the 4-1BB protein and includes any variant, conformation, isoform and species homolog of 4-1BB expressed naturally by cells or expressed by cells transfected with the 4-1BB gene. For example, 4-1BB as described herein may refer to 4-1BB protein derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). Exemplary sequences of human 4-1BB are disclosed in NCBI accession numbers NP-001552.2, NM-001561.5, XP-011539688.1, or XM-011541386.2. Exemplary sequences for non-human 4-1BB include: cynomolgus monkey (monkey) 4-1BB protein (NCBI Ref Seq No. ABY 47575.1); the mouse (mouse) 4-1BB protein (NCBI Ref Seq No. NP-001070977.1 NM_001077509.1, NP_035742.1, NM_011612.2, XP_011248530.1, XM_ 011250228.2); brown rat (rat) 4-1BB protein (NCBI Ref Seq No. NP-852049.1, NP-001020944.1, AAH 97483.1). In certain embodiments, 4-1BB as used herein may also be recombinant 4-1BB, which recombinant 4-1BB may optionally be expressed in the form of a recombinant PD-L1 complex. The recombinant 4-1BB complex may be expressed on the cell surface, or may be expressed in a soluble form that does not bind to the cell surface. In certain embodiments, 4-1BB is human 4-1BB. The terms "4-1BB", "41BB", "CD137", "CD-137", "CD137" and "TNFRSF9" may be used interchangeably throughout this disclosure.

As used herein, "a disease, disorder, or condition associated with PD-L1 and/or 4-1 BB" or "a disease, disorder, or condition associated with PD-L1 and/or 4-1 BB" refers to any disease, disorder, or condition caused by, exacerbated by, or otherwise linked to an increase or decrease in the expression or activity of PD-L1 and/or 4-1 BB. In some embodiments, the disease, disorder, or condition associated with PD-L1 and/or 4-1BB is a disorder associated with excessive cell proliferation, such as cancer. In certain embodiments, the disease, disorder, or condition is characterized by expression or overexpression of PD-L1 and/or 4-1BB or a gene associated with PD-L1 and/or 4-1 BB.

Antibodies to

In one aspect, the disclosure provides antibodies and antigen binding fragments thereof that specifically bind to an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody. In some embodiments, the present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to ATG-101.

As used herein, the term "ATG-101" is a homodimeric bifunctional molecule targeting PD-L1 and 4-1BB, comprising a fully human IgG1 antibody to PD-L1 and an scFv to 4-1BB, wherein the scFv to 4-1BB comprises a VH region to 4-1BB and a VL region to 4-1BB, and the VH regions are linked by a linker (e.g., (GGGGS) ₃ ) Is linked to the VL region and the VH region of the scFv is via a linker (e.g., (GGGGS) ₃ ) Is linked to the C-terminus of the Fc region of a fully human IgG1 antibody directed against PD-L1. In some embodiments, the asparagine at position 297 of ATG-101 (according to the EP numbering system) is mutated to alanine to eliminate binding affinity for the Fcγ receptor. Throughout this specification, the fully human IgG1 antibody to PD-L1 of ATG-101 is referred to as the "PD-L1 binding portion" of ATG-101, and the scFv of ATG-101 to 4-1BB is referred to as the "4-1BB binding portion" of ATG-101. The amino acid sequences of the HCDR, LCDR, VH region, the VL region, the heavy chain and the light chain of ATG-101 are shown in Table 1. The CDR boundaries of ATG-101 in Table 1 are determined according to the IMGT rules.

TABLE 1 amino acid sequences of the HCDR, LCDR, VH region, VL region, heavy chain and light chain of ATG-101

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The binding affinity of an antibody or antigen binding fragment thereof provided herein may be determined by K _D The value represents the ratio (k) of the dissociation rate to the association rate when the binding between antigen and antigen binding molecule reaches equilibrium _off /k _on ). Antigen binding affinity (e.g., K) can be suitably determined using suitable methods known in the art, including, for example, flow cytometry assays _D ). In some embodiments, binding of an antibody or antigen binding fragment thereof to different concentrations of antigen can be determined by flow cytometry, the determined Mean Fluorescence Intensity (MFI) can be first plotted against the antibody concentration, and K can be calculated by fitting the dependence of the specific binding fluorescence intensity (Y) and antibody concentration (X) to one site saturation equation (site saturation equation) using Prism version 5 (San Diego, california) GraphPad software (GraphPad Software, san Diego, CA)) _D Value: y=b _max *X/(K _D +X), wherein B _max Refers to the maximum specific binding of the antibody to be tested to the antigen.

The binding of the antibodies or antigen binding fragments thereof provided herein to ATG-101 may also be performed using "half maximal effective concentrations" (EC ₅₀ ) The values represent the maximum observed for the antibodiesConcentration of 50% of binding. EC (EC) ₅₀ The value may be measured by binding assays known in the art, such as direct or indirect binding assays, e.g., enzyme-linked immunosorbent assays (ELISA), FACS assays, and other binding assays. In certain embodiments, an antibody or antigen binding fragment thereof provided herein is capable of specifically binding to ATG-101 (e.g., as measured by an ELISA assay). In certain embodiments, an antibody or antigen binding fragment thereof provided herein is capable of specifically binding to a PD-L1 binding portion of ATG-101 (e.g., as measured by an ELISA assay). In certain embodiments, an antibody or antigen binding fragment thereof provided herein is capable of specifically binding to the 4-1BB binding portion of ATG-101 (e.g., as measured by an ELISA assay). In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is capable of specifically binding to the PD-L1 binding portion and 4-1BB binding portion of ATG-101 (e.g., as measured by an ELISA assay).

In another aspect, the present disclosure provides antibodies and antigen-binding fragments thereof that compete with ATG-101 for binding to PD-L1 and/or 4-1 BB.

The ability to "compete with ATG-101 for binding to" a target refers to the ability of a primary antibody or antigen-binding fragment thereof to inhibit the interaction of binding between the target (e.g., PD-L1 and/or 4-1 BB) and ATG-101 to any detectable extent. In certain embodiments, an antibody or antigen binding fragment that competes for binding to PD-L1 and/or 4-1BB inhibits the interaction of binding between PD-L1 and/or 4-1BB and ATG-101 by at least 85% or at least 90%. In certain embodiments, the inhibition may be greater than 95% or greater than 99%.

Exemplary antibodies

In certain embodiments, the disclosure provides antibodies or antigen binding fragments thereof comprising:

One or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and/or LCDR 3) comprised within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS.7, 14, 21, 28, 35, 42, 49 and 56.

The CDR boundaries of a VH region or VL region can be defined or determined by methods known to those skilled in the art, provided that the amino acid sequence of the VH region or VL region is known. For example, CDR boundaries of an antibody or antigen binding fragment thereof may be defined or determined by Kabat, IMGT, chothia or Al-Lazikani rules (Al-Lazikani, b., chothia, c., lesk, a.m., journal of molecular biology, 273 (4), 927 (1997); chothia, C.et Al, 12 months 5; 186 (3): 651-63 (1985), chothia, C.and Lesk, A.M., journal of molecular biology, 196,901 (1987), chothia, C.et Al, nature, 12 months 21-28; 342 (6252): 877-83 (1989), kabat E.A. et Al, protein sequences of immunological significance (Sequences of Proteins of immunological Interest), 5 th edition public health agency (Public Health Service), national institutes of health (National Institutes of Health), besseda (Bethesda, md.) (1991), marie-Paule Lefranc et Al, development and comparison immunology (Developmental and Comparative Immunology), 27:55-77 (2003), marie-Paule Leflanc et Al, immune group study (Immunome Research), 1 (3), (2005-Paul) and (2015), leul, mahala, B.35, and (2015). In some embodiments, CDR boundaries of an antibody or antigen binding fragment thereof provided herein are determined according to the Kabat rules. In some embodiments, CDR boundaries of an antibody or antigen binding fragment thereof provided herein are determined according to IMGT rules. In some embodiments, CDR boundaries of an antibody or antigen binding fragment thereof provided herein are determined according to Chothia rules. In some embodiments, the CDR boundaries of an antibody or antigen binding fragment thereof provided herein are determined according to the Al-Lazikani rule.

In certain embodiments, the disclosure provides antibodies or antigen-binding fragments thereof that include one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of antibodies 9D6E11, 10F10G7, 27E8A8, 31B6G8, 58H6F8, 63C9C12, 75H9F7, or 50D6E 10.

As used herein, antibody "9D6E11" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID No. 6 and a light chain variable region having the sequence of SEQ ID No. 7.

As used herein, antibody "10F10G7" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO. 13 and a light chain variable region having the sequence of SEQ ID NO. 14.

As used herein, antibody "27E8A8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO. 20 and a light chain variable region having the sequence of SEQ ID NO. 21.

As used herein, antibody "31B6G8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO. 27 and a light chain variable region having the sequence of SEQ ID NO. 28.

As used herein, antibody "58H6F8" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO. 34 and a light chain variable region having the sequence of SEQ ID NO. 35.

As used herein, antibody "63C9C12" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO. 41 and a light chain variable region having the sequence of SEQ ID NO. 42.

As used herein, antibody "75H9F7" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID No. 48 and a light chain variable region having the sequence of SEQ ID No. 49.

As used herein, antibody "50D6E10" refers to a mouse monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO. 55 and a light chain variable region having the sequence of SEQ ID NO. 56.

Specific amino acid sequences of the heavy chain variable region and the light chain variable region of each of the exemplary antibodies described above are shown in table 4 below.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:6 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 7.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:13 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 14.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:20 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 21.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:27 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 28.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:34 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 35.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:41 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 42.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:48 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 49.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises three heavy chain CDRs (HCDR 1, HCDR2, and HCDR 3) comprising a VH region sequence as set forth in SEQ ID NO:55 and three light chain CDRs (LCDR 1, LCDR2, and LCDR 3) comprising a VL region sequence as set forth in SEQ ID NO: 56.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises at least one (e.g., 1, 2, or 3) heavy or light chain CDR comprising an amino acid sequence selected from the group consisting of: 1, 2, 3, 4, NAK, 5, 8, 9, 10, 11, RAN, 12, 15, 16, 17, 18, AAS, 19, 22, 23, 24, 25, AAS, 26, 29, 30, 31, 32, STS, 33, 36, 37, 38, 39, NAK, 40, 43, 44, 45, 46, NAK, 47, 50, 51, 52, 53, QMS and 54.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a VH region comprising one or two or three of HCDR1, HCDR2, and HCDR3, the HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of: 1, 2, 3, 8, 9, 10, 15, 16, 17, 22, 23, 24, 29, 30, 31, 36, 37, 38, 43, 44, 45, 50, 51 and 52.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a VL region comprising one or two or three of LCDR1, LCDR2, and LCDR3, the LCDR1, LCDR2, and LCDR3 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs 4, NAK, 5, 11, RAN, 12, 18, AAS, 19, 25, AAS, 26, 32, STS, 33, 39, NAK, 40, 46, NAK, 47, 53, QMS and 54.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs 1, 8, 15, 22, 29, 36, 43 and 50; the HCDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs 2, 9, 16, 23, 30, 37, 44 and 51; the HCDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs 3, 10, 17, 24, 31, 38, 45 and 52.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises an LCDR1, an LCDR2, and an LCDR3, the LCDR1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NOS 4, 11, 18, 25, 32, 39, 46 and 53; the LCDR2 comprises an amino acid sequence selected from the group consisting of: NAK, RAN, AAS, AAS, STS, NAK, NAK and QMS; the LCDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NOS 5, 12, 19, 26, 33, 40, 47 and 54.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises:

The heavy chain (denoted "H") variable region, light chain (denoted "L") variable region, HCDR and LCDR SEQ ID NOs of each of the 8 monoclonal antibodies are shown in table 2 below. CDR boundaries as described herein are defined or determined by IMGT rules, unless otherwise specified. The amino acid sequences of each CDR of 8 exemplary monoclonal antibodies are shown in table 3 below. The amino acid sequences of each VH and VL of 8 exemplary monoclonal antibodies are shown in table 4 below.

TABLE 2 VH, VL, HCDR and LCDR SEQ ID NOs of 8 exemplary monoclonal antibodies

TABLE 3 amino acid sequence of each CDR of 8 exemplary monoclonal antibodies

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TABLE 4 amino acid sequences of each VH and VL of 8 exemplary monoclonal antibodies

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It is contemplated that each of the 8 exemplary monoclonal antibodies can bind to ATG-101, and that antigen binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the HCDR1, HCDR2, and HCDR3 sequences and the LCDR1, LCDR2, and LCDR3 sequences of each of the 8 exemplary monoclonal antibodies can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2, and HCDR3, as well as LCDR1, LCDR2, and LCDR 3) to produce an ATG-101 binding molecule of the present disclosure. Binding assays as described above and in the examples can be used to test the binding of such "mixed and matched" antibodies to ATG-101. Preferably, when VH CDR sequences are mixed and matched, HCDR1 sequences, HCDR2 sequences and/or HCDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequences. Likewise, when VL CDR sequences are mixed and matched, LCDR1, LCDR2 and/or LCDR3 sequences from a particular VL sequence are preferably replaced by structurally similar CDR sequences. For example, HCDR1 of antibodies 9D6E11 and 27E8A8 share some structural similarity, and are therefore easy to mix and match. It will be apparent to those skilled in the art that novel VH and VL sequences can be generated by substituting one or more VH and/or VL CDR sequences with structurally similar sequences from the CDR sequences disclosed herein for 8 exemplary monoclonal antibodies.

CDRs are known to be responsible for antigen binding. However, not all 6 CDRs have been found to be indispensable or unchangeable. In other words, one or more CDRs of each of the 8 exemplary monoclonal antibodies can be replaced or altered or modified, but substantially retain specific binding affinity for ATG-101.

In certain embodiments, the antibodies and antigen binding fragments provided herein comprise the heavy chain CDR3 sequence of one of antibodies 9D6E11, 10F10G7, 27E8A8, 31B6G8, 58H6F8, 63C9C12, 75H9F7, or 50D6E 10. In certain embodiments, the antibodies and antigen binding fragments provided herein comprise a heavy chain CDR3 sequence selected from the group consisting of seq id nos: SEQ ID NOs 3, 10, 17, 24, 31, 38, 45 and 52. The heavy chain CDR3 region is centered in the antigen binding site and is therefore considered to be most in contact with the antigen and provides the antibody with the most free energy of affinity for the antigen. It is also believed that heavy chain CDR3 is the CDR of antigen binding sites most diverse so far in length, amino acid composition and conformation by a variety of mechanisms (Tonegawa S., nature) 302:575-81). The diversity of heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, davis MM. immunity 13:37-45) and ideal antigen binding affinities (Schier R et al J Mol biol) 263:551-67).

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a VH region having an amino acid sequence as set forth in SEQ ID No. 6, 13, 20, 27, 34, 41, 48, or 55 or a homologous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID No. 6, 13, 20, 27, 34, 41, 48, or 55.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a VL region having an amino acid sequence as set forth in SEQ ID No. 7, 14, 21, 28, 35, 42, 49, or 56 or a homologous sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID No. 7, 14, 21, 28, 35, 42, 49, or 56.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a VH/VL amino acid sequence pair selected from the group consisting of: SEQ ID NOS 6 and 7, 13 and 14, 20 and 21, 27 and 28, 34 and 35, 41 and 42, 48 and 49, and 55 and 56.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein include suitable Framework Region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof can specifically bind to ATG-101. The CDR sequences provided in table 3 above were obtained from a mouse antibody, but the CDR sequences may be grafted to any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, etc., using suitable methods known in the art, such as recombinant techniques.

In certain embodiments, the antibodies and antigen binding fragments thereof provided herein are humanized. It is desirable for the humanized antibody or antigen-binding fragment thereof to have reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because the non-human CDR sequences are grafted to human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be performed essentially by substituting non-human (e.g., murine) CDR genes for corresponding human CDR genes in human immunoglobulin genes (see, e.g., jones et al (1986) Nature 321:522-525; riechmann et al (1988) Nature 332:323-327; verhoeyen et al (1988) Science 239:1534-1536).

Suitable human heavy and light chain variable domains can be selected using methods known in the art to achieve this. In an illustrative example, a "best fit" approach may be used in which a human sequence closest to the non-human query sequence is identified against a database of known human variable domain sequences or BLAST non-human (e.g., rodent) antibody variable domain sequences, and used as a human framework for grafting non-human CDR sequences (see, e.g., sims et al, (1993) journal of immunology (j. Immunol.)) (151:2296; chothia et al (1987) journal of molecular biology 196:901). Alternatively, frameworks derived from the consensus sequences of all human antibodies can be used to implant non-human CDRs (see, e.g., carter et al (1992) Proc. Natl. Acad. Sci. USA), 89:4285; presta et al (1993) J.Immunol., 151:2623).

In some embodiments, the antibodies or antigen binding fragments thereof provided herein are humanized. In certain embodiments, a humanized antibody or antigen binding fragment thereof provided herein consists of substantially fully human sequences, except for non-human CDR sequences. In some embodiments, the variable region FR and constant region (if present) are derived entirely or substantially from human immunoglobulin sequences. The human FR sequence and the human constant region sequence may be derived from different human immunoglobulin genes, e.g., the FR sequence is derived from one human antibody and the constant region is derived from another human antibody. In some embodiments, the humanized antibody or antigen binding fragment thereof comprises human heavy chains HFR1, HFR2, HFR3 and HFR4 and/or light chains LFR1, LFR2, LFR3 and LFR4.

In some embodiments, the human-derived FR region may include the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are substituted with corresponding residues from the parent non-human antibody. In certain embodiments, this may be desirable to bring the humanized antibody or fragment thereof in close proximity to the non-human parent antibody structure, thereby optimizing binding properties (e.g., increasing binding affinity). In certain embodiments, a humanized antibody or antigen binding fragment thereof provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each of a plurality of human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FR sequences of a heavy or light chain variable domain. In some embodiments, such changes in amino acid residues may be present in only the heavy chain FR region, only the light chain FR region, or in both chains. In certain embodiments, one or more amino acids of the human FR sequence are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are back mutated to the corresponding amino acids of the parent non-human antibody to increase binding affinity.

In some embodiments, antibodies and antigen binding fragments provided herein include all or a portion of a heavy chain variable domain and/or all or a portion of a light chain variable domain. In one embodiment, the antibody or antigen binding fragment thereof provided herein is a single domain antibody consisting of all or a portion of the heavy chain variable domains provided herein. More information about such single domain antibodies is available in the art (see, e.g., U.S. patent No. 6,248,516).

In certain embodiments, an antibody or antigen binding fragment thereof provided herein further comprises an Fc region. In certain embodiments, an antibody or antigen binding fragment thereof provided herein further comprises an Fc region of a human immunoglobulin (Ig). In certain embodiments, an antibody or antigen-binding fragment thereof provided herein further comprises a constant region, optionally further comprising a heavy chain and/or light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and/or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, an anti-drug antibody or antigen-binding fragment thereof provided herein comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4, igA1, igA2, or IgM. In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises a lambda (lambda) light chain or a kappa (kappa) light chain. The constant regions of the antibodies or antigen binding fragments thereof provided herein may be identical to the wild-type constant region sequence or may differ in one or more mutations.

In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. In certain embodiments, an antibody or antigen binding fragment thereof provided herein further comprises an Fc region of a human or mouse immunoglobulin (Ig). In certain embodiments, the antibodies or antigen binding fragments thereof provided herein further comprise an Fc region of a human or mouse IgG. In certain embodiments, the Fc region is derived from IgG1, igG2, igG3, or IgG4.

The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, the Fc regions of IgG1 and IgG3 have been recognized to induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the antibodies and antigen binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype that can induce ADCC or CDC; or alternatively, a constant region of the IgG4 or IgG2 isotype, which has a reduced or depleted effector function. In some embodiments, the Fc region is derived from human IgG1. In some embodiments, the Fc region is derived from human IgG1 having enhanced effector function. In some embodiments, the Fc region includes an amino acid sequence as set forth in SEQ ID NO. 75.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:75)

In some embodiments, the Fc region includes the amino acid sequence shown as SEQ ID NO. 76.

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQS

DLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFP

PKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVS

ELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVS

LTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:76)

In certain embodiments, the antibodies or antigen binding fragments thereof provided herein have a specific binding affinity for ATG-101, which is sufficient to provide diagnostic and/or therapeutic uses.

The antibodies or antigen binding fragments thereof provided herein can be monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, labeled antibodies, bivalent antibodies, anti-idiotype antibodies, or fusion proteins. Recombinant antibodies are antibodies that are produced in vitro, rather than in an animal, using recombinant methods.

In certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof provided herein for binding to ATG-101. In certain embodiments, the disclosure provides antibodies or antigen binding fragments thereof that compete with any of antibodies 9D6E11, 10F10G7, 27E8A8, 31B6G8, 58H6F8, 63C9C12, 75H9F7, and 50D6E10 for binding to ATG-101. In some embodiments, the disclosure provides antibodies or antigen-binding fragments thereof that compete for the same epitope as the antibodies or antigen-binding fragments provided herein.

As used herein, the ability to "block binding" or "compete binding" refers to the ability of an antibody or antigen binding fragment to inhibit the interaction of binding between two molecules (e.g., ATG-101 and an antibody of the disclosure) to any detectable extent. In certain embodiments, an antibody or antigen binding fragment thereof that blocks binding between two molecules inhibits the interaction of binding between two molecules by at least 85% or at least 90%. In certain embodiments, the inhibition may be greater than 95% or greater than 99%.

One of skill in the art will recognize that by determining whether a human monoclonal antibody prevents binding of an antibody of the present disclosure to ATG-101, it can be determined, without undue experimentation, whether the human monoclonal antibody binds to the same epitope as an antibody of the present disclosure (e.g., mouse monoclonal antibody 9D6E11, 10F10G7, 27E8A8, 31B6G8, 58H6F8, 63C9C12, 75H9F7, or 50D6E 10). If the test antibody competes with the antibodies of the disclosure, as indicated by reduced binding of the antibodies of the disclosure to ATG-101, both antibodies bind to the same or closely related epitope. Alternatively, if the binding of the test antibody to ATG-101 is inhibited by an antibody of the present disclosure, both antibodies bind to the same or closely related epitope.

Antibody variants

Antibodies and antigen binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

In certain embodiments, the antibody variants comprise one or more amino acid residue substitutions or modifications, but still retain specific binding affinity for ATG-101. In certain embodiments, at least one of the substitutions or modifications is located in one or more CDR sequences of the VH region or the VL region. In certain embodiments, at least one of the substitutions or modifications is located in one or more non-CDR sequences of the VH region or the VL region. In certain embodiments, the antibodies of the present disclosure, or antigen-binding fragments thereof, further comprise one or more unnatural amino acid (NNAA) substitutions. In certain embodiments, the NNAA is capable of being conjugated.

For example, the antibody variants include one or more of the CDR sequences provided in table 3 above, one or more of the non-CDR sequences of the heavy chain variable region or the light chain variable region provided in table 4 above, and/or one or more amino acid residue substitutions or modifications in the constant region (e.g., fc region). Such variants retain the binding specificity of their parent antibody for ATG-101 but have one or more of the desired properties conferred by the modification or substitution. For example, antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, enhanced effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility with conjugation (e.g., one or more introduced cysteine residues), and the like.

The parent antibody sequences may be screened using methods known in the art, such as "alanine scanning mutagenesis", to identify suitable or preferred residues to be modified or substituted (see, e.g., cunningham and Wells (1989), science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, asp, his, lys and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies generated and screened for a property of interest. If a representation at a particular amino acid position exhibits a functional change of interest, that position can be identified as a potential residue for modification or substitution. The potential residues may be further assessed by substitution with another residue (e.g., a cysteine residue, a positively charged residue, etc.).

Affinity variants

The affinity variant of an antibody may contain one or more CDR sequences, one or more FR sequences provided in table 3 above, or modifications or substitutions in the heavy or light chain variable region sequences provided in table 4 above. The skilled artisan can readily identify FR sequences based on the CDR sequences in table 3 above and the variable region sequences in table 4 above, as it is well known in the art that in the variable region, the CDR regions flank two FR regions. The affinity variant retains the specific binding affinity of the parent antibody for ATG-101 or even has a higher specific binding affinity for ATG-101 than the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences comprise conservative substitutions.

Those skilled in the art will appreciate that one or more amino acid residues may be substituted in the CDR sequences provided in table 3 above and the variable region sequences provided in table 4 above, but the resulting antibody or antigen binding fragment still retains binding affinity or binding capacity for ATG-101, or even has improved binding affinity or binding capacity. Various methods known in the art may be used to achieve this. For example, a library of antibody variants (e.g., fab or scFv variants) can be generated and expressed using phage display techniques, which are then screened for affinity for binding to ATG-101. For another example, computer software may be used to virtually mimic the binding of an antibody to ATG-101 and identify the amino acid residues on the antibody that form the binding interface. Such residues may be avoided in the substitution to prevent binding affinity from decreasing, or may be targeted for substitution to obtain stronger binding.

In certain embodiments, a humanized antibody or antigen-binding fragment thereof provided herein comprises one or more CDR sequences in a CDR sequence and/or one or more amino acid residue substitutions in one or more FR sequences in an FR sequence. In certain embodiments, the affinity variants comprise no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in total in the CDR sequence and/or FR sequence.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises 1, 2, or 3 CDR sequences that have at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequences listed in table 3 above, but still retain specific binding affinity for ATG-101 at a similar or even higher level relative to its parent antibody.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises one or more variable region sequences that have at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequences listed in table 4 above, but still retain specific binding affinity for ATG-101 at a similar or even higher level relative to its parent antibody. In some embodiments, from 1 to 10 amino acids are substituted, inserted, or deleted in total in the variable region sequences listed in table 4 above. In some embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs (e.g., in the FR).

Glycosylation variants

The antibodies or antigen-binding fragments thereof provided herein also encompass glycosylated variants that may be obtained to increase or decrease the degree of glycosylation of the antibodies or antigen-binding fragments thereof.

The antibodies or antigen binding fragments thereof provided herein may include one or more modifications that introduce or remove glycosylation sites. Glycosylation sites are amino acid residues having side chains to which carbohydrate moieties (e.g., oligosaccharide structures) may be attached. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine), where X is any amino acid other than proline. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most commonly serine or threonine. The natural glycosylation site can be conveniently removed, for example by altering the amino acid sequence such that one of the above tripeptide sequences (for an N-linked glycosylation site) or serine or threonine residues (for an O-linked glycosylation site) present in the sequence is substituted. New glycosylation sites can be created in a similar manner by introducing such tripeptide sequences or serine or threonine residues.

Cysteine engineered variants

Antibodies or antigen binding fragments thereof provided herein also encompass cysteine engineered variants comprising one or more introduced free cysteine amino acid residues.

The free cysteine residue is a cysteine residue that is not part of a disulfide bond. Cysteine engineered variants can be used to conjugate, for example, cytotoxic and/or imaging compounds, labels or radioisotopes, etc., at the site of the engineered cysteine via, for example, maleimide or haloacetyl groups. Methods for engineering antibodies or antigen binding fragments thereof to introduce free cysteine residues are known in the art, see, e.g., WO2006/034488.

Fc variants

Antibodies and antigen binding fragments provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions at their Fc region and/or hinge region.

In certain embodiments, the antibody or antigen binding fragment thereof comprises one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptor (FcRn). Such variants may have an extended pharmacokinetic half-life in that it binds to FcRn at acidic pH, freeing it from degradation in lysosomes, and subsequently being transferred and released extracellularly. Methods for engineering antibodies and antigen binding fragments thereof to increase binding affinity to FcRn are well known in the art, see, e.g., vaughn, d. Et al, structure, 6 (1): 63-73,1998; kontermann, R.et al, antibody engineering (Antibody Engineering), volume 1, chapter 27. Fc regions were engineered to improve PK (Engineering of the Fc region for improved PK), published by Springer, 2010; yeung, Y.et al, cancer Research, 70:3269-3277 (2010); and Hinton, P.et al, J.Immunol.176:346-356 (2006).

In certain embodiments, the antibody or antigen binding fragment thereof comprises one or more amino acid substitutions that alter ADCC. Certain amino acid residues at the CH2 domain of the Fc region may be substituted to provide enhanced ADCC activity. Alternatively or additionally, the carbohydrate structure on the antibody may be altered to enhance ADCC activity. Methods for altering ADCC activity by antibody engineering have been described in the art, see for example, shields RL. et al, journal of biochemistry (Jbiol chem.) 2001 276 (9): 6591-604; idusiogie EE et al, J.Immunol. 2000.164 (8): 4178-84; steurer W et al J.Immunology 1995,155 (3): 1165-74; idusibie EE et al J.Immunol.2001, 166 (4): 2571-5; lazar GA. et al, proc. Natl. Acad. Sci. USA (PNAS), 2006,103 (11): 4005-4010; ryan MC. et al, molecular cancer therapy (mol. Cancer ter.), 2007,6:3009-3018; richards JO et al, molecular Cancer therapy (Mol Cancer Ther.) 2008,7 (8): 2517-27; shields R.L. et al, J.Biol.chem., 2002,277:26733-26740; shinkawa T.et al, (J. Biol. Chem.) 2003,278:3466-3473.

In certain embodiments, the antibody or antigen binding fragment thereof comprises one or more amino acid substitutions that alter CDC, e.g., by improving or reducing C1q binding and/or CDC (see, e.g., WO99/51642; duncan and Winter Nature (Science) 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821); and WO94/29351 regarding other examples of variants of Fc regions. One or more amino acids selected from amino acid residues 329, 331 and 322 of the Fc region may be substituted with a different amino acid residue to alter C1q binding and/or enhance CDC (see U.S. patent No. 6,194,551 to Idusogie et al). One or more amino acid substitutions may also be introduced to alter the ability of the antibody to fix complement (see PCT publication WO94/29351 to Bodmer et al).

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid substitutions at positions 234 and/or 235 (according to EP numbering) of a human immunoglobulin (e.g., igG 1). In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises two amino acid substitutions at positions 234 and 235 (numbered according to EP) in a human immunoglobulin (e.g., igG 1). In certain embodiments, an antibody or antigen binding fragment thereof provided herein comprises L234A and L235A (numbering according to EU) amino acid substitutions.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein includes one or more amino acid substitutions in the interface of the Fc region to facilitate promotion and/or promote heterodimerization. These modifications include introducing a protuberance into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to facilitate interaction of the first Fc polypeptide with the second Fc polypeptide to form a heterodimer or complex. Methods of producing antibodies with these modifications are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises an amino acid substitution at position 366 (according to EP numbering) of a first Fc polypeptide and comprises one, two, or three amino acid substitutions at one, two, or three positions 366, 368, and 407 (according to EP numbering) of a second Fc polypeptide. In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a T366W (according to EP numbering) substitution of a first Fc polypeptide and comprises a T366s+l368a+y407V (according to EP numbering) substitution of a second Fc polypeptide.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid substitutions of a first Fc polypeptide and comprises one or more amino acid substitutions of a second Fc polypeptide, thereby introducing a non-native disulfide bond between the two Fc polypeptides. For example, an antibody or antigen-binding fragment thereof provided herein includes an amino acid substitution at position 354 (according to EU numbering) of a first Fc polypeptide and includes an amino acid substitution at position 349 (according to EU numbering) of a second Fc polypeptide. In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises an S354C (according to EP numbering) substitution of a first Fc polypeptide and comprises a Y349C (according to EP numbering) substitution of a second Fc polypeptide.

In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a first Fc polypeptide comprising an amino acid sequence as set forth in SEQ ID No. 77 and a second Fc polypeptide comprising an amino acid sequence as set forth in SEQ ID No. 78.

KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:77)

KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78)

Antigen binding fragments

Antigen binding fragments are also provided herein. Various types of antigen binding fragments are known in the art and can be developed based on the antibodies provided herein, including, for example, exemplary antibodies whose CDRs are shown in table 3 above and whose variable sequences are shown in table 4, and different variants thereof (e.g., affinity variants, glycosylation variants, fc variants, cysteine engineered variants, etc.).

In certain embodiments, the antigen-binding fragments provided herein are bifunctional antibodies (diabodies), fab ', F (ab') ₂ Fd, fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ₂ Bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibody (ds diabody), single chain antibody molecule [ ]scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

Such antigen binding fragments can be produced using a variety of techniques. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., morimoto et al, journal of biochemistry and biophysics methods (Journal of Biochemical and Biophysical Methods) 24:107-117 (1992), and Brennan et al, science 229:81 (1985)), recombinant expression by host cells such as E.coli (e.g., for Fab, fv and ScFv antibody fragments), screening from phage display libraries (e.g., for ScFv) as discussed above, and chemical coupling of two Fab '-SH fragments to form F (ab') ₂ Fragments (Carter et al, biology/Technology (Bio/Technology) 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

In certain embodiments, the antigen binding fragment is an scFv. The production of scFv is described below: for example WO 93/16185; U.S. patent No. 5,571,894; and No. 5,587,458. ScFv can be fused at the amino-or carboxy-terminus to an effector protein to provide a fusion protein (see e.g., antibody engineering, borrebaeck editions).

In certain embodiments, the antibodies or antigen binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule that is more than divalent is considered multivalent and encompasses, for example, trivalent, tetravalent, hexavalent, and the like.

A bivalent molecule may be monospecific if both binding sites specifically bind to the same antigen or the same epitope. In certain embodiments, this provides for stronger binding to an antigen or epitope than the monovalent counterpart. Similarly, multivalent molecules may also be monospecific. In certain embodiments, in a bivalent or multivalent antigen binding portion, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences, but have the same specificity).

Bivalent may also be bispecific if both binding sites are specific for different antigens or epitopes. The same applies to multivalent molecules. For example, a trivalent molecule may be bispecific when two binding sites are monospecific for a first antigen (or epitope) and a third binding site is specific for a second antigen (or epitope).

Bispecific or multispecific antibodies

In certain embodiments, the antibodies or antigen binding fragments thereof provided herein are bispecific or multispecific. In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is further linked to a second functional moiety or antigen-binding fragment thereof having a binding specificity different from that of the antibody. In some embodiments, the bispecific or multispecific antibodies and antigen-binding fragments thereof provided herein have a first specificity for ATG-101 and a second specificity. In some embodiments, the second specificity is for ATG-101 but for a different epitope.

In certain embodiments, the second specificity is for a tumor-associated antigen or epitope thereof. The term "tumor-associated antigen" refers to an antigen that is present or presentable on the surface of tumor cells, as well as on or within tumor cells. In some embodiments, the tumor-associated antigen can only be presented by tumor cells, but not by normal cells (i.e., non-tumor cells). In some other embodiments, the tumor-associated antigen may be expressed exclusively on tumor cells or may represent a tumor-specific mutation compared to non-tumor cells. In some other embodiments, the tumor-associated antigen may be found in both tumor cells and non-tumor cells, but is over-expressed on tumor cells when compared to non-tumor cells, or may be used for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue. In some embodiments, the tumor-associated antigen is located on the vasculature of the tumor.

In certain embodiments, a bispecific antibody or multispecific antibody provided herein, or antigen-binding fragment thereof, is capable of specifically binding to one or more (e.g., 1, 2, 3, 4, 5, or more) additional antigens other than ATG-101, or to a second epitope on ATG-101. In certain embodiments, the one or more additional antigens other than ATG-101 are selected from the group consisting of: KRAS, ERK, XPO1, mTORC1/2, PAK4, NAMPT, ATR, EGFR, FGFR, VEGF, LILRB (e.g., LILRB1, LILRB2, LILRB3, LILRB4, LILRB 5), C-MET, her2, her3, CTLA4, GITA, CD112R, CD2, CD3, CD7, CD16, CD19, CD20, CD24, CD27, CD30, CD34, CD37, CD39, CD70, CD73, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD47, sirpa, CD122, CD137L, OX (CD 134), OX40L (CD 252), BCMA (e.g., BCMA 02), PSMA, CLDN18 (e.g., CLDN 18.2), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS (CD 278), icosolg (CD 275), LAG3 (CD 223), A2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), B7-H5, BTLA (CD 272), CD160, CTLA-4 (CD 152), GPRC5D, IDO (e.g., IDO1, IDO 2), TDO, KIR, LAIR-1, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD 328), SIGLEC-9 (CD 329), SIGLEC-15, git, PVR (CD 155), and tgfβ.

In certain embodiments, a bispecific antibody or multispecific antibody or antigen-binding fragment thereof provided herein is capable of specifically binding to a PD-L1 binding portion and a 4-1BB binding portion of ATG-101.

i. one or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) comprised within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: SEQ ID NOS 6, 13, 20 and 27;

i. HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 2, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 3, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 4, LCDR2 comprising the amino acid sequence shown in NAK, LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 5, LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 29

HCDR1 comprising the amino acid sequence shown as SEQ ID No. 30, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 31, HCDR3 comprising the amino acid sequence shown as SEQ ID NO:

32, an LCDR1 comprising an amino acid sequence as shown in STS, an LCDR2 comprising an amino acid sequence as shown in SEQ ID No. 33;

HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 1, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 2, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 3, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 4, LCDR2 comprising the amino acid sequence shown as NAK, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 5, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 36

HCDR1 comprising the amino acid sequence shown as SEQ ID No. 37, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 38, HCDR3 comprising the amino acid sequence shown as SEQ ID NO:

39, an LCDR1 comprising an amino acid sequence shown as NAK, an LCDR2 comprising an amino acid sequence shown as SEQ ID No. 40, and an LCDR3 comprising an amino acid sequence shown as SEQ ID No. 40;

HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 1, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 2, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 3, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 4, LCDR2 comprising the amino acid sequence shown as NAK, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 5, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 43

HCDR1 of the amino acid sequence shown, HCDR2 comprising the amino acid sequence shown as SEQ ID NO:44, HCDR3 comprising the amino acid sequence shown as SEQ ID NO:45, HCDR3 comprising the amino acid sequence shown as SEQ ID NO:44

LCDR1 of the amino acid sequence shown as NO. 46, LCDR2 comprising the amino acid sequence shown as NAK, and LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 47;

HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 1, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 2, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 3, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 4, LCDR2 comprising the amino acid sequence shown as NAK, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 5, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 50

HCDR1 comprising the amino acid sequence shown as SEQ ID No. 51, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 52, HCDR3 comprising the amino acid sequence shown as SEQ ID NO:

53, LCDR2 comprising an amino acid sequence as shown in QMS, LCDR3 comprising an amino acid sequence as shown in SEQ ID No. 54;

v. HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 9, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 10, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 11, LCDR2 comprising the amino acid sequence shown in RAN, LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 12, HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:

HCDR1 comprising the amino acid sequence shown as SEQ ID No. 8, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 9, HCDR3 comprising the amino acid sequence shown as SEQ ID No. 10, LCDR1 comprising the amino acid sequence shown as SEQ ID No. 11, LCDR2 comprising the amino acid sequence shown as RAN, LCDR3 comprising the amino acid sequence shown as SEQ ID No. 12, HCDR1 comprising the amino acid sequence shown as SEQ ID No. 36, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 37, HCDR3 comprising the amino acid sequence shown as SEQ ID No. 38, HCDR3 comprising the amino acid sequence shown as SEQ ID NO:

HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 8, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 9, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 10, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 11, LCDR2 comprising the amino acid sequence shown in RAN, LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 12, HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 43, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 44, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 45, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 45:

46, LCDR2 comprising an amino acid sequence shown as NAK, LCDR3 comprising an amino acid sequence shown as SEQ ID No. 47;

HCDR1 comprising the amino acid sequence shown in SEQ ID No. 8, HCDR2 comprising the amino acid sequence shown in SEQ ID No. 9, HCDR3 comprising the amino acid sequence shown in SEQ ID No. 10, LCDR1 comprising the amino acid sequence shown in SEQ ID No. 11, LCDR2 comprising the amino acid sequence shown in RAN, LCDR3 comprising the amino acid sequence shown in SEQ ID No. 12, HCDR1 comprising the amino acid sequence shown in SEQ ID No. 50, HCDR2 comprising the amino acid sequence shown in SEQ ID No. 51, HCDR3 comprising the amino acid sequence shown in SEQ ID No. 52, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:

HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 15, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 16, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 17, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 18, LCDR2 comprising the amino acid sequence shown as AAS, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 19

HCDR1 of the amino acid sequence shown as SEQ ID NO. 29, HCDR2 of the amino acid sequence shown as SEQ ID NO. 30, HCDR3 of the amino acid sequence shown as SEQ ID NO. 31, HCDR3 of the amino acid sequence shown as SEQ ID NO:

x. HCDR1 comprising the amino acid sequence shown as SEQ ID NO. 15, HCDR2 comprising the amino acid sequence shown as SEQ ID NO. 16, HCDR3 comprising the amino acid sequence shown as SEQ ID NO. 17, LCDR1 comprising the amino acid sequence shown as SEQ ID NO. 18, LCDR2 comprising the amino acid sequence shown as AAS, LCDR3 comprising the amino acid sequence shown as SEQ ID NO. 19

HCDR1 of the amino acid sequence shown in SEQ ID NO. 36, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 37, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 38, HCDR3 comprising the amino acid sequence shown in SEQ ID NO:

HCDR1 of the amino acid sequence shown as SEQ ID NO. 43, HCDR2 of the amino acid sequence shown as SEQ ID NO. 44, HCDR3 of the amino acid sequence shown as SEQ ID NO. 45:

HCDR1 comprising the amino acid sequence shown as SEQ ID No. 15, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 16, HCDR3 comprising the amino acid sequence shown as SEQ ID No. 17, LCDR1 comprising the amino acid sequence shown as SEQ ID No. 18, LCDR2 comprising the amino acid sequence shown as AAS, LCDR3 comprising the amino acid sequence shown as SEQ ID No. 19, HCDR1 comprising the amino acid sequence shown as SEQ ID No. 50, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 51, HCDR3 comprising the amino acid sequence shown as SEQ ID No. 52, LCDR1 comprising the amino acid sequence shown as SEQ ID No. 53, LCDR2 comprising the amino acid sequence shown as QMS and LCDR3 comprising the amino acid sequence shown as SEQ ID No. 54.

HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 24, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 25, LCDR2 comprising the amino acid sequence shown in AAS, LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 26, HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 29, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 30, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 31, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 32, LCDR2 comprising the amino acid sequence shown in STS and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 33.

HCDR1 comprising the amino acid sequence shown as SEQ ID No. 22, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 23, HCDR3 comprising the amino acid sequence shown as SEQ ID No. 24, LCDR1 comprising the amino acid sequence shown as SEQ ID No. 25, LCDR2 comprising the amino acid sequence shown as AAS, LCDR3 comprising the amino acid sequence shown as SEQ ID No. 26, HCDR1 comprising the amino acid sequence shown as SEQ ID No. 36, HCDR2 comprising the amino acid sequence shown as SEQ ID No. 37, HCDR3 comprising the amino acid sequence shown as SEQ ID No. 38, LCDR1 comprising the amino acid sequence shown as SEQ ID No. 39, LCDR2 comprising the amino acid sequence shown as NAK, and LCDR3 comprising the amino acid sequence shown as SEQ ID No. 40.

xv. comprises an HCDR1 of the amino acid sequence shown in SEQ ID NO. 22, an HCDR2 of the amino acid sequence shown in SEQ ID NO. 23, an HCDR3 of the amino acid sequence shown in SEQ ID NO. 24, an LCDR1 of the amino acid sequence shown in SEQ ID NO. 25, an LCDR2 of the amino acid sequence shown in AAS, an LCDR3 of the amino acid sequence shown in SEQ ID NO. 26, an HCDR1 of the amino acid sequence shown in SEQ ID NO. 43, an HCDR2 of the amino acid sequence shown in SEQ ID NO. 44, an HCDR3 of the amino acid sequence shown in SEQ ID NO. 45:

46, LCDR2 comprising an amino acid sequence shown as NAK, LCDR3 comprising an amino acid sequence shown as SEQ ID No. 47; or (b)

xvi. HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 22, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 23, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 24, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 25, LCDR2 comprising the amino acid sequence shown in AAS, LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 26, HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 50, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 51, HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 52, LCDR1 comprising the amino acid sequence shown in SEQ ID NO. 53, LCDR2 comprising the amino acid sequence shown in QMS and LCDR3 comprising the amino acid sequence shown in SEQ ID NO. 54.

Conjugate(s)

In some embodiments, an antibody or antigen binding fragment thereof provided herein further comprises one or more conjugate moieties. The conjugate moiety may be linked to an antibody or antigen binding fragment thereof. The binding moiety is a moiety that can be linked to an antibody or antigen binding fragment thereof. It is contemplated that a variety of conjugate moieties may bind to the antibodies or antigen binding fragments thereof provided herein (see, e.g., "conjugate vaccine (Conjugate Vaccines)", "contributions to microbiology and immunology (Contributions to Microbiology and Immunology), j.m.cruse and r.e.lewis, jr. (editors), new York Carger Press (Carger Press, new York), (1989)). These conjugate moieties may be linked to the antibody or antigen-binding fragment thereof by covalent binding (e.g., disulfide bonds), affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, etc. In some embodiments, the antibody or antigen binding fragment thereof may be linked to one or more conjugates via a linker or cross-linker. The linker or crosslinker comprises a reactive chemical group that can react with an antibody or antigen binding fragment thereof provided herein. The reactive chemical groups may be N-succinimidyl esters and N-sulfosuccinimidyl esters. In addition, the linker includes a reactive chemical group, which may be a dithiopyridyl group that may react with a drug to form a disulfide bond. Linker molecules include, for example, N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (see, for example, carlsson et al, J. Biol.Chem.173:723-737 (1978)), N-succinimidyl 4- (2-pyridyldithio) butyrate (SPDB) (see, for example, U.S. Pat. No. 4,563,304), N-succinimidyl 4- (2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) (see, for example, U.S. publication No. 2009027413), N-succinimidyl 4- (2-pyridyldithio) valerate (SPP) (see, for example, CAS registry No. 341498-08-6), 2-iminothiolane, or acetyl succinic anhydride. For example, the antibody or cell-binding agent may be modified with a crosslinking reagent, and the resulting antibody or cell-binding agent containing a free or protected thiol group is then reacted with maytansinoid (maytansinoid) containing a disulfide or thiol to produce a conjugate. The conjugate may be purified by chromatography, including but not limited to HPLC, size exclusion, adsorption, ion exchange and affinity capture, dialysis, or tangential flow filtration.

In certain embodiments, the antibodies or antigen binding fragments thereof provided herein may be engineered to contain specific sites other than epitope binding moieties that may be used for binding to one or more conjugate moieties. For example, such sites may include one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent attachment to the conjugate moiety.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein may be linked to a conjugate moiety, either indirectly or through another conjugate moiety. For example, an antibody or antigen binding fragment thereof provided herein can be conjugated to biotin, followed by indirect conjugation to a second conjugate conjugated to avidin. In some embodiments, the conjugate moiety comprises a scavenging modifier (e.g., a half-life extending polymer such as PEG), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, a purification moiety, or other anti-cancer drug (e.g., toll-like receptor 7 (TLR-7), an agonist of TLR-8 and/or TLR-9, an siRNA, an antibody or antigen-binding fragment thereof, a peptide (e.g., a short peptide), etc.).

A "toxin" may be any agent that is harmful to a cell or that can damage or kill a cell. Examples of toxins include, but are not limited to, paclitaxel, CC-1065 and CC-1065 analogs, du Kamei (duocarmycin) and Du Kamei analogs, enediynes such as calicheamicin (calicheamicin), dolastatin (dolastatin) and dolastatin analogs (including auristatin), tolmetin derivatives (tomaymycin derivative), leptomycin derivatives (leptomycin derivative), cisplatin (cislatin), carboplatin (carboplatin), daunorubicin, doxorubicin, vincristine (vincristine), vinblastine (vinblastine), marflange (melphalan), mitomycin C, chlorambucil and morpholino doxorubicin, cytochalasin B ponticin D, ethidium bromide, ipecine, mitomycin, etoposide (etoposide), teniposide (tenoposide), MMAE, MMAF, DM, DM4, vinblastine (vinblastine), colchicine (colchicine), doxorubicin, daunorubicin, dicarboxylanthrone (dihydroxy anthracin dione), mitoxantrone (mitoxantrone), mithramycin (mithramycin), actinomycin D (actinomycin D), 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine, lidocaine (lidocaine), propranolol (puromycin) and analogs thereof (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine (dacarbazine), alkylating agents (e.g., nitrogen mustard, cetylpiperazine chlorambucil (thioepa chlorambucil), melphalan (melphalan), carmustine (carmustine) (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan (busulfan), dibromomannitol, streptozotocin, mitomycin C and dichlorodiamide platinum (II) (DDP cisplatin), anthracyclines (anthracyclines) (e.g., daunorubicin (daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (dactinomycin) (former actinomycin), bleomycin (bleomycin), mithramycin and Anthramycin (AMC)), antimitotics (e.g., vincristine and vinblastine), topoisomerase inhibitors and tubulin binding agents.

Examples of detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or texas red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase, or beta-D-galactosidase), radioisotopes (e.g., ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ³⁵ S、 ³ H、 ¹¹¹ In、 ¹¹² In、 ¹⁴ C、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁸⁶ Y、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁷⁷ Lu、 ²¹¹ At、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹⁵³ Sm、 ²¹² bi and Bi ³² P, other lanthanoid element), luminescent labels, chromophore moieties, digoxin, biotin/avidin, DNA molecules, or gold for detection.

In certain embodiments, the conjugate moiety may be a scavenging modifier that helps increase the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules.

In certain embodiments, the conjugate moiety may be a purification moiety, such as a magnetic bead.

In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as substrates for conjugates.

In certain embodiments, an antibody or antigen binding fragment thereof provided herein is conjugated to a single peptide. Signal peptides (sometimes referred to as signal sequences, leader sequences, or leader peptides) may be used to facilitate secretion and isolation of the antibodies or antigen binding fragments thereof provided herein. Signal peptides are generally characterized as the core of hydrophobic amino acids that are typically cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature protein as it passes through the secretory pathway. Thus, the invention relates to the described polypeptides having a signal sequence and polypeptides (i.e. cleavage products) whose signal sequence has been proteolytically cleaved. In one embodiment, the nucleic acid sequence encoding the signal sequence may be operably linked in an expression vector to a protein of interest, such as a protein that is not normally secreted or otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or simultaneously cleaved. The protein can then be readily purified from the extracellular medium by art-recognized methods. Alternatively, the signal sequence may be linked to the protein of interest using a sequence that facilitates purification, such as using a GST domain.

Chimeric antigen receptor

In certain embodiments, the present disclosure provides a chimeric antigen receptor comprising an antibody or antigen binding fragment thereof provided herein, a transmembrane region, and an intracellular signaling region.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that specifically grafts an antigen to a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, a regulatory T cell, a natural killer cell, or any combination thereof). CARs are also known as artificial cell receptors, chimeric cell receptors or chimeric immune receptors. In some embodiments, the CAR comprises an antigen-specific targeting region (e.g., an antigen-binding fragment of an antibody as provided herein), an extracellular region, a transmembrane region, one or more costimulatory regions, and an intracellular signaling region.

In some embodiments, the antigen-specific targeting region is an scFv. In some embodiments, the transmembrane region comprises a transmembrane region of CD3, CD4, CD8 or CD 28. In some embodiments, the costimulatory region comprises the costimulatory domains of CD28, ICOS, CD27, 4-1BB, OX40, and CD 40L. In some embodiments, the intracellular signaling region is selected from the group consisting of: an intracellular signal region sequence of CD3, fcyri, CD27, CD28, CD137, CD134, myD88, CD40, CD278, TLR, or a combination thereof.

The CAR may be transplanted into a variety of cells, for example, allogeneic, autologous, or xenogeneic cells.

The term "allogeneic cells" as used herein refers to any cells derived from different individuals of the same species.

The term "autologous cells" as used herein refers to any cells derived from the same individual that are subsequently reintroduced into the individual.

The term "heterologous cell" as used herein refers to any cell derived from a different individual of a different species.

In certain embodiments, the CAR is transplanted into an immune effector cell, e.g., a T cell, natural killer cell, macrophage, tumor-infiltrating lymphocyte, or the like.

Polynucleotide and recombination method

The present disclosure provides isolated polynucleotides encoding antibodies or antigen binding fragments thereof provided herein or encoding chimeric antigen receptors provided herein. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single or double stranded form and polymers thereof. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (see Batzer et al, nucleic acids Ind. 19:5081 (1991); ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, molecular and cell probing (mol. Cell. Probes), 8:91-98 (1994)).

DNA encoding an antibody or antigen binding fragment thereof or the chimeric antigen receptor provided herein is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of an antibody). The coding DNA may also be obtained by synthetic methods.

Isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein and/or chimeric antigen receptors provided herein can be inserted into vectors for further cloning (amplification of DNA) or expression using recombinant techniques known in the art. A number of vectors are available. The carrier composition generally includes, but is not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1. Alpha.) and a transcription termination sequence.

The present disclosure provides vectors comprising the isolated polynucleotides provided herein. In certain embodiments, a polynucleotide provided herein encodes an antibody or antigen-binding fragment thereof or chimeric antigen receptor, at least one promoter (e.g., SV40, CMV, EF-1 a) operably linked to a nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40), lambda and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-Gseu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

Vectors comprising polynucleotide sequences encoding antibodies or antigen binding fragments thereof or chimeric antigen receptors provided herein can be introduced into host expression systems (e.g., host cells) for cloning or gene expression. In certain embodiments, the host expression systems provided herein are microbial, yeast, or mammalian cells. In certain embodiments, the microorganism is selected from the group consisting of escherichia coli and bacillus subtilis. In certain embodiments, the yeast is a saccharomyces. In certain embodiments, the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

Suitable host cells for cloning or expressing the DNA in the vectors herein are the above-described prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example Enterobacteriaceae (Enterobacteriaceae), such as Escherichia (e.g. E.coli), enterobacter (Enterobacter), erwinia (Erwinia), klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) (e.g. Salmonella typhimurium (Salmonella typhimurium)), serratia (Serratia) (e.g. Serratia marcescens (Serratia marcescans)) and Shigella (Shigella) and Bacillus (Bacillus) such as Bacillus subtilis and Bacillus licheniformis (B.lichenifermis), pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (P.augusta) and Streptomyces (Streptomyces).

In addition to prokaryotes, eukaryotic microbes (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for vectors encoding antibodies. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Saccharomyces cerevisiae are the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are more common and suitable for use herein, such as schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts (Kluyveromyces host), such as Kluyveromyces lactis (K.lactis), kluyveromyces fragilis (K.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (K.bulgaricus) (ATCC 16,045), kluyveromyces weissei (K.winkerami) (ATCC 24,178), kluyveromyces (K.waiti) (ATCC 56,500), kluyveromyces drosophila (K.drosophila) (ATCC 36,906), kluyveromyces thermotolerans (K.thermophilus) and Kluyveromyces marxianus (K.marxianus); yarrowia (EP 402,226); pichia pastoris (EP 183,070); candida (Candida); trichoderma reesei (Trichoderma reesia) (EP 244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces western (Schwanniomyces occidentalis); and filamentous fungi (filamentous fungi), such as Neurospora (Neurospora), penicillium (Penicillium), curvularia (Tolypocladium), and Aspergillus hosts (Aspergillus host), such as Aspergillus nidulans (A. Nidulans) and Aspergillus niger (A. Niger).

Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells derived from hosts such as: spodoptera frugiperda (Spodoptera frugiperda) (caterpillars), aedes aegypti (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (Drosophila melanogaster) (drosophila melanogaster), and Bombyx mori (Bombyx mori). A variety of viral strains for transfection are publicly available, for example L-1 variants of the NPV of Spodoptera frugiperda (Autographa californica) and Bm-5 variants of the NPV of Bombyx mori, and such viruses may be used as the viruses herein according to the invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

However, the most interesting are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. An example of a useful mammalian host cell line is the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, graham et al, journal of general virology (J.Gen.Virol.)) 36/59 (1977); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, urlaub et al, journal of the national academy of sciences 77:4216 (1980)); mouse Sitertoli cells (TM 4, mather, [ biological reproduction.) ] 23:243-251 (1980) ]; monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat (buffalo rat) hepatocytes (BRL 3a, atcc CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562,ATCC CCL51); TRI cells (Mather et al, "annual book of the New York sciences (Annals N.Y. Acad. Sci.)))" 383:44-68 (1982); MRC 5 cells; FS4 cells; mouse precancerous carcinoma cells (MFC), SNU620 cells, and human hepatoma lines (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, MFC, SNU620, and derivatives thereof.

The host cells are transformed with the expression or cloning vectors described above for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the antibodies may be produced by homologous recombination as known in the art. In certain embodiments, the host cell is capable of producing an antibody or antigen-binding fragment thereof provided herein.

The present disclosure also provides a method of expressing an antibody or antigen-binding fragment thereof or chimeric antigen receptor provided herein, comprising culturing a host expression system provided herein under conditions that express the antibody or antigen-binding fragment thereof or chimeric antigen receptor. The host expression systems for producing the antibodies or antigen binding fragments thereof or chimeric antigen receptors provided herein can be cultured in a variety of media. Commercially available media such as Ham's F (Sigma), minimal essential media (Minimal Essential Medium, MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. In addition, any of the media described below may be used as the medium for the host cells: ham et al, methods of enzymology (meth.Enz.) 58:44 (1979); barnes et al, analytical biochemistry (Anal. Biochem.) 102:255 (1980); U.S. patent No. 4,767,704; 4,657,866; 4,927,762, 4,560,655 or 5,122,469; WO 90/03430; WO 87/00195; or us reissue patent 30,985. Any of these media may be supplemented with hormones and/or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN) ^TM Drug), trace elements (defined as inorganic compounds with final concentrations typically in the micromolar range), and glucoseOr an equivalent energy source. Any other necessary supplements may also be included in suitable concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc., are those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

When recombinant techniques are used, the antibodies may be produced in the cell, in the periplasmic space, or directly secreted into the culture medium. If antibodies are produced intracellularly, as a first step, the host cells or the particulate fragments of the lysed fragments may be removed, for example, by centrifugation or ultrafiltration. Carter et al, bio/technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When antibodies are secreted into the culture medium, supernatants from such expression systems are typically first concentrated using commercially available protein concentration filters (e.g., amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.

Antibodies or antigen binding fragments thereof and/or chimeric antigen receptors produced by the host expression system may be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

In certain embodiments, protein a immobilized on a solid phase is used for immunoaffinity purification of antibodies and antigen binding fragments thereof and/or chimeric antigen receptors. Whether protein a is suitable as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on the human gamma 1, gamma 2 or gamma 4 heavy chain (Lindmark et al J.Immunol.62:1-13 (1983)). Protein G is recommended for all mouse isoforms and human gamma 3 (Guss et al, J.European molecular biology journal (EMBO J.)) 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also useful. Mechanically stable substrates such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody includes a CH3 domain, baker bond ABXTM resin (J. T. Baker, phillips burg, N.J.) may be used for purification other techniques for protein purification are also available depending on the antibody to be recovered, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSEM, chromatography on anion or cation exchange resins such as polyaspartic acid columns, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification steps, the mixture comprising the antibody of interest and the contaminant may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).

Composition and method for producing the same

The present disclosure further provides compositions comprising an antibody or antigen-binding fragment thereof or chimeric antigen receptor provided by the present disclosure, and one or more carriers.

Carriers for the compositions disclosed herein can include, for example, liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/partitioning agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents, emulsifying agents or stabilizing agents, such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole (butylated hydroxanisol), butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen binding fragment and conjugate thereof provided herein reduces oxidation of the antibody or antigen binding fragment thereof. This reduction in oxidation can prevent or reduce loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, there is provided a pharmaceutical composition comprising one or more antibodies or antigen binding fragments thereof as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation, extending shelf life, and/or improving efficacy of an antibody or antigen binding fragment provided herein by mixing the antibody or antigen binding fragment with one or more antioxidants such as methionine.

For further illustration, the carrier may include, for example: aqueous vehicles such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection or dextrose and lactate Ringer's injection; nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil; an antimicrobial agent at a bacteria-inhibiting or fungi-inhibiting concentration; isotonic agents, such as sodium chloride or dextrose; buffers, such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics, such as procaine hydrochloride; suspending and dispersing agents, such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone; emulsifying agents, such as polysorbate 80 (TWEEN-80); sequestering or chelating agents, such as EDTA (ethylene diamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid or lactic acid. Antimicrobial agents useful as carriers may be added to the pharmaceutical composition in the multi-dose container, including phenol or cresol, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

The composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, slow release formulation, or powder. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

In certain embodiments, the composition is formulated as an injectable composition. The injectable composition may be prepared in any conventional form, such as a liquid solution, suspension, emulsion or solid form suitable for producing a liquid solution, suspension or emulsion. Injectable formulations may include sterile and/or pyrogen-free solutions to be injected, sterile dried soluble products (such as lyophilized powders, including subcutaneous injection tablets) to be combined with solvents prior to use, sterile suspensions to be injected, sterile dried insoluble products to be combined with vehicles prior to use, and sterile and/or pyrogen-free emulsions. The solution may be aqueous or non-aqueous.

In certain embodiments, the unit dose parenteral formulation is packaged in an ampoule, vial or syringe with needle. As is known and practiced in the art, all formulations for parenteral administration should be sterile and pyrogen-free.

In certain embodiments, sterile lyophilized powders are prepared by dissolving an antibody or antigen-binding fragment thereof as disclosed herein in a suitable solvent. The solvent may contain excipients that may improve the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art, which in one embodiment is about neutral pH. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an antibody or antigen-binding fragment thereof or a combination thereof. Overfilling the vial with an amount slightly higher than that required for each dose or doses (e.g., about 10%) is acceptable in order to facilitate accurate sampling and accurate dosing. The lyophilized powder may be stored under suitable conditions (e.g., at about 4 ℃ to room temperature).

The lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. In one embodiment, sterile and/or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy administered and can be determined empirically.

Kit for detecting a substance in a sample

In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and/or a composition provided herein and/or a chimeric antigen receptor provided herein. In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and/or a composition provided herein and/or a chimeric antigen receptor provided herein and a second agent. In certain embodiments, the second agent is selected from the group consisting of: detection agents, chemotherapeutic agents, anti-cancer drugs, radiation therapies, immunotherapeutic agents, anti-angiogenic agents, targeted therapies, cell therapies, gene therapies, hormonal therapies, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof and/or chimeric antigen receptor and/or composition provided herein for detecting an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in a sample. In some embodiments, the bispecific PD-L1/4-1BB antibody is ATG-101.

In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof and/or chimeric antigen receptor and/or composition provided herein for detecting an anti-drug antibody (ADA) to an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody.

As used herein, "anti-drug antibody" or "ADA" refers to an antibody that specifically binds to any portion of a drug. For example, an anti-drug antibody may be an antibody or fragment thereof, which may be directed against any region of an antibody drug, such as the variable domain, constant domain, or sugar structure of an antibody. Such anti-drug antibodies may appear as immunogenic responses to the patient during drug therapy. ADA can be any one of the immunoglobulin isotypes (e.g., igM, igE, igA, igG, igD) or subclasses of IgG (IgG 1, 2, 3, and 4). ADA includes ADA from any animal source, including, for example, human or non-human animal (e.g., veterinary) sources.

As will be apparent to those of skill in the art, such kits may further include one or more of a variety of conventional kit components, such as containers with one or more carriers, additional containers, and the like, if desired. Instructions (as an insert or as a label) indicating the amount of the component to be administered, instructions for administration, and/or instructions for mixing the components may also be included in the kit.

Method and use

The present disclosure also provides methods of detecting the presence or amount of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof and/or a composition and/or chimeric antigen receptor provided herein and determining the presence or amount of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in the sample. In some embodiments, the method is a non-diagnostic method. In some embodiments, the methods are used in pharmacokinetic, drug resistance or neutralization assays for anti-PD-L1 antibodies, anti-4-1 BB antibodies, or bispecific PD-L1/4-1BB antibodies.

The present disclosure also provides a method of reducing side effects caused by administration of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody to a subject, the method comprising administering to the subject an antibody of the present disclosure, or an antigen-binding fragment thereof, and/or a composition and/or chimeric antigen receptor. In some embodiments, the side effect is a cytokine storm.

A "cytokine storm", also known as Cytokine Release Syndrome (CRS), is a condition that occurs when the immune system of a subject responds too strongly to infection or immunotherapy. In some embodiments, the cytokine storm is caused by administration of ATG-101 to a subject. Cytokine levels can be monitored by detecting the protein level of the cytokine in a biological sample from the subject. Cytokine levels can also be monitored by detecting nucleic acid levels of cytokines in a biological sample from a subject. In some embodiments, the cytokine is selected from the group consisting of: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra, IL-2R, IFN- α, IFN- γ, MIP-1α, mcPi-1β, MCP-1, TNF α, GM-CSF, G-CSF, CXCL9, CXCL10, CXCR factor, VEGF, RANTES, earthThe gamma ΓN, EGF, HGF, FGF- β, CD40L, ferritin, and any combination thereof.

In some embodiments, the anti-PD-L1 antibody, the anti-4-1 BB antibody, or the bispecific PD-L1/4-1BB antibody is administered to the subject to prevent or treat a disease, disorder, or condition associated with PD-L1 and/or 4-1 BB. In certain embodiments, the subject is a human.

In some embodiments, the PD-L1 and/or 4-1 BB-related disease, disorder, or condition is characterized by the expression or overexpression of PD-L1 and/or 4-1BB.

In certain embodiments, the PD-L1 and/or 4-1BB related disease, disorder or condition is cancer. In certain embodiments, the cancer is a PD-L1 and/or 4-1BB expressing cancer. As used herein, "PD-L1 and/or 4-1BB expressing cancer" refers to a cancer characterized by expressing PD-L1 and/or 4-1BB protein in cancer cells (i.e., tumor-infiltrating immune cells) or by expressing PD-L1 and/or 4-1BB in cancer cells (i.e., tumor-infiltrating immune cells) at a level that is significantly higher than the level expected for normal cells. Various methods can be used to determine the presence and/or amount of PD-L1 and/or 4-1BB in a test biological sample of a subject. For example, a test biological sample may be exposed to an anti-PD-L1 and/or 4-1BB antibody or antigen-binding fragment thereof, which binds and detects the expressed PD-L1 and/or 4-1BB protein. Alternatively, PD-L1 and/or 4-1BB may also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, and the like. In some embodiments, the test sample is derived from a cancer cell or tissue, or a tumor infiltrating immune cell. The reference sample may be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained. For example, the reference sample may be a non-diseased sample adjacent to or in the vicinity of the test sample (e.g., a tumor). In certain embodiments, the cancer is a solid tumor or hematological tumor.

In certain embodiments, the PD-L1 and/or 4-1BB related disease, disorder, or condition is selected from the group consisting of: lung cancer (e.g., non-small cell lung cancer (NSCLC), small Cell Lung Cancer (SCLC) (lung adenocarcinoma or lung squamous cell carcinoma), peritoneal cancer, carcinoid, bone cancer, pancreatic cancer, primitive neuroectodermal tumors, skin cancer, gall bladder cancer, head and neck cancer, squamous cell cancer, uterine cancer, ovarian cancer, rectal cancer, prostate cancer, bladder cancer (e.g., urothelial cancer), anal region cancer (e.g., anal squamous cell cancer), gastric cancer (gastric or stomach cancer) (e.g., gastrointestinal cancer), esophageal cancer, colon cancer, breast cancer, uterine cancer, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma/hepatoma or liver cancer), cholangiocarcinoma, sarcoma, colorectal cancer, fallopian tube cancer, salivary gland cancer, cervical cancer, endometrial or uterine cancer, osteosarcoma, vaginal cancer, vulval cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, nasopharyngeal cancer, soft tissue sarcoma, polycythemia vera, real world cancer, penile cancer, kidney or urinary tract cancer (e.g., renal rhabdoid tumor), cutaneous T-cell lymphoma, medulloblastoma, nephroblastoma, myelodysplastic syndrome, chronic and non-chronic myeloproliferative disorders, chorioallantoic papilloma, renal cell carcinoma, renal pelvis carcinoma, central Nervous System (CNS) neoplasms, soft tissue sarcomas (e.g., rhabdomyosarcoma, fibrosarcoma, kaposi's sarcoma), spinal cord shaft tumors, gliomas (e.g., ependymoma, astrocytoma, anaplastic astrocytoma, oligodendrogliomas), eye cancers (e.g., retinoblastoma), brain stem glioma, or mixed glioma such as oligodendroglioma, brain tumor (e.g., glioblastoma/glioblastoma multiforme (GBM), non-glioblastoma brain tumor or meningioma), melanoma (e.g., cutaneous or intraocular melanoma), thrombocytosis, mesothelioma, mycosis fungoides, szezali syndrome, primary myelofibrosis, solitary plasmacytome, vestibular schwannoma, ewing's sarcoma, chondrosarcoma, MYH-related polyposis, pituitary adenoma, pediatric cancers such as pediatric sarcoma (e.g., neuroblastoma, rhabdomyosarcoma and osteosarcoma), hematologic cancers, hodgkin's lymphoma, non-hodgkin's lymphoma, leukemia (e.g., lymphoblastic/lymphoblastic leukemia), chronic or acute leukemia, mast cell leukemia, lymphocytic lymphoma, primary CNS lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), chronic Myelogenous Leukemia (CML), myelogenous leukemia (cmm), multiple myeloma (dysmyelomas, lymphoblastic leukemia (mcbl), multiple myeloma (lymphomas, lymphomas (dysleukemia), multiple myeloma, lymphomas (lymphomas), or lymphomas (lymphomas), leukemia-relapses) (one of one or multiple myeloma Diffuse large B-cell lymphoma (DLBCL), DLBCL caused by follicular lymphoma, advanced B-cell lymphoma, primary mediastinum large B-cell lymphoma, follicular Lymphoma (FL), and primary mediastinum B-cell lymphoma.

In some embodiments, the subject has been identified as having cancer cells or tumor infiltrating immune cells that express PD-L1 and/or 4-1BB, optionally at a level significantly higher than that typically found on non-cancer cells.

In some embodiments, the antibodies or antigen binding fragments thereof and/or compositions and/or chimeric antigen receptors provided herein are used to neutralize an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody (e.g., ATG-101).

The amount of antibody or antigen binding fragment provided herein will depend on various factors known in the art, such as, for example, body weight, age, prior medical history, current drug treatment, potential for health and cross-reactivity of the subject, allergies, sensitivity and adverse side effects, and the route of administration and the extent of disease progression. Those skilled in the art (e.g., a physician or veterinarian) can scale down or up the dosage according to these and other circumstances or instructions.

In certain embodiments, the antibodies or antigen binding fragments and/or chimeric receptors provided herein can be administered at a dose of about 0.01mg/kg to about 100 mg/kg. In certain embodiments, the dosage administered may vary during the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the dosage administered may vary during the course of treatment according to the subject's response.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose may be administered, or multiple separate doses may be administered over time.

The antibodies or antigen-binding fragments thereof and/or chimeric antigen receptors provided herein can be administered by any route known in the art, for example, by parenteral routes including subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or intradermal injection; or by a non-parenteral route including transdermal, oral, intranasal, intraocular, sublingual, rectal or topical administration.

In some embodiments, an antibody or antigen binding fragment thereof and/or chimeric antigen receptor provided herein can be administered alone or in combination with a therapeutically effective amount of a second agent. For example, the antibodies or antigen binding fragments thereof and/or chimeric antigen receptors disclosed herein can be administered in combination with a second agent, e.g., a detection agent, a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapeutic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, a cytokine, an active agent, an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulatory molecule agonist, a co-inhibitory molecule blocker, an adhesion molecule blocker, an anti-cytokine antibody or functional fragment thereof, a detectable label or reporter, an antibacterial agent, a gene editor, a beta agonist, a viral RNA inhibitor, a polymerase inhibitor, an interferon, or a microrna.

As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system against diseases such as cancer or enhances the immune system in a general manner. Examples of immunotherapy include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

"targeted therapies" are the types of therapies that act on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapies target therapeutic agents to tumors, thereby protecting normal tissue from the therapeutic agent.

In certain of these embodiments, the antibody or antigen-binding fragment thereof provided herein and/or the composition provided herein and/or the chimeric antigen receptor provided herein is administered in combination with one or more additional agents, may be administered concurrently with one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment thereof and/or the composition provided herein and/or the chimeric antigen receptor provided herein and additional agents may be administered as part of the same composition. However, administration of an antibody or antigen binding fragment thereof and/or a composition provided herein and/or a chimeric antigen receptor provided herein in "combination" with another agent need not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment, composition or chimeric antigen receptor thereof administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase is used herein, even if the antibody or antigen-binding fragment, composition or chimeric antigen receptor and the second agent are administered by different routes. Where possible, additional agents administered in combination with the antibodies or antigen binding fragments, compositions or chimeric antigen receptors disclosed herein are administered according to the schedules listed in the product information sheet of additional therapeutic agents or according to physician's Desk Reference manual 2003 (Physicians' Desk Reference), 57 th edition, medical economics company (Medical Economics Company); ISBN:1563634457, 57 th edition (11 2002), or protocols well known in the art.

In another aspect, the present disclosure provides a kit provided herein comprising an antibody or antigen-binding fragment thereof provided herein and/or a composition provided herein and/or a chimeric antigen receptor provided herein, optionally conjugated to a detectable moiety, which can be used to detect ATG-101. The kit may further comprise instructions for use.

In another aspect, the disclosure also provides a use of an antibody provided herein or an antigen-binding fragment thereof and/or a composition provided herein and/or a chimeric antigen receptor provided herein in the preparation of a kit, wherein the kit is used in a method for detecting an anti-drug antibody (ADA) (e.g., ATG-101) of an anti-PD-L1 antibody, an anti-4-1 BB antibody or a bispecific PD-L1/4-1BB antibody.

In some embodiments, the antibodies or antigen-binding fragments thereof used in the methods, kits, or uses as described above include a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof. In some embodiments, the first antibody or antigen binding fragment thereof comprises:

i. one or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) comprised within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: SEQ ID NOS 6, 13, 20 and 27; and

One or two or three light chain complementarity determining regions (LCDR 1, LCDR2 and/or LCDR 3) comprised within any one of the light chain Variable (VL) region sequences selected from the group consisting of: SEQ ID NOS.7, 14, 21 and 28.

HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1 comprising an amino acid sequence as shown in SEQ ID No. 1, said HCDR2 comprising an amino acid sequence as shown in SEQ ID No. 2, said HCDR3 comprising an amino acid sequence as shown in SEQ ID No. 3, said LCDR1 comprising an amino acid sequence as shown in SEQ ID No. 4, said LCDR1 comprising an amino acid sequence as shown in NAK, said LCDR3 comprising an amino acid sequence as shown in SEQ ID No. 5;

i. one or two or three heavy chain complementarity determining regions (HCDR 1, HCDR2 and/or HCDR 3) comprised within any one of the heavy chain Variable (VH) region sequences selected from the group consisting of: 34, 41, 48 and 55; and

In some embodiments, the sample used in the method, kit or use as described above is selected from the group consisting of: tissue sections, biopsies, paraffin embedded tissue, body fluids, colonic exudates, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the bodily fluid is selected from the group consisting of: whole blood, serum, plasma, urine, mucus, saliva, peritoneal fluid, pleural fluid, synovial fluid, cerebrospinal fluid, pleural effusion, peritoneal effusion, and any combination thereof.

In some embodiments, the bispecific PD-L1/4-1BB antibody used in the methods, kits, or uses as described above is ATG-101.

The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials, and methods described below fall within the scope of the invention, in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention but are merely illustrative of specific embodiments that fall within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials and methods without utilizing the inventive capabilities and without departing from the scope of the present invention. It will be appreciated that many variations may be made in the procedure described herein while still remaining within the scope of the invention. It is the intention of the inventors of the present invention that such variations are included within the scope of the invention.

Examples

Example 1 antibody production

1.1Immunization

To generate antibodies against the PD-L1 binding portion and the 4-1BB binding portion of ATG-101, SJL mice were immunized with two immunogenic peptides: YN-035 (described in WO2019196309A1, the entire contents of which are incorporated herein by reference) F (ab') ₂ And scF targeting 4-1BB _V -Fc. F (ab') ₂ Is produced by pepsin digestion of parent antibody YN-035, which provides the PD-L1 binding portion of ATG-101 (pepsin: YN-035 ratio 1:4, reacted overnight at 37 ℃) and by ligating the 4-1BB binding portion of ATG-101 (i.e., the 4-1BB specific scFv domain) with the N-terminus of human IgG1 Fc to produce scFv-Fc targeting 4-1 BB. The primary immunization is followed by several booster immunizations until the animals develop satisfactory antiserum titers suitable for hybridoma development. Gerbu MM was used as an immunoadjuvant. The groupings of animals are shown in table 5 below. Immunization protocols for the two groups of animals are shown in table 6 below.

TABLE 5 grouping of animals

Group of	Immunogens	Pathway	Animal/strain	Group size	Dosage of
						1	PD-L1-targeted YN-035F (ab') ₂	Tar joint	SJL	5/5	10 μg per mouse
2	4-1 BB-targeted scFv-Fc	Tar joint	SJL	5/5	10 μg per mouse

TABLE 6 immunization protocols for groups 1 and 2

1.2 hybridoma Generation and screening

Hybridomas are produced by fusing myeloma with spleen cells of mice assessed by a test blood collection ELISA for the PD-L1 binding portion or 4-1BB binding portion of ATG-101, as determined to be the mice with the best immune response. Lymphocytes from spleen and lymph nodes were fused with Sp2/0-Ag14 cell line using an optimized electrofusion protocol. Multiple fusions are performed to ensure success of the project.

The fusion cells were seeded (1X 10 per well) ⁴ To 10 ⁵ Individual cells) into a stack of 96-well plates. The growth of cells in the plates was monitored and fed once a week. At 10-1Wells with cell growth within 4 days will be picked by ELISA experiments through primary screening. For group 1, F (ab') from the primary screening pair with YN-035 ₂ Positive parent clones positive for human IgG1 and negative for binding were amplified into 24 well plates and subjected to secondary screening. For group 2, positive parental clones from the primary screening that were positive for binding to scFv-Fc targeting 4-1BB and negative for human IgG1 were amplified into 24-well plates and secondary screened.

After the primary screening, a secondary screening was performed, again by ELISA screening to amplify positive parental clones in 24-well plates. For group 1, F (ab') of pair and YN-035 was selected ₂ Positive parental clones positive for ATG-101 binding and negative for human IgG1 were subcloned. For group 2, positive parental clones positive for binding to scFv-Fc, ATG-101 targeting 41BB and negative for human IgG1 were selected for subcloning. Two groups were also subjected to a competition ELISA to assess the blocking ability of antibodies produced by the hybridoma candidates for inhibition of ATG-101 binding to PD-L1 or 4-1BB, including both competitive and non-competitive clones.

Parent hybridomas having the desired reactivity and isotype from the above-described screening funnel are then subcloned by multiple rounds of limiting dilution or single cell sorting until a monoclonal is obtained.

Cells in subclones plates were screened by ELISA as described in the secondary screen, and subclones with good binding capacity were expanded to 24 wells for confirmation testing.

The desired subcloned cell lines were sequenced and further expanded into culture flasks for cryopreservation. Initially at 0.5-1.0X10 ⁷ Individual cells/vials 4-6 vials per cell line were cryopreserved. If desired, a master cell bank and a working cell bank are established for the selected most valuable cell line.

As a result, 8 antibodies having unique sequences were found, which were positive for binding to YN-035 (PD-L1) and ATG-101, or to scFv-Fc targeting 4-1BB and ATG-101, and negative for binding to human IgG1 (as shown in tables 7A and 7B below), indicating that these antibodies specifically recognized the antigen-targeting region of ATG-101.

TABLE 7A binding affinity of selected antibodies to ATG-101 and their ability to compete with ATG-101 for binding to PD-L1 (OD ₄₅₀ Value of

TABLE 7 binding affinity of selected antibodies to ATG-101 and their competitive capacity (OD) ₄₅₀ Value of

Example 2 characterization of antibodies

2.1 antibodies

Hybridoma antibody clones 10F10G7, 9D6E11, 27E8A8, 31B6G8, 50D6E10, 58H6F8, 63C9C12 and 75H9F7 were characterized and their sequences were identified by the following methods.

2.2 hybridoma sequencing

Total RNA was isolated from hybridoma cells according to the technical manual of RNAiso Plus (TAKARA catalog number 9109). The total RNA was then reverse transcribed into cDNA using isotype specific antisense primers or universal primers according to the technical manual of PrimeScript II strand 1 cDNA synthesis kit (TAKARA catalog number 6210A). According to TaKaRa Taq ^TM (catalog number R001A) antibody fragments of VH and VL were amplified. The amplified antibody fragments were cloned individually into standard cloning vectors. Colony PCR was performed to screen clones with inserts of the correct size. Not less than five colonies of inserts of the correct size per fragment were sequenced. The sequences of the different clones were aligned and consensus sequences of these clones were provided.

The variable region sequences of the hybridoma antibodies are provided in table 4 above.

2.3 ELISA binding affinity

The binding affinity of the antibodies to ATG-101 was determined by ELISA.

Cell culture plates were coated with 100. Mu.l/well of 1. Mu.g/ml antigen (ATG-101, fully human IgG) and incubated for 2 hours at 37℃with sealing.

Wash the plate 3 times with a knockout.

Plates were incubated with 300 μl of blocking buffer per well for 2 hours at 37 ℃.

Titration of antibodies by serial dilution, the titrated antibodies will be used for incubation. The solution was prepared by dilution with buffer to achieve final concentration: 10-fold dilutions were performed consecutively, from 100nM (at 8 spots), and incubation with 100. Mu.l/well solution was continued for 1 hour at 37 ℃.

Wash the plate 3 times with a knockout. Secondary antibody (100. Mu.l/well, anti-mouse IgG-HRP at 1:10000 dilution) was added and incubated for 0.5 hours at 37 ℃.

Wash the plate 6 times with a dispenser. 100 μl/well TMB working solution (prepared by mixing solutions A and B at a 1:1 ratio) was added and incubated for 15 minutes at 37deg.C. The reaction was quenched by the addition of 50. Mu.l/well 1N HCl.

The absorbance of each well was read by a microplate reader at a wavelength of 450 nm. Data were analyzed by GraphPad Prism and plotted.

The results are shown in fig. 1A to 1F. As shown in fig. 1A to 1F, selected antibody clones 10F10G7, 9D6E11, 27E8A8, 31B6G8, 50D6E10, 58H6F8, 63C9C12 and 75H9F7 specifically bound to ATG-101, but not IgG1 from negative control mice.

2.4 characterization of antibody Capacity

Antibody competence against ATG-101 and the corresponding antigen was determined by ELISA.

Cell culture plates were coated with 100. Mu.L/well antigen (1. Mu.g/mL PD-L1 or 4-1 BB) and incubated overnight at 4 ℃.

Wash plates 3 times with PBST buffer.

Blocking with a PBST buffer plate pair containing 1% BSA at 37℃for 2 hours.

Wash plates 3 times with PBST buffer.

Add 100. Mu.L/well primary antibody (ATG-101 0.2 nM) and test antibody (100 nm,1:10 serial dilutions, 8 dilution gradients) and incubate at 37℃for 1.5 hours.

Wash plates 3 times with PBST buffer.

mu.L/well secondary antibody (anti-human IgG-HRP) was added and incubated for 1 hour at 37 ℃.

Plates were washed 5 times with PBST buffer and then 100 μl/well TMB was added and incubated for 10 minutes.

Stop the reaction with 50. Mu.L/well 1N HCl.

Absorbance was read with a microplate reader (Molecular device spectra max plus 384) at a wavelength of 450 nm.

The results are shown in fig. 2, table 8A and table 8B below. As shown in fig. 2, table 8A and table 8B, selected antibody clones 10F10G7, 9D6E11, 27E8A8 and 31B6G8 showed different competence for ATG-101 binding to human PD-L1 protein; and selected antibody clones 50D6E10, 58H6F8, 63C9C12 and 75H9F7 showed different competence for ATG-101 binding to human 4-1BB protein.

TABLE 8A binding affinity of selected antibodies to ATG-101 and competitor ability to ATG-101 to bind PD-L1

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TABLE 8 binding affinity of selected antibodies to ATG-101 and competitor ability to ATG-101 to bind 4-1BB

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Claims

1. An anti-drug antibody against a PD-L1/4-1BB bispecific antibody comprising:

i. heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 4, the amino acid sequence of LCDR2 is shown in NAK, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 5;

heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 8, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 9, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 10, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 11, the amino acid sequence of LCDR2 is shown in RAN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 12;

Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 15, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 16, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 17, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 18, the amino acid sequence of LCDR2 is shown in AAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 19;

heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 22, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 23, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 24, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 25, the amino acid sequence of LCDR2 is shown in AAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 26;

v. heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 29, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 30, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 31, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 32, the amino acid sequence of LCDR2 is shown in STS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 33;

Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO:36, the amino acid sequence of HCDR2 is shown as SEQ ID NO:37, the amino acid sequence of HCDR3 is shown as SEQ ID NO:38, the amino acid sequence of LCDR1 is shown as SEQ ID NO:39, the amino acid sequence of LCDR2 is shown as NAK, and the amino acid sequence of LCDR3 is shown as SEQ ID NO: 40;

heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 43, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 44, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 45, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 46, the amino acid sequence of LCDR2 is shown as NAK, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 47; or (b)

Heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 50, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 51, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 52, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 53, the amino acid sequence of LCDR2 is shown as QMS, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 54.

2. The anti-drug antibody of claim 1, comprising a heavy chain Variable (VH) region and a light chain Variable (VL) region amino acid sequence pair selected from the group consisting of: SEQ ID NOS 6 and 7, 13 and 14, 20 and 21, 27 and 28, 34 and 35, 41 and 42, 48 and 49, and 55 and 56.

3. The anti-drug antibody of claim 1 or 2, which is capable of specifically binding to the PD-L1 binding portion and/or the 4-1BB binding portion of ATG-101; or compete with ATG-101 for binding to PD-L1 and/or 4-1BB.

4. The anti-drug antibody of claim 1 or 2, which is a chimeric or humanized antibody.

5. The anti-drug antibody of claim 1 or 2, which is a labeled antibody, a bivalent antibody, an anti-idiotype antibody or a fusion protein.

6. The anti-drug antibody according to claim 1 or 2, which is Fab, fab ', F (ab') ₂ Fv fragments, or single chain antibody molecules (scFv).

7. The anti-drug antibody of claim 1 or 2, further comprising an Fc region.

8. The anti-drug antibody of claim 7, further comprising an Fc region of human IgG.

9. The anti-drug antibody of claim 8, wherein the Fc region is derived from human IgG1, igG2, igG3, or IgG4.

10. The anti-drug antibody of claim 9, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 75-78.

11. The anti-drug antibody of claim 1 or 2, wherein the light chain is a lambda light chain or a kappa light chain.

12. The anti-drug antibody of claim 1, which is a bispecific or multispecific antibody.

13. The anti-drug antibody of claim 12, capable of specifically binding to the PD-L1 binding portion and the 4-1BB binding portion of ATG-101.

14. The anti-drug antibody of claim 13, comprising:

i. HCDR1 of the amino acid sequence shown in SEQ ID NO. 1, HCDR2 of the amino acid sequence shown in SEQ ID NO. 2, HCDR3 of the amino acid sequence shown in SEQ ID NO. 3, LCDR1 of the amino acid sequence shown in SEQ ID NO. 4, LCDR2 of the amino acid sequence shown in NAK and LCDR3 of the amino acid sequence shown in SEQ ID NO. 5; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 29, HCDR2 of the amino acid sequence shown as SEQ ID NO. 30, HCDR3 of the amino acid sequence shown as SEQ ID NO. 31, LCDR1 of the amino acid sequence shown as SEQ ID NO. 32, LCDR2 of the amino acid sequence shown as STS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 33;

HCDR1 of the amino acid sequence shown as SEQ ID No. 1, HCDR2 of the amino acid sequence shown as SEQ ID No. 2, HCDR3 of the amino acid sequence shown as SEQ ID No. 3, LCDR1 of the amino acid sequence shown as SEQ ID No. 4, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID No. 5; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 36, HCDR2 of the amino acid sequence shown as SEQ ID NO. 37, HCDR3 of the amino acid sequence shown as SEQ ID NO. 38, LCDR1 of the amino acid sequence shown as SEQ ID NO. 39, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 40;

HCDR1 of the amino acid sequence shown as SEQ ID No. 1, HCDR2 of the amino acid sequence shown as SEQ ID No. 2, HCDR3 of the amino acid sequence shown as SEQ ID No. 3, LCDR1 of the amino acid sequence shown as SEQ ID No. 4, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID No. 5; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 43, HCDR2 of the amino acid sequence shown as SEQ ID NO. 44, HCDR3 of the amino acid sequence shown as SEQ ID NO. 45, LCDR1 of the amino acid sequence shown as SEQ ID NO. 46, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 47;

HCDR1 of the amino acid sequence shown as SEQ ID No. 1, HCDR2 of the amino acid sequence shown as SEQ ID No. 2, HCDR3 of the amino acid sequence shown as SEQ ID No. 3, LCDR1 of the amino acid sequence shown as SEQ ID No. 4, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID No. 5; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 50, HCDR2 of the amino acid sequence shown as SEQ ID NO. 51, HCDR3 of the amino acid sequence shown as SEQ ID NO. 52, LCDR1 of the amino acid sequence shown as SEQ ID NO. 53, LCDR2 of the amino acid sequence shown as QMS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 54;

v. HCDR1 of the amino acid sequence shown in SEQ ID NO. 8, HCDR2 of the amino acid sequence shown in SEQ ID NO. 9, HCDR3 of the amino acid sequence shown in SEQ ID NO. 10, LCDR1 of the amino acid sequence shown in SEQ ID NO. 11, LCDR2 of the amino acid sequence shown in RAN and LCDR3 of the amino acid sequence shown in SEQ ID NO. 12; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 29, HCDR2 of the amino acid sequence shown as SEQ ID NO. 30, HCDR3 of the amino acid sequence shown as SEQ ID NO. 31, LCDR1 of the amino acid sequence shown as SEQ ID NO. 32, LCDR2 of the amino acid sequence shown as STS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 33;

HCDR1 of the amino acid sequence shown in SEQ ID NO. 8, HCDR2 of the amino acid sequence shown in SEQ ID NO. 9, HCDR3 of the amino acid sequence shown in SEQ ID NO. 10, LCDR1 of the amino acid sequence shown in SEQ ID NO. 11, LCDR2 of the amino acid sequence shown in RAN and LCDR3 of the amino acid sequence shown in SEQ ID NO. 12; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 36, HCDR2 of the amino acid sequence shown as SEQ ID NO. 37, HCDR3 of the amino acid sequence shown as SEQ ID NO. 38, LCDR1 of the amino acid sequence shown as SEQ ID NO. 39, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 40;

HCDR1 of the amino acid sequence shown in SEQ ID NO. 8, HCDR2 of the amino acid sequence shown in SEQ ID NO. 9, HCDR3 of the amino acid sequence shown in SEQ ID NO. 10, LCDR1 of the amino acid sequence shown in SEQ ID NO. 11, LCDR2 of the amino acid sequence shown in RAN and LCDR3 of the amino acid sequence shown in SEQ ID NO. 12; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 43, HCDR2 of the amino acid sequence shown as SEQ ID NO. 44, HCDR3 of the amino acid sequence shown as SEQ ID NO. 45, LCDR1 of the amino acid sequence shown as SEQ ID NO. 46, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 47;

HCDR1 of the amino acid sequence shown as SEQ ID No. 8, HCDR2 of the amino acid sequence shown as SEQ ID No. 9, HCDR3 of the amino acid sequence shown as SEQ ID No. 10, LCDR1 of the amino acid sequence shown as SEQ ID No. 11, LCDR2 of the amino acid sequence shown as RAN and LCDR3 of the amino acid sequence shown as SEQ ID No. 12; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 50, HCDR2 of the amino acid sequence shown as SEQ ID NO. 51, HCDR3 of the amino acid sequence shown as SEQ ID NO. 52, LCDR1 of the amino acid sequence shown as SEQ ID NO. 53, LCDR2 of the amino acid sequence shown as QMS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 54;

HCDR1 of the amino acid sequence shown as SEQ ID NO. 15, HCDR2 of the amino acid sequence shown as SEQ ID NO. 16, HCDR3 of the amino acid sequence shown as SEQ ID NO. 17, LCDR1 of the amino acid sequence shown as SEQ ID NO. 18, LCDR2 of the amino acid sequence shown as AAS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 19; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 29, HCDR2 of the amino acid sequence shown as SEQ ID NO. 30, HCDR3 of the amino acid sequence shown as SEQ ID NO. 31, LCDR1 of the amino acid sequence shown as SEQ ID NO. 32, LCDR2 of the amino acid sequence shown as STS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 33;

x. HCDR1 of the amino acid sequence shown as SEQ ID NO. 15, HCDR2 of the amino acid sequence shown as SEQ ID NO. 16, HCDR3 of the amino acid sequence shown as SEQ ID NO. 17, LCDR1 of the amino acid sequence shown as SEQ ID NO. 18, LCDR2 of the amino acid sequence shown as AAS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 19; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 36, HCDR2 of the amino acid sequence shown as SEQ ID NO. 37, HCDR3 of the amino acid sequence shown as SEQ ID NO. 38, LCDR1 of the amino acid sequence shown as SEQ ID NO. 39, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 40;

HCDR1 of the amino acid sequence shown as SEQ ID NO. 15, HCDR2 of the amino acid sequence shown as SEQ ID NO. 16, HCDR3 of the amino acid sequence shown as SEQ ID NO. 17, LCDR1 of the amino acid sequence shown as SEQ ID NO. 18, LCDR2 of the amino acid sequence shown as AAS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 19; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 43, HCDR2 of the amino acid sequence shown as SEQ ID NO. 44, HCDR3 of the amino acid sequence shown as SEQ ID NO. 45, LCDR1 of the amino acid sequence shown as SEQ ID NO. 46, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 47;

HCDR1 of the amino acid sequence shown as SEQ ID NO. 15, HCDR2 of the amino acid sequence shown as SEQ ID NO. 16, HCDR3 of the amino acid sequence shown as SEQ ID NO. 17, LCDR1 of the amino acid sequence shown as SEQ ID NO. 18, LCDR2 of the amino acid sequence shown as AAS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 19; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 50, HCDR2 of the amino acid sequence shown as SEQ ID NO. 51, HCDR3 of the amino acid sequence shown as SEQ ID NO. 52, LCDR1 of the amino acid sequence shown as SEQ ID NO. 53, LCDR2 of the amino acid sequence shown as QMS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 54;

HCDR1 of the amino acid sequence shown as SEQ ID NO. 22, HCDR2 of the amino acid sequence shown as SEQ ID NO. 23, HCDR3 of the amino acid sequence shown as SEQ ID NO. 24, LCDR1 of the amino acid sequence shown as SEQ ID NO. 25, LCDR2 of the amino acid sequence shown as AAS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 26; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 29, HCDR2 of the amino acid sequence shown as SEQ ID NO. 30, HCDR3 of the amino acid sequence shown as SEQ ID NO. 31, LCDR1 of the amino acid sequence shown as SEQ ID NO. 32, LCDR2 of the amino acid sequence shown as STS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 33;

HCDR1 of the amino acid sequence shown as SEQ ID NO. 22, HCDR2 of the amino acid sequence shown as SEQ ID NO. 23, HCDR3 of the amino acid sequence shown as SEQ ID NO. 24, LCDR1 of the amino acid sequence shown as SEQ ID NO. 25, LCDR2 of the amino acid sequence shown as AAS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 26; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 36, HCDR2 of the amino acid sequence shown as SEQ ID NO. 37, HCDR3 of the amino acid sequence shown as SEQ ID NO. 38, LCDR1 of the amino acid sequence shown as SEQ ID NO. 39, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 40;

xv. HCDR1 of the amino acid sequence shown in SEQ ID NO. 22, HCDR2 of the amino acid sequence shown in SEQ ID NO. 23, HCDR3 of the amino acid sequence shown in SEQ ID NO. 24, LCDR1 of the amino acid sequence shown in SEQ ID NO. 25, LCDR2 of the amino acid sequence shown in AAS and LCDR3 of the amino acid sequence shown in SEQ ID NO. 26; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 43, HCDR2 of the amino acid sequence shown as SEQ ID NO. 44, HCDR3 of the amino acid sequence shown as SEQ ID NO. 45, LCDR1 of the amino acid sequence shown as SEQ ID NO. 46, LCDR2 of the amino acid sequence shown as NAK and LCDR3 of the amino acid sequence shown as SEQ ID NO. 47; or (b)

xvi. HCDR1 of the amino acid sequence shown in SEQ ID NO. 22, HCDR2 of the amino acid sequence shown in SEQ ID NO. 23, HCDR3 of the amino acid sequence shown in SEQ ID NO. 24, LCDR1 of the amino acid sequence shown in SEQ ID NO. 25, LCDR2 of the amino acid sequence shown in AAS and LCDR3 of the amino acid sequence shown in SEQ ID NO. 26; and HCDR1 of the amino acid sequence shown as SEQ ID NO. 50, HCDR2 of the amino acid sequence shown as SEQ ID NO. 51, HCDR3 of the amino acid sequence shown as SEQ ID NO. 52, LCDR1 of the amino acid sequence shown as SEQ ID NO. 53, LCDR2 of the amino acid sequence shown as QMS and LCDR3 of the amino acid sequence shown as SEQ ID NO. 54.

15. The anti-drug antibody of any one of claims 1, 2 or 12 to 14, linked to one or more conjugate moieties.

16. The anti-drug antibody of claim 15, wherein the conjugate moiety comprises a clearance modifier, a toxin, a radioisotope, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer drug.

17. The anti-drug antibody of claim 15, wherein the conjugate moiety is covalently linked directly or through a linker.

18. A chimeric antigen receptor comprising the anti-drug antibody of any one of claims 1 to 14, a transmembrane region, and an intracellular signaling region.

19. The chimeric antigen receptor of claim 18, wherein the transmembrane region comprises a transmembrane region of CD3, CD4, CD8, or CD 28.

20. The chimeric antigen receptor according to claim 18, wherein the intracellular signaling region is selected from the group consisting of: an intracellular signal region sequence of CD3, fcyri, CD27, CD28, CD137, CD134, myD88, CD40, CD278, TLR, or a combination thereof.

21. The chimeric antigen receptor according to any one of claims 18-20, wherein the anti-drug antibody is a scFv.

22. The chimeric antigen receptor according to any one of claims 18 to 20, wherein the chimeric antigen receptor is transplanted into an allogeneic, autologous or xenogeneic cell.

23. The chimeric antigen receptor according to any one of claims 18 to 20, wherein the chimeric antigen receptor is transplanted into an immune effector cell.

24. The chimeric antigen receptor according to any one of claims 18 to 20, wherein the chimeric antigen receptor is transplanted into a T cell, a natural killer cell, a macrophage or a tumor-infiltrating lymphocyte.

25. A composition comprising the anti-drug antibody of any one of claims 1 to 17 or the chimeric antigen receptor of any one of claims 18 to 24, and one or more carriers.

26. An isolated polynucleotide encoding the anti-drug antibody of any one of claims 1 to 14 and/or the chimeric antigen receptor of any one of claims 18 to 24.

27. A vector comprising the isolated polynucleotide of claim 26.

28. A host expression system comprising the vector of claim 27, or having the polynucleotide of claim 27 integrated into its genome.

29. The host expression system of claim 28, which is a microorganism, yeast or mammalian cell, wherein the microorganism is selected from the group consisting of e.coli (e.coli) and b.subtilis; and wherein the mammalian cell is selected from the group consisting of: COS, CHO-S, CHO-K1, HEK-293 and 3T3 cells.

30. A virus comprising the vector of claim 27.

31. A method of expressing the anti-drug antibody of any one of claims 1 to 14 or the chimeric antigen receptor of any one of claims 18 to 24, the method comprising culturing the host expression system of claim 28 under conditions that express the anti-drug antibody of any one of claims 1 to 14 or the chimeric antigen receptor of any one of claims 18 to 24.

32. A method for detecting the presence or amount of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in a sample for non-diagnostic purposes, the method comprising contacting the sample with an anti-drug antibody according to any one of claims 1 to 17, and/or a chimeric antigen receptor according to any one of claims 18 to 24, and/or a composition according to claim 25, and determining the presence or amount of an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody in the sample.

33. The method of claim 32, wherein the method is used in a pharmacokinetic assay, a drug resistance assay, or a neutralization assay for an anti-PD-L1 antibody, an anti-4-1 BB antibody, or a bispecific PD-L1/4-1BB antibody.

34. A kit comprising the anti-drug antibody of any one of claims 1 to 17, and/or the chimeric antigen receptor of any one of claims 18 to 24, and/or the composition of claim 25.

35. Use of an anti-drug antibody according to any one of claims 1 to 17, and/or a chimeric antigen receptor according to any one of claims 18 to 24, and/or a composition according to claim 25 in the preparation of a kit, wherein the kit is used in a method for detecting an anti-PD-L1 antibody, an anti-4-1 BB antibody, or an anti-drug antibody (ADA) of a bispecific PD-L1/4-1BB antibody.

36. The method of claim 32 or 33, wherein the sample is selected from the group consisting of: paraffin-embedded tissues, body fluids, isolated blood cells, and any combination thereof.

37. The method of claim 36, wherein the anti-drug antibody of any one of claims 1 to 17 comprises a first antibody and a second antibody, wherein the first antibody comprises:

heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 8, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 9, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 10, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 11, the amino acid sequence of LCDR2 is shown in RAN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 12;

Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 15, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 16, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 17, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 18, the amino acid sequence of LCDR2 is shown in AAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 19; or (b)

Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 22, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 23, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 24, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 25, the amino acid sequence of LCDR2 is shown in AAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 26;

wherein the second antibody comprises:

i. heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO. 29, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 30, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 31, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 32, the amino acid sequence of LCDR2 is shown in STS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 33;

Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO:36, the amino acid sequence of HCDR2 is shown as SEQ ID NO:37, the amino acid sequence of HCDR3 is shown as SEQ ID NO:38, the amino acid sequence of LCDR1 is shown as SEQ ID NO:39, the amino acid sequence of LCDR2 is shown as NAK, and the amino acid sequence of LCDR3 is shown as SEQ ID NO: 40;

heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 43, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 44, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 45, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 46, the amino acid sequence of LCDR2 is shown as NAK, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 47; or (b)

Heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3, and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown as SEQ ID NO. 50, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 51, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 52, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 53, the amino acid sequence of LCDR2 is shown as QMS, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 54.

38. The method of claim 37, wherein the bodily fluid is selected from the group consisting of: whole blood, serum, plasma, urine, mucus, saliva, peritoneal fluid, pleural fluid, synovial fluid, cerebrospinal fluid, pleural effusion, peritoneal effusion, and any combination thereof.

39. The method of claim 38, wherein the bispecific PD-L1/4-1BB antibody is ATG-101.