CN112745391B - PD-L1 binding molecules - Google Patents

Abstract

The present application provides an isolated PD-L1 binding molecule, in particular an isolated PD-L1 single domain antibody or antigen binding fragment thereof, a nucleic acid encoding said PD-L1 single domain antibody or antigen binding fragment thereof and an expression vector or host cell comprising said nucleic acid, as well as a medicament or kit comprising said PD-L1 single domain antibody or antigen binding fragment thereof.

Description

PD-L1 binding molecule

technical field

This application belongs to the field of biotechnology, and generally relates to PD-L1 binding molecules. More specifically, the present application relates to a single-domain antibody that specifically recognizes PD-L1, its preparation method and its use.

Background technique

Immune checkpoints are regulatory molecules that play an inhibitory role in the immune system, including CTLA-4, PD-1, LAG-3, TIM-3, etc. Currently, there are two immune checkpoints based on CTLA-4 and PD-1 Multiple antibody drugs are on the market. PD-1 (programmed death 1, programmed death receptor 1), is an important immunosuppressive molecule, which was originally cloned from the apoptotic mouse T cell hybridoma 2B4.11. Immunomodulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant survival.

The main ligands of PD-1 are PD-L1 and PD-L2, among which PD-L1 plays an important role in mediating the escape of tumor cells. It is highly expressed in tumor cells and some antigen-presenting cells, and the expression level can be Induced by various cytokines such as IFN-γ, TGF-β, etc. In the tumor microenvironment, the upregulation of PD-L1 expression can directly inhibit the anti-tumor response of T cells and mediate the immune escape of tumor cells through the PD-1 signaling pathway.

Single domain antibody (single domain antibody, sdAb) is an antibody containing a single antibody heavy chain variable region domain. Similar to IgG antibodies, it can selectively bind to specific antigens, but the molecular weight of single domain antibodies is much smaller than that of IgG antibodies. At present, the first single-domain antibody was modified from the heavy chain antibody found in camelids (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993 ) Naturally occurring antibodies devoid of lightchains. Nature 363(6428):446–448.); heavy chain antibodies found in these camelids are also known as VHH fragments. Currently, most studies on single domain antibodies are based on heavy chain variable domains.

Single domain antibodies have many advantages. For example, they have high solubility, good thermal stability and tissue permeability, and some single-domain antibodies can withstand the degradation of papain due to the presence of intramolecular disulfide bonds; in addition, single-domain antibodies can be used in yeast, It can be produced in a variety of expression hosts such as plant and mammalian cells, and the expression level is high, making it extremely cost-effective. (Harmsen MM, De Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77(1):13–22.). With its many advantages, single domain antibodies have good application prospects in various biotechnology and medical fields. At present, Ablynx's first single domain antibody drug has been approved for marketing.

At present, there are not many marketed antibody drugs targeting PD-L1. Therefore, it is still necessary to develop new binding molecules (such as single-domain antibodies) that specifically recognize PD-L1 for cancer immunotherapy, so that it has a lower side effects and better clinical efficacy.

Contents of the invention

The purpose of the present invention is to provide a new PD-L1 binding molecule, specifically, to provide a new single-domain antibody that specifically recognizes PD-L1, and the single-domain antibody that specifically recognizes PD-L1 It has lower toxic and side effects and better clinical efficacy, and can treat cancer more effectively.

In general, the present invention provides a binding molecule or single-domain antibody that specifically recognizes PD-L1, and the PD-L1 single-domain antibody is hereinafter also referred to as PD-L1 single-domain antibody or PD in the form of a nanobody. - L1 antibody, PD-L1 Nanobody or VHH antibody to PD-L1, the above terms may be used interchangeably. The present application also provides methods for constructing and screening the PD-L1 binding molecules or single-domain antibodies, nucleic acid molecules encoding the PD-L1 binding molecules or single-domain antibodies, used for expressing PD-L1 binding molecules or single-domain antibodies Carriers and host cells of domain antibodies, and compositions or kits comprising the PD-L1 binding molecules or single domain antibodies. The PD-L1 binding molecules or single domain antibodies of the present application can treat various cancers by regulating the immune system, and thus can be used to prepare medicaments for treating cancer.

Specifically, the present invention provides PD-L1 antibodies in the form of nanobodies and their derivative molecules after humanization or affinity maturation. While maintaining the advantages of high affinity and low molecular weight, the immunogenicity modification is carried out to improve drug delivery. sex, has a great therapeutic advantage.

In some aspects, the invention provides an isolated PD-L1 binding molecule capable of specifically binding PD-L1 and comprising the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1;

A CDR1 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1; or

CDR1 comprising an amino acid sequence differing from that of SEQ ID NO: 1 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

(ii) CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2;

A CDR2 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2; or

CDR2 comprising an amino acid sequence differing from that of SEQ ID NO: 2 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

and

(iii) CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 12;

A CDR3 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 3 or 12; or

A CDR3 comprising an amino acid sequence differing from that of SEQ ID NO: 3 or 12 with additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

In some embodiments, the PD-L1 binding molecule comprises heavy chain variable region CDR1, CDR2 and CDR3 as follows:

(i) CDR1 as shown in formula RTDSNIX ₁ GMH, wherein X ₁ is H, F or N;

(ii) CDR2 as shown in formula TIFIDX ₂ NTX ₃ , wherein X ₂ is G, L or A, and X ₃ is I or L; and

(iii) CDR3 as shown in formula DVSGYGRX ₄ , wherein X ₄ is A or Y.

In some embodiments, the PD-L1 binding molecule comprises heavy chain variable region CDR1, CDR2 and CDR3 as follows:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 2; and

(iii) CDR3 shown in SEQ ID NO:3.

In some embodiments, the PD-L1 binding molecule comprises heavy chain variable region CDR1, CDR2 and CDR3 as follows:

(i) CDR1 shown in SEQ ID NO: 4;

(ii) CDR2 shown in SEQ ID NO: 5; and

(iii) CDR3 shown in SEQ ID NO:3.

In some embodiments, the PD-L1 binding molecule comprises heavy chain variable region CDR1, CDR2 and CDR3 as follows:

(i) CDR1 shown in SEQ ID NO: 6;

(ii) CDR2 shown in SEQ ID NO: 7; and

(iii) CDR3 shown in SEQ ID NO:3.

In some embodiments, the PD-L1 binding molecule comprises heavy chain variable region CDR1, CDR2 and CDR3 as follows:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 8; and

(iii) CDR3 shown in SEQ ID NO:9.

In some embodiments, the PD-L1 binding molecule comprises heavy chain variable region CDR1, CDR2 and CDR3 as follows:

(i) CDR1 shown in SEQ ID NO: 10;

(ii) CDR2 shown in SEQ ID NO: 11; and

(iii) CDR3 shown in SEQ ID NO:12.

In some embodiments, the PD-L1 binding molecule comprises a heavy chain variable region (VH) comprising any one of the amino acid sequences of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22.

In some embodiments, the heavy chain variable region of the PD-L1 binding molecule consists of any one of the amino acid sequences of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22.

In some embodiments, the heavy chain variable region (VH) of the PD-L1 binding molecule comprises at least 80% of any of SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22, An amino acid sequence that is 85%, 90%, 95% or 99% identical and retains the ability to specifically bind PD-L1.

In some embodiments, the heavy chain variable region (VH) of the PD-L1 binding molecule comprises an An amino acid sequence that has the addition, deletion and/or substitution of multiple amino acids and retains the ability to specifically bind to PD-L1.

In some preferred embodiments, the one or more amino acid additions, deletions and/or substitutions (eg, conservative substitutions) are no more than five, preferably no more than three.

In some embodiments, the PD-L1 binding molecule is an antibody, such as, but not limited to, a camelid antibody, a humanized antibody, or an affinity matured antibody.

In some embodiments, the PD-L1 binding molecule is fused to another molecule, e.g., the Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen-binding fragment of an antibody, an antibody-drug conjugate. conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, or fluorescent proteins.

In some preferred embodiments, the PD-L1 binding molecule is fused to the Fc domain of human IgG (such as human IgG1 or human IgG4).

In some aspects, the PD-L1 binding molecule is a single domain antibody. Accordingly, the present invention also provides an isolated PD-L1 single domain antibody in the form of a Nanobody.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof specifically binds PD-L1.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 as follows:

(i) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1;

A CDR1 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1; or

CDR1 comprising an amino acid sequence differing from that of SEQ ID NO: 1 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

(ii) CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2;

A CDR2 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2; or

CDR2 comprising an amino acid sequence differing from that of SEQ ID NO: 2 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

and

(iii) CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 12;

A CDR3 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 3 or 12; or

A CDR3 comprising an amino acid sequence differing from that of SEQ ID NO: 3 or 12 with additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 as follows:

(i) CDR1 as shown in formula RTDSNIX ₁ GMH, wherein X ₁ is H, F or N;

(ii) CDR2 as shown in formula TIFIDX ₂ NTX ₃ , wherein X ₂ is G, L or A, and X ₃ is I or L; and

(iii) CDR3 as shown in formula DVSGYGRX ₄ , wherein X ₄ is A or Y.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 2; and

(iii) CDR3 shown in SEQ ID NO:3.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising:

(i) CDR1 shown in SEQ ID NO: 4;

(ii) CDR2 shown in SEQ ID NO: 5; and

(iii) CDR3 shown in SEQ ID NO:3.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising:

(i) CDR1 shown in SEQ ID NO: 6;

(ii) CDR2 shown in SEQ ID NO: 7; and

(iii) CDR3 shown in SEQ ID NO:3.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 8; and

(iii) CDR3 shown in SEQ ID NO:9.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising:

(i) CDR1 shown in SEQ ID NO: 10;

(ii) CDR2 shown in SEQ ID NO: 11; and

(iii) CDR3 shown in SEQ ID NO:12.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the heavy chain variable region further comprises a FR region, and the FR region comprises FR1, FR2, FR3 and FR4, and CDR1, CDR2 and CDR3 are arranged at intervals on the heavy chain variable region to form a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region further comprises a FR region, wherein the FR region comprises the following FR1, FR2, FR3 and FR4:

(a) comprising FR1 shown in SEQ ID NO: 24;

FR1 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 24; or

FR1 comprising an amino acid sequence differing from that of SEQ ID NO: 24 with additions, deletions and/or substitutions of no more than 2 (eg, 0, 1, 2) amino acids;

(b) comprising FR2 shown in SEQ ID NO: 25;

FR2 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 25; or

FR2 comprising an amino acid sequence differing from that of SEQ ID NO: 25 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(c) comprising FR3 shown in SEQ ID NO: 26;

FR3 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 26; or

FR3 comprising an amino acid sequence differing from that of SEQ ID NO: 26 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

and

(d) comprising FR4 shown in SEQ ID NO: 27;

FR4 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 27; or

FR4 comprising an amino acid sequence differing from that of SEQ ID NO: 27 by additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region further comprises a FR region, wherein the FR region comprises the following FR1, FR2, FR3 and FR4:

(a) comprising FR1 shown in SEQ ID NO: 28;

FR1 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 28; or

FR1 comprising an amino acid sequence differing from SEQ ID NO: 28 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(b) comprising FR2 shown in SEQ ID NO: 29;

FR2 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 29; or

FR2 comprising an amino acid sequence differing from that of SEQ ID NO: 29 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(c) comprising FR3 shown in SEQ ID NO: 30;

FR3 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 30; or

FR3 comprising an amino acid sequence differing from that of SEQ ID NO: 30 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

and

(d) comprising FR4 shown in SEQ ID NO: 31;

FR4 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 31; or

FR4 comprising an amino acid sequence differing from SEQ ID NO: 31 with additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region further comprises a FR region, wherein the FR region comprises the following FR1, FR2, FR3 and FR4:

(a) comprising FR1 shown in SEQ ID NO: 32;

FR1 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 32; or

FR1 comprising an amino acid sequence differing from that of SEQ ID NO: 32 with additions, deletions and/or substitutions of no more than 2 (eg, 0, 1, 2) amino acids;

(b) comprising FR2 shown in SEQ ID NO: 25;

FR2 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 25; or

FR2 comprising an amino acid sequence differing from that of SEQ ID NO: 25 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(c) comprising FR3 shown in SEQ ID NO: 26;

FR3 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 26; or

FR3 comprising an amino acid sequence differing from that of SEQ ID NO: 26 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

and

(d) comprising FR4 shown in SEQ ID NO: 27;

FR4 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 27; or

FR4 comprising an amino acid sequence differing from that of SEQ ID NO: 27 by additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

In some preferred embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region further comprises a FR region, wherein the FR region comprises FR1, FR2, FR3 and FR4 as follows:

(a) FR1 shown in SEQ ID NO: 24;

(b) FR2 shown in SEQ ID NO: 25;

(c) FR3 shown in SEQ ID NO: 26; and

(d) FR4 shown in SEQ ID NO:27.

In some preferred embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region further comprises a FR region, wherein the FR region comprises FR1, FR2, FR3 and FR4 as follows:

(a) FR1 shown in SEQ ID NO: 28;

(b) FR2 shown in SEQ ID NO: 29;

(c) FR3 shown in SEQ ID NO: 30; and

(d) FR4 shown in SEQ ID NO:31.

In some preferred embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region further comprises a FR region, wherein the FR region comprises FR1, FR2, FR3 and FR4 as follows:

(a) FR1 shown in SEQ ID NO: 32;

(b) FR2 shown in SEQ ID NO: 25;

(c) FR3 shown in SEQ ID NO: 26; and

(d) FR4 shown in SEQ ID NO:27.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising SEQ ID NO: 16, 17, 18, 19, Any one of the amino acid sequences of 20, 21 and 22.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 in any amino acid sequence composition.

In some embodiments, the heavy chain variable region (VH) of the PD-L1 single domain antibody or antigen-binding fragment thereof comprises any of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 An amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical and retains the ability to specifically bind PD-L1.

In some embodiments, the heavy chain variable region (VH) of the PD-L1 single domain antibody or antigen-binding fragment thereof comprises any of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 An amino acid sequence that has one or more amino acid additions, deletions and/or substitutions and retains the ability to specifically bind to PD-L1.

In some preferred embodiments, the one or more amino acid additions, deletions and/or substitutions (eg, conservative substitutions) are no more than five, preferably no more than three.

In some embodiments, the PD-L1 single domain antibody is a camelid antibody, a humanized antibody or an affinity matured antibody.

In some preferred embodiments, the present invention provides a humanized PD-L1 single domain antibody, which comprises the VH of the amino acid sequence shown in SEQ ID NO: 17 or 18.

In some preferred embodiments, the present invention provides an affinity-matured PD-L1 single domain antibody, which comprises a VH of the amino acid sequence shown in any one of SEQ ID NO: 19, 20, 21 and 22.

In some embodiments, the PD-L1 single domain antibody or antigen-binding fragment thereof is fused to another molecule, e.g., an Fc domain of an immunoglobulin (e.g., IgG), an antibody, an antigen of an antibody Binding fragments, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, or fluorescent proteins.

In some preferred embodiments, the antibody or antigen-binding fragment thereof is fused to the Fc domain of a human IgG (eg, human IgGl or human IgG4).

In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PD-L1 binding molecule as disclosed herein or comprising a nucleotide sequence encoding a PD-L1 single domain antibody or an antigen-binding fragment thereof as disclosed herein the nucleotide sequence.

In some aspects, the present invention relates to an expression vector comprising a nucleotide sequence encoding a PD-L1 binding molecule as disclosed herein or comprising a nucleic acid molecule encoding a PD-L1 single domain antibody or an antigen-binding fragment thereof as disclosed herein expression vector.

In some aspects, the invention relates to host cells comprising an expression vector as disclosed herein.

In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

In some aspects, the invention relates to a pharmaceutical composition comprising a PD-L1 binding molecule as disclosed herein or a PD-L1 single domain antibody or antigen-binding fragment thereof as disclosed herein, and a pharmaceutically acceptable carrier.

In some aspects, the invention relates to a method for preparing a PD-L1 binding molecule comprising expressing the PD-L1 binding molecule in a host cell and isolating the PD-L1 binding molecule from the host cell.

In some aspects, the invention relates to a method for preparing a PD-L1 single domain antibody or antigen-binding fragment thereof comprising expressing a PD-L1 single domain antibody or antigen-binding fragment thereof in a host cell and isolating the antibody from the host cell or antigen-binding fragments.

In some aspects, the invention relates to methods of modulating an immune response in a subject comprising administering to a subject a PD-L1 binding molecule as disclosed herein or administering a PD-L1 single domain antibody as disclosed herein, or The antigen-binding fragment such that the immune response in the subject is modulated.

In some embodiments, the subject is a human or mammal with a disease associated with PD-L1. Specifically, the subject may suffer from the following diseases, but not limited thereto: renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, microsatellite unstable solid tumor, and the like.

In some aspects, the present invention relates to a method for treating or preventing a disease associated with PD-L1, comprising administering to a patient suffering from the disease associated with PD-L1 or having the disease associated with PD-L1 A disease-prone subject is administered an effective amount of a PD-L1 binding molecule as disclosed herein, or a PD-L1 single domain antibody or an antigen-binding fragment thereof as disclosed herein, or an effective amount of a PD-L1 binding molecule as disclosed herein. The disclosed pharmaceutical composition of PD-L1 binding molecule, PD-L1 single domain antibody or antigen binding fragment thereof.

In some aspects, the invention relates to methods of treating any disease or condition that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing PD-L1 activity.

In some other aspects, the method of the present invention also relates to a method for treating or preventing tumors through combination therapy, the method comprising administering to a subject an effective amount of the PD-L1 binding molecules and PD-L1 single domain antibodies described herein or an antigen-binding fragment thereof and one or more other drugs.

In some embodiments, the methods disclosed herein further comprise co-administering to the subject an effective amount of a second drug, wherein the PD-L1 binding molecule or PD-L1 single domain antibody or antigen-binding fragment thereof disclosed herein is the first drug. drug. In one embodiment, the second drug is a chemotherapeutic agent, a radiotherapeutic agent or a biomacromolecular drug used to treat the relevant disease. In one embodiment, the biomacromolecular drug is, for example, various monoclonal antibody drugs that attack tumor cells through T cell recognition, such as rituximab, cetuximab, and trastuzumab. The expression "second drug" as used herein does not mean that it refers to the only drug other than the first drug. Thus, the second drug need not be one drug, but may consist of or comprise more than one such drug.

In some embodiments, the subject or individual is a mammal, eg, a mouse or a human, preferably a human.

In some aspects, the invention relates to the use of a PD-L1 binding molecule as disclosed herein in the manufacture of a medicament for the treatment or prevention of a disease associated with PD-L1.

In some aspects, the present invention relates to the use of a PD-L1 single domain antibody or an antigen-binding fragment thereof as disclosed herein in the manufacture of a medicament for treating or preventing a disease associated with PD-L1.

In some embodiments, the PD-L1-related disease is selected from, but not limited to, renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, microsatellite unstable solid tumor, and the like.

In some aspects, the invention relates to kits or devices and related methods for using PD-L1 binding molecules, PD-L1 single domain antibodies or antigen-binding fragments thereof as disclosed herein, and pharmaceutical compositions as disclosed herein, It can be used to treat diseases related to PD-L1, for example, cancer. To this end, the present invention preferably provides an article of manufacture useful in the treatment of such disorders, comprising a container comprising a PD-L1 binding molecule as disclosed herein, a PD-L1 single domain antibody or an antigen-binding fragment thereof and for use as disclosed herein. The disclosed PD-L1 single-domain antibody or antigen-binding fragment thereof is used to treat, improve or prevent PD-L1-related diseases or their progression or recurrence.

The present invention also covers any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof applies to any and all PD-L1 binding molecules, PD-L1 single domain antibodies or antigen-binding fragments thereof, methods and uses of the invention described herein.

In summary, the present invention relates to the following embodiments:

1. An isolated PD-L1 binding molecule, which specifically binds to PD-L1, and comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1;

A CDR1 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1; or

CDR1 comprising an amino acid sequence differing from that of SEQ ID NO: 1 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

(ii) CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2;

A CDR2 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2; or

CDR2 comprising an amino acid sequence differing from that of SEQ ID NO: 2 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

and

(iii) CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 12;

A CDR3 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 3 or 12; or

A CDR3 comprising an amino acid sequence differing from that of SEQ ID NO: 3 or 12 with additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

2. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 as shown in formula RTDSNIX ₁ GMH, wherein X ₁ is H, F or N;

(ii) CDR2 as shown in formula TIFIDX ₂ NTX ₃ , wherein X ₂ is G, L or A, and X ₃ is I or L; and

(iii) CDR3 as shown in formula DVSGYGRX ₄ , wherein X ₄ is A or Y.

3. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 2; and

(iii) CDR3 shown in SEQ ID NO:3.

4. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 shown in SEQ ID NO: 4;

(ii) CDR2 shown in SEQ ID NO: 5; and

(iii) CDR3 shown in SEQ ID NO:3.

5. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 shown in SEQ ID NO: 6;

(ii) CDR2 shown in SEQ ID NO: 7; and

(iii) CDR3 shown in SEQ ID NO:3.

6. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 8; and

(iii) CDR3 shown in SEQ ID NO:9.

7. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises the following heavy chain variable region CDR1, CDR2 and CDR3:

(i) CDR1 shown in SEQ ID NO: 10;

(ii) CDR2 shown in SEQ ID NO: 11; and

(iii) CDR3 shown in SEQ ID NO:12.

8. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule comprises a heavy chain comprising any one of the amino acid sequences of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 variable region.

9. The PD-L1 binding molecule according to embodiment 8, wherein the heavy chain variable region of the PD-L1 binding molecule consists of any one of SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22 composition of amino acid sequences.

10. The PD-L1 binding molecule according to embodiment 1, wherein the heavy chain variable region of the PD-L1 binding molecule comprises any of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 An amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical and retains the ability to specifically bind PD-L1.

11. The PD-L1 binding molecule according to embodiment 1, wherein the heavy chain variable region of the PD-L1 binding molecule comprises any of SEQ ID NO: 16, 17, 18, 19, 20, 21 and 22 An amino acid sequence that has one or more amino acid additions, deletions and/or substitutions and retains the ability to specifically bind to PD-L1.

12. The PD-L1 binding molecule according to embodiment 11, wherein one or more amino acid additions, deletions and/or substitutions (eg conservative substitutions) are no more than five, preferably no more than three.

13. The PD-L1 binding molecule according to embodiment 1, wherein the PD-L1 binding molecule is a camelid antibody, a humanized antibody or an affinity matured antibody.

14. The PD-L1 binding molecule according to any one of embodiments 1-13, wherein the PD-L1 binding molecule is fused to another molecule selected from the group consisting of immunoglobulins (eg IgG) Fc domains, antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, or fluorescent proteins.

15. The PD-L1 binding molecule according to embodiment 14, wherein the PD-L1 binding molecule is fused to the Fc domain of a human IgG (such as human IgG1 or human IgG4).

16. An isolated single domain antibody or an antigen-binding fragment thereof, which specifically binds to PD-L1, said isolated single domain antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), said heavy chain The chain variable region comprises CDR1, CDR2 and CDR3 as follows:

(i) CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1;

A CDR1 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1; or

CDR1 comprising an amino acid sequence differing from that of SEQ ID NO: 1 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

(ii) CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2;

A CDR2 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2; or

CDR2 comprising an amino acid sequence differing from that of SEQ ID NO: 2 by no more than 2 (such as 0, 1, 2) amino acid additions, deletions and/or substitutions;

and

(iii) CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or 12;

A CDR3 comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 3 or 12; or

A CDR3 comprising an amino acid sequence differing from that of SEQ ID NO: 3 or 12 with additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

17. The isolated single domain antibody or antigen-binding fragment thereof of embodiment 16, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 as follows:

(i) CDR1 as shown in formula RTDSNIX ₁ GMH, wherein X ₁ is H, F or N;

(ii) CDR2 as shown in formula TIFIDX ₂ NTX ₃ , wherein X ₂ is G, L or A, and X ₃ is I or L; and

(iii) CDR3 as shown in formula DVSGYGRX ₄ , wherein X ₄ is A or Y.

18. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 2; and

(iii) CDR3 shown in SEQ ID NO:3.

19. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises:

(i) CDR1 shown in SEQ ID NO: 4;

(ii) CDR2 shown in SEQ ID NO: 5; and

(iii) CDR3 shown in SEQ ID NO:3.

20. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises:

(i) CDR1 shown in SEQ ID NO: 6;

(ii) CDR2 shown in SEQ ID NO: 7; and

(iii) CDR3 shown in SEQ ID NO:3.

21. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises:

(i) CDR1 shown in SEQ ID NO: 1;

(ii) CDR2 shown in SEQ ID NO: 8; and

(iii) CDR3 shown in SEQ ID NO:9.

22. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises:

(i) CDR1 shown in SEQ ID NO: 10;

(ii) CDR2 shown in SEQ ID NO: 11; and

(iii) CDR3 shown in SEQ ID NO:12.

23. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region further comprises a FR region comprising FR1, FR2, FR3 and FR4, and associated with CDR1 , CDR2 and CDR3 are alternately arranged on the heavy chain variable region to form a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus.

24. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 23, wherein said FR regions comprise FR1, FR2, FR3 and FR4 as follows:

(a) comprising FR1 shown in SEQ ID NO: 24;

FR1 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 24; or

FR1 comprising an amino acid sequence differing from that of SEQ ID NO: 24 with additions, deletions and/or substitutions of no more than 2 (eg, 0, 1, 2) amino acids;

(b) comprising FR2 shown in SEQ ID NO: 25;

FR2 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 25; or

FR2 comprising an amino acid sequence differing from that of SEQ ID NO: 25 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(c) comprising FR3 shown in SEQ ID NO: 26;

FR3 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 26; or

FR3 comprising an amino acid sequence differing from that of SEQ ID NO: 26 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

and

(d) comprising FR4 shown in SEQ ID NO: 27;

FR4 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 27; or

FR4 comprising an amino acid sequence differing from that of SEQ ID NO: 27 by additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

25. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 23, wherein said FR regions comprise FR1, FR2, FR3 and FR4 as follows:

(a) comprising FR1 shown in SEQ ID NO: 28;

FR1 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 28; or

FR1 comprising an amino acid sequence differing from SEQ ID NO: 28 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(b) comprising FR2 shown in SEQ ID NO: 29;

FR2 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 29; or

FR2 comprising an amino acid sequence differing from that of SEQ ID NO: 29 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(c) comprising FR3 shown in SEQ ID NO: 30;

FR3 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 30; or

FR3 comprising an amino acid sequence differing from that of SEQ ID NO: 30 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

and

(d) comprising FR4 shown in SEQ ID NO: 31;

FR4 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 31; or

FR4 comprising an amino acid sequence differing from SEQ ID NO: 31 with additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

26. The isolated single domain antibody or antigen-binding fragment thereof of embodiment 23, wherein said FR regions comprise FR1, FR2, FR3 and FR4 as follows:

(a) comprising FR1 shown in SEQ ID NO: 32;

FR1 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 32; or

FR1 comprising an amino acid sequence differing from that of SEQ ID NO: 32 with additions, deletions and/or substitutions of no more than 2 (eg, 0, 1, 2) amino acids;

(b) comprising FR2 shown in SEQ ID NO: 25;

FR2 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 25; or

FR2 comprising an amino acid sequence differing from that of SEQ ID NO: 25 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

(c) comprising FR3 shown in SEQ ID NO: 26;

FR3 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 26; or

FR3 comprising an amino acid sequence differing from that of SEQ ID NO: 26 with additions, deletions and/or substitutions of no more than 2 (such as 0, 1, 2) amino acids;

and

(d) comprising FR4 shown in SEQ ID NO: 27;

FR4 comprising an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 27; or

FR4 comprising an amino acid sequence differing from that of SEQ ID NO: 27 by additions, deletions and/or substitutions of no more than 2 (eg 0, 1, 2) amino acids.

27. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, 21 and Any one of the 22 amino acid sequences.

28. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises any of SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22 An amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical and retains the ability to specifically bind PD-L1.

29. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said heavy chain variable region comprises any of SEQ ID NOs: 16, 17, 18, 19, 20, 21 and 22 An amino acid sequence that has one or more amino acid additions, deletions and/or substitutions and retains the ability to specifically bind to PD-L1.

30. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 16, wherein said isolated antibody is a camelid antibody, a humanized antibody or an affinity matured antibody.

31. The isolated single domain antibody or antigen-binding fragment thereof according to any one of embodiments 16-30, fused to another molecule selected from an immunoglobulin Fc domain of a protein (eg IgG), antibody, antigen-binding fragment of an antibody, antibody-drug conjugate, antibody-like molecule, antigen-binding fragment of an antibody-like molecule, or fluorescent protein.

32. The isolated single domain antibody or antigen-binding fragment thereof according to embodiment 31 fused to the Fc domain of a human IgG, such as human IgGl or human IgG4.

33. An isolated nucleic acid molecule comprising a single domain antibody encoding an isolated PDL1 binding molecule as defined in any one of embodiments 1-15 or a single domain antibody as defined in any one of embodiments 16-32, or Nucleotide sequence of the antigen-binding fragment.

34. A vector comprising the nucleic acid molecule of embodiment 33.

35. A host cell comprising the vector of embodiment 34.

36. A pharmaceutical composition comprising at least one PDL1 binding molecule as defined in any one of embodiments 1-15 or a single domain antibody or an antigen thereof as defined in any one of embodiments 16-32 A combination fragment, and a pharmaceutically acceptable carrier.

37. A method of preparing a PDL1 binding molecule as defined in any one of embodiments 1-15 or a single domain antibody or antigen-binding fragment thereof as defined in any one of embodiments 16-32, comprising the following step:

- expressing in the host cell of embodiment 35 a PDL1 binding molecule as defined in any one of embodiments 1-15 or a single domain antibody or an antigen-binding fragment thereof as defined in any one of embodiments 16-32; and

- isolating said PDL1 binding molecule or single domain antibody or antigen-binding fragment thereof from said host cell.

38. A PDL1-binding molecule as defined in any one of embodiments 1-15 or a single-domain antibody or an antigen-binding fragment thereof as defined in any one of embodiments 16-32 is used in preparation for prevention or treatment of a subject Use in medicine for diseases related to PD-L1 in patients.

39. The use according to embodiment 38, wherein the subject is a mouse or a human, preferably a human.

40. The use according to embodiment 38, wherein the PD-L1-related disease is selected from renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, and microsatellite unstable solid tumors.

41. A kit for preventing or treating a disease associated with PD-L1 in a subject, comprising a container comprising at least one PDL1-binding compound as defined in any one of embodiments 1-15 A molecule or an antibody or an antigen-binding fragment thereof as defined in any one of embodiments 16-32.

Description of drawings

Figure 1 shows the results of binding affinity screening of PD-L1-binding VHH antibody lysate samples.

Figure 2 shows the results of the blocking assay screening of anti-PD-L1 candidate antibody molecules.

Figure 3 shows the results of cell binding experiments for candidate antibody molecules: (A) isotype control, (B) NB22D-21, (C) NB22gb-10, (D) positive control KN035.

Figure 4 shows the experimental verification results of the specific binding reaction of NB22D-21 molecules.

Figure 5 shows the experimental verification results of mixed lymphocyte reaction of NB22D-21 molecule.

Fig. 6 shows the verification results of the binding experiment of NB22D-21 molecule and its derivative molecule after humanization transformation.

Fig. 7 shows the results of human-mouse cross-reaction experiments of NB22D-21 molecule and its derivative molecule after humanization transformation.

Figure 8 shows the results of flow cytometry of the human-mouse cross-reaction experiment of the derivative molecule NB22D-21-huVH1 after humanization: (A) control molecule KN035, (B) NB22D-21-huVH1.

Fig. 9 shows the results of binding and blocking experiments of NB22D-21 molecule and its humanized modified molecule.

Figure 10 shows the affinity detection results of the molecules after affinity maturation on PD-L1-CHO.

Figure 11 shows that affinity matured molecules block PD-1 binding to PD-L1-CHO cells.

Figure 12 shows a sequence alignment of candidate antibody molecules with boxed CDR sequences.

Sequence Listing Overview

Accompanying this application is a Sequence Listing containing numerous nucleotide and amino acid sequences. Tables A, B and C below provide a summary of the sequences included.

Detailed ways

It will be understood by those skilled in the art that this invention is not limited to the particular methodology, embodiments and reagents described herein, as these are illustrative. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be defined only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Further, unless otherwise required by context, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes multiple antibodies.

definition

For a better understanding of the present invention, definitions and explanations of related terms are provided below.

The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated numerical value.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they It is sufficient that the desired antigen-binding activity is exhibited. A whole antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may in some cases comprise fewer chains, eg naturally occurring antibodies in camelids may comprise only heavy chains.

The term "single domain antibody" is referred to as a single domain antibody (single domain antibody, sdAb), which is an antibody containing a single antibody heavy chain variable region domain. Similar to IgG antibodies, it can selectively bind to specific antigens, but the molecular weight of single domain antibodies is much smaller than that of IgG antibodies.

The term "antigen-binding portion" as used herein refers to a portion that specifically binds to a target antigen. The term includes antibodies and other natural molecules (eg, receptors, ligands) or synthetic molecules (eg, DARPins) that are capable of specifically binding to a target antigen. In a preferred embodiment, the antigen-binding portion of an antibody of the invention is an antibody fragment.

The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy Fc region-containing antibody. chain.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules having a single amino acid composition, and not to the method by which they are produced. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or combinations of these or other techniques known in the art.

As used herein, the term "PD-1" refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and functions as a co-inhibitory receptor to negatively regulate the immune system. PD-1 is a member of the CD28/CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. Alternative names or synonyms for PD-1 include PDCD1, PD1, CD279, and SLEB2, among others. A representative amino acid sequence of human PD-1 is disclosed under NCBI accession number: NP_005009.2, and a representative nucleic acid sequence encoding human PD-1 is shown under NCBI accession number NM_005018.3.

As used herein, the term "PD-L1" refers to programmed cell death ligand 1 (PD-L1, see eg Freeman et al. (2000) J. Exp. Med. 192:1027). Alternative names or synonyms for PD-L1 include PDCD1L1, PDL1, B7H1, CD274, and B7-H, among others. A representative amino acid sequence of human PD-L1 is disclosed in NCBI Accession No. NP_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is shown under NCBI Accession No.: NM_014143.4. PD-L1 is expressed in the placenta, spleen, lymph nodes, thymus, heart, fetal liver, and is also found in many tumors or cancer cells. PD-L1 binds its receptor PD-1 or B7-1, which is expressed on activated T cells, B cells and myeloid cells. Binding of PD-L1 and its receptor induces signal transduction to inhibit TCR-mediated activation of cytokine production and T cell proliferation. Thus, PD-L1 plays a major role in suppressing the immune system during specific events (e.g., pregnancy, autoimmune disease, tissue allografts) and is thought to allow tumor or cancer cells to bypass immune checkpoints and escape immune responses.

As used herein, the terms "bind" and "specifically bind" refer to the binding of an antibody or antigen-binding portion to an antigenic epitope in an in vitro assay, preferably in bioluminescent interferometry (ForteBio) using purified wild-type antigen. In certain embodiments, an antibody or antigen-binding portion is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

Depending on the amino acid sequence of the constant region of their heavy chains, antibodies are divided into "classes": IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses, eg, IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The heavy-chain constant regions that correspond to the different antibody classes are called [image] delta, epsilon, gamma, and mu, respectively. The light chain constant regions (CL) found in all five antibody classes are called kappa and lambda. Within full-length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, eg, Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd ed., Raven Press, N.Y. (1989)) (which is hereby incorporated by reference in its entirety for all purposes). The variable region of each light chain/heavy chain pair generally forms the antigen binding site.

The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions. (See, eg, Kindt et al. Kuby Immunology, 6 ^th ed., WH Freeman and Co. p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind a particular antigen can be used to isolate antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

Variable regions generally exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from the two chains of each pair, which allow the antibody to bind a specific epitope, are usually aligned by the framework regions. Both light and heavy chain variable regions generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.

"Antibody fragment" refers to a molecule other than an intact antibody that comprises the portion of an intact antibody that binds the antigen to which the intact antibody binds.

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, unless otherwise stated, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y is usually expressed in terms of a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

As used herein, the term " _EC50 ", also known as "half effective concentration", refers to the concentration of a drug, antibody or poison that induces a 50% response between baseline and maximum after a specified exposure time. In the context of this application, the units of _EC50 are "nM".

"Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

"Human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from among subtypes of variable domain sequences. Generally, the subtypes of the sequences are as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subtype is subtype κI as in Kabat et al. (supra). In one embodiment, for VH, the subtype is subgroup III as in Kabat et al. (supra).

A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The term "conservative substitution" refers to the substitution of one amino acid by another amino acid within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another Neutral amino acid substitutions. Exemplary substitutions are shown in Table D below:

Table D. Exemplary Substitutions

original residue exemplary substitution conservative substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids can be grouped by common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidity: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues affecting chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail exchanging a member of one of these classes for another.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parental antibody (eg, a humanized antibody). Typically, the resulting variant or variants selected for further study have certain improvements (e.g., improvements) in biological properties relative to the parental antibody (e.g., increased affinity, reduced immunogenicity), and and/or will have the particular biological properties substantially retained by the parental antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, for example, using phage-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as aligning the sequences (and introducing gaps, if necessary) for the greatest percent sequence identity, and not considering any conservative substitutions as Following the portion of sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Where percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. Full-length calculations relative to longer sequences apply to both nucleic acid and polypeptide sequences.

The terms "effective amount" and "therapeutically effective amount" refer to the amount or dose of the antibody or antigen-binding fragment of the present invention, which, after single or multiple doses administered to a patient, produces the desired effect in a treated subject, including subject Improvement of the subject's condition (eg, improvement of one or more symptoms) and/or delay in the progression of symptoms, etc. "Effective amount" and "therapeutically effective amount" may also refer to an amount sufficient to reduce PD-L1 signaling.

An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as the species of the mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease; the individual patient's response; the particular antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.

The term "blocking" as used herein means reducing signaling of PD-L1 in the presence of an antibody of the invention. PD-L1-mediated signal transmission block refers to the PD-L1 signal transmission level in the presence of the PD-L1 single domain antibody of the present invention is lower than the control level of PD-L1 (i.e. the PD-L1 signal transmission level in the absence of the antibody) ), the reduction is greater than or equal to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. The level of PD-L1 signaling can be measured using a variety of standard techniques, such as, by way of non-limiting example, measuring downstream gene activation and/or luciferase reporter assays in response to PD-L1 activation. Those of skill in the art will appreciate that PD-L1 signaling levels can be measured using a variety of assays, including, for example, commercially available kits.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, alpacas, and rodents Animals (eg, mice and rats). In some embodiments, the individual or subject is a human.

Example

The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and not intended to be limiting of the invention.

Those skilled in the art should understand that unless otherwise noted, the reagents, plasmids, cells, etc. used in the following examples are all commercially available products.

Example 1 Construction of cell lines stably expressing PD-L1

In this example, we respectively constructed CHO-s cells expressing human PD-L1, monkey PD-L1, and mouse PD-L1, and prepared control antibodies from Roche.

1.1 Control antibody preparation

The light chain and heavy chain gene sequences of the whole gene synthesis control antibody-Atezolizumab (Roche) (gene synthesis supplier: General Biology), the expression of the control antibody was expressed using the ExpiCHO transient expression system (purchased from Thermo Fisher), cultured The base is ExpiCHO ^™ Expression Medium (Gibco, A29100-01), and the transfection kit is ExpiFectamine ^™ CHOTransfection Kit (Gibco, A29129).

The specific method is as follows: construct the ExpiCHO expression plasmids of the light chain and heavy chain genes of the atezolizumab antibody by molecular cloning, pass the ExpiCHO cells (purchased from Gibco A29127) one day before transfection, and put the constructed plasmids in a 25ml cell culture system 25μg (plasmid mixture containing light chain and heavy chain genes with a mass ratio of 2:1) was mixed with transfection reagent and added dropwise to 25ml ExpiCHO cell culture, mixed well, expressed at 37°C for 18-22 hours, according to The kit indicates to add feeding medium. After feeding, the cells are cultured at 32°C. On the 5th day after transfection, the second feed is added, and the cells are cultured at 32°C. After 10-12 days, The expressed cell suspension was centrifuged at high speed to obtain the supernatant, and the resulting supernatant was filtered through a 0.22 μm filter membrane and then purified by using the Protein A/G affinity chromatography column affinity purification method, using 100mM glycine salt (pH3.0) The protein of interest was eluted, followed by neutralization to pH 7.0 with 1M Tris-HCl. After a small amount of sampling was identified by SDS-PAGE, it was subpackaged and put into storage for frozen storage.

The preparation method of the control antibody KN035 (see the sequence listing SEQ ID NO: 23 for the amino acid sequence) is as follows:

The entire gene sequence of the control antibody KN035 was synthesized with the whole gene, and the ExpiCHO expression plasmid of the KN035 antibody gene was constructed by molecular cloning. After mixing 25 μg of the plasmid with the transfection reagent, transfection was carried out according to the method for the preparation of the above-mentioned control antibody - Atezolizumab, and the antibody was expressed.

1.2 Construction of stable cell lines

Recombinant vector plasmids expressing the full-length proteins of human PD-L1 (gi number: NP_054862.1), mouse PD-L1 (gi number: NP_068693) and rhesus monkey PD-L1 (gi number: ABO33163.1) were respectively constructed, using The constructed plasmid was introduced into CHO-s cells (purchased from Thermo Fisher) and A375 melanoma cell line (ATCC, CRL-1619) by electroporation. Through screening, the CHO-s cell lines with high expression of PD-L1 protein in the above three species and the A375 cell line with high expression of human PD-L1 (PD-L1-A375) were respectively obtained.

1.2.1 Construction of plasmids expressing human PD-1 and PD-L1 extracellular domain proteins

The expression vectors containing the full-length protein gene sequences of human PD-L1, mouse PD-L1 and rhesus monkey PD-L1 were synthesized by gene synthesis respectively, and then introduced into E. coli after ligation, and the correct plasmid was obtained by sequencing after picking a single clone of E. coli Cloned, plasmid extracted and sequenced again.

1.2.2 Construction of CHO-s cell lines expressing PD-1 and PD-L1 proteins

1.2.2.1 Electric transfer

Use CD-CHO serum-free medium (Gibco, 10743029) to culture and maintain CHO-s cells, pass the cells to 5×10 ⁶ /mL one day before electroporation, and use electroporation kit (Invitrogen, Neon ^TM Kit, MPK10096) the next day and an electroporator (Invitrogen, NeonTM Transfection System, MP922947) to introduce the constructed plasmids into CHO-s cells respectively. The electroporated cells were added to 3 mL of CD-CHO medium, and placed in a 37°C carbon dioxide incubator for 48 hours.

1.2.2.2 Cell plating and culture

The CHO-s cells after electroporation were plated on 96-well cell culture plates at 2000 cells/well, and L-methionine sulfoximine (L-Methionine sulfoximine, MSX) (Millipore, GSS-1015-F ), maintain a volume of 100 μL/well for cell culture and 1×GS supplement (Sigma, 58672C), place it in a 37°C carbon dioxide incubator for culture, and add a medium containing 30 μM MSX and 1×GS supplement after 10 days 50 μL.

1.2.2.3 Cloning identification and cell expansion

The grown clones were picked and transferred to 24-well cell culture plates for culture. The cell lines were identified by FACS method, and clones with high expression levels were selected for expansion and cryopreservation. The relevant FACS identification methods are as follows:

1) First collect empty CHO-s cells and 2×10 ⁵ cloned CHO-s cells, centrifuge at 300g to remove the supernatant, and resuspend the cells in 200 μL of prepared FACS buffer (1×PBS+2% FBS) in 96-well round bottom plate;

2) Centrifuge the 96-well round bottom plate at 300 g for 5 min, and remove the supernatant;

3) Add anti-PD-L1 antibody diluent or negative control antibody diluent to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200 μL of FACS buffer to the corresponding well and resuspend the cells using a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abcam, ab98596), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

1.2.3 Preparation of A375 cell line expressing PD-L1

The PD-L1 high-expressing cell line (PD-L1-A375) of the A375 cell line was prepared in the same way as the electroporation of CHO-s cells in 1.2.2.1, which was used for the construction of the animal model of the A375 cell line. Example 2 Animal immunity and serum titer detection

2.1 Animal immunity

Purchase PD-L1 (NP_054862.1) ectodomain protein (Shenzhou, 10084-H05H) to immunize 2 alpacas (Nanchang Dajia Technology). Each alpaca was immunized with 500 μg each time, once every 2 weeks, and immunized 4 times in total.

2.2 Serum titer detection

After the alpaca immunization, the alpaca serum was taken for immune titer detection.

The immunopotency determination is to measure the binding ability of the immune serum to the recombinant protein PD-L1 (Shenzhou, 10084-H05H) by ELISA method, and judge the immune effect according to the titer of the binding antibody.

The specific method is as follows:

2.2.1 Antigen coating: On the day before the immunopotency determination, the antigenic recombinant protein PD-L1 was diluted with PBS to a final concentration of 2 μg/mL to obtain a dilution. 30 μl of the obtained dilution was added to the ELISA plate and coated overnight at 4°C. On the day of immunopotency determination, rinse with PBS three times, then block with PBST containing 5% skimmed milk at room temperature for two hours, and then rinse with PBS three times.

2.2.2 Serum dilution: On another dilution plate, dilute the non-immunized negative serum and post-immunization serum with PBS, dilute 200-fold in the first well, and then use 3-fold gradient dilution in the subsequent 7 wells.

2.2.3 Antibody reaction: Add the diluted serum to the first ELISA plate, incubate at 37°C for 1 hour, wash twice with PBS, and then add the secondary antibody (purchased from GenScript, Nanjing) at 1:5000. .

2.2.4 Color development reading: After washing the above secondary antibody with PBS for 3 times, add color development solution for 5 minutes, add stop solution, and read the plate at OD450 with a microplate reader (Molecular Devices, SpecterMax 190). The results are shown in the table 1.

Table 1.

Dilution ratio negative serum NSY004 NSY005 1:2000 0.209 2.582 2.163 1:4000 0.133 2.286 2.216 1:8000 0.093 1.923 2.131 1:16000 0.052 1.817 1.868 1:32000 0.054 1.337 1.218 1:64000 0.048 1.048 0.792 1:128000 0.042 0.761 0.587 1:256000 0.048 0.404 0.473

Among them, the two columns of NSY004 and NSY005 are the results of ELISA chromogenic experiment of two alpaca immunized sera after dilution of different times, and the negative serum is the result of ELISA experiment of unimmunized alpaca serum. According to the results in Table 1, it can be seen that the immune titer IgG titer of the two alpacas both reached 256,000, and the immune effect is good, which can be used for the next step of peripheral blood immune antibody library construction.

Example 3 Alpaca immune library construction and preliminary screening

After animal immunization, 50 mL of alpaca fresh blood was taken, and peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) were separated by Ficoll-Paque density gradient separation medium (GE, 17144003S), and anti-human PD-L1 antibody phage alpaca Immunity library construction.

The specific method is as follows:

Dilute the collected alpaca blood with PBS at a ratio of 1:1, take 15ml of Ficoll-Paque density gradient separation solution and slowly add it to a 50ml centrifuge tube, tilt the centrifuge tube and slowly add 30mL of diluted alpaca blood along the tube wall , so that the two liquids maintain a clear separation interface. Centrifuge at 4°C for 20 min, maintaining an acceleration of 3 down to 0. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is plasma mixture, the lower layer is red blood cells and granulocytes, the middle layer is Ficoll-Paque PLUS, and at the junction of the upper and middle layers there is a white narrow band mainly composed of PBMC, that is, the PBMC cell layer . Carefully use a pipette to transfer the middle PBMC cells to a new 50mL centrifuge tube. Rinse twice with PBS, centrifuge horizontally at 1500rpm for 10min at 4°C, and finally resuspend with 1.5ml PBS, and count through a microscope.

RNA was extracted from the isolated PBMC cells, and the extracted RNA was reverse-transcribed into cDNA by a reverse transcription kit (TaKaRa, 6210A). Since the molecular form of the alpaca antibody is different from ordinary antibodies, it does not contain the light chain and the heavy chain does not contain CH1. Therefore, firstly, common primers are designed on the front end of VH and CH2, and two fragments of different sizes are obtained by PCR. Small purpose fragments. Then, by comparing the amino acid sequences of all Germlines of common VHHs, Germline-specific degenerate primers containing NcoI and NotI restriction sites at both ends were designed to amplify all VHH genes using the recovered products as templates, and finally through double Digestion and ligation Insert the target antibody gene fragment into the phage display vector. It should be noted that the C-terminal of the VHH gene on the expression vector is fused to the GIII gene in the phage expression vector. The ligation product was recovered by a recovery kit (Omega, D6492-02), and finally transformed into competent Escherichia coli SS320 by an electroporator (Bio-Rad, MicroPulser), and spread on an ampicillin-resistant 2-YT solid plate. In order to calculate the storage capacity, take 10 μl of the bacterial solution of the library for 10-fold serial dilution, take 2 μl of each dilution gradient and spot on the plate, and count the clones formed on the plate to calculate the total number of clones formed by all electroporations, that is, the library capacity. The capacity of this immune library is 1×10 ⁹ .

Based on this storage capacity, take 10 times the amount of bacteria (about 20 OD) of the storage capacity and add it to fresh 2-YT liquid medium, adjust the amount of medium added so that the initial OD value of the bacterial liquid dilution is 0.05. Place at 37°C and culture at 220rpm until the logarithmic growth phase. At this time, add VSCM13 helper phage in an amount 50 times the number of bacteria, mix well, let stand for 30min, then culture at 220rpm for 1h, centrifuge at 10000rpm for 5min and replace Put into carbenicillin/kanamycin double-resistant 2-YT medium, and cultivate overnight at 30°C and 220rpm. The next day, centrifuge at 13,000g for 10 minutes, and add 20% PEG/NaCl solution to the supernatant to precipitate the phage corresponding to the alpaca immune antibody library. After washing once with PBS, it is used for target PD-L1 antibody screening.

Phage screening uses recombinant PD-L1 protein, using two methods of magnetic bead screening and immune tube screening, the specific methods are as follows.

3.1 Magnetic bead screening

Based on the binding of biotin-labeled recombinant PD-L1 protein to avidin-coupled magnetic beads (purchased from Thermo Fisher, catalog number: 11205D) for screening, the recombinant human PD-L1 protein (Bio For the biotin labeling method, see Roche’s biotin protein labeling kit instructions, product number: 11418165001), incubate the biotin-labeled PD-L1 protein with the magnetic beads, so that the PD-L1 protein binds to the magnetic beads. Incubate the magnetic beads bound with PD-L1 antigen and the phage library with nanobody display at room temperature for 2 hours, wash 6-8 times with PBST, remove non-specifically adsorbed phage, add trypsin (Gibco) and mix gently for 20 minutes , to display phages by eluting Nanobodies that specifically bind to human PD-L1 protein. Then, the eluted phages were used to infect logarithmic phase SS320 cells (Lucigen, MC1061 F), and the phage-infected SS320 cells were spread on a carbenicillin-resistant plate, cultured overnight at 37°C, and collected the next day. bacteria. Use SS320 bacteria to prepare phage, and the preparation method is detailed in the above library phage preparation method. The finally obtained phages continue to be used for the second round of screening, and trypsin is used for elution at the end of the second round of screening. The phages obtained in the second round of screening were used for the third round of screening, and eluted with trypsin at the end of the third round of screening. Repeatedly, 10 clones were randomly selected in each round for sequence analysis. The results showed that the monoclonal phage obtained after 3 rounds of screening, the sequenced monoclonal, and the gene sequences of different clones were repeated, which proved that the sequence enrichment was obvious.

3.2 Immunotube screening

The immunotube screening is based on coating the antigen on the surface of the immunotube, and screening the antibody display phages that bind to the target antigen. The day before the screening, the recombinant human PD-L1 protein was used to coat the immune tube in advance, and the immune tube bound to the PD-L1 antigen was incubated with the phage library with nanobody display for 2 hours at room temperature, washed 6-8 times with PBST, and the nonspecific protein was removed. Heteroadsorbed phages were added with trypsin (Gibco) and gently mixed for 20 min to elute the nanobody that specifically binds to human PD-L1 protein to display the phages. Then, the eluted phages were used to infect logarithmic phase SS320 cells (Lucigen, MC1061 F), and the phage-infected SS320 cells were spread on a carbenicillin-resistant plate, cultured overnight at 37°C, and collected the next day. bacteria. Use SS320 bacteria to prepare phage, and the preparation method is detailed in the above library phage preparation method. The final phages were then used for the second round of screening. At the end of the second round of screening, trypsin was used for elution. The phages obtained in the second round of screening were used for the third round of screening, and the trypsin method was used for elution at the end of the third round of screening. Repeatedly, 10 clones randomly selected in each round were sequenced and sequenced. The results showed that after 3 rounds of screening, the gene sequences of different clones were repeated in the single clone after sequencing, which proved that the sequence enrichment was obvious.

Monoclonal screening was performed on the phage libraries obtained by two different screening methods, and the positive clones in the third-round products of magnetic bead screening and immune tube screening were picked respectively. The specific method is as follows:

The day before the screening, the recombinant human PD-L1 protein was coated on a 96-well ELISA plate, and the induced phage supernatant was prepared in the 96-well plate on the second day, and positive clones targeting human PD-L1 recombinant protein were screened by phage ELISA, and then picked All positive clones were sequenced and analyzed, and the lysate was prepared from the clone with the only sequence. The preparation method was as follows: inoculate 50mL of the bacterial solution of the clone at a ratio of 1:100 the day before, shake and culture at 37°C for 14h, centrifuge at 10000g for 5min at room temperature, and use Resuspend the bacteria in 1 mL of pH 9.0 Tris-HCl buffer containing benzonase nuclease, lyse on ice for 30 min, centrifuge at 10,000 g at 4°C for 10 min, and collect the supernatant to obtain a positive clone lysate.

The lysates of the prepared positive clones were further verified at the flow cytometric level, and candidate antibodies that specifically recognized human PD-L1 were screened. The streaming level verification method is as follows:

1) First collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody lysate and control antibody dilution to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300g to remove the supernatant, add 200 μL of FACS buffer to the corresponding well and resuspend the cells using a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled flow cytometry antibody (GenScript), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The results of binding affinity screening of anti-PD-L1 VHH lysate samples are shown in Figure 1.

It can be seen from Figure 1 that the VHH molecules of the indicated clone numbers all have certain affinity to CHO cells expressing PD-L1. Since this detection experiment is only qualitative and semi-quantitative, it is impossible to confirm which clone number has a better affinity for the VHH antibody molecule, and further experimental confirmation is required.

The prepared lysate of positive clones was further subjected to blocking screening at the flow cytometry level, and candidate antibodies that specifically recognized human PD-L1 and blocked its binding to PD-1 protein were screened. The streaming level verification method is as follows:

1) First collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add candidate antibody lysates and control antibody dilutions with concentration gradients of 1:1, 1:5, and 1:25 to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C to incubate for 30 minute;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of PD-1-Fc protein dilution solution (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a row gun;

8) Repeat step 7) twice, centrifuge at 300g to remove the supernatant;

9) Add PE-labeled anti-human IgG Fc flow antibody (Abcam), blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The screening results of the blocking experiment for anti-PD-L1 antibody candidate molecules are shown in Figure 2. Through preliminary screening by methods such as ELISA and FACS, it can be seen that the antibody lysate prepared from the screened antibody sequence contains the anti-PD-L1 binding to PD1. Reacting to antibodies with blocking effect, we screened 10 candidate molecules with good affinity and blocking activity.

Example 4 Production and Expression of Chimeric VHH-Fc Antibody

The positive VHH candidate antibody obtained by screening was fused with the Fc segment of human IgG1, and the fusion expression vector was constructed by linking the C-terminus of the positive VHH gene sequence to the N-terminus of the Fc segment gene sequence of human IgG1, and the fusion expression vector plasmid was transformed into ExpiCHO cells, The VHH-Fc chimeric antibody protein fused with the Fc fragment was induced to express.

The antibody was expressed using the ExpiCHO transient expression system, the medium was (Gibco, A29100-01), and the transfection kit was (Gibco, A29129). The specific method is as follows: Passage the ExpiCHO cells one day before transfection, mix 25 μg of the constructed plasmid with the transfection reagent in a 25ml system, drop into 25ml ExpiCHO cell culture, mix well, and express 18- After 22 hours, add feed medium according to the instructions in the kit. After feeding, the cells were cultured at 32°C. On the 5th day after transfection, a second feed was added, and the cells were cultured at 32°C for 10 - After 12 days, the expressed cell suspension was centrifuged at high speed to obtain the supernatant, which was filtered at 0.22 μm and purified by Protein A/G affinity purification method, and eluted with 100 mM glycine salt (pH 3.0) The protein of interest was then neutralized with 1M Tris-HCl.

Example 5 Verification of Affinity Activity at the Cell Level of Chimeric VHH-Fc Antibody

The obtained VHH-Fc candidate antibody was evaluated, and its binding activity to the PD-L1 protein on cells was detected by FACS method, the specific method is as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abcam, ab98596), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 2, through FACS experiments, we screened two nanobody candidate molecules with high affinity, and the affinity was higher than or similar to that of the control antibody.

Table 2. Antibody EC50

clone number EC50(μg/mL) NB22D-21 0.39 NB22gb-10 0.39 KN035 0.39 Atezolizumab 0.83

Example 6 Verification of the activity of chimeric VHH-Fc antibody blocking PD-1

The obtained VHH-Fc candidate antibody was evaluated, and its blocking activity on PD-1/PD-L1 was detected by FACS method. The specific method is as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL FACS to the corresponding well and resuspend the cells using a row gun;

8) Repeat step 7) twice, centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (eBioscience, 12-4317-87), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect on a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 3, through FACS experiments, we verified that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 in Example 5 have high blocking activity at the same time, and their blocking activity is higher than that of the control antibody Or similar to the control antibody.

Table 3. Antibody IC50

clone number IC50(μg/mL) NB22D-21 0.41 NB22gb-10 0.54 KN035 0.39 Atezolizumab 0.86

Example 7 Verification of the activity of chimeric VHH-Fc antibody blocking CD80

The obtained VHH-Fc candidate antibody was evaluated, and its blocking activity on CD80/PD-L1 was detected by FACS method, the specific method is as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled CD80 protein dilution (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a row gun;

8) Repeat step 7) twice, centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (eBioscience, 12-4317-87), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

As shown in Table 4, through FACS experiments, we verified that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 in Example 5 have high blocking activity at the same time, and their blocking activity is higher than that of the control antibody Or similar to the control antibody.

Table 4. IC50 of antibodies

clone number IC50(μg/mL) NB22D-21 0.7081 NB22gb-10 0.7651 KN035 0.6307 Atezolizumab 0.9077

Example 8 Binding Activity of Chimeric VHH-Fc Antibody on Tumor Cells

In order to evaluate the binding activity of the VHH-Fc candidate antibody on the human melanoma cell line A375 cells (ATCC, CRL-1619), the binding activity to the PD-L1 protein on the cells was detected by FACS method, the specific method is as follows:

1) Digest A375 cells with Trypsin containing 0.25% EDTA, collect the cells and centrifuge at 300 g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add A375 cells into 96-well round bottom plate at 100 μL per well, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of each candidate antibody and control antibody dilution (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a row gun;

8) Repeat step 7) twice, centrifuge at 300g to remove the supernatant;

9) Add PE-labeled anti-biotin flow antibody (Abcam), blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102). The detection results are shown in Figure 3.

It can be seen from FIG. 3 that the binding activity of the two nanobody candidate molecules whose clone numbers are NB22D-21 and NB22gb-10 on the human melanoma cell line A375 cells is equivalent to that of the control antibody.

Example 9 Verification of the activity of chimeric VHH-Fc antibody binding to mouse and monkey PD-L1

In order to evaluate the cross-binding activity of the VHH-Fc candidate antibody with monkey and mouse PD-L1, the FACS method was used to detect its binding activity to the PD-L1 protein on cells, the specific method is as follows:

1) Collect the cultured mouse PD-L1-CHO cells and monkey PD-L1-CHO cells, centrifuge at 300g to remove the medium, resuspend the cells with the prepared FACS buffer, count and adjust the density of the cell suspension to 2 ×10 ⁶ /mL;

2) Add mouse PD-L1-CHO cells and monkey PD-L1-CHO cells to a 96-well round bottom plate at 100 μL per well, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells using a row gun;

8) Repeat step 7) twice, centrifuge at 300g to remove the supernatant;

9) Add PE-labeled anti-biotin flow antibody (Abcam), blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The human-mouse cross-reaction test results showed that the nanobody candidate molecule with the clone number NB22D-21 had certain mouse PD-L1 binding activity.

The detection results of human-monkey cross-reactivity are shown in Table 5, from which it can be seen that the two nanobody candidate molecules with clone numbers NB22D-21 and NB22gb-10 have good activity in recognizing monkey PD-L1.

Table 5. EC50 values

clone number EC50(μg/mL) NB22D-21 0.25 NB22gb-10 0.20 KN035 0.21 Atezolizumab 0.33

Example 10 Specific detection of chimeric VHH-Fc antibody binding to PD-L1

In order to confirm the specificity of the candidate molecule binding to the PD-L1 protein, the ELISA method was used to detect the activity of the candidate molecule binding to other proteins of the B7 family. The specific method is as follows:

The day before the experiment, add 30 μL of B7-H1, B7-H2, B7-H3, B7-H4, B7-DC with a final concentration of 2 μg/mL to the ELISA plate (the above proteins were all purchased from Yiqiao Shenzhou Company, the catalog number is 10084 -HNAH, 11559-H08H, 11188-H08H, 10738-H08H, 10292-H08H-B) and other protein dilutions, incubate overnight at 4°C; wash the ELISA plate 3 times with PBST the next day, then add 150 μL 5% PBSM to block Incubate the ELISA plate at room temperature for 2 hours; then wash the plate 3 times with PBST, then add 30 μL of the dilution of candidate antibody and control antibody to the ELISA plate, and incubate for 1 hour at room temperature; wash the plate 3 times with PBST, add 1:7000 diluted Anti-human IgG Fc-HRP secondary antibody, 30 μL per well, incubated at room temperature for 30 minutes; washed the plate with PBST 6 times, added TMB for color development, and finally added 2M HCl to terminate the reaction, and passed the microplate reader (Molecular Devices, SpecterMax 190) on Read at 450nM wavelength, the results are shown in Table 6 and Figure 4.

The experimental verification results of the specific binding reaction of antibody molecules are shown in Table 6 and FIG. 4 .

Table 6.

From the results in Table 6 and Figure 4, it can be seen that the two nanobody candidate molecules whose clone numbers are NB22D-21 and NB22gb-10 have no binding activity to B7 family molecules other than B7-H1, and only have binding activity to B7-H1 , this binding specificity was consistent with that of the control antibody.

Example 11 In vitro biological activity verification of chimeric VHH-Fc antibody

In order to confirm the ability of candidate molecules to stimulate T cell activation in vitro, we established a mixed lymphocyte reaction system in vitro, the specific method is as follows:

CD14-positive monocytes and CD4-positive T cells were sorted in vitro using Miltenyi sorting kit (Miltenyibiotec, 130-050-201), and cultured with 100 ng/mL IL-4 and 100 ng/mL GM-CSF in vitro. On the first day, monocytes were induced to become dendritic cells (Dendritic cells, DCs), and then CD4 positive T cells were mixed with DCs at a cell ratio of 10:1, and serially diluted candidate antibodies and control antibodies were added to the cells, 37 Cultivate in a cell incubator at ℃ for 5 days, take the cell supernatant after 72 hours of culture, and use the IL-2ELISA kit to detect the secretion of IL-2 in the culture supernatant after dilution in PBS; The IFN-γ secretion in the culture supernatant was detected using the IFN-γELISA kit.

By detecting the secretion of IL-2, we obtained the verification results of the mixed lymphocyte reaction test of the antibody molecule, which is shown in FIG. 5 . It can be seen from Figure 5 that the nanobody candidate molecule of the present invention can activate an immune response.

Example 12 Antibody Humanization Transformation

Antibody Modeling，采用同源建模方法建模，选取5-10个最优结构解，Loop区域一般使用同源建模方法建模，如CDR氨基酸序列比对结果显示低于50％同一性，则使用从头建模方法搭建CDR3结构模型。使用PDB BLAST调取序列最接近的10个抗体晶体结构模型(结构分辨率高于2.5埃)，对比自动建模模型，选取最优的结构模型。我们对NB22D-21分子进行了抗体人源化改造，得到了两个人源化的分子，这两个人源化改造后的分子编号分别为NB22D-21-huVH1和NB22D-21-huVH2。In order to reduce the immunogenicity of the molecule in vivo, we performed a humanization design on the candidate molecule. Using Discovery Studio and

Antibody Modeling, using the homology modeling method to model, select 5-10 optimal structural solutions, the Loop region is generally modeled using the homology modeling method, if the CDR amino acid sequence alignment results show less than 50% identity, then The CDR3 structural model was constructed using the ab initio modeling method. Use PDB BLAST to retrieve the 10 antibody crystal structure models with the closest sequence (structural resolution higher than 2.5 angstroms), compare the automatic modeling model, and select the optimal structure model. We carried out antibody humanization transformation on NB22D-21 molecule, and obtained two humanized molecules.

Example 13 Derivative Molecules After Humanization Transformation Bind to Human PD-L1-CHO Cells

In order to detect the effect of humanization on the binding activity of molecules to human PD-L1 antigen, we used FACS to detect the candidate molecules and their humanized derivatives.

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abcam, ab98596), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102), the results are shown in Figure 6.

It can be seen from Figure 6 that the derived molecules obtained after humanization have a similar binding affinity to human PD-L1 as their parent molecules.

Example 14 Derivative Molecules After Humanization Transformation Bind to Murine PD-L1-CHO Cells

To test the effect of humanization on the cross-reactivity of molecules binding to the murine PD-L1 antigen, we tested the candidate molecules by FACS.

1) Collect the cultured mouse PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ cells/mL;

2) Add mouse PD-L1-CHO cells to a 96-well round bottom plate at 100 μL per well, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add PE-labeled anti-human IgG Fc flow antibody (Abcam, ab98596), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

7) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

8) Repeat step 7) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102), the results are shown in Figure 7 and Figure 8.

It can be seen from Figure 7 that among the derivative molecules obtained after humanization, the binding affinity of NB22D-21-huVH1 to mouse PD-L1 is better than that of the parent molecule.

Specifically, it can be seen from Figure 7 that, compared with the parent molecule NB22D-21 and the control molecule KN035, the derivative molecule NB22D-21-huVH1 obtained after humanization has a very good binding activity to the mouse PD-L1 protein ; Select the flow peak diagrams of KN035 and NB22D-21-huVH1 (as shown in Figure 8), from which it can be clearly seen that using 20 μg/mL of KN035 and NB22D-21-huVH1 combined with mouse PD-L1 overexpression Cells, FACS detection data showed that KN035 could not recognize mouse PD-L1 on the surface of CHO cells (Figure 8(A)), while NB22D-21-huVH1 could well recognize mouse PD-L1 on the surface of CHO cells (Figure 8(B) ) (Note: The dividing line in the middle of each figure is the set fluorescence intensity threshold position).

Example 15 Verification of the PD-1-blocking activity of the derived molecule after humanization transformation

The derived molecules obtained after humanization were evaluated, and their blocking activity on PD-1/PD-L1 was detected by FACS method. The specific method is as follows:

1) Collect the cultured human PD-L1-CHO cells, centrifuge at 300g to remove the supernatant, resuspend the cells in the prepared FACS buffer, count and adjust the density of the cell suspension to 2×10 ⁶ /mL.

2) Add 100 μL of PD-L1-CHO cells to each well of a 96-well round bottom plate, and centrifuge at 300 g to remove the supernatant;

3) Add serially diluted candidate antibody dilutions and control antibody dilutions to the corresponding wells, blow the cells evenly with a row gun and place them at 4°C for 30 minutes;

4) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL to the corresponding well and resuspend the cells with a row gun;

5) Repeat step 4) twice, and centrifuge at 300g to remove the supernatant;

6) Add 100 μL of biotin-labeled PD-1-Fc protein dilution (1 μg/mL) to the corresponding well, resuspend the cells and incubate the cells at 4°C for 30 minutes;

7) Centrifuge the incubated cell mixture at 300 g to remove the supernatant, add 200 μL FACS to the corresponding well and resuspend the cells using a row gun;

8) Repeat step 7) twice, centrifuge at 300g to remove the supernatant;

9) Add PE-labeled streptavidin (eBioscience, 12-4317-87), blow the cells evenly with a row gun and incubate at 4°C for 30 minutes;

10) Centrifuge at 300g to remove the supernatant, add FACS buffer and resuspend the cells;

11) Repeat step 10) twice, add FACS buffer to the wells, 200 μL per well, resuspend the cells, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102), the results are shown in Figure 9.

It can be seen from Figure 9 that the derivative molecule obtained after humanization is equivalent to its parent molecule in blocking PD-1 protein binding to PD-L1.

Example 16 Affinity Maturation of Humanized Molecules

In order to improve the affinity and blocking activity of antibody molecules binding to human and mouse PD-L1 antigens, we designed affinity maturation for the antibody-derived molecules obtained after humanization.

Affinity maturation uses methods such as single-point saturation mutation, multi-point combination mutation, and strand replacement. Trim primer technology is used to design primers for the CDR region, and an affinity matured antibody library is constructed, and the molecules after affinity maturation are screened using phage display technology. The VHH lysate was prepared after the monoclonal was obtained, and the affinity and blocking activity of the clone were detected by FACS. Through screening, we obtained 4 clones that may be better than the mother parent, namely SY01-201, SY01-208, SY01-NB-004, SY01-NB-027-M.

The specific experimental results are shown in Figure 10 and Figure 11. It can be roughly seen from the qualitative/semi-quantitative results of the cell lysate that SY01-201, SY01-208, SY01-NB-004, SY01-NB-027-M The binding and blocking activities of the four molecules were similar to those of the parent NB22D-21 molecule.

Example 17 Drug efficacy verification of NB22D-21 molecule in A375 mouse tumor model

In order to confirm the activity of candidate antibody molecules in inhibiting tumor growth in vivo, we established a mouse model based on the human melanoma A375 cell line overexpressing human PD-L1, the specific method is as follows:

Select NOD-SCID mice about 6 weeks old, with similar size and weight, and divide the mice into 3 groups: control group, candidate antibody group and positive control antibody group, with 8 mice in each group. Human melanoma cell line PD-L1-A375 (prepared in Example 1) was cultured in vitro, 1×10 ⁷ PD-L1-A375 cells were mixed with 5×10 ⁶ PBMC cells, and injected into mice through the tail vein. Recorded as day 0. On the second day, 5 mg/mL or 10 mg/mL of candidate antibody or control antibody was injected into each group of mice, and then administered once every 7 days for 6 consecutive administrations. The body weight and tumor size of the mice were recorded weekly from day 7 until the tumor grew to 1500 mm ³ .

Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention disclosed only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not to be limited to the particular embodiments described in detail herein. Instead, reference should be made to the appended claims as indicating the scope and content of the invention.

SEQUENCE LISTING

<110> Sanyou Biopharmaceutical (Shanghai) Co., Ltd.

<120> PD-L1 binding molecules

<130> IDC196019

<160> 36

<170> PatentIn version 3.5

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Arg Thr Asp Ser Asn Ile Asn Gly Met His

1 5 10

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Thr Ile Phe Ile Asp Gly Asn Thr Ile

1 5

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<213> Artificial Sequence

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Asp Val Ser Gly Tyr Gly Arg Ala

1 5

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Arg Thr Asp Ser Asn Ile His Gly Met His

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Thr Ile Phe Ile Asp Leu Asn Thr Ile

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Arg Thr Asp Ser Asn Ile Phe Gly Met His

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Thr Ile Phe Ile Asp Ala Asn Thr Ile

1 5

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Thr Ile Phe Ile Asp Gly Asn Thr Leu

1 5

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Asp Val Ser Gly Tyr Gly Arg Tyr

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Arg Thr Asp Ser Asn Ile Asn Ser Met His

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<223> CDR2

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Thr Trp Phe Ile Asp Gly Asn Thr Ile

1 5

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<220>

<223> CDR3

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Asp Glu Trp Met Tyr Gly Arg Ala

1 5

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<223> CDR1

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Gly Phe Thr Phe Ser Arg Arg Cys Lys Ala

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<223> CDR2

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Lys Ile Leu Thr Thr Ser Gly Ser Thr Tyr

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<223> CDR3

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Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser Gly Ala

1 5 10 15

Phe Gln Tyr

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<211> 116

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<213> Artificial Sequence

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<223> VH

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Glu Val Gln Val Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser Arg Thr Asp Ser Asn Ile Asn

20 25 30

Gly Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Trp Val

35 40 45

Gly Thr Ile Phe Ile Asp Gly Asn Thr Ile Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 17

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Ser Asn Ile Asn

20 25 30

Gly Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val

35 40 45

Gly Thr Ile Phe Ile Asp Gly Asn Thr Ile Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 18

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser Arg Thr Asp Ser Asn Ile Asn

20 25 30

Gly Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val

35 40 45

Gly Thr Ile Phe Ile Asp Gly Asn Thr Ile Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 19

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser Arg Thr Asp Ser Asn Ile His

20 25 30

Gly Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val

35 40 45

Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 20

<211> 116

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<213> Artificial Sequence

<220>

<223> VH

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser Arg Thr Asp Ser Asn Ile Phe

20 25 30

Gly Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val

35 40 45

Gly Thr Ile Phe Ile Asp Ala Asn Thr Ile Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 21

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser Arg Thr Asp Ser Asn Ile Asn

20 25 30

Gly Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val

35 40 45

Gly Thr Ile Phe Ile Asp Gly Asn Thr Leu Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Val Ser Gly Tyr Gly Arg Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 22

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser Arg Thr Asp Ser Asn Ile Asn

20 25 30

Ser Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val

35 40 45

Gly Thr Trp Phe Ile Asp Gly Asn Thr Ile Val Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Glu Trp Met Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 23

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> VH

<400> 23

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Arg

20 25 30

Cys Lys Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Lys Ile Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 25

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<213> Artificial Sequence

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<223>FR1

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser

20 25

<210> 25

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 25

Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val Gly

1 5 10

<210> 26

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 26

Val Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 27

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>FR4

<400> 27

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

1 5 10

<210> 28

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223>FR1

<400> 28

Glu Val Gln Val Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala His Ser

20 25

<210> 29

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 29

Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Trp Val Gly

1 5 10

<210> 30

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 30

Val Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

1 5 10 15

Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Phe Cys Ala Ala

35

<210> 31

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>FR4

<400> 31

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 32

<211> 25

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<220>

<223>FR1

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 33

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223>FR1

<400> 33

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 34

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> FR2

<400> 34

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ala

1 5 10

<210> 35

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> FR3

<400> 35

Leu Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

1 5 10 15

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys Ala Ala

35

<210> 36

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223>FR4

<400> 36

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

Claims (24)

1. An isolated single domain antibody or an antigen-binding fragment thereof, which specifically binds to PD-L1, said single domain antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain The variable region contains: (i) CDR1 shown in SEQ ID NO: 1; (ii) CDR2 shown in SEQ ID NO: 2; and (iii) CDR3 shown in SEQ ID NO:3. 2. An isolated single domain antibody or an antigen-binding fragment thereof, which specifically binds to PD-L1, said single domain antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain The variable region contains: (i) CDR1 shown in SEQ ID NO: 4; (ii) CDR2 shown in SEQ ID NO: 5; and (iii) CDR3 shown in SEQ ID NO:3. 3. An isolated single domain antibody or an antigen-binding fragment thereof, which specifically binds to PD-L1, said single domain antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain The variable region contains: (i) CDR1 shown in SEQ ID NO: 6; (ii) CDR2 shown in SEQ ID NO: 7; and (iii) CDR3 shown in SEQ ID NO:3. 4. An isolated single domain antibody or an antigen-binding fragment thereof, which specifically binds to PD-L1, said single domain antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain The variable region contains: (i) CDR1 shown in SEQ ID NO: 1; (ii) CDR2 shown in SEQ ID NO: 8; and (iii) CDR3 shown in SEQ ID NO:9. 5. An isolated single domain antibody or an antigen-binding fragment thereof, which specifically binds to PD-L1, said single domain antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH), wherein said heavy chain The variable region contains: (i) CDR1 shown in SEQ ID NO: 10; (ii) CDR2 shown in SEQ ID NO: 11; and (iii) CDR3 shown in SEQ ID NO:12. 6. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the heavy chain variable region further comprises a FR region comprising FR1, FR2, FR3 and FR4 , and CDR1, CDR2 and CDR3 are arranged at intervals on the heavy chain variable region to form a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus. 7. The single domain antibody or antigen-binding fragment thereof according to claim 6, wherein the FR regions comprise FR1, FR2, FR3 and FR4 as follows: (a) FR1 shown in SEQ ID NO: 24; (b) FR2 shown in SEQ ID NO: 25; (c) FR3 shown in SEQ ID NO: 26; and (d) FR4 shown in SEQ ID NO:27. 8. The single domain antibody or antigen-binding fragment thereof according to claim 6, wherein the FR regions comprise FR1, FR2, FR3 and FR4 as follows: (a) FR1 shown in SEQ ID NO: 28; (b) FR2 shown in SEQ ID NO: 29; (c) FR3 shown in SEQ ID NO: 30; and (d) FR4 shown in SEQ ID NO:31. 9. The single domain antibody or antigen-binding fragment thereof according to claim 6, wherein the FR regions comprise FR1, FR2, FR3 and FR4 as follows: (a) FR1 shown in SEQ ID NO: 32; (b) FR2 shown in SEQ ID NO: 25; (c) FR3 shown in SEQ ID NO: 26; and (d) FR4 shown in SEQ ID NO:27. 10. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 20, Any amino acid sequence in 21 and 22. 11. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the heavy chain variable region is identical to SEQ ID NO: 16, 17, 18, 19, 20, 21 and Any of the 22 are at least 80%, 85%, 90%, 95% or 99% identical and retain the ability to specifically bind PD-L1. 12. The isolated single domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the heavy chain variable region is identical to SEQ ID NO: 16, 17, 18, 19, 20, Any one of 21 and 22 has one or more amino acid additions, deletions and/or substitutions and retains the ability to specifically bind PD-L1. 13. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the isolated antibody is a camelid antibody, a humanized antibody or an affinity matured antibody. 14. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1-5, fused to another molecule selected from the group consisting of immunoglobulins Fc domains, antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, or fluorescent proteins. 15. The single domain antibody or antigen-binding fragment thereof of claim 14 fused to an Fc domain of a human IgG. 16. The single domain antibody or antigen-binding fragment thereof of claim 15, wherein said antibody or antigen-binding fragment thereof is fused to an Fc domain of human IgGl or human IgG4. 17. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the single domain antibody or antigen-binding fragment thereof of any one of claims 1-16. 18. A vector comprising the nucleic acid molecule of claim 17. 19. A host cell comprising the vector of claim 18. 20. A pharmaceutical composition comprising at least one single domain antibody or antigen-binding fragment thereof according to any one of claims 1-16, and a pharmaceutically acceptable carrier. 21. A method for preparing the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-16, comprising the steps of: - expressing a single domain antibody or an antigen-binding fragment thereof according to any one of claims 1-16 in a host cell according to claim 19; and - isolating said single domain antibody or antigen-binding fragment thereof from said host cell. 22. Use of the single domain antibody or antigen-binding fragment thereof according to any one of claims 1-16 in the preparation of a medicament for preventing or treating a disease associated with PD-L1 in a subject, wherein the The disease associated with PD-L1 is selected from renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, microsatellite unstable solid tumor. 23. The use according to claim 22, wherein the subject is a mouse or a human. 24. A kit for preventing or treating a disease associated with PD-L1 in a subject, comprising a container comprising at least one single domain according to any one of claims 1-16 An antibody or an antigen-binding fragment thereof, wherein the PD-L1-related disease is selected from renal cell carcinoma, non-small cell lung cancer, bladder cancer, urothelial carcinoma, and microsatellite unstable solid tumors.

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