EA044684B1 - ANTI-PD-L1 ANTIBODY AND ITS APPLICATION - Google Patents

Description

Field of technology

The invention relates to the field of biomedicine, in particular to an anti-PD-L1 antibody, and further relates to a method for treating malignant neoplasms by using such an antibody in combination with an anti-CD137 antibody, and further relates to a bispecific antibody formed by such an antibody and an anti-CD137 antibody.

State of the art

Programmed cell death protein-1 (PD-1, also known as CD279) and its ligands PD-L1 (also known as B7-H1, CD274) and PD-L2 (also known as B7-DC, CD273) interact, leading to to the emergence of a negative costimulatory signal to regulate T cell activation. In the absence of a costimulatory signal, T cells have difficulty sensing antigenic stimulation and cannot mount an effective immune response, and this can also lead to exhaustion or tolerance to heterogeneous antigens. PD-1 can be expressed in T cells, B cells, natural killer T cells, activated mononuclear cells and dendritic cells (DC). PD-1 can be expressed by activated but not stimulated CD4+ and CD8+ T cells, B cells, and human bone marrow cells. PD-L1 and PD-L2 differ from each other in their expression profiles. PD-L1 can be expressed not only in hematopoietic cells, but also in various non-hematopoietic cells, where the expression of PD-L2 is mainly limited to dendritic cells and macrophages. B7.1 (CD80) is also a PD-L1 receptor expressed on activated B cells, T cells, macrophages and dendritic cells. As a ligand of PD-1 and B7.1, PD-L1 is also selectively expressed in various tumor cells. Inhibition of PD-L1 signaling involves blocking the interaction between PD-L1 and PD-1 and B7.1 or both, and thus preventing PD-L1 from sending a negative costimulatory signal to T cells and other antigen-presenting cells. This may enhance the immune response to infection (eg, acute or chronic) and antitumor immunity.

CD137 (also known as 4-1BB, TNFRSF9, etc.) is a transmembrane protein of the tumor necrosis factor superfamily (TNFRS). Studies show that CD137-activating monoclonal antibody increases the expression of costimulatory molecules in a variety of models, which significantly enhances cytolytic T-lymphocyte responses and plays an antitumor role. The antitumor response of CD137-targeted therapies has been demonstrated in studies involving the antitumor therapeutic effect of an activating anti-mouse CD137 monoclonal antibody in mice.

Anti-PD-L1 antibodies are currently approved for use in the treatment of several malignancies, and existing anti-CD137 antibodies have potential use in the treatment of various tumors. The antitumor therapeutic effect of an anti-PD-1 antibody in combination with an anti-CD137 antibody is reported to have been investigated using a tumor-bearing mouse model. The results showed that the two antibodies used in combination exhibited significantly enhanced tumor inhibitory activity compared to their use alone, but in terms of compliance and pain, the combination treatment was inconvenient for patients and increased treatment costs. Development of a bispecific antibody (BsAb) from two monoclonal antibodies can help solve the above problem due to the presence of two specific antigen binding sites in the bispecific antibody. Therefore, there is an urgent need to develop a new anti-PD-L1 antibody with better pharmaceutical efficacy and lower immunogenicity and a PDL1/CD137 bispecific antibody to achieve better antitumor effect.

The essence of the invention

The present invention provides an anti-PD-L1 antibody, an antigen binding fragment or variant thereof, having one or more of the following properties: 1) can bind to the PD-L1 protein with high affinity and specificity; 2) can inhibit the binding of the PD-1 protein to the PDL1 protein; and 3) may lead to relief of symptoms or treatment of tumors. The present invention also provides a bispecific antibody having one or more of the following properties: 1) can bind to the PD-L1 protein with high affinity and specificity; 2) can bind to the CD137 protein with high affinity and specificity; 3) can enhance the functions of T cells; and 4) may lead to relief of symptoms or treatment of tumors. The present invention also relates to a method for producing and using an anti-PD-L1 antibody, an antigen binding fragment or variant thereof, a bispecific antibody.

In one aspect, the present invention provides an antibody, antigen binding fragment or variant thereof that binds to the PD-L1 protein with a KD of 3x10'9 M or less.

In some embodiments, the antibody comprises an antibody light chain or fragment thereof comprising LCDR1, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53: TGTX1SX2VGGYX3X4VS; where X1 is S, R or V; X2 is D, E or S; X3 is N or R; X4 is Y or E, and where LCDR1 is defined according to the Kabat antibody numbering index.

- 1 044684

In some embodiments, LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-58. In some embodiments, the antibody light chain or fragment thereof comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59: X1NSX2RPS, wherein X1 is G or E; X2 is N or I, and where LCDR2 is defined according to the Kabat antibody numbering index. In some embodiments, LCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 60-61. In some embodiments, the antibody light chain or fragment thereof comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62: QSYDSSLSGX1V, wherein X1 is S or T, and wherein LCDR3 is defined in accordance with the Kabat antibody numbering index.

In some embodiments, the antibody light chain or fragment thereof further includes the L-FR1, L-FR2, L-FR3, and L-FR4 framework regions. In some embodiments, the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences. In some embodiments, the C terminus of LFR1 is linked directly or indirectly to the N terminus of LCDR1, and L-FR1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 71. In some embodiments, LFR2 is located between LCDR1 and LCDR2, and L-FR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 72. In some embodiments, L-FR3 is between LCDR2 and LCDR3, and L-FR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 73-75. In some embodiments, the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of LCDR3, and L-FR4 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 76.

In some embodiments, the antibody light chain or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33 , SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41.

In some embodiments, the antibody light chain or fragment thereof further includes a human constant region, and the human constant region includes a human Igλ constant region.

In some embodiments, the antibody light chain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42.

In some embodiments, the antibody includes an antibody heavy chain or fragment thereof including HCDR1, and HCDR1 includes an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 45: X1YAIS, where X1 is S or T, and where HCDR1 determined according to the Kabat antibody numbering index. In some embodiments, the antibody heavy chain or fragment thereof comprises HCDR2, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 48, and wherein HCDR2 is defined in accordance with the Kabat antibody numbering index. In some embodiments, the antibody heavy chain or fragment thereof comprises HCDR3, and HCDR3 comprises an amino acid sequence selected from the group consisting of those set forth in SEQ ID NO: 49: TMX1X2YX3X4GNX5DY, wherein X1 is D, E or G, X2 is G or E, X3 is S or G, X4 is Y or F, X5 is F or Y, and where HCDR3 is defined according to the Kabat antibody numbering index. In some embodiments, HCDR3 includes the amino acid sequence set forth in SEQ ID NO: 5052.

In some embodiments, the antibody heavy chain or fragment thereof further includes the H-FR1, H-FR2, H-FR3, and H-FR4 framework regions. In some embodiments, the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences. In some embodiments, the C terminus of H-FR1 is directly or indirectly linked to the N terminus of HCDR1, and H-FR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65-67. In some embodiments, H-FR2 is between HCDR1 and HCDR2, and H-FR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 68. In some embodiments, HFR3 is between HCDR2 and HCDR3, and H-FR3 includes an amino acid a sequence selected from the group consisting of SEQ ID NO: 69. In some embodiments, the N-terminus of HFR4 is linked directly or indirectly to the C-terminus of HCDR3, and H-FR4 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70.

In some embodiments, the antibody heavy chain or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31 and SEQ ID NO: 35.

In some embodiments, the antibody heavy chain or fragment thereof is further

- 2044684 includes a human constant region, and the human constant region includes a human IgG1 constant region.

In some embodiments, the antibody heavy chain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 32, and SEQ ID NO: 36.

In some embodiments, the PD-L1 protein is selected from the group consisting of human PD-L1 protein, monkey PD-L1 protein, and mouse PD-L1 protein.

In another aspect, the present invention provides an antibody, antigen-binding fragment or variant thereof that binds to the CD137 protein with a KD of 5x10 ^-9 M or less.

In some embodiments, the antibody, antigen binding fragment or variant thereof binds to the CD137 protein with a KD of 3x10 ^-9 M or less.

In some embodiments, an antibody, antigen-binding fragment, or variant thereof may result in the amelioration or treatment of tumors. In some embodiments, an antibody, antigen-binding fragment, or variant thereof may result in the alleviation or treatment of colon cancer.

In some embodiments, the antibody includes an antibody light chain or fragment thereof. In some embodiments, the light chain or fragment thereof comprises LCDR1-3, and the amino acid sequences of LCDR1-3 are sequentially set forth in SEQ ID NOs: 80-82. In some embodiments, the antibody light chain or fragment thereof further includes the LFR1, L-FR2, L-FR3, and L-FR4 framework regions. In some embodiments, the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences. In some embodiments, the C terminus of L-FR1 is directly or indirectly linked to the N terminus of LCDR1, and L-FR1 includes the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, L-FR2 is located between LCDR1 and LCDR2, and LFR2 includes the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, L-FR3 is located between LCDR2 and LCDR3, and L-FR3 includes the amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the N-terminus of L -FR4 is directly or indirectly linked to the C terminus of LCDR3, and L-FR4 includes the amino acid sequence set forth in SEQ ID NO: 92.

In some embodiments, the antibody light chain or fragment thereof includes a VL light chain variable region, and the VL light chain variable region includes the amino acid sequence set forth in SEQ ID NO: 20.

In some embodiments, the antibody light chain or fragment thereof further includes a human constant region, and the human constant region includes a human Igλ constant region.

In some embodiments, the antibody light chain or fragment thereof includes the amino acid sequence set forth in SEQ ID NO: 23.

In some embodiments, the antibody comprises an antibody heavy chain or fragment thereof, wherein the heavy chain or fragment thereof comprises HCDR1-3, and the amino acid sequences of HCDR1-3 are sequentially set forth in SEQ ID NOs: 77-79. In some embodiments, the antibody heavy chain or fragment thereof further includes the H-FR1, H-FR2, HFR3, and H-FR4 framework regions. In some embodiments, the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences. In some embodiments, the C terminus of H-FR1 is linked directly or indirectly to the N terminus of HCDR1, and H-FR1 includes the amino acid sequence set forth in SEQ ID NOs: 84-85. In some embodiments, H-FR2 is between HCDR1 and HCDR2, and H-FR2 includes the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, H-FR3 is between HCDR2 and HCDR3, and H-FR3 includes the amino acid sequence, set forth in SEQ ID NO: 87. In some embodiments, the N terminus of H-FR4 is linked directly or indirectly to the HCDR3 C terminus, and H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 88.

In some embodiments, the antibody heavy chain or fragment thereof includes a VH heavy chain variable region, and the VH heavy chain variable region includes an amino acid sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 25.

In some embodiments, the antibody heavy chain or fragment thereof further includes a human constant region, and the human constant region includes a human IgG constant region.

In some embodiments, the antibody heavy chain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 27.

In some embodiments, the CD137 protein includes a human CD137 protein.

In another aspect, the present invention provides a bispecific antibody that binds to the PD-L1 protein with a KD of 2x10 ^-9 M or less and binds to the CD137 protein with a KD of 8x10' ⁹ M or less.

- 3 044684

In some embodiments, the bispecific antibody includes a first targeting moiety that specifically binds to a PD-L1 protein, wherein the first targeting moiety comprises an antibody, an antigen-binding fragment, or a variant thereof.

In some embodiments, the bispecific antibody includes a second targeting moiety that specifically binds to the CD137 protein, wherein the second targeting moiety comprises the antibody, an antigen-binding moiety, or a variant thereof that binds to the CD137 protein. In some embodiments, the antibody that binds to the CD137 protein includes a scFv, and the scFv includes the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the bispecific antibody includes a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes a variable region an antibody heavy chain that binds to the PD-L1 protein, an antibody heavy chain variable region that binds to the CD137 protein, and an antibody light chain variable region that binds to the CD137 protein; and the second polypeptide chain includes an antibody light chain variable region that binds to the PD-L1 protein.

In some embodiments, the first polypeptide chain of the heavy chain variable region of the PD-L1 protein-binding antibody is located at the N-terminus of the heavy chain variable region of the CD137 protein-binding antibody, and the heavy chain variable region of the CD137 protein-binding antibody is at the N-terminus of the variable region of the light chain of an antibody that binds to the CD137 protein; alternatively, the heavy chain variable region of the PD-L1 protein-binding antibody is at the N-terminus of the light chain variable region of the CD137 protein-binding antibody, and the light chain variable region of the CD137 protein-binding antibody is at the N-terminus of the variable region the heavy chain of an antibody that binds to the CD137 protein.

In some embodiments, the CD137 protein-binding antibody light chain variable region and the CD137 protein-binding antibody heavy chain variable region in the first polypeptide chain constitute a scFv.

In some embodiments, the first polypeptide chain further includes a human IgG constant region, and the human IgG constant region is located at the C-terminus of the heavy chain variable region of the antibody binding to the PD-L1 protein and is located at the N-terminus of the light chain variable region of the antibody binding with protein CD137; alternatively, the human IgG constant region is located at the C-terminus of the heavy chain variable region of the antibody binding to the PD-L1 protein and is located at the N-terminus of the heavy chain variable region of the antibody binding to the CD137 protein.

In some embodiments, the first polypeptide chain includes an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 43, and SEQ ID NO: 44. In some embodiments, the second polypeptide chain includes an amino acid sequence selected from the group consisting of consisting of SEQ ID NO: 15 and SEQ ID NO: 34.

In another aspect, the present invention provides one or more nucleic acid molecules encoding an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody.

In another aspect, the present invention relates to a vector comprising a nucleic acid molecule.

In another aspect, the present invention relates to a cell including a nucleic acid molecule or vector.

In another aspect, the present invention provides a method for producing an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, comprising culturing cells under conditions allowing expression of the antibody, an antigen binding fragment or variant thereof, or the bispecific antibody.

In another aspect, the present invention provides a pharmaceutical composition comprising an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, a nucleic acid molecule, a vector and/or a cell, and optionally a pharmaceutically acceptable excipient.

In another aspect, the present invention relates to the use of an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, nucleic acid molecule, vector, cell and/or pharmaceutical composition in the manufacture of a medicament for the relief or treatment of tumors.

In another aspect, the present invention relates to a method of inhibiting the binding of a PDL1 protein to a PD-1 protein, comprising administering an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, a nucleic acid molecule, a vector, a cell, and/or a pharmaceutical composition.

In the present invention, the position of a CDR in an antibody is determined by the Kabat method.

Other aspects and advantages of the present invention will become apparent to those skilled in the art from the detailed description below. The following detailed description represents exclusively

- 4 044684 examples of embodiments of the present invention. For example, those skilled in the art will appreciate that the contents of the present specification will allow those skilled in the art to make changes to the described embodiments without departing from the spirit and scope of the invention to which the present invention relates. Accordingly, the accompanying drawings and description in the present invention are merely examples and not limitations.

Brief description of drawings

Specific features of the invention described in the present invention are given in the claims. The features and advantages of the invention described in the present invention will be better understood in view of the detailed examples of embodiments and accompanying drawings presented later in the present description. A brief description of the accompanying drawings is as follows:

In fig. 1 shows an alignment of the amino acid sequences of the heavy chain variable regions and light chain variable regions of an anti-PD-L1 antibody of the present invention and the corresponding germline sequences. CDRs determined by the Kabat method are underlined.

In fig. 2 shows the results regarding the inhibition of binding of human PD-L1 to human PD-1 by the anti-PD-L1 antibody of the present invention.

In fig. 3 shows the results regarding the inhibition of the binding of Macaca fascicularis PD-L1 to Macaca fascicularis PD-1 by the anti-PD-L1 antibody of the present invention.

In fig. 4 shows the results regarding the inhibition of the binding of mouse PD-L1 to mouse PD-1 by the anti-PD-L1 antibody of the present invention.

In fig. 5 shows the effect of various antibodies of the present invention on Mc38 tumor growth in a C57BL/6 mouse (*, P < 0.05; ***, P < 0.001; unpaired t test).

In fig. 6 shows an alignment of the amino acid sequences of the heavy chain variable regions and light chain variable regions of the anti-CD137 antibody of the present invention and the corresponding germline sequences, where the CDRs determined by the Kabat method are underlined.

In fig. 7 shows the effect of the anti-CD137 antibody of the present invention on the growth of MC38 tumor in a female C57BL/6 human CD137 gene knockin mouse (*, P < 0.05; **, P < 0.01; unpaired t test).

In fig. 8 shows an alignment of the amino acid sequences of the heavy chain variable regions and light chain variable regions of the anti-PD-L1 antibody of the present invention and the corresponding germline sequences, with the CDRs determined by the Kabat method underlined and the CDRs determined by the IMGT method shaded. Amino acid residues in various CDR regions of YN-003 (including some amino acid residues in the CDR regions and several amino acid residues in the FR regions adjacent to the CDR regions) that are randomly affected when performing affinity maturation are indicated in italics.

In fig. 9 shows an alignment of the amino acid sequences of the heavy chain variable regions of the anti-PD-L1 antibody of the present invention with the corresponding germline sequences. The CDRs determined by the Kabat method are underlined and the CDRs determined by the IMGT method are shaded.

In fig. 10 shows an alignment of the amino acid sequences of the light chain variable regions of an anti-PD-L1 antibody of the present invention with the corresponding germline sequences. The CDRs determined by the Kabat method are underlined and the CDRs determined by the IMGT method are shaded.

In fig. 11 shows the binding of an anti-PD-L1 antibody of the present invention to CHO cells stably expressing human PD-L1.

In fig. 12 shows that the anti-PD-L1 antibody of the present invention inhibits the binding of human PD-1 to human PD-L1.

In fig. 13 shows that the anti-PD-L1 antibody of the present invention binds to CHO cells stably expressing murine PD-L1.

In fig. 14 shows that the anti-PD-L1 antibody of the present invention inhibits the binding of mouse PD-1 to mouse PD-L1.

In fig. 15 shows that the anti-PD-L1 antibody of the present invention binds to MDA-MB-231 cells.

In fig. 16 shows that the anti-PD-L1 antibody and anti-PD-L1/CD137 bispecific antibody of the present invention bind to CHO cells stably expressing human PD-L1.

In fig. 17 shows that the anti-PD-L1 antibody and the anti-PD-L1/CD137 bispecific antibody of the present invention inhibit the binding of human PD-1 to human PD-L1.

In fig. 18 shows that the anti-PD-L1 antibody and anti-PD-L1/CD137 bispecific antibody of the present invention bind to CHO cells stably expressing murine PD-L1.

- 5 044684

In fig. 19 shows that the anti-PD-L1 antibody and the anti-PD-L1/CD137 bispecific antibody of the present invention inhibit the binding of mouse PD-1 to mouse PD-L1.

In fig. 20 shows that the CD137 antibody and anti-PD-L1/CD137 bispecific antibody of the present invention bind to 293T cells stably expressing human CD137.

In fig. 21 shows that the molecular weight of the antibody of the present invention is analyzed by SDS-PAGE. a, non-reducing conditions; and b, reducing conditions.

In fig. 22 shows results regarding stimulation of luciferase activity by various antibodies of the present invention in 293T cells expressing human CD137 and having a stably integrated luciferase reporter gene.

In fig. 23 shows the effect of antibodies of the present invention on the growth of MC38 tumors in female C57BL/6 mice with the human CD137 gene (*, P < 0.05; unpaired t test).

Detailed Description of Embodiments

Hereinafter, in the present description, embodiments of the invention presented in the present invention are illustrated by means of specific examples. Other advantages and effects of the invention presented herein will become apparent to those skilled in the art from the following description.

In the present invention, the term antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains linked by a disulfide bond, and includes any molecule comprising an antigen-binding portion thereof. The term antibody includes a monoclonal antibody, antibody fragment, or antibody derivative, including, but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv), and antigen binding antibody fragments (e.g., Fab-, Fab'-, and Fab) ₂ -fragments). The term antibody further includes all recombinant antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies and any antigen binding fragment of an antibody as provided herein, and derivatives thereof. Each heavy chain may consist of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may consist of light chain variable regions (VL) and light chain constant regions. The VH and VL regions can be further divided into highly variable regions called complementarity determining regions (CDRs), distributed over more conserved regions called framework regions (FRs). Each VH and VL may consist of three CDRs and four FRs, which may be arranged from amino terminus to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Variable regions in the heavy chains and light chains include binding domains that interact with antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).

In the present invention, the term PD-L1 protein generally refers to the programmed death protein-1 (PD-1) ligand. PD-1 is an Ig superfamily receptor that interacts with a specific ligand (PD-L) and negatively regulates T-cell antigen receptor signaling and is thought to play a role in the maintenance of self-tolerance. In the absence of a costimulatory signal, T cells have difficulty recognizing antigen stimulation, cannot effectively produce an immune response, and this may further cause exhaustion or tolerance to the heterologous antigen. PD-L1 can be expressed in hematopoietic cells or various non-hematopoietic cells. B7.1 (CD80) is also a PD-L1 receptor expressed on activated B cells, T cells, macrophages and dendritic cells. As a ligand of PD-1 and B7.1, PD-L1 is further selectively expressed in various tumor cells. Inhibition of PD-L1 signaling involves blocking the interactions of PD-L1 with PD-1 and B7.1 or both, and thus preventing PD-L1 from sending a negative costimulatory signal to T cells and other antigen-presenting cells. This may enhance the immune response to infection (eg, acute and chronic) and antitumor immunity.

In the present invention, the term CD137 protein, also known as 4-1BB or TNFRS9, generally refers to a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), which is an activation-inducible costimulatory molecule and an important regulator of immune responses, studies have shown that CD137-activating monoclonal antibodies increase the expression of costimulatory molecules in many models, and significantly enhance the response of cytolytic T lymphocytes and have antitumor effects. The antitumor effect of CD137-targeted therapy can be demonstrated in an antitumor efficacy study in mice using an activating anti-mouse CD137 monoclonal antibody. CD137 has emerged as a potent activator of immune cells and an important candidate antigen for the treatment of various diseases (see Vinay, Dass S., and Byoung S. Kwon. 4-1BB (CD137), an inducible costimulatory Receptor,

- 6 044684 as a specific target for cancer therapy. BMB reports 47.3 (2014): 122.).

As used herein, the term NFkB refers to nuclear factor kappa light chain enhancer of activated B cells (NFkB). It is a protein complex that controls DNA transcription. NFkB exists in almost all animal cell types and is involved in cellular responses to many stimuli, including stress, cytokines, free radicals, UV radiation, LDL oxidation, and microbial or viral antigens. In the immune response to infection, NFkB plays an important regulatory role (κ-light chain is an important part of immunoglobulin). Abnormal regulation of NFkB is associated with malignancies, inflammation and autoimmune diseases, septic shock, viral infections and abnormal development of the immune system. NFkB is also closely associated with synaptic plasticity and memory processes. The main cell targets of NFkB are chemokines, immune receptors, adhesion molecules, stress response genes, apoptosis regulators, transcription factors, growth factors, enzymes and cell cycle regulators. In addition, NFkB influences the transcription of several viral promoters/enhancers (for example, HIV-1 and CMV) (see Tergaonkar, Vinay. NFkB pathway: A good signaling paradigm and therapeutic target. The international journal of biochemistry & cell biology 38, 10 (2006): 16471653.).

In the present invention, the term K _D can be used interchangeably with the term KD, and generally refers to the equilibrium dissociation constant of a particular antibody-antigen interaction in units of M (mol/L). KD can be calculated from the concentrations of substance AB and substances A and B obtained through dissociation of AB: KD=c(A)*c(B)/c(AB). From this formula it can be seen that the higher the KD value, the higher the dissociation and weaker the affinity between substances A and B; conversely, the lower the KD value, the lower the dissociation, and the higher the affinity between substances A and B.

In the present invention, the term monoclonal antibody generally refers to a group of antibodies that are substantially homologous, i.e. Each antibody included in the group is identical, except for possible natural mutations in minor quantities. Monoclonal antibodies are highly specific and directed against one site of an antigen. In addition, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody directed against a single determinant on an antigen is not interpreted as requiring any particular method of producing the antibodies. For example, monoclonal antibodies can be produced using hybridoma technology or produced in bacteria, eukaryotic animal or plant cells by recombinant DNA techniques. Alternatively, monoclonal antibodies can also be produced from a phage antibody library using technology as described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

In the present invention, the term single chain antibody (scFv) generally refers to a molecule formed by linking the heavy chain variable region of an antibody to the light chain variable region using a short peptide linker (linker).

In the present invention, the term chimeric antibody generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to the corresponding amino acid sequence in an antibody of a particular biological species or belongs to a particular class, and the remainder of the chain is homologous to the corresponding sequence of another biological kind. For example, the light chain and heavy chain variable regions are derived from the variable region of an antibody of one species (eg, mouse, rat, etc.), while the constant portion is homologous to the sequence of an antibody of another species (eg, human). For example, in the case of producing chimeric antibodies, non-human B cells or hybridoma cells can be used to produce the variable regions, and the constant regions combined with them are obtained from humans. The variable region has the advantage of being easy to obtain and its specificity is not affected by the source of the constant region combined with it. At the same time, because Since the constant region of a chimeric antibody can be obtained from humans, the possibility of the chimeric antibody inducing an immune response upon injection is lower than in the case of an antibody whose constant region is obtained from a non-human source.

As used herein, the term humanized antibody generally refers to an engineered antibody produced by reducing the immunogenicity of antibodies, immunoglobulin binding proteins, and polypeptides derived from non-human species (e.g., mice or rats) for humans while retaining the antigen-binding properties of the original antibody. . For example, techniques that can be used include CDR grafting (Jones et al., Nature 321:522 (1986)) and its variant involving remodeling (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al. , 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), hyperchimerization (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al. ., 1991 Proc Natl

- 7 044684

Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154) and venation (Mark, et al., Derivation of therapeutically active humanized and veneered anti-CD18 antibodies. in: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312) or the like, to humanize the non-human binding domain. If other regions, such as the hinge region and constant region domains, are also obtained from non-human sources, these regions may also be humanized.

In the present invention, the term fully human antibody generally means that all parts of the antibody (including the constant regions, CH and CL regions of the antibody) are encoded by human-derived genes. The fully human antibody can significantly reduce the immune side effects caused by heterogeneous antibodies on the human body.

In the present invention, the term bispecific antibody (BsAb) generally refers to an antibody or antibody construct with dual specificity in its binding arm. Natural antibodies are monospecific and have the same specificity in their antigen-binding arms. Bispecific antibodies are obtained from two different sources and are constructed through recombinant DNA technology or cell fusion in such a way that they retain the two specificities of the original antibody. Bispecific antibodies carry two different antigen-binding sites. The Fc portion of a bispecific antibody is different from the Fc portion of any parent monospecific antibody. Because Bispecific antibodies have dual specificity for drugs and tumor cells, and could theoretically cause drug accumulation in tumors to be higher than in non-tumor parts of the body. Most of the new bispecific antibodies can redirect cytotoxic effector cells (eg, Tc cells, NK cells, neutrophils) to target cells (eg, tumor cells). Bispecific antibodies can simultaneously block two different mediators/pathways that play unique or overlapping roles in pathogenesis, and can also increase binding specificity by interacting with two, instead of one, different cell surface antigens (Fanger, Michael W., and Paul M. Guyre. Bispecific antibodies for targeted cellular cytotoxicity. Trends in biotechnology, 1991: 375-380), (Fan, Gaowei, et al. Bispecific antibodies and their applications. Journal of hematology & oncology, 2015: 130).

In the present invention, the term sequence homology generally means that the amino acid sequences of homologous proteins have significant similarity.

In the present invention, the term epitope generally refers to an antigenic determinant, i.e. part of a molecule recognized by the immune system (eg, recognized by an antibody). For example, epitopes are discrete three-dimensional regions on an antigen that are recognized by the immune system. An epitope typically consists of chemically active surface groups of molecules (such as amino acids or sugar side chains) and has specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be classified into conformational epitopes and non-conformational epitopes (linear epitopes) depending on their structure. The difference between conformational epitopes and non-conformational epitopes is that the former will lose their binding in the presence of denaturing solvents, while the latter will not. Epitopes that are found only on the surface of antigenic substances and readily bind to antigen recognition receptors or antibodies can be called functional epitopes; and epitopes that are located within the molecule and are not immunogenic can be called masked epitopes. Epitopes may consist of continuous residues or may be formed by discontinuous residues close to each other as a result of folding of the antigenic polymer. Epitopes formed by consecutive amino acids in proteins are generally retained when exposed to denaturing solvents, but epitopes formed by discontinuous amino acids are generally lost after such exposure.

In the present invention, the term antigen binding fragment generally refers to a portion of an intact antibody, eg, the antigen binding region and/or variable region of an intact antibody. In some embodiments, the antigen binding fragment may include Fab, Fab', F(ab) ₂ , F(ab') ₂ , and Fv fragments. In some embodiments, the antibodies of the present invention may include double-chain antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed by antibody fragments. Two identical antigen-binding fragments obtained by digestion of an antibody having an intact structure with papain (eg, by removing the Fc region and the hinge region) are called Fab fragments. The Fab fragment consists of an intact light chain, a heavy chain variable region (VH), and a first constant domain (CH1) of the heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e. it contains one antigen-binding site. F(ab)2 antibody fragments were initially obtained as pairs of Fab fragments connected through a cysteine. One large F(ab') ₂ fragment, obtained by cleavage of an antibody having an intact structure with pepsin, is approximately equal to two Fab fragments connected by a disulfide bond and having different antigen-binding activity, and is still able to cross-link antigens.

- 8 044684 gen. Fab' fragments differ from Fab fragments in that they have several additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. The Fv fragment consists of the VL and VH domains of one arm of the antibody.

In the present invention, the term variant generally refers to an amino acid sequence having substantially the same function (e.g., the ability to specifically bind to PDL1) and at least 85% (e.g., at least 85, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99% or at least 100 %) sequence identity. In some embodiments, the amino acid sequence variant is an amino acid sequence having substantially the same function (e.g., the ability to specifically bind to PD-L1), and includes one or more (e.g., 1-2, 1-3, 1-4 , 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid substitutions, deletions or additions.

In the present invention, the term identity generally refers to the percentage of identical amino acid residues among all amino acid residues when comparing a candidate sequence with a particular peptide or polypeptide sequence.

In the present invention, the term IgG generally refers to immunoglobulin G. IgG is one of the human immunoglobulins, and others include Igλ IgM, IgD and IgE. IgG is the major antibody component of serum, accounting for approximately 75% of serum Ig. According to the differences in antigenicity of the y-chain of the IgG molecule, human IgG has four subtypes: IgG1, IgG2, IgG3 and IgG4. IgG plays an important role in immunity. In the present invention, the term IgG1 generally refers to the subtype with the highest proportion of IgG having a higher affinity for Fc receptors.

In the present invention, the term nucleic acid molecule generally refers to the isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, isolated from their natural environment or synthesized artificially.

In the present invention, the term vector generally refers to a nucleic acid molecule capable of self-replication in a suitable host organism, which transfers the integrated nucleic acid molecule into and/or between host cells. A vector may include a vector primarily used for the insertion of DNA or RNA into cells, a vector primarily used for DNA or RNA replication, and a vector primarily used for the expression, transcription and/or translation of DNA or RNA. The vector also includes a vector having many of the functions described above. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when inserted into a suitable host cell. In general, when a suitable host cell containing the vector is cultured, the vector can produce the desired expression product.

As used herein, the term host cell generally refers to a plasmid or vector that can or does include a nucleic acid molecule of the present invention, or a single cell, cell line, or cell culture that can express an antibody or antigen binding fragment thereof of the present invention. . A host cell may include progeny of one host cell. Due to natural, random and intentional mutations, the progeny cells may not be exactly the same as the original parent cells in morphology or genome, provided that they can express the antibody or antigen binding fragment thereof of the present invention. The host cell can be obtained by transfecting cells in vitro using the vector of the present invention. The host cell may be a prokaryotic cell (eg, E. coli) and may also be a eukaryotic cell (eg, such as yeast cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells , NS0 cells or myeloma cells). In some embodiments, the host cell is a mammalian cell. For example, a mammalian cell may be a CHO-Ki cell. In the present invention, the term recombinant host cell generally refers to the cell into which the recombinant expression vector is inserted. A recombinant host cell not only includes a particular cell, but also includes the progeny of such cells.

As used herein, the term malignancy generally refers to the physiological status of a mammal, typically characterized by abnormalities in cell proliferation or survival. Non-limiting examples of malignancies include malignancies, lymphoma, maternal cell tumor, sarcoma, leukemia and lymphoid malignancy. For example, it may be lymphoma.

In the present invention, the term between generally means that the C-terminus of an amino acid fragment is directly or indirectly linked to the N-terminus of a first amino acid fragment, and its N-terminus is directly or indirectly linked to the C-terminus of a second amino acid fragment. In the light chain, for example, the N-terminus of L-FR2 is directly or indirectly linked to the C-terminus of LCDR1, and the C-terminus of L-FR2 is directly or indirectly linked to the N-terminus of LCDR2. As another example, the N-terminus of L-FR3 is directly or indirectly linked to the C-terminus of LCDR2, and the C-terminus of L-FR3 is directly or indirectly linked to the N-terminus of LCDR3. In a heavy price

- 9 044684 pi, for example, the N-terminus of H-FR2 is directly or indirectly linked to the C-terminus of HCDR1, and the C-terminus of H-FR2 is directly or indirectly linked to the N-terminus of HCDR2. As another example, the N terminus of H-FR3 is directly or indirectly linked to the C terminus of HCDR2, and the C terminus of H-FR3 is directly or indirectly linked to the N terminus of HCDR3.

In the present invention, the term approximately generally refers to fluctuations in the range of the specified value ±0.5% - ±10%, such as fluctuations in the range of the specified value ±0.5, ±1, ±1.5, ±2, ± 2.5, ±3, ±3.5, ±4, ±4.5, ±5, ±5.5, ±6, ±6.5, ±7, ±7.5, ±8, ±8, 5, ±9, ±9.5 or ±10%.

In the present invention, the term includes generally means include, add up, have or contain. In some cases it also means is or consists of.

Antibody or antigen-binding fragment.

In one aspect, the present invention provides an antibody or antigen binding fragment or variant thereof that binds to the PD-L1 protein with a KD value of 3x10'9 M or less (e.g., a KD value of no more than about 3x10'9, no more than about 2x10', no more than approximately 1x10'9, no more than approximately 9x10'10, no more than approximately 8x10'10, no more than approximately ^7x10'10 , no more than approximately ^6x10'10 , no more than approximately ^5x10'10 , no more than approximately ^4x10'10 , not more than approximately 3x10' ¹⁰ , no more than approximately 2x10' ¹⁰ , no more

^1x10'10 , not more than about 9x10'11, not more than about 8x10'11, not more than about 7x10'11, not more than about 6x10'11, not more than about 1x10'11 M or less). For example, an antibody, antigen binding fragment or variant thereof may bind to the PD-L1 protein with a KD value of less than 8x10'11 M.

An antibody or antigen binding fragment or variant thereof of the present invention can inhibit the binding of PD-L1 to PD-1.

An antibody or antigen-binding fragment or variant thereof of the present invention may lead to the amelioration or treatment of tumor-associated diseases. Tumors include colon cancer.

In the present invention, the antibody may be selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

In the present invention, the antigen binding fragment may be selected from the group consisting of Fab-, Fab'-, F(ab)2- and Fv-fragment.

In the present invention, a variant may be an amino acid sequence having substantially the same function (e.g., may specifically bind to PD-L1), and has at least 85% or more (e.g., at least about 85, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99% or more) sequence identity. In some embodiments, the amino acid sequence variant has substantially the same function (e.g., the ability to specifically bind to PD-L1) and includes an amino acid sequence obtained by addition, deletion, or substitution of one or more (e.g., 1-2, 1- 3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acids. As another example, a variant of the present invention may be selected from the following group: a protein or polypeptide subjected to the substitution, deletion, or addition of one or more amino acids in an antibody or antigen binding fragment thereof; and a protein or polypeptide having more than 85% (e.g., at least about 85, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99% or more ) sequence identity with respect to the antibody or its antigen-binding fragment.

In some embodiments, an antibody of the present invention may include an antibody light chain or fragment thereof comprising LCDR1, and LCDR1 includes the amino acid sequence set forth in SEQ ID NO: 53: TGTX1SX2VGGYX3X4VS; where X1 is S, R or V; X2 is D, E or S; X3 is N or R; X4 is Y or E, and where LCDR1 is defined according to the Kabat antibody numbering index. For example, an antibody light chain or fragment thereof may include LCDR1, and LCDR1 includes the following amino acid sequence or variant thereof: SEQ ID NO: 54-58.

In some embodiments, the antibody light chain or fragment thereof may comprise LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59: X1NSX2RPS, wherein X1 is G or E; X2 is N or I, and where LCDR2 is defined according to the Kabat antibody numbering index. For example, an antibody light chain or fragment thereof may include LCDR2 comprising the following amino acid sequence or variant thereof: SEQ ID NO: 6061.

In some embodiments, the antibody light chain or fragment thereof may comprise LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62: QSYDSSLSGX1V, wherein X1 is S or T, and wherein LCDR3 is defined in accordance with the index number

- 10 044684 tion of antibodies Kabat. For example, an antibody light chain or fragment thereof may include LCDR3 comprising an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 63 and SEQ ID NO: 64.

In some embodiments, the antibody light chain or fragment thereof may further include the L-FR1, L-FR2, L-FR3, and L-FR4 framework regions. For example, the C-terminus of L-FR1 can be directly or indirectly linked to the N-terminus of LCDR1, and L-FR1 includes the amino acid sequence set forth in SEQ ID NO: 71. L-FR2 is located between LCDR1 and LCDR2, and L-FR2 includes the amino acid sequence the sequence shown in SEQ ID NO: 72. L-FR3 is between LCDR2 and LCDR3, and L-FR3 includes the amino acid sequence shown in SEQ ID NO: 73-75. The N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and L-FR4 includes the amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the framework regions are selected from the group consisting of human consensus framework sequences and sequences human germ line.

In some embodiments, a light chain or fragment thereof from an antibody of the present invention includes a VL light chain variable region, and the VL light chain variable region includes an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41.

In an antibody or antigen binding fragment or variant thereof of the present invention, the antibody light chain or fragment thereof may further include a human constant region, and the human constant region includes a human Igλ constant region.

In the present invention, the antibody light chain or fragment thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42.

In the present invention, the antibody of the present invention may comprise an antibody heavy chain or a fragment thereof, wherein the antibody heavy chain or fragment thereof may comprise HCDR1, and HCDR1 may comprise an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 45: X1YAIS, where X1 is S or T, and where HCDR1 is defined according to the Kabat antibody numbering index. For example, the antibody heavy chain or fragment thereof may comprise HCDR1, and HCDR1 comprises an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 47.

In the present invention, an antibody heavy chain or a fragment thereof may include HCDR2, and HCDR2 includes the following amino acid sequence or a variant thereof: SEQ ID NO: 48, and wherein HCDR2 is defined according to the Kabat antibody numbering index.

In the present invention, the antibody heavy chain or fragment thereof may comprise HCDR3, and HCDR3 comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 49: TMX1X2YX3X4GNX5DY, wherein X1 is D, E or G, X2 is G or E, X3 is S or G, X4 is Y or F, X5 is F or Y, and where HCDR3 is defined according to the Kabat antibody numbering index. For example, the antibody heavy chain or fragment thereof may include HCDR3, and HCDR3 includes the following amino acid sequence or variant thereof: SEQ ID NO: 50-52.

In some embodiments, the antibody heavy chain or fragment thereof further includes the H-FR1, H-FR2, H-FR3, and H-FR4 framework regions. For example, the C terminus of H-FR1 is directly or indirectly linked to the N terminus of HCDR1, and H-FR1 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67 H-FR2 is between HCDR1 and HCDR2, and H-FR2 may include the amino acid sequence set forth in SEQ ID NO: 68. H-FR3 is between HCDR2 and HCDR3, and H-FR3 may include the amino acid sequence set forth in SEQ ID NO: : 69. The N terminus of H-FR4 is directly or indirectly linked to the C terminus of HCDR3, and H-FR4 may include the amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the framework regions are selected from the group consisting of consensus human framework sequences and human germline sequences.

The heavy chain of an antibody or a fragment thereof of the present invention may include a VH heavy chain variable region, and the VH heavy chain variable region may include an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31, and SEQ ID NO: 35. In an antibody, an antigen binding fragment thereof, or a variant of the present invention, the antibody heavy chain or fragment thereof may further comprise a human constant region. For example, the human constant region may include the human IgG constant region. For example, the human IgG constant region may include the human IgG1 constant region.

In some embodiments, an antibody heavy chain or fragment thereof may include an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 32, and SEQ ID NO: 36.

- 11 044684

In some embodiments, an antibody of the present invention comprises an antibody light chain or fragment thereof comprising LCDR1, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53: TGTX1SX2VGGYX3X4VS; where X1 is S, R or V; X2 is D, E or S; X3 is N or R; X4 is Y or E, and where LCDR1 is defined according to the Kabat antibody numbering index. For example, an antibody light chain or fragment thereof may comprise LCDR1, and LCDR1 comprises an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NOs: 54-58. The antibody light chain or fragment thereof includes LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59: X1NSX2RPS, where X1 is G or E; X2 is N or I, and where LCDR2 is defined according to the Kabat antibody numbering index. For example, an antibody light chain or fragment thereof may include LCDR2 comprising an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NOs: 60-61. The antibody light chain or fragment thereof includes LCDR3, comprising the amino acid sequence set forth in SEQ ID NO: 62: QSYDSSLSGX1V, where X1 is S or T, and where LCDR3 is defined in accordance with the Kabat antibody numbering index. For example, an antibody light chain or fragment thereof may include LCDR3 comprising an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NOs: 63-64. In addition, the antibody of the present invention includes an antibody heavy chain or fragment thereof comprising HCDR1, and HCDR1 includes an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 45: X1YAIS, where X1 is S or T, and where HCDR1 is defined according to the Kabat antibody numbering index. For example, the antibody heavy chain or fragment thereof may include HCDR1, and HCDR1 includes an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 47. For example, the antibody heavy chain or fragment thereof may include HCDR2 and HCDR2 includes the following amino acid sequence or a variant thereof: SEQ ID NO: 48, and wherein HCDR2 is defined in accordance with the Kabat antibody numbering index. As another example, the antibody heavy chain or fragment thereof includes HCDR3, and HCDR3 includes an amino acid sequence selected from SEQ ID NO: 49: TMX1X2YX3X4GNX5DY, where X1 is D, E or G, X2 is G or E, X3 is S or G , X4 is Y or F, X5 is F or Y, and where HCDR3 is defined according to the Kabat antibody numbering index. The antibody heavy chain or fragment thereof may include HCDR3, and HCDR3 includes the following amino acid sequence or variant thereof: SEQ ID NO: 50-52.

The amino acid sequence of an LCDR1 antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 54 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 63 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 46; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may include SEQ ID NO: 50 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-002 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, an antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 73 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of FIFR1 may include SEQ ID NO: 65 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include an antibody having the same LCDR1-3, HCDR13, L-FR1-4 and H-FR1-4 as the YN-002 antibody. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 4 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 2 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-002 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 8, and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 6. For example, an antibody , its antigen binding fragment or variant may include the YN-002 antibody or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to the PD-L1 protein (on

- 12 044684 example, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 54 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 63 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 46 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may include SEQ ID NO: 50 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-002 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 73 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 65 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-002 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 4 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 2 or a variant thereof. For example, the reference antibody may include antibody YN-002 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 8, and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 6. For example, the reference antibody may include an antibody YN-002 or have the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 54 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 63 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 46 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may include SEQ ID NO: 50 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-003 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 74 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 66 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-003 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 11 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 9 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-003 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 15 and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 13. For example, an antibody its antigen binding fragment or variant may include the YN-003 antibody or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a PD-L1 protein (eg, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 54 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; amino acid sequence

- 13 044684 LCDR3 activity may include SEQ ID NO: 63 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 46 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may include SEQ ID NO: 50 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-003 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 74 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 66 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-003 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 11 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 9 or a variant thereof. For example, the reference antibody may include antibody YN-003 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 15, and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 13. For example, the reference antibody may include an antibody YN-003 or have the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 55 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may include SEQ ID NO: 51 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-035 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, an antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 74 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-035 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 33 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 31 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-035 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 34, and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 32. For example, an antibody , its antigen binding fragment or variant may include the YN-035 antibody or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a PD-L1 protein (eg, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 55 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may

- 14 044684 include SEQ ID NO: 51 or a variation thereof. In some embodiments, the reference antibody may include antibody YN-035 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 74 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-035 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 33 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 31 or a variant thereof. For example, the reference antibody may include antibody YN-035 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 34, and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 32. For example, the reference antibody may include an antibody YN-035 or have the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 55 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-036 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 74 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-036 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 33 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-036 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 34 and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 36. For example, an antibody its antigen binding fragment or variant may include antibody YN-036 or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a PD-L1 protein (eg, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 55 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 60 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-036 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence

- 15 044684 L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of LFR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 74 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-036 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 33 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the reference antibody may include antibody YN-036 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 34 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 36. For example, the reference antibody may include a YN antibody -036 or have the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 56 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-037 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, an antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 75 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-037 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 37 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-037 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 38 and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 36. For example, an antibody its antigen binding fragment or variant may include antibody YN-037 or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a PD-L1 protein (eg, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 56 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-037 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 75 or a variant thereof; the amino acid sequence of L-FR4 may include

- 16 044684

SEQ ID NO: 76 or variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-037 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 37 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the reference antibody may include antibody YN-037 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 38 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 36. For example, the reference antibody may include a YN antibody -037 or have the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 57 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-038 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, an antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 75 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-038 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 39 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-038 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 40 and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 36. For example, an antibody its antigen binding fragment or variant may include the YN-038 antibody or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a PD-L1 protein (eg, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 57 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-038 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 75 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof;

- 17 044684 and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-038 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 39 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the reference antibody may include antibody YN-038 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 40 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 36. For example, the reference antibody may include a YN antibody -038 or have the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 58 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-039 or an antibody with the same LCDR1-3 and HCDR1-3. In some cases, the amino acid sequence of an L-FR1 antibody, an antigen binding fragment or variant thereof may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of L-FR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 75 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-039 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the light chain of an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 41 or a variant thereof; and wherein the heavy chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-039 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain being set forth in SEQ ID NO: 42 and the amino acid sequence of the heavy chain being set forth in SEQ ID NO: 36. For example, an antibody its antigen binding fragment or variant may include the YN-039 antibody or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a PD-L1 protein (eg, human PD-L1 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 58 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 52 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-039 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody includes L-FR1-4 and H-FR1-4, and the amino acid sequence of L-FR1 may include SEQ ID NO: 71 or a variant thereof; the amino acid sequence of LFR2 may include SEQ ID NO: 72 or a variant thereof; the amino acid sequence of L-FR3 may include SEQ ID NO: 75 or a variant thereof; the amino acid sequence of L-FR4 may include SEQ ID NO: 76 or a variant thereof; and the amino acid sequence of H-FR1 may include SEQ ID NO: 67 or a variant thereof; the amino acid sequence of H-FR2 may include SEQ ID NO: 68 or a variant thereof; the amino acid sequence of H-FR3 may include SEQ ID NO: 69 or a variant thereof; and the amino acid sequence of H-FR4 may include SEQ ID NO: 70 or a variant thereof. For example, the antibody or antigen binding fragment thereof may include antibody YN-039 or an antibody with the same LCDR1-3, HCDR1-3, L-FR1-4 and H-FR1-4. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 41 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 35 or a variant thereof. For example, the reference antibody may include antibody YN-039 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 42 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 36. For example, the reference antibody may include a YN antibody -039 or have the same light chain and heavy chain.

In the present invention, the PD-L1 protein is selected from the group consisting of human PD-L1 protein, monkey PD-L1 protein and mouse PD-L1 protein.

In another aspect, the present invention also provides an antibody, antigen binding fragment or variant thereof that binds to the CD137 protein with a K _D less than 5x10'9 M (e.g., a K _D of no more than about 5x10'9, no more than about 4x10'9, no more than about 3x10'9, no more than about 2x10'9, no more than about 1x10'9, or no more than about 1x10'10 M or less). For example, it binds to the CD137 protein with a K _D below 3x10'9 M.

An antibody, antigen binding fragment or variant thereof of the present invention may result in the amelioration or treatment of tumors. For example, the tumor may include colon cancer.

In the present invention, the antibody may be selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

In the present invention, the antigen binding fragment may be selected from the group consisting of Fab-, Fab'-, F(ab)2- and Fv-fragment.

In the present invention, a variant may be an amino acid sequence having substantially the same function (e.g., the ability to specifically bind to CD137), and have at least 85% or more (e.g., at least about 85, about 90, about 91 , about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99% or more) sequence identity. In some embodiments, an amino acid sequence variant is an amino acid sequence having substantially the same function (e.g., the ability to specifically bind to CD137), and essentially further includes the addition, deletion, or substitution of one or more (e.g., 1-2 , 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acids.

In some embodiments, the antibody may include an antibody light chain or fragment thereof. For example, an antibody light chain or fragment thereof may include LCDR1, and LCDR1 may include the following amino acid sequence or a variant thereof: SEQ ID NO: 80. For example, an antibody light chain or fragment thereof may include LCDR2 comprising the following amino acid sequence or variant thereof: SEQ ID NO: 81. As another example, an antibody light chain or fragment thereof includes LCDR3 or a variant thereof, and LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 82.

In some embodiments, with respect to an antibody, an antigen binding fragment or variant thereof, the antibody light chain or fragment thereof further includes the L-FR1, L-FR2, L-FR3 and L-FR4 framework regions. For example, the C terminus of L-FR1 is directly or indirectly linked to the N terminus of LCDR1, and LFR1 may include the amino acid sequence set forth in SEQ ID NO: 89. L-FR2 is located between LCDR1 and LCDR2, and L-FR2 may include the amino acid sequence set forth in SEQ ID NO: 90. L-FR3 is located between LCDR2 and LCDR3, and L-FR3 may include the amino acid sequence set forth in SEQ ID NO: 91. The N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3 , and L-FR4 may include the amino acid sequence set forth in SEQ ID NO: 92.

In some embodiments, with respect to an antibody, an antigen binding fragment or variant thereof, the antibody light chain or fragment thereof includes a VL light chain variable region, and the VL light chain variable region may include the following amino acid sequence or variant thereof: SEQ ID NO: 20.

In the present invention, the antibody light chain or fragment thereof may further include a human constant region, and the human constant region includes a human Igλ constant region.

In the present invention, a light chain or fragment thereof from an antibody may comprise the amino acid sequence set forth in SEQ ID NO: 23.

In some embodiments, the antibody may include an antibody heavy chain or fragment thereof. The antibody heavy chain or fragment thereof includes HCDR1, and HCDR1 may include the following amino acid sequence or a variant thereof: SEQ ID NO: 77. The antibody heavy chain or fragment thereof includes HCDR2, and HCDR2 may include the following amino acid sequence or variant thereof: SEQ ID NO: 78. The antibody heavy chain or fragment thereof includes HCDR3, and

HCDR3 may include the following amino acid sequence or a variant thereof: SEQ ID NO:

79.

In some embodiments, an antibody heavy chain or fragment thereof may further include the H-FR1, H-FR2, H-FR3, and H-FR4 framework regions. The C terminus of H-FR1 is directly or indirectly linked to the N terminus of HCDR1, and H-FR1 may comprise the amino acid sequence set forth in SEQ ID NOs: 84-85. H-FR2 is between HCDR1 and HCDR2, and H-FR2 may include the amino acid sequence set forth in SEQ ID NO: 86. H-FR3 is between HCDR2 and HCDR3, and H-FR3 may include the amino acid sequence set forth in SEQ ID NO: 87. The N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and H-FR4 may include the amino acid sequence set forth in SEQ ID NO: 88.

The heavy chain of an antibody or fragment thereof of the present invention includes a VH heavy chain variable region, and the VH heavy chain variable region may include an amino acid sequence or a variant thereof selected from the group consisting of SEQ ID NO: 18 or SEQ ID NO: 25.

In an antibody, antigen binding fragment or variant thereof, the antibody heavy chain or fragment thereof may further comprise a human constant region. For example, the human constant region may include the human IgG constant region. For example, the human IgG constant region may include the human IgG1 constant region.

In the present invention, an antibody heavy chain or fragment thereof may include an amino acid sequence or variant thereof selected from the group consisting of SEQ ID NO: 21 or SEQ ID NO: 27.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 80 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 81 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 82 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 77 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 78 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 79 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-005 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the amino acid sequence of the light chain variable region of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 20 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 18 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-005 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 23 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 21. For example, the antibody, antigen binding fragment or variant thereof may include the YN-005 antibody or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a CD137 protein (eg, human CD137 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 80 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 81 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 82 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 77 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 78 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 79 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-005 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 20 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 18 or a variant thereof. For example, the reference antibody may include antibody YN-005 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 23 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 21. For example, the reference antibody may include a YN antibody -005 or have

- 20 044684 the same light chain and heavy chain.

In some embodiments, the LCDR1 amino acid sequence of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 80 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 81 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 82 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 77 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 78 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 79 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-006 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the amino acid sequence of the light chain variable region of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 20 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 25 or a variant thereof. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-006 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 23 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 27. For example, the antibody, antigen binding fragment or variant thereof may include antibody YN-006 or have the same light chain and heavy chain.

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention competes with a reference antibody for binding to a CD137 protein (eg, human CD137 protein). The reference antibody may include LCDR1-3 and HCDR1-3, and the amino acid sequence of LCDR1 may include SEQ ID NO: 80 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 81 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 82 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ ID NO: 77 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 78 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 79 or a variant thereof. In some embodiments, the reference antibody may include antibody YN-006 or an antibody with the same LCDR1-3 and HCDR1-3. In some embodiments, the reference antibody may include a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region may include SEQ ID NO: 20 or a variant thereof; and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 25 or a variant thereof. For example, the reference antibody may include antibody YN-006 or an antibody having the same light chain variable region and heavy chain variable region. In some embodiments, a reference antibody may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 23 and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 27. For example, the reference antibody may include a YN antibody -006 or have the same light chain and heavy chain.

In the present invention, the CD137 protein may include human CD137 protein.

In another aspect, the present invention also provides a bispecific antibody that binds to PD-L1 protein with a K _D of less than 2x10'9 M (e.g., a K _D of no more than about 2x10'9, no more than about 1x10'9, no more than about ^1x10'10 or no more than about 8x10'11 M or less) and binding to a CD137 protein with a K _D less than 8x10'9 M (e.g., a K _D no more than about 8x10'9, no more than about 7x10'9, no more than about 6x10'9, no more than about 5x10'9, no more than about 4x10'9, no more than about 3x10'9, no more than about 2x10'9, no more than about 1x10'9 or no more than about ^1x10'10 M or less).

In the present invention, the PD-L1 protein is selected from the group consisting of human PD-L1 protein, monkey PD-L1 protein and mouse PD-L1 protein; and CD137 protein includes human CD137 protein.

In the present invention, the bispecific antibody includes a first targeting moiety that specifically binds to a PD-L1 protein, where the first targeting moiety may include the antibody, an antigen-binding moiety, or a PD-L1-binding variant thereof.

For example, the antigen binding fragment in the first targeting fragment may be selected from the group consisting of Fab, Fab', F(ab) ₂ and Fv fragment. As another example, the variant in the first targeting fragment may be selected from the group consisting of a protein or polypeptide subjected to substitution, deletion, or addition of one or more amino acids in the antibody or antigen binding fragment thereof; and a protein or polypeptide having 85% or more (e.g., at least about 85, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99%, or more) sequence identity with respect to the antibody or its

- 21 044684 antigen-binding fragment. The antibody, antigen binding fragment or variant thereof may be an antibody, antigen binding fragment or variant thereof that binds to the PDL1 protein.

In some embodiments, the PD-L1 protein binding antibody in the bispecific antibody may include a light chain or a fragment thereof. For example, the light chain or fragment thereof includes LCDR1-3, and the amino acid sequence of LCDR1 is SEQ ID NO: 5458; the amino acid sequence of LCDR2 is SEQ ID NO: 60-61; and the amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO: 63-64. In some embodiments, the light chain or fragment thereof may comprise a light chain variable region, and the amino acid sequence of the light chain variable region may be selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41. In some embodiments, a light chain or fragment thereof from an antibody may include a human constant region, and the human constant region includes a human Igλ constant region.

In some embodiments, the light chain or fragment thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42.

In some embodiments, the antibody that binds to the PD-L1 protein may include a heavy chain or fragment thereof. For example, the heavy chain or fragment thereof includes HCDR1-3, and the amino acid sequence of HCDR1 is selected from the group consisting of SEQ ID NO: 46-47; the amino acid sequence of HCDR2 is SEQ ID NO: 48; and the amino acid sequence of HCDR3 is SEQ ID NO: 50-52.

In some embodiments, the heavy chain or fragment thereof may comprise a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may be selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31, and SEQ ID NO: 35.

In some embodiments, the heavy chain or fragment thereof may further include a human constant region, and the human constant region may include a human IgG constant region. For example, the human IgG constant region is the human IgG1 constant region.

In some embodiments, the heavy chain or fragment thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:32, and SEQ ID NO:36.

In some embodiments, the bispecific antibody includes a second targeting moiety that specifically binds to the CD137 protein, where the second targeting moiety may include the antibody, an antigen-binding moiety, or a variant thereof that binds to the CD137 protein. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab) ₂ and Fv fragment. In some embodiments, the variant is selected from the group consisting of a protein or polypeptide subjected to substitution, deletion, or addition of one or more amino acids in the antibody or antigen-binding fragment thereof; and a protein or polypeptide having 85% or more (e.g., at least about 85, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99%, or more) sequence identity with respect to the antibody or its antigen-binding fragment.

In some embodiments, an antibody that binds to the CD137 protein may include a light chain or a fragment thereof. For example, the light chain or fragment thereof includes LCDR1-3, and the amino acid sequences of LCDR1-3 may sequentially be SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82. In some embodiments, the light chain or fragment thereof may include a light chain variable region, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20.

In some embodiments, an antibody that binds to the CD137 protein may include a heavy chain or fragment thereof. In some embodiments, the heavy chain or fragment thereof from an antibody may include HCDR1-3, and the amino acid sequences of HCDR1-3 may sequentially be SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 79. In some embodiments, the heavy chain or a fragment thereof from an antibody may comprise a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may be selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 25.

In some embodiments, an antibody that binds to the CD137 protein may comprise a scFv, and the scFv may comprise the amino acid sequence set forth in SEQ ID NO: 83.

In the present invention, the bispecific antibody may include a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain may include a heavy chain variable region of a PD-L1 protein-binding antibody, a heavy chain variable region of a CD137 protein-binding antibody, and a light chain variable region of an antibody that binds to the CD137 protein; and the second polypeptide chain may include a light chain variable region of an antibody that binds to the PD-L1 protein.

For example, in the first polypeptide chain, the heavy chain variable region of an antibody binding to PD-L1 protein may be located at the N-terminus of the heavy chain variable region of an antibody binding to CD137 protein, and the heavy chain variable region of an antibody binding to CD137 protein may be located at the N-terminus of the variable region of the light chain of an antibody that binds to the CD137 protein. As another example, in the first polypeptide chain, the heavy chain variable region of a PD-L1 protein-binding antibody may be at the N-terminus of the CD137 protein-binding antibody light chain variable region and the CD137 protein-binding antibody light chain variable region. , may be located at the N-terminus of the variable region of the heavy chain of an antibody that binds to the CD137 protein.

For example, in the first polypeptide chain, the CD137 protein-binding antibody light chain variable region and the CD137 protein-binding antibody heavy chain variable region constitute a scFv. For example, the scFv may include the amino acid sequence set forth in SEQ ID NO: 83.

The first polypeptide chain may further include a human IgG constant region. For example, the human IgG constant region may be a human IgG1 constant region.

In some embodiments, the human IgG constant region may be at the C-terminus of the heavy chain variable region of an antibody binding to the PD-L1 protein and may be at the N-terminus of the light chain variable region of an antibody binding to the CD137 protein; alternatively, the human IgG constant region may be located at the C-terminus of the heavy chain variable region of an antibody binding to the PD-L1 protein and may be located at the N-terminus of the heavy chain variable region of an antibody binding to the CD137 protein.

In some embodiments, the first polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO: 30, SEQ ID NO: 43, and SEQ ID NO: 44.

In some embodiments, the second polypeptide chain may further comprise a human Igλ constant region.

In the present invention, the second polypeptide chain may include the following amino acid sequences: SEQ ID NO: 15 and SEQ ID NO: 34.

For example, in the first targeting fragment of the bispecific antibody of the present invention, the amino acid sequence of LCDR1 may be selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58 or its variant; the amino acid sequence of LCDR2 may be selected from the group consisting of SEQ ID NO: 60 and SEQ ID NO: 61 or a variant thereof; the amino acid sequence of LCDR3 may be selected from the group consisting of SEQ ID NO: 63 and SEQ ID NO: 64 or a variant thereof; and the amino acid sequence of HCDR1 may be selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 47 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 48 or a variant thereof; and the amino acid sequence of HCDR3 may be selected from the group consisting of SEQ ID NO: 50-52 or a variant thereof. In some embodiments, the amino acid sequence of the light chain variable region of an antibody, antigen binding fragment, or variant thereof of the present invention may be selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41 or variations thereof; and the amino acid sequence of the heavy chain variable region may be selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31 and SEQ ID NO: 35 or a variant thereof. In some embodiments, an antibody, antigen binding fragment or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain may be an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42, and the heavy chain amino acid sequence may be an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 13 , SEQ ID NO: 32 and SEQ ID NO: 36.

For example, in the second targeting fragment of the bispecific antibody of the present invention, the amino acid sequence of LCDR1 may include SEQ ID NO: 80 or a variant thereof; the amino acid sequence of LCDR2 may include SEQ ID NO: 81 or a variant thereof; the amino acid sequence of LCDR3 may include SEQ ID NO: 82 or a variant thereof; and the amino acid sequence of HCDR1 may include SEQ GO NO: 77 or a variant thereof; the amino acid sequence of HCDR2 may include SEQ ID NO: 78 or a variant thereof; and the amino acid sequence of HCDR3 may include SEQ ID NO: 79 or a variant thereof. In some embodiments, the amino acid sequence of the light chain variable region of an antibody, antigen binding fragment or variant thereof of the present invention may include SEQ ID NO: 20 or a variant thereof; and the amino acid sequence of the heavy chain variable region may be a sequence selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 25 or variants thereof.

- 23 044684

In some embodiments, an antibody, antigen binding fragment, or variant thereof of the present invention may include a light chain and a heavy chain, the amino acid sequence of the light chain may be set forth in SEQ ID NO: 23, and the amino acid sequence of the heavy chain may be set forth in SEQ ID NO: 21 or SEQ ID NO: 27.

For example, in the bispecific antibody of the present invention, the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 30, and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 15. The bispecific antibody may include antibody YN-007 or antibody the same first polypeptide and a second polypeptide, wherein the first polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 25, the amino acid sequence set forth in SEQ ID NO: 20, and the amino acid sequence set forth in SEQ ID NO: 13; and the second polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 15.

As another example, in the bispecific antibody of the present invention, the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 43 and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 34. The bispecific antibody may include antibody YN-051 or an antibody with the same first polypeptide and second polypeptide. Where the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 25, the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence shown in SEQ ID NO: 32; and the second polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 34.

As another example, in the bispecific antibody of the present invention, the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 44, and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 34. The bispecific antibody may include antibody YN-052 or an antibody with the same first polypeptide and second polypeptide. Where the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 25, the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence shown in SEQ ID NO: 36; and the second polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 34.

For example, in the bispecific antibody of the present invention, the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 30, and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 15. The bispecific antibody may include antibody YN-007 or antibody the same first polypeptide and second polypeptide. Where the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 27, the amino acid sequence shown in SEQ ID NO: 23, and the amino acid sequence shown in SEQ ID NO: 13; and the second polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 15.

As another example, in the bispecific antibody of the present invention, the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 43 and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 34. The bispecific antibody may include antibody YN-051 or an antibody with the same first polypeptide and second polypeptide. Where the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 32, the amino acid sequence set forth in SEQ ID NO: 27, the amino acid sequence set forth in SEQ ID NO: 23; and the second polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 34.

As another example, in the bispecific antibody of the present invention, the first polypeptide may include the amino acid sequence set forth in SEQ ID NO: 44 and the second polypeptide may include the amino acid sequence set forth in SEQ ID NO: 34. The bispecific antibody may include antibody YN-052 or an antibody with the same first polypeptide and second polypeptide. Where the first polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 36, the amino acid sequence set forth in SEQ ID NO: 27, the amino acid sequence set forth in SEQ ID NO: 23; and the second polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 34.

Nucleic acid, vector, host cell and production method.

In another aspect, the present invention also provides one or more isolated nucleic acid molecules encoding an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody of the present invention. For example, each nucleic acid molecule of one or more nucleic acid molecules may encode a whole antibody or an antigen binding fragment thereof, or encode a portion thereof (e.g., one or more of HCDR1-3, LCDR1-3, VL, VH, light chain, or heavy chain) .

The nucleic acid molecule of the present invention may be isolated. For example, it can be produced or synthesized by the following methods: (i) in vitro amplification such as polymerase chain reaction (PCR) amplification, (ii) clonal recombination, (iii)

- 24 044684 purification, such as fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis, such as chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced using recombinant DNA technology.

In the present invention, the nucleic acid encoding an antibody or antigen binding fragment thereof can be produced by a variety of methods known in the art, including, but not limited to, extension PCR using restriction fragments or synthetic oligonucleotides. For specific steps, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube, et al., Current Protocols in Molecular biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.

In another aspect, the present invention provides one or more vectors and includes one or more nucleic acid molecules of the present invention. Each vector may include one or more nucleic acid molecules. In addition, the vector may further include other genes, such as marker genes, allowing selection of the vector in a suitable host cell and under suitable conditions. In addition, the vector may further include an expression control element to allow proper expression of the coding region in a suitable host cell. Such control elements are well known to those skilled in the art, for example, they may include promoters, ribosome binding sites, enhancers and other control elements for regulating gene transcription or mRNA translation, etc. In some embodiments, the expression control sequence is a regulatory element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically includes a 5' untranscribed sequence and 5' and 3' untranslated sequences involved in the initiation of transcription and translation, such as the TATA box, cap sequence, CAAT sequence, etc. For example, the 5'untranscribed expression control sequence may include a promoter region, which may include a promoter sequence for transcriptional control of an operably linked nucleic acid. One or more nucleic acid molecules described in the present invention can be operably linked to an expression control element.

The vector may include, for example, a plasmid, cosmid, virus, phage, or other vectors commonly used in genetic engineering, for example, the vector is an expression vector.

In another aspect, the present invention relates to a cell, which may include a nucleic acid molecule of the present invention and/or a vector described in the present invention. The cell may be a host cell. In some embodiments, each host cell may include one nucleic acid molecule or vector described in the present invention. In some embodiments, each host cell may include multiple (eg, two or more) nucleic acid molecules or vectors described in the present invention. For example, the vector of the present invention can be inserted into a host cell, such as a eukaryotic cell, such as a plant cell, a fungal cell, or a yeast cell, etc. The vector of the present invention can be introduced into a host cell by methods known in the art, such as electroporation, lipofectin transfection, lipofectamine transfection, and the like.

In another aspect, the present invention relates to one or more host cells comprising a nucleic acid molecule or vector.

In another aspect, the present invention relates to a method for producing an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody. The method may include culturing a host cell of the present invention under conditions allowing expression of the antibody, antigen binding fragment or variant thereof, or bispecific antibody, and optionally collecting the antibody, antigen binding fragment or variant thereof. For example, a suitable medium, suitable temperature, culture time, etc. can be used, and these methods will be understood by those skilled in the art.

Pharmaceutical composition and use.

In another aspect, the present invention provides a pharmaceutical composition that may include an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, a nucleic acid molecule, a vector and/or a host cell, and optionally a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelators, counterions, metal complexes and/or nonionic surfactant, and the like.

In the present invention, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration via

- 25 044684 subcutaneous depot.

In another aspect, the present invention relates to the use of an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, nucleic acid molecule, vector, host cell and/or pharmaceutical composition in the manufacture of a medicament for the relief or treatment of tumors. Tumors include, but are not limited to, colon cancer.

In another aspect, the present invention provides a method of enhancing T cell function, comprising administering to an individual in need thereof a PD-L1 binding antibody, an antigen binding fragment or variant thereof, a CD137 binding antibody, an antigen binding fragment or variant thereof, or a bispecific antibody , nucleic acid molecules, vector, host cell and/or pharmaceutical composition. Among them, T cells are tumor-associated dysfunctional T cells.

In another aspect, the present invention provides a method of inhibiting the binding of PDL1 protein to PD-1 protein, comprising administering to an individual in need thereof an antibody, an antigen binding fragment or variant thereof, or a bispecific antibody, a nucleic acid molecule, a vector, a host cell, and/or a pharmaceutical composition.

In the present invention, the CDR position of an antibody can be determined by the Kabat method.

The present invention further includes the following embodiments.

1. An antibody, antigen-binding fragment or variant thereof that binds to the PD-L1 protein with a KD of 3x10'9 M or less.

2. An antibody, antigen binding fragment or variant thereof of Embodiment 1 that binds to the PD-L1 protein with a KD of 8x10'11 M or less.

3. The antibody, antigen binding fragment or variant thereof of any one of embodiments 1-2, inhibiting the binding of PD-L1 to PD-1.

4. The antibody, antigen-binding fragment or variant thereof according to any one of embodiments 1-3, capable of leading to the alleviation or treatment of tumor-associated diseases.

5. The antibody, antigen binding fragment or variant thereof of embodiment 4, wherein the tumor includes colon cancer.

6. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 1-5, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody and a fully human antibody.

7. The antibody, antigen-binding fragment or variant thereof according to any one of embodiments 1-6, wherein the antigen-binding fragment is selected from the group consisting of Fab-, Fab'-, F(ab)2- and Fv-fragment.

8. The antibody, antigen-binding fragment or variant thereof according to any one of embodiments 1-7, wherein the variant is selected from the group consisting of:

1) a protein or polypeptide obtained by substitution, deletion or addition of one or more amino acids in an antibody or an antigen-binding fragment thereof; And

2) a protein or polypeptide having 90% or more sequence identity to an antibody or antigen-binding fragment thereof.

9. An antibody, an antigen binding fragment, or a variant thereof as in any one of embodiments 1-8 that competes with a reference antibody that binds to the PD-L1 protein, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody includes LCDR1-3, the light chain variable region of the reference antibody includes LCDR1-3, and LCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 54-58; LCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 60-61; and LCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 63-64; and the heavy chain variable region of the reference antibody includes HCDR1-3, HCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 46-47; HCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 48; and HCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 50-52.

10. The antibody, antigen binding fragment or variant thereof of Embodiment 9, wherein the light chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41, and the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31 and SEQ ID NO: 35.

11. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 9-10, wherein the light chain of the reference antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 34 , SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42; and the heavy chain of the reference antibody includes the amino acid sequence

- 26 044684 activity selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 32 and SEQ ID

NO: 36.

12. The antibody, antigen binding fragment or variant thereof of embodiments 1-11, wherein the antibody comprises an antibody light chain or fragment thereof comprising LCDR1, and LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53: TGTX1SX2VGGYX3X4VS; where X1 is S, R or V; X2 is D, E or S; X3 is N or R; X4 is Y or E, and where LCDR1 is defined according to the Kabat antibody numbering index.

13. The antibody, antigen binding fragment or variant thereof of embodiment 12, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-58.

14. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 11-13, wherein the light chain of the antibody or fragment thereof comprises LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59: X1NSX2RPS, wherein X1 is G or E; X2 is N or I, and where LCDR2 is defined according to the Kabat antibody numbering index.

15. The antibody, antigen binding fragment or variant thereof of embodiment 14, wherein LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-61.

16. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 11-15, wherein the light chain of the antibody or fragment thereof comprises LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 62: QSYDSSLSGX1V, wherein X1 is S or T, and where LCDR3 is defined according to the Kabat antibody numbering index.

17. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 11-16, wherein the antibody light chain or fragment thereof further comprises the LFR1, L-FR2, L-FR3, and L-FR4 framework regions.

18. The antibody, antigen binding fragment or variant thereof of embodiment 17, wherein the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences.

19. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 17-18, wherein the C terminus of L-FR1 is linked directly or indirectly to the N terminus of LCDR1, and L-FR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 71.

20. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 17-19, wherein L-FR2 is between LCDR1 and LCDR2, and LFR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 72.

21. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 17-20, wherein L-FR3 is between LCDR2 and LCDR3, and LFR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 73-75.

22. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 17-21, wherein the N-terminus of L-FR4 is linked directly or indirectly to the C-terminus of LCDR3, and L-FR4 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 76.

23. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 12-22, wherein the antibody light chain or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41.

24. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 12-23, wherein the antibody light chain or fragment thereof further includes a human constant region, and the human constant region includes a human Igλ constant region.

25. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 12-24, wherein the light chain of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID no: 8, SEQ ID NO: 15, SEQ ID NO : 34, SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42.

26. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 1-25, wherein the antibody comprises an antibody heavy chain or fragment thereof comprising HCDR1, and HCDR1 comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO : 45: X1YAIS, where X1 is S or T, and where HCDR1 is defined according to the Kabat antibody numbering index.

27. The antibody, antigen binding fragment or variant thereof of Embodiment 26, wherein the antibody heavy chain or fragment thereof comprises HCDR2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 48, and wherein HCDR2 is defined in accordance with the Kabat antibody numbering index.

28. The antibody, antigen-binding fragment, or variant thereof of any one of embodiments 26-27, wherein the antibody heavy chain or fragment thereof includes HCDR3, and HCDR3 includes an amino

- 27 044684 acid sequence selected from SEQ ID NO: 49: TMX1X2YX3X4GNX5DY, where X1 is D, E or G, X2 is G or E, X3 is S or G, X4 is Y or F, X5 is F or Y, and where HCDR3 is defined according to the Kabat antibody numbering index.

29. The antibody, antigen binding fragment or variant thereof of embodiment 28, wherein HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-52.

30. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 26-29, wherein the antibody heavy chain or fragment thereof further comprises the HFR1, H-FR2, H-FR3, and H-FR4 framework regions.

31. The antibody, antigen binding fragment, or variant thereof of embodiments 30, wherein the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences.

32. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 30-31, wherein the C terminus of H-FR1 is linked directly or indirectly to the N terminus of HCDR1, and H-FR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 65-67.

33. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 30-32, wherein H-FR2 is between HCDR1 and HCDR2, and HFR2 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 68.

34. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 30-33, wherein H-FR3 is between HCDR2 and HCDR3, and HFR3 includes an amino acid sequence selected from the group consisting of SEQ ID NO: 69.

35. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 30-34, wherein the N terminus of H-FR4 is linked directly or indirectly to the C terminus of HCDR3, and H-FR4 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 70.

36. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 26-35, wherein the antibody heavy chain or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31 and SEQ ID NO: 35.

37. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 26-36, wherein the antibody heavy chain or fragment thereof further includes a human constant region, and the human constant region includes a human IgG1 constant region.

38. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 26-37, wherein the antibody heavy chain or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO : 32 and SEQ ID NO: 36.

39. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 1-38, wherein the PD-L1 protein is selected from the group consisting of human PDL1 protein, monkey PD-L1 protein and mouse PD-L1 protein.

40. An antibody, antigen-binding fragment or variant thereof that binds to the CD137 protein with a KD of 5x10'9 M or less.

41. An antibody, antigen binding fragment, or variant thereof of embodiment 40 that binds to the CD137 protein with a KD of 3x10'9 M or less.

42. The antibody, antigen-binding fragment or variant thereof of any one of embodiments 40-41, having CD137 agonist activity.

43. The antibody, antigen-binding fragment or variant thereof of any one of embodiments 40-42, capable of ameliorating or treating tumors.

44. The antibody, antigen binding fragment, or variant thereof of embodiment 43, wherein the tumor includes colon cancer.

45. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 40-44, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

46. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 40-45, wherein the antigen binding fragment is selected from the group consisting of Fab-, Fab'-, F(ab) ₂ - and Fv fragment.

47. The antibody, antigen binding fragment, or variant thereof according to any one of embodiments 40-46, wherein the variant is selected from the group consisting of:

1) a protein or polypeptide obtained by substitution, deletion or addition of one or more amino acids in an antibody or an antigen-binding fragment thereof; And

2) a protein or polypeptide having 90% or more sequence identity to an antibody or antigen-binding fragment thereof.

48. An antibody, an antigen-binding fragment, or a variant thereof according to any one of embodiments 40-47, competing with a reference antibody for binding to the CD137 protein, wherein the reference

- 28 044684 antibody includes a light chain variable region and a heavy chain variable region, the light chain variable region of the reference antibody includes LCDR1-3, and the amino acid sequences of LCDR1-3 are sequentially shown in SEQ ID NO: 80-82, and the heavy chain variable region of the reference antibody includes HCDR1-3, and the amino acid sequences of HCDR1-3 are sequentially shown in SEQ ID NO: 77-79.

49. The antibody, antigen binding fragment or variant thereof of Embodiment 48, wherein the light chain variable region of the reference antibody comprises an amino acid sequence selected from SEQ ID NO: 20 and the heavy chain variable region of the reference antibody comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 18 and SEQ ID NO: 25.

50. The antibody, antigen-binding fragment or variant thereof according to any one of embodiments 48-49, wherein the light chain of the reference antibody comprises an amino acid sequence selected from SEQ ID NO: 23; and the heavy chain of the reference antibody comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 27.

51. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 40-50, wherein the antibody comprises an antibody light chain or fragment thereof.

52. The antibody, antigen binding fragment or variant thereof of embodiment 51, wherein the light chain of the antibody or fragment thereof comprises LCDR1, and LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 80.

53. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 51-52, wherein the antibody light chain or fragment thereof comprises LCDR2, and LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 81.

54. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 51-53, wherein the antibody light chain or fragment thereof comprises LCDR3, and LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 82.

55. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 51-54, wherein the antibody light chain or fragment thereof further comprises the LFR1, L-FR2, L-FR3, and L-FR4 framework regions.

56. The antibody, antigen binding fragment or variant thereof of embodiment 55, wherein the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences.

57. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 55-56, wherein the C terminus of L-FR1 is linked directly or indirectly to the N terminus of LCDR1, and L-FR1 includes an amino acid sequence selected from SEQ ID NO: 89 .

58. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 55-57, wherein L-FR2 is between LCDR1 and LCDR2, and LFR2 includes an amino acid sequence selected from SEQ ID NO: 90.

59. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 55-58, wherein L-FR3 is between LCDR2 and LCDR3, and LFR3 comprises an amino acid sequence selected from SEQ ID NO: 91.

60. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 55-59, wherein the N terminus of L-FR4 is linked directly or indirectly to the C terminus of LCDR3, and L-FR4 includes an amino acid sequence selected from SEQ ID NO: 92 .

61. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 51-60, wherein the antibody light chain or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 20.

62. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 51-61, wherein the antibody light chain or fragment thereof further includes a human constant region, and the human constant region includes a human Igλ constant region.

63. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 51-62, wherein the antibody light chain or fragment thereof comprises an amino acid sequence selected from SEQ ID NO: 23.

64. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 40-63, wherein the antibody comprises an antibody heavy chain or fragment thereof.

65. The antibody, antigen binding fragment or variant thereof of embodiment 64, wherein the antibody heavy chain or fragment thereof comprises HCDR1, and HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 77.

66. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 64-65, wherein the antibody heavy chain or fragment thereof comprises HCDR2, and HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 78.

67. An antibody, an antigen-binding fragment or a variant thereof according to any of the embodiments

- 29 044684 leniya 64-66, wherein the antibody heavy chain or fragment thereof includes HCDR3, and HCDR3 includes an amino acid sequence selected from SEQ ID NO: 79.

68. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 64-67, wherein the antibody heavy chain or fragment thereof further comprises the HFR1, H-FR2, H-FR3, and H-FR4 framework regions.

69. The antibody, antigen binding fragment, or variant thereof of embodiment 68, wherein the framework regions are selected from the group consisting of human consensus framework sequences and human germline sequences.

70. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 68-69, wherein the C terminus of H-FR1 is linked directly or indirectly to the N terminus of HCDR1, and H-FR1 includes an amino acid sequence selected from SEQ ID NO: 84 -85.

71. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 68-70, wherein H-FR2 is between HCDR1 and HCDR2, and HFR2 includes an amino acid sequence selected from SEQ ID NO: 86.

72. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 68-71, wherein H-FR3 is between HCDR2 and HCDR3, and HFR3 comprises an amino acid sequence selected from SEQ ID NO: 87.

73. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 68-72, wherein the N terminus of H-FR4 is linked directly or indirectly to the C terminus of HCDR3, and H-FR4 comprises an amino acid sequence selected from SEQ ID NO: 88 .

74. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 68-73, wherein the antibody heavy chain or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 18 or SEQ ID NO: 25.

75. The antibody, antigen binding fragment, or variant thereof as in any one of embodiments 64-74, wherein the antibody heavy chain or fragment thereof further includes a human constant region, and the human constant region includes a human IgG constant region.

76. The antibody, antigen binding fragment, or variant thereof of embodiment 75, wherein the IgG constant region includes a human IgG1 constant region.

77. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 64-76, wherein the heavy chain of the antibody or fragment thereof comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 21 or SEQ ID NO: 27.

78. The antibody, antigen binding fragment or variant thereof as in any one of embodiments 40-77, wherein the CD137 protein includes a human CD137 protein.

79. A bispecific antibody that binds to the PD-L1 protein with a K _D of 2x10'9 M or less and binds to the CD137 protein with a K _{D of} 8x10'9 M or less.

80. The bispecific antibody of embodiment 30, comprising a first targeting moiety that specifically binds to the PD-L1 protein, wherein the first targeting moiety comprises the antibody, an antigen-binding moiety, or a variant thereof of any one of embodiments 1-39.

81. The bispecific antibody of any one of embodiments 30-31, comprising a second targeting moiety that specifically binds to the CD137 protein, wherein the second targeting moiety comprises the antibody, an antigen-binding fragment, or a variant thereof of any of embodiments 40-78.

82. The bispecific antibody of any one of embodiments 79-81, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes a PD-L1 protein-binding antibody heavy chain variable region, a CD137 protein-binding antibody heavy chain variable region , and a light chain variable region of an antibody that binds to the CD137 protein; and the second polypeptide chain includes a light chain variable region of an antibody that binds to the PD-L1 protein.

83. The bispecific antibody of embodiment 82, wherein in the first polypeptide chain, the heavy chain variable region of an antibody binding to the PD-L1 protein is at the N-terminus of the heavy chain variable region of the antibody binding to the CD137 protein, and the heavy chain variable region of the antibody, binding to the CD137 protein, located at the N-terminus of the variable region of the light chain of the antibody binding to the CD137 protein; alternatively, the heavy chain variable region of the PD-L1 protein-binding antibody is at the N-terminus of the light chain variable region of the CD137 protein-binding antibody, and the light chain variable region of the CD137 protein-binding antibody is at the N-terminus of the variable region the heavy chain of an antibody that binds to the CD137 protein.

84. The bispecific antibody of any one of embodiments 82-83, wherein the light chain variable region of the CD137 protein-binding antibody and the heavy chain variable region of the CD137 protein-binding antibody in the first polypeptide chain constitute a scFv.

- 30 044684

85. The bispecific antibody of any one of embodiments 82-84, wherein the first polypeptide chain further comprises a human IgG constant region, and the human IgG constant region is located at the C-terminus of the heavy chain variable region of the antibody binding to the PDL1 protein and is located at the N- the end of the variable region of the light chain of an antibody that binds to the CD137 protein; alternatively, the human IgG constant region is located at the C-terminus of the heavy chain variable region of the antibody binding to the PD-L1 protein and is located at the N-terminus of the heavy chain variable region of the antibody binding to the CD137 protein.

86. The bispecific antibody of any one of embodiments 82-85, wherein the first polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 43, and SEQ ID NO: 44.

87. The bispecific antibody of any one of embodiments 82-86, wherein the second polypeptide chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 34.

88. One or more isolated nucleic acid molecules encoding an antibody, antigen binding fragment or variant thereof as in any one of embodiments 1-39, an antibody, antigen binding fragment or variant thereof as in any one of embodiments 40-78 or a bispecific antibody as in any one of embodiments 79-87.

89. A vector comprising the nucleic acid molecule of embodiment 88.

90. A cell comprising the nucleic acid molecule of embodiment 88 or the vector of embodiment 89.

91. A method for producing an antibody, antigen-binding fragment or variant thereof according to any one of embodiments 1-39, an antibody, antigen-binding fragment or variant thereof according to any one of embodiments 40-78 or a bispecific antibody according to any one of embodiments 79-87, comprising culturing a cell in embodiment 90, under conditions allowing expression of the antibody, antigen binding fragment or variant thereof of any one of embodiments 1-39, the antibody, antigen binding fragment or variant thereof of any one of embodiments 40-78, or the bispecific antibody of any one of embodiments 79 -87.

92. A pharmaceutical composition comprising an antibody, an antigen binding fragment or variant thereof according to any of embodiments 1-39, an antibody, an antigen binding fragment or variant thereof according to any one of embodiments 40-78 or a bispecific antibody according to any one of embodiments 79-87, a molecule the nucleic acid of embodiment 88, the vector of embodiment 89 and/or the cell of embodiment 90, and optionally a pharmaceutically acceptable excipient.

93. Use of an antibody, antigen-binding fragment or variant thereof as in any one of embodiments 1-39, an antibody, antigen-binding fragment or variant thereof as in any one of embodiments 40-78 or a bispecific antibody as in any one of embodiments 79-87, a nucleic acid molecule as in embodiment 88, the vector of embodiment 89, the cell of embodiment 90, and/or the pharmaceutical composition of embodiment 92 in the manufacture of a medicament for the relief or treatment of tumors.

94. Use according to embodiment 93, wherein the tumor includes colon cancer.

95. A method of inhibiting the binding of a PD-L1 protein to a PD-1 protein, comprising administering an antibody, an antigen binding fragment or variant thereof according to any one of embodiments 1-39, an antibody, an antigen binding fragment or variant thereof according to any one of embodiments 40-78 or a bispecific the antibodies of any of embodiments 79-87, the nucleic acid molecule of embodiment 88, the vector of embodiment 89, the cell of embodiment 90, and/or the pharmaceutical composition of embodiment 92.

96. A method of enhancing T cell function, comprising administering an antibody, antigen binding fragment or variant thereof as in any one of embodiments 1-39, an antibody, antigen binding fragment or variant thereof as in any one of embodiments 40-78 or a bispecific antibody as in any one of embodiments 79-87, the nucleic acid molecule of embodiment 88, the vector of embodiment 89, the cell of embodiment 90, and/or the pharmaceutical composition of embodiment 92.

Without being limited by any theory, the following examples are presented solely to illustrate operational methods in the case of the device, method and system of the present invention, but do not limit the scope of the present invention.

Examples

Example 1 Anti-PD-L1 Antibody Screening Using a Phage Antibody Library.

Human PD-L1-Fc protein (Origincell Therapeutics Co., Ltd.) was used as an antigen to sort a human natural antibody phage library (Origincell Therapeutics Co., Ltd.). The ELISA tube was coated with CBS buffer containing 20 μg/ml (first and second rounds) or 10 μg/ml (third and fourth rounds) of PD-L1 protein at 4°C overnight. The tube was then washed with buffer

- 31 044684

PBS. Add 10% skim milk powder to block the ELISA tube, and then add 1 ml of blocked phage and incubate at room temperature (20±5°C) for 1 hour. After thorough washing with PBS, add 800 µl of Gly-HCl pH buffer solution 2.2, for elution, and then immediately added 400 μl of Tris-HCl buffer solution, pH 8.0, for neutralization. The mixture was then added to 20 ml of E. coli SS320 in logarithmic growth phase with an OD value of approximately 0.8, mixed thoroughly and left at 37°C for 1 hour. 500 µl of the microbial solution was taken to measure the phage titer and glycerol was used to protect the microorganisms , the remaining microbial solution was used to coat the plate and cultured at 37°C overnight. The next day, the bacteria on the plate were proportionally inoculated into 80 ml of 2YT-Amp medium such that the microbial solution had an OD value of 0.2 and cultured for several hours. When the OD value reached 0.8, 160 μl of helper phage was added, mixed thoroughly and left at 37°C for 1 hour. Then IPTG and the antibiotic kanamycin were added and cultured with shaking at 250 rpm at 30°C overnight . The supernatant was then collected for phage precipitation with PEG/NaCl solution, which was resuspended in 1.5 ml PBS enrichment screening buffer.

96-well ELISA plates were coated with solutions containing 1 μg/ml human PD-L1-Fc protein (Origincell Therapeutics Co., Ltd.) and mouse PD-L1-Fc protein (M5251, purchased from ACROBiosystems Inc.) at 4°C. C overnight. The plate was then blocked with 10% nonfat powdered milk at nonspecific binding sites. After thorough washing, the monoclonal phage supernatant was collected and added to 96-well plates and incubated at 37°C for 2 hours. After thorough washing, HRP-labeled anti-M13 antibody (27-9421-01, GE Healtcare) was added to each well and the reaction was carried out at 37°C for 45 minutes. After thorough washing, TMB was added to each well to develop color. After reacting at room temperature (20 ± 5°C) for 5–10 min, sulfuric acid was added to each well to stop the reaction. A microplate reading spectrophotometer was used to measure the OD value of each well at 450 nm.

Phage with antibody clone 1B10, which can specifically bind to human PD-L1 and mouse PD-L1, was identified by ELISA. According to the sequencing results, the nucleotide sequence encoding the VH heavy chain variable region of phage antibody 1B10 is shown in SEQ ID NO: 1, and the amino acid sequence of the VH heavy chain variable region of phage antibody 1B10 is shown in SEQ ID NO: 2; the nucleotide sequence encoding the VL light chain variable region of phage antibody 1B10 is shown in SEQ ID NO: 3, and the amino acid sequence of the VL light chain variable region of phage antibody 1B10 is shown in SEQ ID NO: 4.

Example 2 Expression and purification of a fully human intact anti-PD-L1 antibody. A primer was designed for PCR amplification of the VH phage antibody.

1B10 and the PCR product were cloned by recombination into the pCMV-IgG1NDL vector, doubly digested with AgeI and SalI. A primer for PCR amplification of VL phage antibody 1B10 was designed and the PCR product was cloned by recombination into the pCMV-λ vector double-digested with AgeI and BsiWI. After sequencing, 293F cells were cotransfected with heavy chain and light chain transient expression vectors and purified using a protein A column to generate intact IgG1,λ antibody from phage antibody 1B10. This fully human anti-PD-L1 antibody was designated YN-002.

According to the sequencing results, the nucleotide sequence of VH encoding antibody YN-002 is shown in SEQ ID NO: 1, and the amino acid sequence of VH antibody YN-002 is shown in SEQ ID NO: 2. The nucleotide sequence of VL encoding antibody YN002 is shown in SEQ ID NO: 3, and the amino acid sequence of the VL antibody YN-002 is shown in SEQ ID NO: 4. The nucleotide sequence of the heavy chain encoding the antibody YN-002 is shown in SEQ ID NO: 5, and the amino acid sequence of the heavy chain of YN-002 is shown in SEQ ID NO: 6. The nucleotide sequence encoding the light chain of the YN-002 antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain of the YN-002 antibody is shown in SEQ ID NO: 8.

Example 3 Germline version of fully human anti-PD-L1 antibody YN-002.

By comparing the heavy chain sequence of the YN-002 antibody with the known human germline immunoglobulin heavy chain sequence, it was confirmed that the heavy chain of the YN-002 antibody used the VH segment from the human germline IGHV1 -69*09, the D segment from the IGHD5-18*01 germline human lineage and the JH segment from the human germline IGHJ4*02.

By comparing the light chain sequence of the YN-002 antibody with the known human germline immunoglobulin light chain sequence, it was confirmed that the VL segment from the human germline IGLV2-14*01 and the JL segment from the human germline IGLJ2*01 were used in the light chain of the YN-002 antibody .

The sequence of the CDR region of antibody YN-002 was analyzed using the Kabat system (see Fig. 1).

- 32 044684

According to the sequencing results, the amino acid sequences of LCDR1-3 antibody YN-002 are shown in SEQ ID NO: 54, SEQ ID NO: 60 and SEQ ID NO: 63, respectively; and the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 50, respectively.

To minimize the immunogenicity of the YN-002 antibody, certain amino acid residues can be reverse mutated to the germline sequence. Antibody YN-003 is a germline version of antibody YN-002, produced by mutating one amino acid in the FR1 region of the heavy chain variable region of YN-002 back to the germline sequence and mutating 1 amino acid in the FR2 region and 6 amino acids in the FR3 region of the light variable region YN-002 chains back into the germline sequence (see Fig. 1). The fully human anti-PD-L1 antibody heavy chain expression vector YN-003 is produced by site-directed mutagenesis using a mutagenesis kit (Tiangen point mutation kit, KM101) based on the constructed heavy chain expression plasmid YN-002. The light chain expression vector YN-003 is produced by site-directed mutagenesis using a mutagenesis kit (Tiangen point mutation kit, KM101) based on the light chain expression plasmid YN-002.

The amino acid sequence of the VH antibody YN-003 is shown in SEQ ID NO: 9, which can be obtained by mutation of one amino acid residue in the amino acid sequence of the VH antibody YN-002. The nucleotide sequence encoding the VH of YN-003 is shown in SEQ ID NO: 10. The amino acid sequence of the VL antibody YN-003 is shown in SEQ ID NO: 11, which can be obtained by mutation of 7 amino acid residues in the amino acid sequence of the VL antibody YN-002, and the nucleotide sequence corresponding to VL YN-003 is shown in SEQ ID NO: 12. The amino acid sequence of the heavy chain of the antibody YN-003 is shown in SEQ ID NO: 13; the nucleotide sequence encoding the heavy chain of YN-003 is shown in SEQ ID NO: 14; the amino acid sequence of the light chain of antibody YN-003 is shown in SEQ ID NO: 15; and the nucleotide sequence encoding the light chain of the antibody YN-003 is shown in SEQ ID NO: 16.

For the expression and purification of the YN-003 antibody, see the steps for the expression and purification of the YN-002 antibody in Example 2.

Example 4 Anti-PD-L1 Antibody Binding Affinity Testing.

The binding affinities of antibody YN-002 and antibody YN-003 to human, Macas fascicularis, mouse and dog recombinant PD-L1 proteins were measured using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.). The following proteins were labeled with biotin (EZ-Link sulfo-NHS-LC-biotin, Pierce, 21327): recombinant human PD-L1-Fc, recombinant Masasa fascicularis PD-L1-Fc (90251-C02H, purchased from Sino Biological Inc.), recombinant mouse PD-L1-Fc (M5251, purchased from ACROBiosystems Inc.), recombinant canine PD-L1-Fc (70110-D02H, purchased from Sino Biological Inc.). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. Biotin-bound antigen protein at a concentration of 50 nM was fixed on the SA sensor (Pall ForteBio Analytics Co., Ltd.) and bound at 1500 rpm for 10 min; it was then coupled to a doubly diluted antibody solution at 1500 rpm for 10 min; and finally subjected to dissociation at 1500 rpm for 10 min. The obtained results were analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to obtain K _D , K _on (1/Ms) and Kof (1/s).

The results of measuring the binding affinity of antibodies against PD-L1 YN-002 and YN-003 are shown in table. 1-4.

Table 1

Binding affinity of anti-PD-L1 antibody to human PD-L1-Fc

Antibody iO-Ll-Fc human-biotin Con(1/Ms) Coff (1/c) Kd(M) YN-002 5.69x10 5.94xl0 ^{4 1.88x10'w} YN-003 l.50x 10 ⁶ 5.11x10’4 ^1.08xl0'w

- 33 044684

table 2

Binding affinity of anti-PD-L1 antibody to mouse PD-L1-Fc

Antibody PD-Ll-Fc mouse-biotin kopeck (1/Ms) _kOff (1/s) K _D (M) YN-002 9.77x10 1.68x10' ³ 1.72x10 ^-9 YN-003 1.03x10 ⁶ 9.97x10 ^-4 9.96χ10' ^ιυ

Table 3

Binding affinity of anti-PD-L1 antibody to PD-L1-Fc Macaca Fascicularis

Antibody PD-Ll-Fc Masasa Fascicularis-biotin kopeck (1/Ms) _kOff (1/s) K _D (M) YN-002 1.77x10 ⁶ 1.90x10 ^-3 1.07x10 ^-9 YN-003 2.08x10 ⁶ 1.36x10' ³ 6.54χ10' ^ιυ

Table 4

Binding affinity of anti-PD-L1 antibody to canine PD-L1-Fc_______

Antibody PD-Ll-Fc dogs-biotin kopeck (1/Ms) _kOff (1/s) Kd(M) YN-002 1.02x10 ⁶ 7.59x10' ⁴ 7.43χ10' ^ιυ YN-003 8.64x10 5.14x10' ⁴ 5.94χ10' ^ιυ

Example 5 Anti-PD-L1 antibody inhibits the binding of human PD-L1 and human PD-1.

Human PD-1-Fc protein (ACROBiosystems Inc., H5257) was labeled with biotin (Sulfo-NHS-LCbiotin EZ-Link, Pierce, 21327). Human PD-1-Fc, labeled with a subsaturated concentration of biotin, was added to 1x10 ⁶ /ml CHO cells stably expressing human PD-L1, and then each antibody was added, diluted fivefold, mixed thoroughly and incubated (4°C, 1 h ). After washing the cells, streptavidin R-PE conjugate (Life Technology, SA10041) was added and incubated (4°C, 30 min). After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results are shown in Fig. 2: YN-002 and YN-003 can effectively inhibit the binding of human PD-1 to CHO cells expressing human PD-L1.

Example 6 An anti-PD-L1 antibody inhibits the binding of Macasa Fascicularis PD-L1 to Macasa fascicularis PD-1.

The ability of anti-PD-L1 antibodies YN-002 and YN-003 to block the binding of Masaka fascicularis PD-L1-Fc (Sino Biological Inc., 90251-C02H) to Macasa fascicularis PD-1-Fc (Sino Biological Inc., 90311C02H) was assessed with using an Octet RED384 device ((Pall ForteBio Analytics Co., Ltd.). First, PD-1Fc of Macasa fascicularis was labeled with biotin (Sulfo-NHS-LC-biotin EZ-Link, Pierce, 21327). Analysis of the binding kinetics of anti-PD-L1 antibody in In case of inhibition of the binding of PD-L1 of Macasa fascicularis to PD-1 of Macasa fascicularis, it was carried out using biolayer interferometry (BLI) technology using a Fortebio Octet RED384 (PALL) device to analyze molecular interactions (antigen and antibody diluted with 0.1% BSA and 0.02 % Tween 20 in PBS buffer) 100 nM biotin-bound recombinant human PD-1-Fc was fixed on the SA sensor at 1500 rpm and bound for 10 min. Macasa fascicularis PD-L1 at a final concentration of 75 nM was mixed with diluted three times the antibody solution, incubated for 60 min, and then combined in the device for 10 min at 1500 rpm, and finally dissociated for 10 min at 1500 rpm. The obtained results were analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.). The results are shown in Fig. 3: YN-002 and YN-003 can effectively inhibit the binding of Macaca fascicularis PD-L1-Fc to Macaca fascicularis PD-1-Fc.

Example 7 Anti-PD-L1 antibody inhibits the binding of mouse PD-L1 to mouse PD-1.

Mouse PD-1-Fc (ACROBiosystems Inc., M5259) was labeled with biotin (sulfo-NHS-LC-biotin EZLink, Pierce, 21327), mouse PD-1-Fc labeled with a subsaturated concentration of biotin was added to 1x10 ⁶ /ml CHO cells , stably expressing PD-L1 mice, and then each antibody was added, diluted fivefold, mixed thoroughly and incubated (4°C, 1 h). After washing the cells, streptavidin R-PE conjugate (Life Technology, SA10041) was added and incubated (4°C, 30 min). After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results are shown in Fig. 4: YN-002 and YN-003 can effectively inhibit the binding of mouse PD-1 to CHO cells expressing mouse PD-L1.

Example 8: Studies of the antitumor effect of a monoclonal antibody against PD-L1 in mice bearing colon cancer.

On day 0, female C57BL/6 mice, 6–8 weeks old, were inoculated subcutaneously with 3 x 10 ⁶ co

- 34 044684 mouse lorectal cancer MC38 (Shanghai Linyuan Biological Technology Co., Ltd.). On day 3, mice were evenly distributed into 7 groups with 8-9 mice in each group. Tumor-bearing mice in each group were intraperitoneally injected with human IgG-Fc antibody protein (10 mg/kg, twice a week, for a total of two weeks), YN-003 antibody (10 mg/kg, twice a week, for a total of two weeks), atezolizumab ( 10 mg/kg, twice a week, for a total of two weeks), respectively. Mice in each group were monitored regularly for changes in body weight and tumor size. The experimental results indicate that, compared with control IgG1-Fc, YN-003 and atezolizumab can effectively inhibit tumor growth, where the antitumor effect of YN-003 is more significant (Figure 5).

Example 9: Anti-CD137 Antibody Screening Using a Phage Antibody Library.

CD137 protein (purchased from Sino Biological Inc.) was used as an antigen to sort a human natural antibody phage library (Origincell Therapeutics Co., Ltd.). The ELISA tube was coated with CBS buffer containing 20 μg/ml (first and second rounds) or 10 μg/ml (third and fourth rounds) of CD137 protein at 4°C overnight. The tube was then washed with PBS buffer. Add 10% skim milk powder to block the ELISA tube, and then add 1 ml of blocked phage and incubate at room temperature (20±5°C) for 1 hour. After thorough washing with PBST, add 800 µl Gly-HCl pH buffer solution 2.2, for elution, and then immediately added 400 μl of Tris-HCl buffer solution, pH 8.0, for neutralization. The mixture was then added to 20 ml of E. coli SS320 in logarithmic growth phase with an OD value of approximately 0.8, mixed thoroughly and left at 37°C for 1 hour. 500 µl of the microbial solution was taken to measure the phage titer and glycerol was used to protect the microorganisms , the remaining microorganism solution was used to coat the plate and cultured at 37°C overnight. The next day, the bacteria on the plate were proportionally inoculated into 80 ml of 2YT-Amp medium such that the microorganism solution had an OD value of 0.2 and cultured for several hours. When the OD value reached 0.8, 160 μl of helper phage was added, mixed thoroughly and left at 37°C for 1 hour. Then IPTG and the antibiotic kanamycin were added and cultured with shaking at 250 rpm at 30°C overnight . The supernatant was then collected for phage precipitation with PEG/NaCl solution, which was resuspended in 1.5 ml PBS enrichment screening buffer.

96-well ELISA plates were coated with solutions containing 1 μg/ml human CD137 protein at 4°C overnight. The plate was then blocked with 10% nonfat powdered milk at nonspecific binding sites. After thorough washing, monoclonal phage supernatant was collected, added to 96-well plates and incubated at 37°C for 2 hours. After thorough washing, HRP-labeled anti-M13 antibody (27-9421-01, GE Healtcare) was added to each well and reaction at 37°C for 45 minutes. After thorough washing, TMB was added to each well to develop color. After reacting at room temperature (20 ± 5°C) for 5–10 min, sulfuric acid was added to each well to stop the reaction. A microplate reading spectrophotometer is used to measure the OD value in each well at 450 nm.

A phage antibody clone 1A6 that can specifically bind to human CD137 was identified by ELISA, and the VH and VL gene sequences were obtained by sequencing. According to the sequencing results, the nucleotide sequence encoding the VH of the phage antibody 1A6 is shown in SEQ ID NO: 17, the amino acid sequence of the VH of the phage antibody 1A6 is shown in SEQ ID NO: 18; the nucleotide sequence encoding the VL of phage antibody 1A6 is shown in SEQ ID NO: 19, and the amino acid sequence of the VL of phage antibody 1A6 is shown in SEQ ID NO: 20.

Example 10 Expression and purification of a fully human intact anti-CD137 antibody.

A primer was designed for PCR amplification of VH phage antibody 1A6. The PCR product was cloned by recombination into the pCMV-IgG2 vector, double-digested with AgeI and SalI. A primer for PCR amplification of VL phage antibody 1A6 was designed, and the PCR product was cloned by recombination into the pCMV-λ vector digested with AgeI and BsiWI. After proper sequencing, 293F cells were cotransfected with heavy chain and light chain transient expression vectors and purified using a protein A column. The intact IgG2,λ,1A6 antibody, a fully human anti-CD137 antibody, was designated YN-005.

According to the sequencing results, the amino acid sequence of the VH antibody YN-005 is shown in SEQ ID NO: 18, the nucleotide sequence encoding the VH antibody YN005 is shown in SEQ ID NO: 17; and the amino acid sequence of the VL antibody YN-005 is shown in SEQ ID NO: 20, and the nucleotide sequence encoding the VL antibody YN-005 is shown in SEQ ID NO: 19. The amino acid sequence of the heavy chain of the YN-005 antibody is shown in SEQ ID NO: 21 , the nucleotide sequence encoding the heavy chain of the antibody YN-005 is shown in SEQ ID NO: 22; the amino acid sequence of the light chain of the YN-005 antibody is shown in SEQ ID NO: 23, and the nucleotide sequence encoding the light chain of the YN-005 antibody is shown in SEQ ID NO: 24.

- 35 044684

Example 11: Germline version of fully human anti-CD137 antibody YN-005.

By comparing the heavy chain sequence of the YN-005 antibody with the known human germline immunoglobulin heavy chain sequence, it was confirmed that the YN-005 antibody heavy chain used the VH segment from human germline IGHV3-23*04, the D segment from

human germline IGHD7-27*01 and the JH segment of human germline IGHJ3*02.

By comparing the YN-005 immunoglobulin light chain sequence with the known human germline immunoglobulin light chain sequence, it was confirmed that the YN-005 antibody light chain used the VL segment from human germline IGLV1-44*01 and the JL segment from human germline IGLJ1*01 .

The Kabat system was used to analyze the sequence of the CDR region of antibody YN-005 (see FIG. 6), and according to the sequencing results, the amino acid sequence of HCDR1-3 of antibody YN-005 is shown in SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, respectively. The amino acid sequence LCDR1-3 of antibody YN-005 is shown in SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

To minimize the immunogenicity of the YN-005 antibody, certain amino acid residues can be reverse mutated to the germline sequence. Antibody YN-006 is a germline version of antibody YN-005 produced by mutating one amino acid in the FR1 region of the heavy chain variable region of YN-005 back to the germline sequence (see FIG. 6). The heavy chain expression vector of the fully human anti-CD137 antibody YN-006 is prepared by site-directed mutagenesis using a mutation kit (Tiangen point mutation kit, KM101) based on the constructed YN-005 heavy chain expression plasmid.

According to the sequencing results, the amino acid sequence of VH antibody YN-006 is shown in SEQ ID NO: 25, and the nucleotide sequence encoding VH YN-006 is shown in SEQ ID NO: 26. The amino acid sequence of VL antibody YN-006 is shown in SEQ ID NO: 20, and the nucleotide sequence encoding VL YN-006 is shown in SEQ ID NO: 19. The amino acid sequence of the heavy chain of the antibody YN-006 is shown in SEQ ID NO: 27; the nucleotide sequence encoding the heavy chain of the antibody YN-006 is shown in SEQ ID NO: 28; and the amino acid sequence of the light chain of the YN-006 antibody is shown in SEQ ID NO: 23, and the nucleotide sequence encoding the light chain of YN-006 is shown in SEQ ID NO: 24.

In the case of expression and purification of the YN-006 antibody, see the specific steps for the expression and purification of the YN-005 antibody in Example 9.

Example 12 Determination of binding affinity of anti-CD137 antibody.

The binding affinity of antibody YN-005 and antibody YN-006 to recombinant human CD137-His protein (Origincell Therapeutics Co., Ltd.) was measured using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. The antibody at a concentration of 50 nM was fixed on the ANS sensor and bound at 1500 rpm. Within 5 minutes; then it was associated with a two-fold diluted solution of the recombinant antigenic protein CD137-His (100, 50, 25, 12.5, 6.25, 3.125, 1.56 nM) at 1500 rpm for 5 min; and finally subjected to dissociation at 1500 rpm for 10 min. The ANS sensor was regenerated by pulsed injection of glycine and then reused. The obtained results were analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to obtain _KD , _Kon (1/Mc) and _Koff (1/c). The results of binding affinity measurements of anti-CD137 antibodies YN-005 and YN-006 are shown in table. 5.

Table 5

Affinity of anti-CD137 antibody to human CD137-His

Antibody CD137-His K _on (1/Mc) K _Off (1/s) _KD (M) YN-005 9.38χ10 ⁴ 4.55x10’ ^4.85xl0'w YN-006 9.68χ10 ⁴ 1.33x10’ 1.37χ10' ^ιυ

Example 13: Studies of the antitumor effect of an anti-CD137 monoclonal antibody in mice bearing colon cancer.

On day 0, 6-8 week old female human CD137 C57BL/6 mice (BhTNFRSF9 (4-1BB) mice purchased from Beijing BioCytogen Co., Ltd.) were subcutaneously inoculated with 1.5 x 10 ⁶ MC38 mouse colorectal cancer cells. On day 3, mice were evenly divided into 3 groups with 7 mice in each group. Tumor-bearing mice in each group were injected intraperitoneally with human IgG-Fc antibody protein (3 mg/kg, twice a week, for a total of two weeks), anti-CD137 antibody YN-006 (3 mg/kg, twice a week, for a total of two weeks), and anti-CD137 antibody utomilumab (3 mg/kg,

- 36 044684 twice a week, two weeks in total), respectively. Mice in each group were monitored regularly for changes in body weight and tumor size. The experimental results showed that, compared with control IgG1-Fc, YN-006 and utomilumab can effectively inhibit tumor growth, and it is worth noting that the antitumor effect of YN-006 is more significant than that of utomilumab (for example, as shown in Fig. 7).

Example 14: Construction of bispecific anti-PD-L1/CD137 antibody YN-007.

The first polypeptide gene of the anti-PD-L1/CD137 bispecific antibody YN-007 was constructed by a molecular cloning method such as overlap extension PCR, site-directed mutagenesis or the like. using the heavy chain gene of the anti-PD-L1 antibody YN003 and the heavy chain and light chain genes of the anti-human CD137 antibody YN-006, and cloned by combination into the pCMVIgG1 AEM vector double-digested with AgeI and BamHI to construct the expression vector of the first YN polypeptide -007.

The second polypeptide gene of YN-007 is the same as the light chain gene of YN-003. The expression vector of the second polypeptide YN-007 is the constructed light chain expression vector YN-003 described above.

After proper sequencing, 293F cells were cotransfected with the first polypeptide and second polypeptide transient expression vectors and purified using a protein A column to generate the anti-PD-L1/CD137 bispecific antibody YN-007.

The nucleotide sequence encoding the first polypeptide of the YN-007 antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the first polypeptide of the YN-007 antibody is shown in SEQ ID NO: 30; the nucleotide sequence encoding the second polypeptide of the YN-007 antibody is shown in SEQ ID NO: 16, and the amino acid sequence of the second polypeptide of the YN007 antibody is shown in SEQ ID NO: 15.

Example 15 Determination of binding affinity of anti-PD-L1/CD137 bispecific antibody YN-007.

The binding affinity of anti-PD-L1/CD137 bispecific antibody YN-007 to recombinant human PD-L1-Fc protein and recombinant mouse PD-L1-Fc protein was measured using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd. ). Recombinant human PD-L1-Fc protein and recombinant mouse PD-L1-Fc protein were labeled with biotin (SulfoNHS-LC-biotin EZ-Link, Pierce, 21327). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. Biotin-bound antigen protein at a concentration of 50 nM was fixed on the SA sensor (Pall ForteBio Analytics Co., Ltd.) and bound at 1500 rpm for 10 min; then it was combined with a two-fold diluted solution of the YN-007 antibody at 1500 rpm for 10 min; and finally subjected to dissociation at 1500 rpm for 10 min. The obtained results will be analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to obtain K _D , K _on (1/Mc) and K _off (1/c).

The results of measuring the binding affinity of YN-007 to human PD-L1-Fc are shown in table. 6.

The results of measuring the binding affinity of YN-007 to mouse PD-L1-Fc are shown in table. 7.

Table 6 Binding affinity of anti-PD-L1/CD137 bispecific antibody YN-007 to human PD-L1-Fc

Antibody Human PD-Ll-Fc-biotin kon(1/Mc) _kOff (1/s) Kd(M) YN-007 1.13x10 ⁶ 5.92χ10' ⁴ 5.25χ10' ^ιυ

Table 7

Binding affinity of anti-PD-L1/CD137 bispecific antibody YN-007 to mouse PDL1-Fc

Antibody PD-Ll-Fc mouse-biotin k _on (1/Mc) _kOff (1/s) _KD (M) YN-007 8,lxl0 ⁶ l,09xl0' ³ l, ^34xl0'y

The binding affinity of the anti-PD-L1/CD137 bispecific antibody YN-007 to recombinant human CD137-His protein was measured using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. Antibody at a concentration of 50 nM was fixed on the ANS sensor and bound at 1500 rpm for 5 min; then he was associated with a divorced man

- 37,044,684 times with a solution of recombinant human antigenic protein CD137-His (100, 50, 25, 12.5, 6.25, 3.125, 1.56 nM) at 1500 rpm. within 5 minutes; and finally subjected to dissociation at 1500 rpm for 10 min. The ANS sensor was regenerated by pulsing glycine and then reused. The obtained results will be analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to calculate the binding strength between antigen and antibody to obtain _KD , _Kon (1/Ms) and _Koff (1/s) .

The results of measuring the binding affinity of the anti-PD-L1/CD137 bispecific antibody YN-007 to human CD137 are shown in table. 8.

Table 8

Binding affinity of anti-PD-L1/CD137 bispecific antibody YN-007 to human CD137-His

Antibody Human CD137-His k _on (1/Ms) _kOff (1/s) K _D (Μ) YN-007 8.96^10 ⁴ 9.83 χ ΙΟ' ^{5 1.1x10'y}

Example 16: Affinity maturation of anti-PD-L1 antibody YN-003.

A primer was designed to construct a single-chain antibody gene (ScFv) from anti-PD-L1 antibody YN-003 by overlap extension PCR, cloned into the phagemid vector pDF and designated ScFv pDF-YN-003. ScFv pDF-YN-003 was used as a template to design a degenerate primer to randomize 6 CDR regions (including some amino acid residues in the CDR regions and individual amino acid residues in the FR regions adjacent to the CDR regions) of the YN-003 antibody by PCR with the completion of overlapping fragments , respectively (as shown in Fig. 8).

ScFv gene fragments randomized by CDR region, obtained by overlapping extension PCR, were double-digested with BssHII and NheI and then ligated with the phagemid vector pDF, double-digested with BssHII and NheI. 1 μg of ligation product was electroporated into electrocompetent TG1 cells and coated on a 2YT+AG plate after multiple dilutions. The next day, the plate was scraped off and transferred to 300 ml of 2YT+Amp medium. Cells were cultured at 37°C to an OD of approximately 0.8. Helper phage was added and mixed thoroughly, allowed to stand for 1 h and IPTG at a final concentration of 1 mM and 50 μg/ml kanamycin was added and shaken at 30°C overnight. The next day, the supernatant was collected by centrifugation and filtered through a 0.45 filter. 1/5 volume of PEG-NaCl was added to the precipitated phage and centrifuged to collect the precipitate. A 1/10 volume of PBS was used to resuspend the pellet and OD260 was measured to calculate the phage PFU. The product was stored at 4°C. The phage antibody library can be used directly for subsequent panning.

Human PD-L1-His protein (Origincell Therapeutics Co., Ltd.) was used as the antigen to sort the above phage antibody library. In ELISA tubes, CBS buffer was used to coat PD-L1 antigen (1 ml) at a concentration of 100 nM (first and second round) or 5 nM (third round) at 4°C overnight. The next day, 2 ml of PBS buffer containing 10% skim milk powder was used to block the tube. Add 1 ml of blocked phage to a test tube and incubate for 1 hour at room temperature; washed with PBST 20 times (first round), 50 times (second round), or 100 times (third round). 800 μl of Gly-HCl buffer (pH 2.2) was added for elution, and 400 μl of Tris-HCl buffer (pH 8.0) was immediately added for neutralization. It was added to 20 ml of E. coli TGI in logarithmic growth phase with an OD of approximately 0.8, mixed thoroughly and left at 37°C for 1 hour. 500 µl was taken to determine the phage titer and glycerol was used to protect the microorganisms. The remaining microbial fluid was distributed onto the plate and incubated at 37°C in an incubator overnight. The next day, the microorganisms were plated and inoculated into 80 ml of 2YTAmp medium in some proportion to achieve an OD of 0.2. The mixture was incubated for several hours until the OD reached 0.8. 160 μl of helper phage was added, mixed thoroughly and left at 37°C for 1 hour. IPTG and the antibiotic kanamycin were added and incubated with shaking at 250 rpm and 30°C overnight. The supernatant was collected and treated with PEG/NaCl solution to precipitate the phage, which was resuspended in 1.5 ml PBS buffer. The resuspended phage was used for the next round of enrichment screening. After 3 rounds of sorting, significant enrichment was observed. Sorted phage antibody clones were identified by ELISA: human PD-L1-His protein was coated on a 96-well ELISA plate at a concentration of 1 μg/ml at 4°C overnight. Nonspecific binding sites were then blocked with 10% skim milk powder. After sufficient washing, the monoclonal phage supernatant was added to a 96-well plate and incubated at 37°C for 2 hours. After thorough washing, HRP-labeled anti-M13 antibody (GE Healtcare, 27-9421-01) was added and reacted at 37°C within 45 min. After thorough washing, TMB was added to develop color and the reaction was carried out at room temperature for 5-10 minutes. And finally, the reaction remains

- 38 044684 poured sulfuric acid. The OD value in each well was measured at 450 nm and the phage antibody clone with the higher OD450 value was selected for sequencing. After obtaining the variable heavy chain and light chain gene sequences of each phage antibody clone, each phage antibody was reconstructed as a full-length IgG1,λ antibody: a primer was designed to perform PCR amplification of the VH of each phage antibody clone, and the PCR product was cloned by recombination into a heavy chain expression vector. pCMVIgG1NDL antibody chains, double digested with AgeI and SalI. A primer was designed to PCR amplify the VL of each phage antibody clone, and the PCR product was cloned into the antibody light chain expression vector pCMV-λ, doubly digested with AgeI and BsiWI. After proper sequencing, 293F cells were cotransfected with expression vectors for the heavy chain and light chain of each antibody for transient expression. After 7 days of culture in serum-free medium, the cell culture supernatant was collected and purified using a protein A column to obtain antibody protein. The purified antibody was dialyzed with PBS and ultimately quantified using a BCA protein assay kit (Pierce, 23225). Binding affinity of the indicated full-length antibody to recombinant human PDL1-His protein (Origincell Therapeutics Co., Ltd.) using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. Antibody at a concentration of 50 nM was fixed on the ANS sensor and bound at 1500 rpm for 5 min; then associated with a twofold diluted antigen solution (50, 25, 12.5, 6.25, 3.125, 1.56 nM) with 7 concentration gradients at 1500 rpm for 5 min, and finally subjected to dissociation at 1500 rpm ./min for 10 min. The ANS sensor was regenerated by pulsing glycine and then reused. The obtained results will be analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to obtain K _D , K _on (1/Ms) and K _off (1/s). After screening the resulting high-affinity mutant clones, point mutations of high-affinity amino acids in different CDR regions were combined by methods such as overlap extension PCR, and then the antibody protein was expressed and purified in the above manner and the affinity was determined. After multiple rounds of screening and combination, the top 5 high affinity mutant clones of YN-003 antibody were designated as YN-035 antibody, YN-036 antibody, YN-037 antibody, YN-038 antibody, and YN-039 antibody, respectively.

The affinity values of 5 antibodies obtained by the above method are given in table. 9. The results showed that the affinity of YN-035 antibody, YN-036 antibody, YN-037 antibody, YN038 antibody and YN-039 antibody was higher than that of the anti-PD-L1 antibodies atezolizumab and avelumab.

- 39 044684

Table 9

Binding affinity of anti-PD-L1 antibody to human PD-L1-His

Antibody Human PD-Ll-His _KD (M) Error K _D K _on (1/Mc) Error Kon Kof _f (l/c) Error K ₀ ff Full R ^A 2 Atezol izumab 9.16xlO' ¹⁰ 8.79xl0' ¹² 3.78x10 1.95x10 3.46x10' ⁴ 2.80xl0' ⁶ 0.9984 Awelu mab l,03xl0 ^-y 9.20* 10' ¹² 2.96x10 1.31x10 3.05 x 10 ^-4 2.37xl0' ⁶ 0.9991 YN- 035 4.52^10 ^-10 3.54xl0' ¹² 4.27x10 1.16x10 1.93 x 10 ^-4 1.42x1 O' ⁶ 0.9995 YN- 036 4.74xlO' ¹⁰ 4.95xl0' ¹² 3.69x10 1.22x10 1.75x10' ⁴ 1.73x1 O' ⁶ 0.9994 YN- 037 3.82xlO' ¹⁰ 5.66xl0' ¹² 3.55x10 1.36x10 l,36xl0 ^-4 1.94x1 O' ⁶ 0.9991 YN- 038 4.47 x 10 ^-10 4.61xl0' ¹² 3.50x10 1.05x10 l.56xl0 ^-4 l,54xl0' ⁶ 0.9995 YN- 039 ^4.85xl0'lu 4.41 x 10 ^-12 3.56x10 1.02x10 1.73 x 10 ^-4 1.49x1 O' ⁶ 0.9995

The amino acid sequence of the heavy chain variable region of the antibody YN-035 (VH YN-035) is shown in SEQ ID NO: 31, the amino acid sequence of the heavy chain of the YN035 antibody is shown in SEQ ID NO: 32, the amino acid sequence of the light chain variable region of the YN-035 antibody (YN -035 VL) is shown in SEQ ID NO: 33, and the amino acid sequence of the YN-035 antibody light chain is shown in SEQ ID NO: 34.

The amino acid sequence of the heavy chain variable region of the antibody YN-036 (YN036 VH) is shown in SEQ ID NO: 35, the amino acid sequence of the heavy chain of the YN036 antibody is shown in SEQ ID NO: 36, the amino acid sequence of the variable light chain region of YN-036 antibody (YN-036VL ) is shown in SEQ ID NO: 33, and the amino acid sequence of the light chain of the antibody YN-036 is shown in SEQ ID NO: 34.

The amino acid sequence of the heavy chain variable region of the antibody YN-037 (YN037 VH) is shown in SEQ ID NO: 35, the amino acid sequence of the heavy chain of the YN037 antibody is shown in SEQ ID NO: 36, the amino acid sequence of the variable light chain region of YN-037 antibody (YN-037VL ) is shown in SEQ ID NO: 37, and the amino acid sequence of the light chain of the antibody YN-037 is shown in SEQ ID NO: 38.

The amino acid sequence of the variable region heavy chain of the antibody YN-038 (YN038 VH) is shown in SEQ ID NO: 35, the amino acid sequence of the heavy chain of the antibody YN038 is given in SEQ ID NO: 36, the amino acid sequence of the variable region light chain of the YN-038 antibody (YN-038VL ) is shown in SEQ ID NO: 39, and the amino acid sequence of the YN-038 antibody light chain is shown in SEQ ID NO: 40.

The amino acid sequence of the variable region heavy chain of the antibody YN-039 (YN039VH) is given in SEQ ID NO: 35, the amino acid sequence of the heavy chain of the antibody YN-039 is given in SEQ ID NO: 36, the amino acid sequence of the variable region of the light chain of the antibody YN-039 (YN- 039 VL) is shown in SEQ ID NO: 41, and the amino acid sequence of the YN-039 antibody light chain is shown in SEQ ID NO: 42.

In fig. 9 shows an alignment of the amino acid sequences of the heavy chain variable regions and the corresponding germline sequences of the antibodies YN-003 and YN-035YN-039, where the CDRs determined by the Kabat method are underlined. In fig. 10 shows an alignment of the amino acid sequences of the light chain variable regions and the corresponding germline sequences of antibodies YN-003 and YN-035-YN-039, where the CDRs determined by the Kabat method are underlined. Of these, the amino acid sequence of HCDR1 antibody YN-003 is shown in SEQ ID NO: 46, the amino acid sequence of HCDR1 YN-035-YN-039 is shown in SEQ ID NO: 47; the amino acid sequence of the HCDR2 antibody YN-003 and the amino acid sequence of the HCDR2 antibody YN-035-YN-039 are shown in SEQ ID NO: 48; amino acid sequence

- 40 044684

HCDR3 YN-003 is shown in SEQ ID NO: 50; the amino acid sequence of HCDR3 YN-035 is shown in SEQ ID NO: 51; and the amino acid sequence of HCDR3 antibodies YN-036-YN-039 is shown in SEQ ID NO: 52.

The amino acid sequence of LCDR1 antibody YN-003 is shown in SEQ ID NO: 54, the amino acid sequence of LCDR1 antibodies YN-035-YN-036 is shown in SEQ ID NO: 55, the amino acid sequence of LCDR1 antibody YN-037 is shown in SEQ ID NO: 56, amino acid the LCDR1 sequence of antibody YN-038 is shown in SEQ ID NO: 57, the amino acid sequence of LCDR1 antibody YN-039 is shown in SEQ ID NO: 58; the amino acid sequence of LCDR2 antibodies YN-003 and antibodies YN-035-YN-036 is shown in SEQ ID NO: 60, the amino acid sequence of LCDR2 antibodies YN-037-YN-039 is shown in SEQ ID NO: 61; the amino acid sequence of LCDR3 antibody YN-003 is shown in SEQ ID NO: 63; and the LCDR3 amino acid sequence of antibodies YN-035-YN-039 is shown in SEQ ID NO: 64.

The binding activity of antibodies against PD-L1 YN-035, YN-036, YN-037, YN-038, YN-039 with human PDL1 was determined by flow cytometry: 1x10 ⁶ /ml CHO cells stably expressing human PD-L1 were added separate 2-fold diluted anti-PD-L1 antibodies. The mixture was mixed thoroughly and incubated at 4°C for 1 h. After washing the cells, goat anti-human IgG-Fc F(ab') ₂ (DyLight 650) (ab98593, Abcam) was added and incubated at 4°C for 30 min. After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 11: YN-035, YN-036, YN-037, YN-038, YN-039 can all effectively bind to CHO cells expressing human PD-L1.

The inhibitory ability of anti-PD-L1 antibodies YN-035, YN-036, YN-037, YN-038, YN-039 against human PD-L1/human PD-1 was determined by flow cytometry: human PD-1-Fc protein ( Origincell Therapeutics Co., Ltd.) were labeled with biotin (Sulfo-NHS-LC-biotin EZ-Link, Pierce, 21327). Human PD-1-Fc, labeled with a subsaturated concentration of biotin, was added to 1x10 ⁶ /ml CHO cells stably expressing human PD-L1, and then individual 2-fold diluted antibodies were immediately added, mixed thoroughly and incubated (4°C, 1 h ). After washing the cells, streptavidin R-PE conjugate (Life Technology, SA10041) was added and incubated (4°C, 30 min). After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 12: All of the anti-PD-L1 antibodies YN-035, YN-036, YN-037, YN-038, YN-039 can effectively inhibit the binding of human PD-1 to CHO cells expressing human PD-L1.

The binding activity of antibodies against PD-L1 YN-035, YN-036, YN-037, YN-038, YN-039 with mouse PDL1 was determined by flow cytometry: 1x10 ⁶ /ml CHO cells stably expressing mouse PD-L1 were added separate 2-fold diluted anti-PD-L1 antibodies. The mixture was mixed thoroughly and incubated at 4°C for 1 hour. After washing the cells, goat anti-human IgG-Fc F(ab')2 (DyLight 650) (ab98593) was added and incubated at 4°C for 30 minutes. After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 13: YN-035, YN-036, YN-037, YN-038, YN-039 can all effectively bind to CHO cells expressing mouse PD-L1.

The inhibitory ability of anti-PD-L1 antibodies YN-035, YN-036, YN-037, YN-038, YN-039 against mouse PD-L1/mouse PD-I was determined by flow cytometry: mouse PD-1-Fc (Origincell Therapeutics Co., Ltd.) were labeled with biotin (Sulfo-NHS-LC-biotin EZ-Link, Pierce, 21327). Mouse PD-1-Fc, labeled with a subsaturated concentration of biotin, was added to 1x10 ⁶ /ml CHO cells stably expressing mouse PD-L1, and then individual 2-fold diluted antibodies were immediately added, mixed thoroughly and incubated (4°C, 1 h ). After washing the cells, streptavidin R-PE conjugate (Life Technology, SA10041) was added and incubated (4°C, 30 min). After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results are shown in Fig. 14: All of the anti-PD-L1 antibodies YN-035, YN-036, YN-037, YN038, YN-039 can effectively inhibit the binding of mouse PD-1 to CHO cells stably expressing mouse PD-L1.

Example 17. Construction and identification of bispecific antibodies against PD-L1/CD137 YN-051 and YN-052.

The first polypeptide gene of the anti-PD-L1/CD137 bispecific antibody YN-051 was constructed by a molecular cloning method such as overlap extension PCR, site-directed mutagenesis or the like using the heavy chain gene of the anti-PD-L1 antibody YN-035 and the heavy chain and light chain genes of the anti-human CD137 antibody YN-006 and cloned by recombination into the vector pCMV-IgG1AEM, doubly digested with AgeI and BamHI, to construct the expression vector of the first polypeptide YN-051. The second polypeptide gene of YN-051 is the same as the light chain gene of YN-035. The YN-035 second polypeptide expression vector is the YN-035 light chain expression vector.

- 41 044684

After proper sequencing, 293F cells were cotransfected with the first polypeptide and second polypeptide anti-PD-L1/CD137 YN-051 bispecific antibody expression vectors for transient expression. After culture in serum-free medium for 7 days, the cell culture supernatant was collected and purified using a protein A column to obtain antibody protein. The purified antibody was dialyzed with PBS and ultimately quantified using a BCA protein assay kit (Pierce, 23225).

The amino acid sequence of the first YN-051 polypeptide is shown in SEQ ID NO: 43, and the amino acid sequence of the second YN-051 polypeptide is shown in SEQ ID NO: 34.

The first polypeptide gene of the anti-PD-L1/CD137 bispecific antibody YN-052 was constructed by a molecular cloning method such as overlap extension PCR, site-directed mutagenesis or the like using the heavy chain gene of the anti-PD-L1 antibody YN-036 and the heavy chain and light chain genes of the anti-human CD137 antibody YN-006 and cloned by recombination into the pCMV-IgG1AEM vector, doubly digested with AgeI and BamHI, to construct the first polypeptide expression vector YN-052. The second polypeptide gene of YN-052 is the same as the light chain gene of YN-036. The second polypeptide expression vector YN-052 is the light chain expression vector YN-036.

After proper sequencing, 293F cells were cotransfected with the first polypeptide and second polypeptide anti-PD-L1/CD137 YN-052 bispecific antibody expression vectors for transient expression. After culture in serum-free medium for 7 days, the cell culture supernatant was collected and purified using a protein A column to obtain antibody protein. The purified antibody was dialyzed with PBS and ultimately quantified using a BCA protein assay kit (Pierce, 23225).

The amino acid sequence of the first YN-052 polypeptide is shown in SEQ ID NO: 44, and the amino acid sequence of the second YN-052 polypeptide is shown in SEQ ID NO: 34.

The binding affinity of anti-PD-L1/CD137 bispecific antibodies YN-051 and YN-051 to recombinant human PD-L1-His protein (Origincell Therapeutics Co., Ltd.) was measured using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. Antibody at a concentration of 50 nM was fixed on the ANS sensor and bound at 1500 rpm for 5 min; then it was associated with a two-fold diluted antigen solution (50, 25, 12.5, 6.25, 3.125, 1.56 nM) with 7 concentration gradients at 1500 rpm for 5 min; and finally subjected to dissociation at 1500 rpm. within 10 min.

The ANS sensor was regenerated by pulsing glycine and then reused. The obtained results will be analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to obtain K _D , K _on (1/Ms) and K _off (1/s). The results of measuring the binding affinity of YN-007 to human PD-L1-Fc are shown in table. 10.

Table 10

Binding affinity of anti-PD-L1/CD137 bispecific antibody to human PD-L1-His

Antibody Human PD-Ll-His K _D (M) Error K _D Cop (1/Ms) Error Cop Kof _f (l/c) Error K ₀ ff Full R ^A 2 YN-051 5.17χ1Ο' ¹⁰ 4.97хУ' ¹² 5.32x10^ 2.54x10 ³ 2.75x10' ⁴ 2.29x10' ⁶ 0.9981 YN-052 5.72* 10' ¹⁰ 5.69x10' ¹² 4.48 xYu ⁵ 2.06x10 ³ 2.57x10' ⁴ 2.26x10 ^-6 0.9984

The binding affinity of anti-PD-L1/CD137 bispecific antibodies YN-051 and YN-052 to recombinant human CD137-His protein (Origincell Therapeutics Co., Ltd.) was measured using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co. , Ltd.). Antigen-antibody binding kinetics were analyzed by biolayer interferometry (BLI) technology using a molecular interaction analyzer (Octet RED384, purchased from Pall ForteBio Analytics Co., Ltd.) and PBS buffer containing 0.1% BSA and 0.02% Tween 20. Antibody at a concentration of 50 nM was fixed on the ANS sensor and bound at 1500 rpm for 5 min; then it was coupled with a two-fold diluted solution of the recombinant human CD137-His protein antigen (100, 50, 25, 12.5, 6.25, 3.125, 1.56 nM) at 1500 rpm. within 5 minutes; and finally subjected to dissociation at 1500 rpm for 10 min. The ANS sensor was regenerated using a pulsed

- 42 044684 injection of glycine and then reused. The obtained results will be analyzed using Octet Data Analysis 9.0 software (Pall ForteBio Analytics Co., Ltd.) to obtain K _D , K _on (1/Ms) and K _off (1/s).

Results of binding affinity measurements of bispecific antibodies against PD-L1/CD137

YN-051 and YN-052 with human CD137 are shown in table. eleven.

Table 11

Binding affinity of anti-PD-L1/CD137 bispecific antibody to human CD137-His

Antibody 4-lBB-His person K _D (Μ) Error K _D Cop (1/Ms) Error Cop kdis(ΙΑ) Error kdis Full R ^A 2 YN-051 ^1.64x10 ' 3.93χ10 ^-11 8.99χ10 ⁴ 7.25x10 ² 1.47 xlO ^-4 3.33χ10' ⁶ 0.9981 YN-052 2.08 xlO ^-9 4.36χ10 ^-11 8.37χ10 ⁴ 6.93 xYu ² 1.74χ10 ^-4 3.35χ10 ^-6 0.9982

The human mammary cell line MDA-MB-231 (Cell Library of the Chinese Academy of Sciences in Shanghai) highly expresses the human PD-L1 molecule. The binding of antibodies against PD-1 YN035, YN-036 and bispecific antibodies against PD-L1/CD137 YN- ⁰⁵¹ and YN-052 to MDA-MB231 cells was determined by flow cytometry: separate The two-fold diluted antibodies were mixed thoroughly and incubated at 4°C for 1 hour. After washing the cells, goat anti-human IgG-Fc F(ab') ₂ (DyLight 650) (ab98593, Abcam) was added and incubated at 4 °C for 30 min. After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 15: All of YN-035, YN-036, YN-051 and YN-052 can effectively bind to MDA-MB-231 cells.

The binding activity of antibodies against PD-1 YN-035, YN-036 and bispecific antibodies against PDL1/CD137 YN-051 and YN-052 with human PD-L1 was determined by flow cytometry: to 1x10 ⁶ /ml CHO cells stably expressing PD- L1 human, individual 2-fold diluted antibodies were added, mixed thoroughly and incubated at 4°C for 1 hour. After washing the cells, goat anti-human IgG-Fc F(ab') ₂ (DyLight 650) was added (ab98593, Abcam ) and incubated at 4°C for 30 min. After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 16: YN-035, YN-036, YN-051, YN-052, YN-007 can all effectively bind to CHO cells expressing human PD-L1.

The inhibitory ability of anti-PD-1 antibodies YN-035, YN-036 and anti-PDL1/CD137 bispecific antibodies YN-051, YN-052, YN-007 with human PD-L1/human PD-t was determined by flow cytometry: PD- protein Human l -Fc was labeled with biotin (Sulfo-NHS-LC-biotin EZ-Link, Pierce, 21327). Human PD-1-Fc, labeled with a subsaturated concentration of biotin, was added to 1x10 ⁶ /ml CHO cells stably expressing human PD-L1, and then individual 2-fold diluted antibodies were immediately added, mixed thoroughly and incubated (4°C, 1 h ). After washing the cells, streptavidin R-PE conjugate (Life Technology, SA10041) was added and incubated (4°C, 30 min). After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 17: All anti-PD-L1 antibodies YN-035, YN-036, YN-051, YN-052, YN-007 can effectively inhibit the binding of human PD-1 to CHO cells expressing human PD-L1.

The binding activity of antibodies against PD-1 YN-035, YN-036 and bispecific antibodies against PDL1/CD137 YN-007, YN-051 and YN-052 with mouse PD-L1 was determined by flow cytometry: to 1x10 ⁶ /ml CHO cells, stably expressing PD-L1 mice, individual 2-fold diluted antibodies were added, mixed thoroughly and incubated at 4°C for 1 hour. After washing the cells, goat anti-human IgG-Fc F(ab') ₂ was added (DyLight 650) (ab98593, Abcam) and incubated at 4°C for 30 min. After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 18: All of YN-035, YN-036, YN-051, YN-052, YN007 can effectively bind to CHO cells expressing mouse PD-L1.

The inhibitory ability of anti-PD-1 antibodies YN-035, YN-036 and anti-PDL1/CD137 bispecific antibodies YN-007, YN-051 and YN-052 against mouse PD-L1/mouse PD-1 was determined by flow cytometry: PD- Mouse 1-Fc labeled with biotin (sulfo-NHS-LC-biotin EZ-Link, Pierce, 21327). Mouse PD-1-Fc labeled with a subsaturated concentration of biotin was added to 1x10 ⁶ /ml

- 43 044684 CHO cells stably expressing mouse PD-L1, and then individual two-fold diluted antibodies were immediately added, mixed thoroughly and incubated (4°C, 1 h). After washing the cells, streptavidin R-PE conjugate (Life Technology, SA10041) was added and incubated (4°C, 30 min). After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results are shown in Fig. 19: All anti-PD-L1 antibodies YN-035, YN-036, YN-051, YN052, YN-007 can effectively inhibit the binding of mouse PD-1 to CHO cells expressing mouse PD-L1.

The binding activity of the anti-CD137 antibody YN-006 and the anti-PDL1/CD137 bispecific antibodies YN- ⁰⁰⁷ , YN-051 and YN-052 towards human CD137 was determined by flow cytometry: separate The two-fold diluted antibodies were mixed thoroughly and incubated at 4xC for 1 hour. After washing the cells, F(ab') ₂ goat anti-human IgG-Fc (DyLight 650) (ab98593, Abcam) was added and incubated at 4°C within 30 min. After washing the cells, fluorescence intensity was determined using a flow cytometer (Intellicyt iQue Screener). The results of the experiment are shown in Fig. 20: YN-006, YN-007, YN-051 and YN-052 can all effectively bind to 293T cells expressing human CD137.

Example 18 Polyacrylamide gel electrophoresis (PAGE) analysis in the presence of SDS.

After purification as described above, the molecular weights of antibodies YN-003, YN-006, YN007, YN-035, YN-036, YN-051, YN-052 were measured by SDS-PAGE under reducing and non-reducing conditions. The results are shown in Fig. 21: Under non-reducing conditions (as shown in Fig. 21a), all of the monoclonal antibodies YN-003, YN006, YN-035 and YN-036 appeared as a band with a molecular mass of approximately 150 kDa, while all of the bispecific antibodies YN- 007, YN -051, YN-052 appeared as a band with a molecular mass of approximately 200 kDa. Under reducing conditions (as shown in Fig. 21b), all of the monoclonal antibodies YN-003, YN006, YN-035 and YN-036 appeared as two bands with a molecular weight of approximately 55 kDa and 25 kDa, while all of the bispecific antibodies YN- 007, YN-051, YN-052 appeared as two bands with molecular weights of approximately 75 and 25 kDa.

Example 19 Agonist activity of anti-PD-L1/CD137 bispecific antibody (Luciferase activity assay).

293T cells expressing human CD137 and into which the NFKB-luciferase reporter gene was stably inserted were obtained. Cells were harvested, washed, and resuspended in complete phenol red-free medium (DMEM containing 10% fetal bovine serum, HEPES buffer, nonessential amino acids, and L-glutamine) at a density of 6 x 10 ⁵ cells/ml. 96-well plates (purchased from PerkinElmer) were used and 50 μl of cell suspension was added to each well. Cross-linked antibody (goat anti-human IgG Fc antibody) was added at a ratio of 2.5:1, then YN-006 (10 μg/ml), YN-035 (10 μg/ml), YN-035+YN-006 was added (10 µg/ml+10 µg/ml), YN-051 (10 µg/ml), YN-003+YN006 (10 µg/ml+10 µg/ml), YN-007 (10 µg/ml) and control IgG Fc antibody (10 μg/ml), respectively, and incubated at 37°C for 5 h. Then, 75 μl of Bright-Glo luciferase reagent (purchased from Promega) was added and luciferase activity was measured using a microplate reader spectrophotometer (purchased from Tesap). The ratios of fluorescence values from each group of antibody-treated cells were compared with the group of untreated cells (see FIG. 22). The results showed that compared with the control IgG Fc, all of YN-006, YN035+YN006, YN003+YN006, YN-007, YN-051 had obvious agonist activity, while anti-PD-L1 antibody YN-035 did not had it (see Fig. 22).

Example 20: Studies of the antitumor effect of a bispecific antibody against PDL1/CD137 in mice bearing colon cancer.

On day 0, 6-8 week old female C57BL/6 mice with 4-1 human BB (B-hTNFRSF9 (41BB) mice purchased from Beijing Biocytogen Co., Ltd.) were subcutaneously inoculated with 3 × 10 ⁶ MC38 mouse colorectal cancer cells (Shanghai Linyuan Biotechnology Co., Ltd.). On day 6, mice were evenly divided into 8 groups. Tumor-bearing mice in each group were injected intraperitoneally with PBS (twice a week for a total of two weeks), control human IgG-Fc antibody (7.5 mg/kg, twice a week for a total of two weeks), anti-CD137 antibody YN-006 (3 mg/kg, twice a week, for a total of two weeks), anti-PD-L1 antibody YN-035 (7.5 mg/kg, twice a week, for a total of two weeks), anti-CD137 antibody YN-006 (3 mg/kg, twice a week, for a total of two weeks) + anti-PD-L1 antibody YN-035 (7.5 mg/kg, twice a week, for a total of two weeks), bispecific anti-PDL1/CD137 antibody YN-051 (7.5 mg/kg , twice a week, for a total of two weeks), the anti-PD-L1 antibody atezolizumab (7.5 mg/kg, twice a week, for a total of two weeks) and the anti-PD-L1 antibody avelumab (7.5 mg/kg, twice a week , only two weeks), respectively. With the exception of the PBS group, which included 5 mice, each of the other 7 groups included 6 mice. Mice in each group were monitored regularly for changes in body weight and tumor size. The results of the experiment are shown in Fig. 23 and in table. 12. In FIG. 23 showed that YN006, YN035, YN006+YN035, YN051, atezolizumab and avelumab all had significant antitumor activity

- 44 044684 compared to the PBS group or the human IgG-Fc control antibody group. Among them, the antitumor activity of YN006+YN035 and YN051 was significantly higher than that of YN006, YN035, atezolizumab and avelumab. In table Figure 12 shows that by the end of the animal experiment, 5 mice in the YN051 group had completely regressed their tumors, while only 1 mouse in the YN006+YN035 group had completely regressed their tumors, indicating that the therapeutic effect of YN051 was better than YN006+YN035.

Table 12 Number of mice in each group whose tumors completely disappeared

Antibody Number of mice whose tumors completely disappeared/total number of experimental animals in each group PBS 0/5 Human IgGl-Fc 0/6 Atezolizumab 0/6 Avelumab 0/6 YN-006 0/6 YN-035 1/6 YN-006+YN-035 1/6 YN-051 5/6

The foregoing detailed description is presented by way of explanation and examples and is not intended to limit the scope of the claims. At this time, various modifications to the embodiments set forth herein will be apparent to those skilled in the art and are intended to be within the scope of the claims and their equivalents.

Claims (11)

(1) LCDR1: SEQ ID NO: 54, LCDR2: SEQ ID NO: 60, LCDR3: SEQ ID NO: 63, HCDR1: SEQ ID NO: 46, HCDR2: SEQ ID NO: 48 and HCDR3: SEQ ID NO: 50 ; 1. An antibody or antigen-binding fragment thereof capable of binding to the PD-L1 protein, containing a light chain variable region VL, which contains LCDR1-3, and a heavy chain variable region VH, which contains HCDR1-3, where the antibody or antigen-binding fragment thereof contains amino acids sequences selected from any of the following groups: 2. The antibody or antigen binding fragment thereof according to claim 1, wherein said VH heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 31 and SEQ ID NO : 35, and said VL light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41. (2) LCDR1: SEQ ID NO: 55, LCDR2: SEQ ID NO: 60, LCDR3: SEQ ID NO: 64, HCDR1: SEQ ID NO: 47, HCDR2: SEQ ID NO: 48 and HCDR3: SEQ ID NO: 51 ; 3. A bispecific antibody containing a first targeting fragment that specifically binds to the PD-L1 protein, and a second targeting fragment that specifically binds to the CD137 protein, where said first targeting fragment contains an antibody or an antigen-binding fragment according to any one of claims 1, 2 . (3) LCDR1: SEQ ID NO: 55, LCDR2: SEQ ID NO: 60, LCDR3: SEQ ID NO: 64, HCDR1: SEQ ID NO: 47, HCDR2: SEQ ID NO: 48 and HCDR3: SEQ ID NO: 52 ; 4. The bispecific antibody of claim 3, wherein said second targeting fragment comprises an antibody or an antigen binding fragment thereof that comprises a VL light chain variable region that comprises LCDR1-3, wherein the amino acid sequences of said LCDR1-3 are sequentially set forth in SEQ ID NO: 80 -82. (4) LCDR1: SEQ ID NO: 56, LCDR2: SEQ ID NO: 61, LCDR3: SEQ ID NO: 64, HCDR1: SEQ ID NO: 47, HCDR2: SEQ ID NO: 48 and HCDR3: SEQ ID NO: 52 ; 5. The bispecific antibody according to claim 4, wherein said antibody or an antigen-binding fragment thereof from the second targeting fragment comprises a VH heavy chain variable region containing - 45 044684 pressing HCDR1-3, where the amino acid sequences of said HCDR1-3 are sequentially given in SEQ ID NO: 77-79. (5) LCDR1: SEQ ID NO: 57, LCDR2: SEQ ID NO: 61, LCDR3: SEQ ID NO: 64, HCDR1: SEQ ID NO: 47, HCDR2: SEQ ID NO: 48 and HCDR3: SEQ ID NO: 52 ; and (6) LCDR1: SEQ ID NO: 58, LCDR2: SEQ ID NO: 61, LCDR3: SEQ ID NO: 64, HCDR1: SEQ ID NO: 47, HCDR2: SEQ ID NO: 48 and HCDR3: SEQ ID NO: 52. 6. The bispecific antibody according to any one of claims 4, 5, wherein said antibody that binds to the CD137 protein contains a scFv, and said scFv contains the amino acid sequence set forth in SEQ ID NO: 83. 7. The bispecific antibody according to any one of claims 5, 6, containing a first polypeptide chain and a second polypeptide chain, wherein said first polypeptide chain contains said heavy chain variable region of an antibody binding to the PD-L1 protein, said heavy chain variable region of an antibody binding with the CD137 protein, and the specified variable region of the light chain of an antibody that binds to the CD137 protein; and said second polypeptide chain comprises said light chain variable region of an antibody that binds to the PD-L1 protein. 8. The bispecific antibody of claim 7, wherein in said first polypeptide chain, said heavy chain variable region of an antibody binding to the PD-L1 protein is located at the N-terminus of said heavy chain variable region of an antibody binding to the CD137 protein, and said variable region the CD137 protein-binding antibody heavy chain is located at the N-terminus of the CD137 protein-binding antibody light chain variable region; alternatively, said PD-L1 protein-binding antibody heavy chain variable region is located at the N-terminus of said CD 137 protein-binding antibody light chain variable region, and said CD137 protein-binding antibody light chain variable region is located at the N-terminus of said CD137 protein-binding antibody light chain variable region. region of the heavy chain of the antibody that binds to the CD137 protein. 9. The bispecific antibody according to any one of claims 3 to 8, wherein said first polypeptide chain contains the amino acid sequence set forth in SEQ ID NO: 43, and said second polypeptide chain contains the amino acid sequence set forth in SEQ ID NO: 34. 10. A pharmaceutical composition containing an antibody or an antigen-binding fragment thereof according to any one of claims 1, 2 or a bispecific antibody according to any of claims 3-9 and a pharmaceutically acceptable excipient. 11. Use of an antibody, or an antigen-binding fragment thereof according to any one of claims 1, 2 or a bispecific antibody according to any of claims 3-9, or the specified pharmaceutical composition according to claim 10 in the production of a medicinal product for the treatment of tumors.