Anti-PD-l1 and CD40 bispecific antibody and use thereof

Abstract

Provided in the present invention are a bispecific antibody that specifically binds to PD-L1 and CD40 and the use thereof. The bispecific antibody comprises: (1) a PD-L1-binding domain, which is VHH, wherein sequences of CDR1-3 in the VHH are as set forth in SEQ ID NOs: 2-4, respectively; and (2) a CD40-binding domain, comprising VH and VL, wherein sequences of HCDR1-3 in the VH are as set forth in SEQ ID NOs: 21-23, respectively, and sequences of LCDR1-3 in the VL are as set forth in SEQ ID NOs: 18-20, respectively.

Description

Anti-PD-L1 and CD40 bispecific antibodies and uses thereof Technical Field

[0001] The present invention relates to the field of antibody drugs, and in particular to bispecific antibodies that specifically bind to PD-L1 and CD40, and pharmaceutical compositions and uses thereof, especially their application in tumor treatment. Background Art

[0002] PD-L1 (programmed death ligand 1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a type I transmembrane protein expressed on immune cells such as T cells and B cells, as well as tumor cells. When PD-L1 on the tumor cell membrane binds to PD-1 on immune cells such as T cells, the tumor cell sends an inhibitory signal, preventing the T cell from recognizing and killing the tumor cell (Chamoto, K., Al-Habsi, M., & Honjo, T. (2017). “Role of PD-1 in Immunity and Diseases”. Current Topics in Microbiology and Immunology, 410, 75–97). Therefore, anti-PD-1 / PD-L1 monoclonal antibodies (e.g., atezolizumab) have been proposed for the treatment of various solid tumors, including NSCLC, head and neck squamous cell carcinoma, and melanoma (Radvanyi, et al, P. (2013). “Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer”--letter. Clinical cancer research, 19(19), 5541). However, existing clinical trials have shown that the response rate of PD-1 / PD-L1 antibodies is affected by multiple factors such as the level of PD-L1 expression and the quantity and quality of T cells in the tumor. Currently, less than 40% of solid tumors are responsive to treatment.

[0003] CD40 is a type I transmembrane glycoprotein that is a member of the tumor necrosis factor receptor superfamily. It is widely expressed on the surface of immune cells, including B cells, monocytes, macrophages, and dendritic cells. It is also expressed on the surface of non-immune cells, such as epithelial cells, endothelial cells, adipocytes, stromal cells, and platelets. The ligand for human CD40 is also called CD40L or CD154. Current evidence suggests that after activation, CD40 can allow dendritic cells to promote the activation of anti-tumor T cells and affect the tumor microenvironment, converting so-called "cold tumors" into hot tumors (i.e., tumors with significant T cell infiltration), thereby increasing the sensitivity of tumors to immune checkpoint inhibition therapy. Clinically developed agonist CD40 antibodies based on this mechanism of action include selicrelumab (RO7009789) and CDX-1140 with IgG2 isotypes, and sotigalimab (APX005M), ChiLob7 / 4, ADC-1013, Dacetuzumab, and SEA-CD40 with IgG1 isotypes. The activation potency of these CD40 mAbs varies, ranging from strong efficacy (sotigalimab and selicrelumab) to weak efficacy (Dacetuzumab). In addition, different CD40 mAbs have different requirements and abilities for Fc receptor (FcR) cross-linking. Currently, there is no consensus on the optimal CD40 mAb format. See, for example, Vonderheide, RH CD40 Agonist Antibodies in Cancer Immunotherapy. Annu. Rev. Med. 71, 47–58 (2020).

[0004] Given that anti-CD40 agonist antibodies have the effect of activating antigen-presenting cells, drug combinations of anti-CD40 agonist antibodies and anti-PD-1 / PD-L1 antibodies, as well as bispecific antibodies targeting PD-1 / PD-L1 and CD40, have been proposed as a promising anti-cancer development direction (see, for example, WO2023232036A1 and WO2023186113A1). However, based on current research (see, for example, Padrón, LJ et al. Sotigalimab and / or nivolumab with chemotherapy in first-line metastatic pancreatic cancer: clinical and immunologic analyses from the randomized phase 2 PRINCE trial. Nat Med 28, 1167–1177 (2022). https: / / doi.org / 10.1038 / s41591-022-01829-9), finding suitable drug combinations and bispecific antibody formats to maximize the efficacy of anti-tumor drugs while limiting the toxic side effects of the molecules remains a challenge in this field.

[0005] SUMMARY OF THE INVENTION

[0006] In view of the above, the present inventors, through intensive research, constructed a novel bispecific antibody against PD-L1 and CD40, further explored preferred embodiments with optimized antibody configurations and Fc regions, and confirmed the anti-tumor therapeutic efficacy of the constructed bispecific antibody in multiple cold tumor models. Based on these studies, the present inventors established the antibodies of the present invention and their use in anti-tumor therapy.

[0007] Therefore, in a first aspect, the present invention provides anti-PD-L1 and CD40 bispecific antibodies and pharmaceutical compositions and uses thereof, wherein the antibody comprises at least one antigen-binding site that specifically binds to PD-L1 and at least one antigen-binding site that specifically binds to CD40. In some embodiments, the PD-L1 antigen-binding site comprises a VHH domain comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 2-4, respectively. In some embodiments, the CD40 antigen-binding site comprises a VH domain comprising HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 21-23, respectively, and a VL domain comprising LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 18-20, respectively. In some preferred embodiments, the antibody is a trivalent bispecific antibody comprising one PD-L1 antigen-binding site and two CD40 antigen-binding sites per molecule.

[0008] In a second aspect, the present invention provides an anti-PD-L1 antibody and a pharmaceutical composition and use thereof.

[0009] In a third aspect, the present invention provides an immunoconjugate or immunofusion comprising the antibody of the present invention, and pharmaceutical compositions and uses thereof.

[0010] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the present invention, and changes that are readily apparent to those skilled in the art will be included within the spirit of the present invention and the protection scope of the appended claims.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the reporter experiment of Dace-hz03C06.g6-2+1 and Selicrelumab molecules on MC38 cells (PD-L1-Negative).

[0013] Figure 2 shows the reporter experiment of Dace-hz03C06.g6-2+1 and Selicrelumab molecules on MC38 PD-L1 cells (PD-L1-positive).

[0014] Figure 3 shows the activation of DCs by Selicrelumab, Dace-hz03C06.g6-2+1, and Dace-hz03C06.g6-2+2 molecules under conditions of high PD-L1 expression.

[0015] Figure 4 shows the anti-tumor efficacy of the Dace-hz03C06.g6-2+1 antibody drug in the B16F10-hPD-L1 tumor-bearing mouse model.

[0016] FIG5 shows the effect of the Dace-hz03C06.g6-2+1 antibody drug on the body weight changes of mice.

[0017] Figure 6 shows the anti-tumor efficacy of the Dace-hz03C06.g6-2+1 antibody drug in the B16F10-hPD-L1 tumor-bearing mouse model.

[0018] FIG7 shows the effect of the Dace-hz03C06.g6-2+1 antibody drug on the body weight changes of mice.

[0019] Figure 8 shows the anti-tumor efficacy of the Dace-hz03C06.g6-2+1 antibody drug in the KPC-hPD-L1 tumor-bearing mouse model.

[0020] FIG9 shows the effect of the Dace-hz03C06.g6-2+1 antibody drug on the body weight changes of mice.

[0021] FIG10 shows the effects of Dace-hz03C06.g6-2+1 and Selicrelumab antibody drugs on ALT levels in mouse serum.

[0022] FIG11 shows the effects of Dace-hz03C06.g6-2+1 and Selicrelumab antibody drugs on AST levels in mouse serum.

[0023] FIG12 shows the effects of Dace-hz03C06.g6-2+1 and Selicrelumab antibody drugs on the TNF-α level in mouse serum.

[0024] FIG13 shows the effects of Dace-hz03C06.g6-2+1 and Selicrelumab antibody drugs on IL-6 levels in mouse serum.

[0025] FIG14 shows an exemplary molecular schematic diagram of the 2+1 anti-PD-L1 and CD40 bispecific antibody of the present invention.

[0026] Detailed Description of the Invention

[0027] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0028] definition

[0029] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0030] As used herein, the term "and / or", when used in conjunction with a plurality of alternatives, means any one of the alternatives or any two or more or all of the alternatives.

[0031] In this document, when the terms "comprising" or "including" are used, unless otherwise specified, it also covers the situation consisting of the recited elements, integers or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0032] As used herein, the term "antigen binding molecule" refers to a molecule comprising an antigen binding domain or antigen binding site that can bind to a target antigen, such as a protein or polypeptide or a molecule derived therefrom. In the present invention, when the target antigen is PD-L1 and / or CD40, the antigen binding molecule that binds to PD-L1 and / or CD40 is also referred to as a PD-L1 binding molecule, a CD40 binding molecule, or a PD-L1 / CD40 binding molecule. Antigen binding molecules include, for example, antibodies and antigen binding fragments thereof, and various fusions and conjugates constructed based on antibodies or antigen binding fragments, such as VHH-Fc antibodies, immunoconjugates, antibody drug conjugates (ADC), multi- / bispecific antibodies, chimeric antigen receptors (CAR). As will be appreciated by those skilled in the art, the antigen binding site of an antibody typically comprises amino acid residues from a "complementary determining region" or "CDR".

[0033] As used herein, the term "antibody" refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term encompasses various antibody structures, including but not limited to monoclonal antibodies, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy-chain antibodies, chimeric or humanized antibodies, complete antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0034] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to a natural antibody structure or having a heavy chain comprising an Fc-region as defined herein. In some embodiments, a "full-length antibody" is an antibody comprising an antigen-binding variable region and an Fc region. In other embodiments, a "full-length antibody" is an antibody comprising an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. Preferably, a "full-length antibody" comprises two antibody light chains (each comprising a light chain variable domain and a light chain constant domain) and two antibody heavy chains (each comprising a heavy chain variable domain, a hinge region and heavy chain constant domains CH1, CH2 and CH3). The two antibody heavy chains of the full-length antibody may or may not have C-terminal amino acid residues K or GK independently of each other. The constant domains contained in the antibody may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Accordingly, herein, the expression "full-length anti-CD40 antibody" refers to an antibody comprising a CD40 antigen-binding variable region and an Fc region. In some embodiments, preferably, the full-length anti-CD40 antibody consists of two Fab or scFv domains that specifically bind to CD40 and a dimerizing Fc region located at its C-terminus.

[0035] As used herein, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably to refer to molecules other than intact antibodies that comprise a portion of an intact antibody and are capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, crossFab, Fab', Fab'-SH, F(ab')2; diabodies; triabodies; linear antibodies; single-chain antibodies (e.g., scFv or scFab); single-domain antibodies (sdAb); camelid antibodies (heavy chain antibodies) or fragments thereof (e.g., VHH); and monospecific, bispecific, or multispecific antibodies formed from antibody fragments. Unless otherwise specified herein or clearly contradicted by the context, the term "antibody" herein is equivalent to an "antibody or an antibody fragment thereof."

[0036] As used herein, the terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region of an antibody molecule that actually binds to an antigen, which may include, but is not limited to, the variable domain from a heavy chain antibody (i.e., "VHH") and the heavy and light chain variable domain pairs from conventional antibodies (i.e., "VH" and "VL").

[0037] As used herein, the term "bispecific" refers to an antigen binding molecule (e.g., an antibody) having at least two antigen binding sites, wherein at least one of the at least two antigen binding sites binds to a first antigen or a first antigenic epitope, while the remaining antigen binding sites bind to a different second antigen or a second antigenic epitope, e.g., different epitopes on different antigens or different epitopes on the same antigen. In contrast, "monospecific" refers to the ability to bind only one epitope.

[0038] As used herein, the expression "valence" or "valency" in relation to antibodies refers to the total number of antigen binding sites in an antibody molecule, or the number of antigen binding sites with the same antigen binding specificity. For example, a quadrivalent antibody means that the antibody molecule comprises a total of four antigen binding sites; the antibody molecule may be a "2+2" type bispecific antibody, i.e., the antibody has two different antigen binding specificities, wherein two identical antigen binding sites are present for each antigen binding specificity. For another example, a trivalent antibody means that the antibody molecule comprises a total of three antigen binding sites; the antibody molecule may be a "2+1" type bispecific antibody, i.e., the antibody has two different antigen binding specificities, wherein two identical antigen binding sites are present for one antigen binding specificity and only one antigen binding site is present for the other antigen binding specificity.

[0039] In this article, the term "PD-L1" refers to programmed death ligand-1. Unless otherwise indicated, the term includes any variant of human PD-L1, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers species homologs thereof. In addition, it should be understood that the term covers not only PD-L1 naturally or recombinantly expressed by cells or PD-L1 expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins comprising the extracellular domain of PD-L1. An example of PD-L1 is a human PD-L1 protein comprising the amino acid sequence under UniProtKB accession number Q9NZQ7, or a recombinant protein comprising the extracellular domain of the protein (especially, the amino acid sequence of amino acids 19-238). Another example of PD-L1 is monkey PD-L1 protein, or a recombinant protein comprising the extracellular domain of the protein. In this article, unless otherwise specified, the term "PD-L1" refers to PD-L1 derived from humans. Herein, “antigen binding specificity for PD-L1”, that is, “an antigen binding site that specifically binds to PD-L1”, is preferably provided by a VHH domain.

[0040] As used herein, the term "PD-L1-positive" cells refers to cells that express PD-L1 on their cell surface, such as dendritic cells, cancer cells, or modified non-tumor cells. The level of PD-L1 expression on the cell surface can be determined by any conventional method known in the art for determining the level of cell surface antigen expression, such as FACS detection, immunofluorescence staining, or immunohistochemical staining.

[0041] As used herein, the term "CD40" refers to the B-cell surface antigen CD40, also known as tumor necrosis factor receptor superfamily member 5. Unless otherwise indicated, the term encompasses any variant of human CD40, including sequence variants, particularly naturally occurring variants, allelic variants, as well as post-translational modification variants and conformational variants, and encompasses species homologs thereof. Furthermore, it should be understood that the term encompasses not only CD40 naturally or recombinantly expressed by cells or expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins comprising the extracellular domain of CD40. An example of CD40 is a human CD40 protein comprising the amino acid sequence of UniProtKB-P25942, or a recombinant protein comprising the extracellular domain of said protein. Another example of CD40 is a monkey CD40 protein, or a recombinant protein comprising the extracellular domain of said protein. As used herein, unless otherwise indicated, the term "CD40" refers to CD40 of human origin. In the antibodies of the present invention, "antigen-binding specificity for CD40", that is, "an antigen-binding site that specifically binds to CD40", is preferably provided by a pair of VH and VL domains.

[0042] As used herein, the term "CD40-positive" cells refers to cells that express CD40 on their cell surface, such as dendritic cells, B cells, and engineered non-tumor cells. The level of CD40 expression on the cell surface can be determined by any conventional method known in the art for determining the level of cell surface antigen expression, such as FACS detection or immunofluorescence staining.

[0043] In this article, the term "combined positive score" (CPS) related to PD-L1 scoring in tumors is defined as the percentage of viable tumor cells that are PD-L1 positive (partial or complete membrane staining of any intensity) and lymphocytes and macrophages that are PD-L1 positive (membrane or cytoplasmic staining of any intensity) relative to all viable tumor cells. The CPS score can be calculated using the following formula: (sum of PD-L1-stained tumor cells + tumor-associated immune cells) / total number of tumor cells x 100, and is expressed as a value from 0 to 100.

[0044] As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, for example, by detecting the binding ability of an antibody to an antigen by an ELISA assay, or by detecting the binding ability of an antibody to cells expressing the antigen on their surface by a FACS assay, or by detecting the affinity constant KD by SPR technology or BLI technology.

[0045] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigenic epitope). In this context, "binding affinity" reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity can generally be expressed in terms of the binding dissociation equilibrium constant (KD) and can be measured by common methods known in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI). In contrast, as used herein, the term "avidity" or "binding affinity" refers to the combined strength of the interactions of multiple binding sites of a molecule (antibody) with the same target.

[0046] As used herein, the term "high affinity" refers to an antigen binding domain having a binding affinity for its target epitope with a KD value of less than about 100 x 10 -9 M, preferably less than about 50x10 -9 M, about 40x10 -9 M, about 30x10 -9 M or approximately 20x10 -9 M, more preferably less than about 10x10 -9 M.

[0047] As used herein, "blocking activity" of an antibody against PD-L1 refers to the ability of the antibody to block the binding of PD-L1 to the receptor PD-1 and / or reduce PD-L1 / PD-1 signaling. An assay for determining this blocking activity can be, for example, a reporter gene-based signaling pathway blocking assay, such as the reporter gene-based assay described in Example 2. The blocking activity of the test antibody against PD-L1 can be determined by reference to the levels of PD-L1 / PD-1 binding or PD-L1 / PD-1 signaling in the absence of the antibody and / or in the presence of a positive antibody.

[0048] As used herein, the "activation activity" of an antibody on CD40 refers to the ability of the antibody to activate the CD40 signaling pathway. An assay for determining this activation activity can be, for example, a reporter gene-based signaling pathway activation assay, such as the reporter gene-based assay described in Example 6. The CD40 activation activity of a test antibody can be determined by reference to the level of CD40 signaling in the absence of the antibody and / or in the presence of a positive antibody.

[0049] As used herein, the expression "selectivity" or "PD-L1-dependence" in relation to the CD40 activation activity of an antibody means that the antibody preferentially activates the CD40 signaling pathway in the presence of PD-L1-mediated antibody cross-linking (or with high levels of PD-L1-mediated antibody cross-linking) relative to the absence of PD-L1-mediated antibody cross-linking (or with only low levels of PD-L1-mediated antibody cross-linking). This selectivity of an antibody can be characterized by a selectivity index (i.e., the ratio of the EC50 values ​​for activation of CD40-expressing cells by the antibody under these two conditions).

[0050] As used herein, the term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also known as a light chain variable domain, followed by a light chain constant domain (CL). The heavy chains of immunoglobulins can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant region, some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be assigned to one of two classes, called κ and λ, based on the amino acid sequence of their constant domains.

[0051] In this article, the term "isotype" refers to the antibody type determined by the constant region of the heavy chain of the antibody. For example, the antibody according to the present invention can be IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM and IgD antibodies, and has a heavy chain constant region of the immunoglobulin type. In this article, it should be understood that when referring to an antibody having a certain isotype, not only antibodies with a native sequence constant region of the isotype are encompassed, but also antibodies that contain a few mutations in the constant region and still belong to the isotype.

[0052] As used herein, the term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in binding an antibody to an antigen. In the case of conventional antibodies, the heavy chain variable domain (VH) is paired with the light chain variable domain (VL) to confer antigen binding specificity. In the case of heavy chain antibodies, such as heavy chain antibodies from Camelidae, a single VH domain (also referred to herein as a VHH domain) may be sufficient to confer antigen binding specificity. The VHH domain, like the heavy and light chain variable regions of conventional IgG antibodies, comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs), and is arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CD3-FR4. In some aspects of the present invention, one or more residues in the variable region of an antibody may be modified, for example, by residue modification in one or more CDR regions and / or by substitution of conserved residues in one or more framework regions to obtain an antibody variant that substantially retains at least one biological property (e.g., antigen binding ability) of the parent antibody. In further aspects, the antibody variable region may be modified by CDR transplantation. Since CDR sequences are responsible for most antibody-antigen interactions, antibody variants that mimic the properties of known antibodies can be constructed. In such antibody variants, CDR sequences from known antibodies are transplanted onto the framework regions of different antibodies with different properties, and one to several residue mutations, such as back mutations, can be performed as needed to refine the desired properties of the antibody. The properties of the mutated and / or modified antibody can be evaluated in in vitro or in vivo assays, such as target antigen binding properties or other desired functional properties, such as CD40 activation activity, and / or in vivo tumor killing activity. Therefore, the present invention also contemplates variants of any variable region (e.g., VHH, VH / VL region) given herein. In some cases, for therapeutic applications of antibodies or their derivative molecules, it is desirable to reduce their immunogenicity. Therefore, in some embodiments, preferably, the antibody comprises a humanized variable domain (e.g., VHH domain and / or VH / VL domain).

[0053] As used herein, "complementarity determining region" is used interchangeably with "CDR region," "CDR," and "hypervariable region" and refers to regions in an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. In a VHH domain, CDRs are numbered sequentially from the N-terminus and are typically referred to as CDR1, CDR2, and CDR3. In a VH domain and a VL domain, CDRs are numbered sequentially from the N-terminus and are typically referred to as HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. The CDR sequences in a defined variable region (e.g., a VHH domain or a VH / VL domain) can be determined using methods well known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes to define the regional extent of CDRs and their combined extents. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-described ways and combinations thereof. In addition, it is known in the art that, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. Such a minimum binding unit can be a sub-portion of a CDR. The residues of the rest of the CDR sequence, as will be appreciated by those skilled in the art, can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues can be replaced.

[0054] Unless otherwise indicated, throughout the present invention, references to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues) are to positions numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0055] Herein, the terms "VHH" and "VHH domain" are used interchangeably to refer to heavy chain variable domains derived from heavy chain antibodies lacking light chains, sometimes also referred to as single variable domain fragments (sVD) or single domain antibodies (sdAb). Therefore, VHH is different from the conventional VH of four-chain immunoglobulins in that it does not need to be paired with a light chain variable domain to form an antigen binding site. Such VHH molecules can be derived from antibodies produced in Camelidae species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains, and VHHs from such heavy chain antibodies are also within the scope of the present invention.

[0056] Herein, the term "Fab domain" is used to refer to a structure similar to that formed by the pairing of the heavy chain variable region VH and the heavy chain constant region CH1 (VH-CH1) with the complementary light chain variable region VL and the light chain constant region CL (VL-CL) in conventional four-chain IgG antibodies. The term also encompasses a structure in which CH1 and CL are exchanged, that is, a structure formed by pairing VH-CL with VL-CH1. In some embodiments, as shown in Figure 14, the Fab domain can be fused to the N-terminus of the immunoglobulin Fc region.

[0057] As used herein, the term "scFv domain" refers to a single-chain polypeptide comprising a VH domain and a VL domain connected by a flexible linker, wherein the VH domain and the VL domain on the polypeptide chain pair to form an antigen-binding domain responsible for antigen binding. In some embodiments, the scFv domain can be fused to the N-terminus or C-terminus of the Fc region of an immunoglobulin.

[0058] Herein, the term "immunoglobulin Fc region" is used interchangeably with "Fc region" and "Fc domain" to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the heavy chain constant region. Herein, the term "Fc region" or "Fc domain" excludes the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin, as well as the heavy chain constant region (CH1) and light chain constant region (CL); however, it may include the CH2 and CH3 domains, and may or may not include the immunoglobulin hinge region. For example, in some instances, the Fc region may include or consist of the CH2 and CH3 domains from the N-terminus to the C-terminus. In other examples, the Fc region may comprise, from the N-terminus to the C-terminus, an immunoglobulin hinge region or a portion of an immunoglobulin hinge region (or a connecting peptide that can replace the hinge region), a CH2 domain, and a CH3 domain, or may be composed of the immunoglobulin hinge region or a portion of the hinge region (or a connecting peptide that can replace the hinge region), a CH2 domain, and a CH3 domain. The sequence constituting the Fc region may be a native sequence or a variant sequence. Therefore, the term "Fc region" encompasses both native sequence Fc regions and variant Fc regions herein. The Fc region that can be used in the antibodies of the present invention includes, but is not limited to, an Fc region of IgG1, IgG2, IgG3, or IgG4 having a native sequence or a variant sequence.

[0059] Unless otherwise indicated herein, amino acid residue numbering in the Fc region and the heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., SEQuences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0060] In this article, the term "native sequence Fc region" encompasses naturally occurring various immunoglobulin Fc region sequences, such as various Ig subtypes and the Fc region sequences of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the heavy chain carboxyl terminus. However, the lysine (Lys447) or glycine-lysine (Gly446Lys447) at the C-terminal end of the Fc region may be present or absent. In some embodiments, human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminal end, such as, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225.

[0061] As used herein, the term "variant sequence Fc region" refers to an Fc region polypeptide that comprises modifications relative to a native sequence Fc region polypeptide. The modifications may be additions, deletions, and / or substitutions of amino acid residues. Substitutions may include naturally occurring amino acid substitutions and non-naturally occurring amino acid substitutions. The purpose of the modifications may be to alter the binding of the Fc region to its receptor and the effector functions thereby elicited, or to prevent undesirable heavy chain mispairing, or to introduce site-specific amino acid mutations that may contribute to interchain disulfide bond formation.

[0062] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include Fc receptor binding, C1q binding, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Depending on the intended use of the antibody molecule, the Fc region of the antibody can be modified to have altered effector functions, such as reduced or abolished Fcγ receptor binding, relative to an antibody molecule having a wild-type Fc region.

[0063] Herein, when an antibody comprises an Fc region, the antibody polypeptide chain comprising the Fc region is also referred to as a heavy chain, and the antibody polypeptide chain not comprising the Fc region is also referred to as a light chain. Accordingly, the term "heavy chain" in relation to a full-length antibody refers to the antibody chain comprising the Fc region in the full-length antibody, while "light chain" refers to the antibody chain not comprising the Fc region in the full-length antibody. In some cases, the full-length antibody may or may not have a covalently linked antigen-binding domain, such as a VHH domain, at the C-terminus of the heavy chain Fc region.

[0064] As used herein, the terms "flexible linker peptide" or "linker" or "connector peptide" are used interchangeably to refer to a short amino acid sequence consisting of amino acids, such as peptides of glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, or from the hinge region of an immunoglobulin.

[0065] As used herein, the "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identified as identical to the amino acid residues at corresponding positions in the specific amino acid sequence, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and, if necessary, introducing gaps to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of sequence identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules specifically disclosed herein. The variants may comprise conservative modifications.

[0066] For polypeptide sequences, "conservative modifications" include substitutions of the polypeptide sequence that result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservative replacements for each other: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M). In some embodiments, the term "conservative modification" is particularly used to refer to amino acid modifications that do not significantly affect or change the desired properties (e.g., binding characteristics and / or CD activation characteristics) of the parent polypeptide (e.g., parent antibody) to which the modification is to be introduced.

[0067] Herein, a "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs in the humanized antibody correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. Herein, in some embodiments, the humanized antibodies of the present invention have a framework region sequence that is "derived from" a specific human germline sequence. Here, "derived from" means that the amino acid sequence of the antibody framework region has at least 90%, 93%, 95%, 96%, 97%, 98% or higher identity to the corresponding framework region amino acid sequence encoded by the human germline immunoglobulin gene, and that the antibody maintains antigen binding activity.

[0068] As used herein, "isolated" antibodies refer to artificial antibodies, recombinantly produced antibodies, and antibodies that have been at least partially separated from components of the natural environment in which they were produced. In some embodiments, the antibodies of the present invention are "isolated" antibodies. In some embodiments, the isolated antibodies are purified to greater than 90%, 95%, or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0069] Herein, if the variable region amino acid sequence (e.g., VHH or VH / VL pair) is specific for two different antigens or antigenic determinants (e.g., PD-L1 or CD40 from different mammalian species, such as human PD-L1 and cynomolgus monkey PD-L1, or human CD40 and cynomolgus monkey CD40), it is said to be "cross-reactive" to these two different antigens or antigenic determinants. It would be advantageous for the antibody to have human-monkey species cross-reactivity, especially having similar human-monkey antigen binding affinity, as this property can facilitate preclinical drug development of the antibody. In some embodiments, the antibodies of the present invention preferably have human-monkey PD-L1 cross-reactivity and / or human-monkey CD40 cross-reactivity.

[0070] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0071] As used herein, the term "expression vector" refers to a vector capable of directing the expression of a nucleotide sequence to which it is operably linked. An expression vector typically contains cis-acting elements for expression of the nucleotide sequence; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include, for example, but are not limited to, cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0072] As used herein, the term "immunoconjugate" or "immunoconjugate" generally refers to a molecule formed by conjugating or fusing one or more immunoglobulin-related molecules or fragments thereof (e.g., antibodies or fragments thereof) to one or more other molecules. In some cases, the other molecules may be proteinaceous molecules, such as peptides, polypeptides, or proteins. In some cases, the other molecules may also be non-proteinaceous molecules, for example, chemical toxins. In some cases, the other molecules may be immunoglobulin-related molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-related molecules or fragments thereof. The one or more other molecules may be the same or different from each other. For example, the other molecules may be target binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules, etc.

[0073] As used herein, the terms "individual" or "subject" are used interchangeably to refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, an individual particularly refers to a human individual.

[0074] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving prognosis. In the context of tumor or cancer treatment, "treatment" encompasses anti-tumor biological effects that can be induced by human intervention (e.g., by the administration of a drug), including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0075] As used herein, the term "prevention" refers to a medical intervention performed before the onset of at least one symptom of a disease to inhibit, delay, or prevent the onset or development of a disease or a particular disease symptom. Thus, in some embodiments, prevention includes administering a drug to a subject before the onset of a disease or symptom.

[0076] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors. Specific implementation plan

[0077] With the introduction of new immunotherapeutic drugs, the treatment of cancer has undergone revolutionary changes. However, the design and development of successful anti-cancer therapies still face many challenges. In the process of exploring antibody drugs targeting inhibitory immune checkpoints and co-stimulatory checkpoints, the inventors constructed new anti-PD-L1 and CD40 bispecific antibodies. Therefore, in some aspects, the present invention provides novel anti-PD-L1 and CD40 bispecific antibodies. In some embodiments, the antibody comprises a high-affinity PD-L1 antigen-binding domain, such as a PD-L1 antigen-binding domain having a CDR sequence of SEQ ID NOs: 2-4; and a CD40 antigen-binding domain with weak agonistic activity in the absence of cross-linking, such as a CD40 antigen-binding domain having a CDR sequence of SEQ ID NOs: 18-23. In some embodiments, the antibodies of the present invention exhibit PD-L1-dependent CD40 activation and / or enhanced ability to selectively activate antigen-presenting cells (such as dendritic cells) compared to non-PD-L1-dependent agonist CD40 antibodies.

[0078] Based on the above antibodies, in some aspects of the present invention, the inventors further explored and established preferred embodiments of the antibodies of the present invention with different antibody configurations. According to previous reports, CD40 and PD-L1 bispecific antibodies mostly adopt a 2+2 type molecular design. In order to further optimize the antibodies, the inventors explored the effects of different antibody configurations on the efficacy of the bispecific antibodies of the present invention. Studies have shown that compared with the reported 2+2 type molecular configuration, the antibodies of the present invention with a 2+1 configuration (ie, each molecule contains 2 anti-CD40 antigen binding sites and 1 anti-PD-L1 antigen binding site) can better achieve full utilization of the PD-L1 anchoring molecules in the target tissue at the same PD-L1 abundance and antibody molecule dose, thereby maximizing the efficacy. Therefore, in some preferred embodiments of the present invention, the present invention provides a 2+1 type anti-PD-L1 and CD40 bispecific antibody of the present invention. In some more preferred embodiments, the antibody comprises 2 anti-CD40 Fabs and 1 anti-PD-L1 VHH. In some more preferred embodiments, the bispecific antibody comprises or consists of an anti-CD40 full-length IgG antibody and an anti-PD-L1 VHH domain conjugated thereto (e.g., conjugated to the C-terminus of one of its antibody heavy chains), for example, having the structure shown in FIG14 .

[0079] On the basis of the above antibodies, in other aspects, the inventors have further explored and established preferred embodiments of the antibodies of the present invention with different Fc regions. A major problem faced by antibodies with CD40 agonistic activity is large systemic toxic side effects and low tolerance doses. It has been reported that Selicrelumab with an IgG2 isotype can cause cytokine release syndrome and hepatotoxicity and other side effects in some patients when the dosage exceeds 0.2 mg / kg in clinical trials (WO2023 / 232036). It is speculated that one of the reasons for this large toxic side effect may come from the Fc region of the antibody. Previous literature (Xiaojie Yu et al, Isotype Switching Converts Anti-CD40 Antagonism to Agonism to Elicit Potent Antitumor Activity, Cancer Cell. 2020 Jun 8; 37(6): 850-866.e7.doi: 10.1016 / j.ccell.2020.04.013) suggests that the Fc region with the IgG2 isotype has the potential to promote the agonistic activity of CD40 antibodies, but because it can induce CD40 clustering and activation independently of the Fc-γ receptor, it is also more likely to induce non-tumor-specific immune activation in vivo, resulting in greater toxic side effects, thereby limiting the tolerable dosage and corresponding efficacy of the antibody in clinical practice (Xiaojie Yu et al, Cancer Cell. 2020 Jun 8; 37(6): 850-866.e7.doi: 10.1016 / j.ccell.2020.04.013). In view of this, the inventors have investigated the effects of different Fc regions on the efficacy and safety of the bispecific antibody molecules of the present invention, and proposed an optimized Fc region design that is beneficial to the antibodies of the present invention. In some embodiments, the optimized Fc region has an IgG1 isotype and has modifications (such as amino acid mutations or glycosylation modifications) that reduce or eliminate its binding activity to Fcγ receptors. Compared to the IgG2 Fc region previously reported in the literature, studies have shown that the optimized Fc region of the present invention can, on the one hand, eliminate the interaction between the antibody Fc and the Fcγ receptor, and on the other hand, does not have the non-specific agonistic activity of CD40 brought by IgG2, thereby greatly reducing the non-specific toxicity outside the tumor of the bispecific antibody molecule of the present invention, improving its safety and tolerable dose and the subsequent antibody effectiveness. Therefore, in some preferred embodiments of the present invention, the present invention provides a bispecific antibody with an optimized Fc region design, wherein the Fc region is an IgG1 Fc region with a mutation that reduces or eliminates Fcγ receptor binding activity, wherein the mutation can especially be a LALA mutation (L234A, L235A).

[0080] In the efficacy confirmation study of the antibody molecule of the present invention, a variety of different cold tumor models were used to match the potential clinical application scenarios of the antibody molecule, including B16F10-hPDL1 (Urska Kamensek et al., Bioelectrochemistry 140 (2021) 107831, doi.org / 10.1016 / j.bioelechem.2021.107831) and KPC-hPDL1 cold tumor models (doi.org / 10.3390%2Fcancers13030440), to test the bispecific antibody of the present invention. The results of the study showed that the antibody of the present invention has good pharmacodynamic efficacy on tumors that are resistant to PD-1 / L1 immune checkpoint inhibitors and is an anti-cancer drug candidate with broad clinical application prospects.

[0081] The components of the bispecific antibodies of the present invention are described in detail below. Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of any technical features of these components is contemplated by the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, the antibodies of the present invention (including any form of antibody) may include any such combination of features.

[0082] Antigen binding site

[0083] In the study, the inventors found that, in some cases, combining a PD-L1 antigen binding site with high binding affinity with a CD40 antigen binding site that has only weak agonistic activity in the absence of cross-linking can advantageously confer advantages in efficacy and safety to the bispecific antibodies of the present invention.

[0084] Therefore, in some embodiments according to the present invention, the present invention provides a bispecific antibody comprising a PD-L1 antigen-binding site that binds to PD-L1 with high affinity and a CD40 antigen-binding site that has only weak agonistic activity in the absence of cross-linking. Preferably, the CD40 antigen-binding site is a CD40 antigen-binding site that selectively activates the CD40 signaling pathway in a PD-L1-dependent manner.

[0085] In some embodiments, the PD-L1 antigen binding site is less than about 30x10 -9 M, less than approximately 20x10 -9 M, less than approximately 15x10 -9 M, or less than 10x10 -9 In some embodiments, the PD-L1 antigen binding site binds to PD-L1 with a KD value of approximately 10×10 -9 M to 0.1x10 -9The KD value of M is, for example, approximately 9x10 -9 M, about 8x10 -9 M, about 7x10 -9 M, about 6x10 -9 M, about 5x10 -9 M, about 4x10 -9 M, about 3x10 -9 M, about 2x10 -9 M or approximately 1x10 -9 In some embodiments, the PD-L1 antigen binding site preferably binds to PD-L1 with a KD value of approximately 5x10 -9 M to 0.5x10 -9 The KD value of the present invention is about 1 nM to about 5 nM, and preferably about 1 nM to about 5 nM, for binding to PD-L1. In some embodiments, the KD value is determined using a biofilm interferometry technique, such as described in Example 8.

[0086] In some embodiments, the CD40 antigen binding site selectively activates the CD40 signaling pathway in a PD-L1-dependent manner, preferably, the selectivity index, defined as the ratio of the EC50 values ​​of the antibody in the absence and presence of PD-L1-expressing cells, is greater than 100, for example, greater than 150, greater than 200, greater than 300, and for example, about 300-1000, 500-1000, or 700-1000. In some embodiments, the EC50 value is determined and the selectivity index is calculated using a fluorescent reporter assay in the presence and absence of PD-L1 (high) expressing cells, for example, as described in Example 6.

[0087] In some embodiments, the PD-L1 antigen binding site is cross-reactive with human and monkey PD-L1. In other embodiments, the CD40 antigen binding site is cross-reactive with human and monkey CD40.

[0088] In some embodiments, the PD-L1 antigen binding site is provided by an antigen binding domain that specifically binds to PD-L1, and the antigen binding domain can be selected from, for example but not limited to, a VHH domain, a single domain antibody, a VH / VL domain pair, a Fab domain, and a scFv domain. Preferably, the antigen binding domain comprises or consists of a VHH domain.

[0089] In some embodiments, the CD40 antigen binding site is provided by an antigen binding domain that specifically binds to CD40, which can be selected from, for example but not limited to, a VHH domain, a single domain antibody, a VH / VL domain pair, a Fab domain, and a scFv domain. Preferably, the antigen binding domain comprises or consists of a Fab domain or a scFv domain.

[0090] In some embodiments, the PD-L1 antigen binding site is provided by a VHH domain; and the CD40 antigen binding site is provided by a Fab domain.

[0091] Exemplary PD-L1 antigen binding sites

[0092] Herein, the PD-L1 antigen binding site according to the present invention is also referred to as the anti-PD-L1 domain according to the present invention. In the bispecific antibodies according to the present invention, in some embodiments, preferably, the PD-L1 antigen binding site according to the present invention is provided by a VHH domain (or simply referred to as an "anti-PD-L1 VHH domain").

[0093] In some embodiments, the anti-PD-L1 VHH domain according to the present invention comprises a CDR1, CDR2, and CDR3 sequence in the variable region having the amino acid sequence shown in SEQ ID NOs: 5 or 6. Preferably, the CDR sequences in the variable region are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDRs are defined according to Kabat. However, it should be understood that the CDRs may also be defined in any other manner known in the art.

[0094] In some embodiments, the anti-PD-L1 VHH domain according to the present invention comprises CDR1, CDR2 and CDR3 sequences, wherein

[0095] (i) the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 2;

[0096] (ii) the CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 3;

[0097] (iii) the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0098] In some embodiments, an anti-PD-L1 VHH domain according to the present invention comprises a VHH variable region sequence of any of the exemplary anti-PD-L1 antibodies of the present invention, or a variant thereof. For example, the VHH domain can have the same CDR sequences as one of the exemplary antibodies, and the same or different framework region sequences, such as a VHH domain with a humanized sequence. The VHH domain can be evaluated in in vitro or in vivo assays to determine whether it maintains or improves its PD-L1 antigen-binding properties or other functional properties.

[0099] In some embodiments, the anti-PD-L1 VHH domain according to the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 5 or 6. In further embodiments, the VHH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 5 or 6 and retains the ability to specifically bind to PD-L1. In further embodiments, the VHH domain comprises an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions (e.g., conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5 or 6 and retains the ability to specifically bind to PD-L1. Preferably, the amino acid additions, deletions and / or substitutions do not occur in the CDR region.

[0100] In some preferred embodiments, the anti-PD-L1 VHH domain according to the present invention comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0101] Exemplary CD40 antigen binding sites

[0102] Herein, the CD40 antigen binding site according to the present invention is also referred to as the anti-CD40 domain according to the present invention. In some embodiments, preferably, the CD40 antigen binding site according to the present invention is provided by a paired VH domain and VL domain. In some embodiments, the CD40 antigen binding site according to the present invention is provided by a Fab domain (or simply referred to as an "anti-CD40 Fab domain") or a scFv domain (or simply referred to as an "anti-CD40 scFv domain").

[0103] In some embodiments, the anti-CD40 domain according to the present invention is derived from an anti-CD40 antibody, and preferably comprises a VH / VL domain pair from such an antibody. Examples of anti-CD40 antibodies that can be used in the present invention include, for example, but are not limited to, the S2C6 antibody (U.S. Patent No. 6,946,129) or a humanized version thereof (see, for example, WO 2006 / 128103 and WO 2008 / 091954).

[0104] In some embodiments, the anti-CD40 antibody has one or more or all of the following properties:

[0105] - Cross-immunoreactivity with human CD40 protein and cynomolgus macaque CD40 protein;

[0106] -Activates the CD40 signaling pathway.

[0107] In some embodiments, the anti-CD40 domain according to the present invention comprises a VH and a VL domain, wherein the VH domain comprises the HCDR1, HCDR2, and HCDR3 sequences of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 17; and the VL domain comprises the LCDR1, LCDR2, and LCDR3 sequences of the light chain variable region having the amino acid sequence of SEQ ID NO: 16. Preferably, the CDR sequences in the variable region are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDRs are defined according to Kabat. However, it should be understood that the CDRs may also be defined using any other method known in the art.

[0108] In some embodiments, an antigen binding site that specifically binds CD40 according to the present invention comprises:

[0109] (i) HCDR1 comprising or consisting of SEQ ID NO: 21;

[0110] (ii) a HCDR2 comprising or consisting of SEQ ID NO: 22;

[0111] (iii) a HCDR3 comprising or consisting of SEQ ID NO: 23;

[0112] (iv) LCDR1 comprising or consisting of SEQ ID NO: 18;

[0113] (v) LCDR2 comprising or consisting of SEQ ID NO: 19; and

[0114] (vi) LCDR3 comprising or consisting of SEQ ID NO: 20.

[0115] In some embodiments, a CD40 antigen-binding site according to the present invention comprises the VH / VL variable region sequence of any of the exemplary anti-CD40 antibodies of the present invention, or a variant thereof. For example, the CD40 antigen-binding site can have the same CDR sequences as one of the exemplary antibodies and the same or different framework region sequences, such as an anti-CD40 antibody fragment having a humanized sequence. The CD40 antigen-binding site can be evaluated for retention or improvement of antigen-binding properties or other functional properties in in vitro or in vivo assays.

[0116] In some embodiments, the CD40 antigen-binding site according to the present invention comprises a heavy chain variable region VH and a light chain variable region VL. In some embodiments, the VH comprises or consists of the amino acid sequence of SEQ ID NO: 17. In other embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 17 and retains the ability to specifically bind to CD40. In other embodiments, the VH comprises an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions (e.g., conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 17 and retains the ability to specifically bind to CD40. Preferably, the amino acid additions, deletions and / or substitutions do not occur in the CDR region. In some embodiments, the VL comprises or consists of the amino acid sequence of SEQ ID NO: 16. In other embodiments, the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 16 and retains the ability to specifically bind to CD40. In other embodiments, the VL comprises an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions (e.g., conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16 and retains the ability to specifically bind to CD40, preferably, the amino acid additions, deletions and / or substitutions do not occur in the CDR regions.

[0117] In some preferred embodiments, the CD40 antigen-binding site according to the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 17, or consists of the amino acid sequence shown in SEQ ID NO: 17; and the VL comprises the amino acid sequence of SEQ ID NO: 16, or consists of the amino acid sequence shown in SEQ ID NO: 16.

[0118] Immunoglobulin Fc region

[0119] In some embodiments, the bispecific antibodies of the present invention may comprise an immunoglobulin Fc region. The native Fc region is the C-terminal constant domain of an immunoglobulin that interacts with cell surface Fc receptors and some proteins of the complement system. The immunoglobulin Fc region typically comprises two or three heavy chain constant domains (referred to as CH2, CH3, and CH4) and optionally a hinge region, and typically exists in a dimerized form.

[0120] The immunoglobulin Fc region used in the bispecific antibodies of the present invention can be an Fc region from any immunoglobulin. In some embodiments, the immunoglobulin Fc region comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a hinge region or a portion of a hinge region. In some embodiments, the immunoglobulin Fc region comprises, or consists of, an immunoglobulin hinge region or a portion of a hinge region, a CH2 domain, and a CH3 domain from N-terminus to C-terminus. In some cases, an immunoglobulin Fc region comprising a hinge region sequence is preferred, as it can, for example, promote dimerization of antibody polypeptide chains.

[0121] The immunoglobulin Fc region can be fused to the C or N terminus of other domains (i.e., PD-L1 or CD40 binding domains). The immunoglobulin Fc can be fused to other domains through a linker, or directly fused to other domains. In embodiments where the immunoglobulin Fc region is fused to other domains at its N terminus, the fusion is preferably performed through an immunoglobulin hinge region sequence. In embodiments where the immunoglobulin Fc region is fused to other domains at its C terminus, the fusion is preferably performed through a flexible connecting peptide (such as the connecting peptide of SEQ ID NO: 30).

[0122] The immunoglobulin Fc region used in the bispecific antibodies of the present invention can be a native Fc region sequence. Alternatively, the Fc region can comprise mutations relative to the native Fc sequence. Mutations include substitutions, insertions and / or deletions. Such mutations can be performed for the purpose of introducing desired therapeutic properties. For example, to promote heterodimerization, a Knob-into-Hole (KiH) mutation can be introduced into the CH3 domain. In this case, one Fc chain is designed to include a large protruding residue (i.e., Knob), while the other Fc chain is designed to include a complementary pocket (i.e., Hole). Suitable positions for KiH mutations are known in the art. Exemplary KiH mutations include, but are not limited to, a combination of Knob mutation T366W and Hole mutations T366S, L368A, Y407V; and a combination of Knob mutation T366Y and Hole mutation Y407T. For dimerized Fc with KiH mutations and their use in engineered antibodies, see, for example, Carter P.; Ridgway JBB; Presta LG: Immunotechnology, Vol. 2, February 1, 1996, pp. 73-73. When the bispecific antibody of the present invention comprises an asymmetric two-chain structure, it is preferred that the Fc region comprises a KiH mutation that promotes correct heterodimerization of the antibody polypeptide chains.

[0123] The immunoglobulin Fc region used in the bispecific antibodies of the present invention may include a cysteine ​​residue portion for forming an interchain disulfide bond between heavy chains, for example, a cysteine ​​residue mutation introduced into the Fc region. Exemplary cysteine ​​residue mutations include, but are not limited to, introducing a Y359C mutation on one Fc chain of the dimerized Fc region and introducing an S354C mutation on the other Fc chain.

[0124] Depending on the specific application of the antibody or antibody-based molecule, the immunoglobulin Fc region used in the bispecific antibody of the present invention may include mutations that alter effector function. For example, where effector function is not required, the Fc region may include mutations that reduce or eliminate effector function. Such mutations can be found, for example, in Ian Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished, PLoS ONE 16(12), https: / / doi.org / 10.1371 / journal.pone.0260954. In humans, three types of FcγRs have been characterized, which are:

[0125] - FcγRI (CD64), a receptor that binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modification of the IgG Fc-region at least at one of amino acid residues E233-G236, P238, D265, N297, A327, and P329 (numbering according to the EU index of Kabat) can reduce binding to FcγRI.

[0126] -FcγRII (CD32), a receptor that binds complexed IgG with moderate to low affinity and is widely expressed. This receptor can be divided into two subclasses, FcγRIIA and FcγRIIB. FcγRIIA is expressed on many cells involved in cytotoxicity (e.g., macrophages, monocytes, neutrophils) and is able to activate the killing process. FcγRIIB plays a role in the inhibitory process and is found on B-cells, macrophages, as well as mast cells and eosinophils. It has been found that mutating the IgG Fc-region at least at one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414 (numbering according to the EU index of Kabat) can reduce binding to FcγRIIA.

[0127] -FcγRIII (CD16), this receptor binds IgG with medium to low affinity and exists in two forms, FcγRIIIA and FcγRIIIB. FcγRIIIA is found on NK cells, macrophages, eosinophils, and some monocytes and T cells, which mediates ADCC. FcγRIIIB is highly expressed on neutrophils. It has been found that IgG Fc-regions with mutations in at least one of amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (numbering according to the EU index of Kabat) have reduced binding to FcγRIIIA.

[0128] In some cases, preferably, the immunoglobulin Fc region used in the bispecific antibody of the present invention comprises a mutation that reduces or eliminates the binding of the Fc region to the Fcγ receptor, such as a LALA mutation in which lysine (L) at positions 234 and 235 of the Fc region is converted to alanine (A). Additionally or alternatively, mutations can be introduced into the Fc region to increase binding to FcRn and / or remove amino acid modifications at protease sites. Additionally or alternatively, the Fc region can be mutated for antibody production reasons, such as removing or replacing amino acids that may undergo post-translational modification (e.g., glycosylation) to provide improved drugability and developability of therapeutic antibodies.

[0129] In some embodiments, bispecific antibodies according to the present invention comprise an Fc region derived from IgG. In some embodiments, preferably, the immunoglobulin Fc region is of the IgG1 isotype or a subtype thereof. In some embodiments, preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from a human. Such Fc region sequences that can be used in antibodies of the present invention include, but are not limited to, amino acid sequences selected from SEQ ID NOs: 25-27, or amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0130] In some embodiments, the bispecific antibody according to the present invention comprises a dimerized Fc region, wherein the dimerized Fc region consists of a first and a second Fc region. In some embodiments, one or both of the first and second Fc regions comprise a mutation that reduces or eliminates binding to Fcγ receptors, preferably, an L234AL235A mutation (numbered according to the Kabat EU index).

[0131] In some preferred embodiments, the first and second Fc regions are identical.In some embodiments, the first and second Fc regions are of the IgG1 isotype.

[0132] In other preferred embodiments, the first and second Fc regions are different from each other. In some embodiments, the first and second Fc regions comprise KiH mutations, and optionally mutations of cysteine ​​residues for forming interchain disulfide bonds between the first and second Fc regions. In some embodiments, the KiH mutation is a combination of Knob mutation T366W and Hole mutation T366S, L368A, Y407V (numbered according to the Kabat EU index). In some preferred embodiments, the first and second Fc regions independently comprise a combination of mutations selected from the following: (a) T366W; and (b) T366S, L368A, Y407V. In other preferred embodiments, the first and second Fc regions independently comprise a combination of mutations selected from the following: (a) S354C and T366W; and (b) Y349C and T366S, L368A, Y407V. In other preferred embodiments, the first and second Fc regions independently comprise a combination of mutations selected from the group consisting of: (a) S354C and T366W and L234AL235A; and (b) Y349C and T366S, L368A, Y407V and L234AL235A mutations. In some embodiments, the first and second Fc regions have an IgG1 isotype.

[0133] In some embodiments, the bispecific antibody according to the present invention comprises a dimeric Fc region formed by two different Fc domains, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto; preferably, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 25 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 26. Preferably, the bispecific antibody is a trivalent bispecific antibody having an asymmetric structure, in particular a trivalent bispecific antibody having the structure shown in Figure 14.

[0134] In other embodiments, the bispecific antibody according to the present invention comprises a dimeric Fc region formed by two identical Fc domains, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 27. Preferably, the bispecific antibody has a symmetrical structure, such as a "2+2 type" tetravalent bispecific antibody.

[0135] Connector peptide

[0136] In the bispecific antibody according to the present invention, the antibody components (ie, the antigen binding domain and optionally the immunoglobulin Fc region) can be connected using a linker peptide.

[0137] There are no specific restrictions on the connecting peptides that can be used in the antibodies of the present invention. The connecting peptide sequence is generally flexible. It can be mainly composed of amino acids such as glycine, alanine and serine that do not have large side chains that may limit flexibility. Alternatively, it can be composed of sequences from the hinge region of an immunoglobulin. Depending on the connection position and the components to be connected, those skilled in the art can easily determine the sequence or optimal length of the available connecting peptide.

[0138] Suitable connecting peptide length can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid lengths, or longer. In some cases, the length of the connecting peptide sequence can be shorter, for example, less than about 20 or 15 amino acid lengths, for example 2-15 amino acid lengths or 5-10 amino acid lengths.

[0139] Suitable connecting peptide sequences include, but are not limited to, G4S (SEQ ID NO: 32); (G4S)2 (SEQ ID NO: 33); (G4S)3 (SEQ ID NO: 34); GGGSG (SEQ ID NO: 35); GGSGG (SEQ ID NO: 36); GSGGG (SEQ ID NO: 37); GSGGGP (SEQ ID NO: 38); GGEPS (SEQ ID NO: 39); GGEGGGP (SEQ ID NO: 40) and GGEGGGSEGGGS (SEQ ID NO: 41); and (G4S)n (SEQ ID NO: 42), wherein n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO: 43), wherein n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO: 44), wherein n is an integer equal to or greater than 1; (G4)n (SEQ ID NO: 45); NO:45), wherein n is an integer equal to or greater than 1; (GRPGS)n(SEQ ID NO:46), wherein n is an integer equal to or greater than 1. The connecting peptide that can be used in the antibody molecule of the present invention can also be, for example, but not limited to, the following amino acid sequences: (G3S)2 (SEQ ID NO:47), (G4S)2 (SEQ ID NO:48), (G3S)3 (SEQ ID NO:49), (G4S)3 (SEQ ID NO:50), (G3S)4 (SEQ ID NO:51), (G4S)4 (SEQ ID NO:52), (G3S)5 (SEQ ID NO:53), (G4S)5 (SEQ ID NO:54), (G3S)6 (SEQ ID NO:55), (G4S) (SEQ ID NO:56), GGG (SEQ ID NO:57), DGGGS (SEQ ID NO:58), TGEKP (SEQ ID NO:59), GGRR (SEQ ID NO:60), EGKSSGSGSESKVD (SEQ ID NO:61), KESGSVSSEQLAQFRSLD (SEQ ID NO:62), GGRRGGGS (SEQ ID NO:63), NO: 63), LRQRDGERP (SEQ ID NO: 64), LRQKDGGGSERP (SEQ ID NO: 65) and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 66). Alternatively, computer programs can be used to simulate the three-dimensional structure of proteins and peptides, or phage display methods can be used to rationally design suitable flexible linker peptides.

[0140] In some embodiments, the connecting peptide used in the antibody of the present invention is a flexible connecting peptide of 5-50 amino acid, preferably a connecting peptide comprising glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 5, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In some embodiments, the connecting peptide comprises an amino acid sequence (G) n, wherein n is an integer equal to or greater than 5, for example, n is an integer of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In further embodiments, the connecting peptide is a hinge region from an immunoglobulin.

[0141] In some preferred embodiments, the bispecific antibody according to the present invention comprises a connecting peptide having an amino acid sequence of SEQ ID NO: 30. In some embodiments, the connecting peptide is used to conjugate the anti-PD-L1 VHH domain according to the present invention to the C-terminus of the Fc region, more preferably, to the C-terminus of one or two (preferably one) heavy chains of an anti-CD40 full-length antibody according to the present invention comprising an Fc region.

[0142] Bispecific antibody formats

[0143] The bispecific antibodies according to the present invention can be in any suitable form, such as single-chain, double-chain, triple-chain or quadruple-chain, symmetrical or asymmetrical. Preferably, the bispecific antibodies according to the present invention are 2+2 antibodies, each molecule comprising two PD-L1 binding sites and two CD40 binding sites. More preferably, the bispecific antibodies according to the present invention are 2+1 antibodies, each molecule comprising one PD-L1 binding site and two CD40 binding sites. In some embodiments, the bispecific antibodies according to the present invention further comprise an Fc region.

[0144] In some embodiments, the present invention provides a bispecific antibody comprising:

[0145] (a) an anti-CD40 full-length antibody comprising a dimerizing Fc region and two CD40 antigen-binding sites according to the present invention connected thereto; and

[0146] (b) one or two (preferably one) PD-L1 antigen binding sites according to the present invention,

[0147] wherein the PD-L1 antigen binding site is conjugated to the full-length antibody, optionally via a linker peptide,

[0148] Preferably, the CD40 antigen binding site is connected to the N-terminus of the dimerized Fc region, and the PD-L1 antigen binding site is connected to the C-terminus of the dimerized Fc region.

[0149] More preferably, the CD40 antigen binding site is selected from Fab, VH / VL pair, scFab, scFv and crossFab, preferably a Fab or scFv domain, more preferably a Fab domain; and the PD-L1 antigen binding site is a VHH domain.

[0150] In some preferred embodiments, the full-length antibody comprises two heavy chains, and the anti-PD-L1 VHH domain is fused to the C-terminus of one heavy chain of the full-length antibody.

[0151] a) the full-length antibody heavy chain fused to an anti-PD-L1 VHH domain has the tripeptide LSP as the C-terminal amino acid residue, wherein a proline thereof is fused to the VHH domain via a linker peptide, and

[0152] b) The full length antibody heavy chain not fused to the VHH domain has the tripeptide LSP or SPG or PGK as the C-terminal amino acid residue.

[0153] Exemplary bispecific antibodies

[0154] In some aspects, the present invention provides a bispecific antibody comprising a first, a second, and a third polypeptide chain, wherein:

[0155] - the first polypeptide chain comprises, from N-terminus to C-terminus, a VH domain, a CH1 domain, an Fc region, a connecting peptide and a VHH domain;

[0156] - the second polypeptide chain comprises a VH domain, a CH1 domain and an Fc region from N-terminus to C-terminus;

[0157] - the third polypeptide chain comprises a VL domain and a CL domain from N-terminus to C-terminus;

[0158] wherein the VHH domain specifically binds to PD-L1, and wherein the VH domain is paired with the VL domain and specifically binds to CD40. Preferably, the VHH domain is an anti-PD-L1 domain according to the present invention, and the VH and VL domains are paired with an anti-CD40 domain according to the present invention.

[0159] In some embodiments, the VH domain comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 21, 22, and 23, respectively, and the VL domain comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0160] Preferably, the VH domain comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 17, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 17; and

[0161] The VL domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 16, or an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16;

[0162] Most preferably, the VH domain comprises the amino acid sequence shown in SEQ ID NO: 17, and the VL domain comprises the amino acid sequence shown in SEQ ID NO: 16.

[0163] In some embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively. Preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 5 or 6, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 5 or 6. Most preferably, the VHH domain comprises, or consists of, the amino acid sequence of SEQ ID NO: 6.

[0164] In some embodiments, the CH1 domain is a human IgG CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 29. Most preferably, the CH1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0165] In some embodiments, the CL domain is a human immunoglobulin kappa or lambda light chain constant domain. In some embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 24. Most preferably, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 24, or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0166] In some embodiments, the Fc regions of the first and second polypeptide chains each comprise a knob mutation and a hole mutation that promote heterodimerization of the first and second polypeptide chains, and optionally, a cysteine ​​residue mutation for forming an interchain disulfide bond between the first and second polypeptide chains. In some embodiments, the knob mutation is T366W; and the hole mutation is T366S, L368A, and Y407V. In some embodiments, the cysteine ​​residue mutation is Y349C introduced into the Fc region of the first polypeptide chain and S354C introduced into the Fc region of the second polypeptide chain, or is S354C introduced into the Fc region of the first polypeptide chain and Y349C introduced into the Fc region of the second polypeptide chain. Furthermore, preferably, the Fc regions of the first and second polypeptide chains each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 25 or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 26 or an amino acid sequence at least 95% identical thereto. More preferably, the Fc region of the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 25 and the Fc region of the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26; or vice versa.

[0167] Still more preferably, the bispecific antibody comprises a first, a second and a third polypeptide chain, wherein:

[0168] The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0169] The second polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8; and

[0170] The third polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:7.

[0171] More preferably, the bispecific antibody comprises a first, a second and a third polypeptide chain having the amino acid sequences shown in SEQ ID NOs: 9, 8 and 7, respectively. Most preferably, the bispecific antibody is a trivalent bispecific antibody consisting of a first polypeptide chain, a second polypeptide chain and two light chains.

[0172] In other aspects, the present invention provides a bispecific antibody or antigen-binding fragment thereof, wherein the bispecific antibody comprises a first and a second polypeptide chain, wherein:

[0173] - the first polypeptide chain comprises, from N-terminus to C-terminus, a VH domain, a CH1 domain, an Fc region, a connecting peptide and a VHH domain;

[0174] - the second polypeptide chain comprises a VL domain and a CL domain from N-terminus to C-terminus;

[0175] wherein the VHH domain specifically binds to PD-L1, and wherein the VH domain is paired with the VL domain and specifically binds to CD40. Preferably, the VHH domain is an anti-PD-L1 domain according to the present invention, and the VH and VL domains are paired with an anti-CD40 domain according to the present invention.

[0176] In some embodiments, the VH domain comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 21, 22, and 23, respectively, and the VL domain comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 18, 19, and 20, respectively.

[0177] Preferably, the VH domain comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 17, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 17; and

[0178] The VL domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 16, or an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16;

[0179] Most preferably, the VH domain comprises the amino acid sequence shown in SEQ ID NO: 17, and the VL domain comprises the amino acid sequence shown in SEQ ID NO: 16.

[0180] In some embodiments, the VHH domain comprises CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively. Preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 5 or 6, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 5 or 6. Most preferably, the VHH domain comprises, or consists of, the amino acid sequence of SEQ ID NO: 6.

[0181] In some embodiments, the CH1 domain is a human IgG CH1 domain. In some embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 29. Most preferably, the CH1 domain comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0182] In some embodiments, the CL domain is a human immunoglobulin kappa or lambda light chain constant domain. In some embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 24. Most preferably, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 24, or consists of the amino acid sequence shown in SEQ ID NO: 24.

[0183] In some embodiments, the Fc region of the first polypeptide chain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 27, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 27. Most preferably, the Fc region comprises, or consists of, the amino acid sequence of SEQ ID NO: 27.

[0184] In some embodiments, preferably, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and

[0185] The second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0186] More preferably, the bispecific antibody comprises a first and a second polypeptide chain having the amino acid sequences shown in SEQ ID NOs: 11 and 10, respectively. Most preferably, the bispecific antibody is assembled into a tetravalent bispecific antibody by two first polypeptide chains and two second polypeptide chains.

[0187] Properties of the antibodies of the present invention

[0188] The bispecific antibodies of the present invention may have one or more of the following properties:

[0189] (i) binds with high affinity to cells expressing PD-L1 (preferably tumor cells or antigen-presenting cells, such as DCs);

[0190] (ii) having species cross-reactivity with cynomolgus monkey PD-L1;

[0191] (iii) having species cross-reactivity with cynomolgus monkey CD40; and

[0192] (iv) It can selectively activate the CD40 signaling pathway and activate DC cells depending on PD-L1.

[0193] In some aspects, according to the bispecific antibodies of the present invention, the PD-L1 binding site is used as a medium for cross-linking of the antibody, giving the antibody the ability to selectively activate CD40-expressing cells (e.g., DC cells) in the presence of PD-L1-expressing cells. This selective feature of the antibody of the present invention can allow the possibility of the antibody inducing CD40 activation in normal tissues and thereby causing toxic side effects to be reduced. At the same time, the reduction in toxic side effects can increase the tolerable dosage of the antibody of the present invention in the human body. The increase in dosage can lead to an increase in the antibody concentration in the blood and its enrichment in the tumor microenvironment, thereby enhancing the efficacy of the antibody.

[0194] In other aspects, the bispecific antibodies of the present invention reduce the Fc region-mediated effector functions such as ADCC and CDC by removing the binding of the Fc region to the Fcγ receptor, thereby avoiding the killing effect of the effector functions on antigen-presenting cells (such as DC cells) to which the antibody targets, thereby enhancing the efficacy of the antibody.

[0195] In still other aspects, the antibodies according to the present invention adopt a unique 2+1 design, using a monovalent PD-L1 binding site as an anchoring site. Compared with the design using a bivalent PD-L1 binding site, this not only can achieve the maximum possible utilization of PD-L1 in target tumor tissues, but also can reduce the antibody's PD-L1 binding avidity, thereby reducing the antibody's binding to normal tissue cells with low PD-L1 expression levels, and increasing the antibody's ability to selectively bind to PD-L1 high-expressing cells in the tumor environment, which is beneficial to the enrichment of the antibody in the tumor microenvironment and promotes the safety and efficacy of the antibody.

[0196] II. Anti-PD-L1 Antibodies of the Present Invention

[0197] In a second aspect, the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a VHH domain that specifically binds to PD-L1, and wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences contained in SEQ ID NO: 5 or 6;

[0198] Preferably, the CDR1, CDR2 and CDR3 sequences comprise or consist of the amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively;

[0199] Still more preferably, the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 6, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 5 or 6, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 5 or 6,

[0200] Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0201] The anti-PD-L1 antibodies according to the present invention may have any suitable antibody structure, including, but not limited to, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), linear antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, or humanized antibodies. In some embodiments, the anti-PD-L1 antibodies according to the present invention comprise an immunoglobulin Fc region connected to the VHH domain (i.e., having a VHH-Fc format). In some embodiments, the anti-PD-L1 antibodies according to the present invention are heavy chain antibodies. As used herein, the term "heavy-chain antibody (hcAb)" refers to an antibody without a light chain, which may comprise VHH-CH2-CH3 from N-terminus to C-terminus, or may comprise VHH-CH1-CH2-CH3; it may constitute a homodimer, such as a heavy chain dimer antibody without a light chain.

[0202] The VHH domains of the anti-PD-L1 antibodies according to the present invention have excellent tumor targeting properties and exhibit cross-reactivity between human and monkey species. Therefore, the anti-PD-L1 antibodies or their VHH domains according to the present invention can be used as targeting modules in conjugates or coupled to agents such as chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes, or signaling molecules to provide applications such as improved tumor killing, immune regulation, or disease detection.

[0203] III. Production and Purification of Antibodies of the Invention

[0204] In a third aspect, the present invention provides a method for producing an antibody of the present invention. To produce an antibody of the present invention, the polypeptide chains of the antibody of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production and assembled under suitable conditions.

[0205] For recombinant production, the polynucleotide encoding any one polypeptide chain and / or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Using conventional methods, the polynucleotide can be easily isolated and sequenced. In one embodiment, a polynucleotide encoding one or more polypeptide chains of an antibody of the present invention is provided. In another embodiment, the present invention provides a vector comprising one or more polynucleotides of the present invention, preferably an expression vector. Therefore, in one embodiment, the present invention provides a method for producing an antibody of the present invention, the method comprising: culturing a host cell comprising encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the antibody; and optionally assembling the polypeptide chains to produce the antibody under conditions suitable for the assembly of the polypeptide chains into the antibody.

[0206] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0207] In one embodiment, the present invention also provides a host cell comprising one or more polynucleotides of the present invention. In some embodiments, a host cell comprising an expression vector of the present invention is provided. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney line (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

[0208] The antibodies prepared by the methods described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, the purity of the antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibodies provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0209] In a preferred embodiment, the antibodies of the present invention exhibit good production properties when recombinantly produced in mammalian host cells, such as CHO cells, in particular, good expression yields and a good by-product profile.

[0210] IV. Immunofusions and Immunoconjugates

[0211] In a fourth aspect, the invention provides immunofusions or immunoconjugates produced by fusing or conjugating an antibody of the invention to a heterologous molecule.

[0212] In one embodiment, in an immunofusion, an antibody of the invention (or an antigen-binding fragment thereof) is linked directly or via an amino acid linker to a heterologous peptide or polypeptide molecule. Examples of heterologous peptides or polypeptides include, but are not limited to, proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate purification or detection of the immunofusion.

[0213] In one embodiment, in an immunoconjugate, an antibody of the present invention (or its Fab) is conjugated to a therapeutic agent, a diagnostic agent, or a detectable agent. In a conjugate, a joint can be used to covalently link the different entities of the conjugate. Suitable joints include chemical joints or peptide joints. Advantageously, the joint is a "cleavable joint" that is beneficial to the release of the polypeptide after delivery to the target site. For example, an acid-labile joint, a peptidase-sensitive joint, a light-labile joint, a dimethyl joint, or a disulfide-containing joint can be used.

[0214] In embodiments where conjugated to a therapeutic agent, therapeutic agents suitable for use in the conjugate include, but are not limited to, cytotoxins (eg, cytostatic or cell-killing agents), drugs, or radioisotopes.

[0215] In embodiments conjugated to diagnostic or detectable agents, such conjugates can be used as part of a clinical test method (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnosis and detection can be achieved by coupling the antibody to a detectable agent, including but not limited to a variety of enzymes, such as horseradish peroxidase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0216] V. Pharmaceutical Compositions, Drug Combinations, and Kits

[0217] In a fifth aspect, the present invention provides compositions, e.g., pharmaceutical compositions comprising an antibody of the present invention or an immunoconjugate of the present invention or an immune fusion formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, an antibody of the present invention or an immunoconjugate of the present invention or an immune fusion is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody of the present invention or an immunoconjugate of the present invention or an immune fusion and one or more other therapeutic agents.

[0218] In another aspect, the present invention also provides a pharmaceutical combination comprising an antibody as described herein or an immunoconjugate or immunofusion of the invention and one or more other therapeutic agents.

[0219] The other therapeutic agents suitable for use in the pharmaceutical compositions and drug combinations of the present invention can be selected from any one of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; and (iv) drugs that have tumor inhibitory effects.

[0220] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody of the present invention or an immunoconjugate or immunofusion of the present invention. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount may vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is an amount in which any toxic or deleterious effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to an untreated subject. The ability of the antibodies of the present invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system predictive of efficacy in human tumors. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic outcome at the desired dosage and for the desired period of time. Generally, because prophylactic doses are used in subjects before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0221] Kits comprising the antibodies described herein are also within the scope of the present invention. The kits may include one or more other elements, including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0222] VI. Use and Methods

[0223] In a sixth aspect, based on the excellent targeting properties of the antibodies or immunoconjugates or fusions of the present invention for tumor cells and tumor microenvironment cells and the other excellent properties described above, the present invention also provides applications and methods of the antibodies or immunoconjugates or fusions of the present invention in the treatment and prevention of tumors. In such applications, the antibodies or antigen-binding fragments of the present invention, or the immunoconjugates or conjugates of the present invention, or pharmaceutically acceptable salts or solvates thereof, can be administered to a subject as the sole active agent, or can be administered to a subject in combination with other therapies or therapeutic agents. Such other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells and eliminate tumors by binding to and / or blocking these molecules; and drugs that activate the subject's immune system to promote its spontaneous elimination of tumors.

[0224] In yet another aspect, the present invention also provides a method for preventing or treating a tumor in a subject, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof of the present invention, or administering to a subject in need thereof an immunoconjugate or coupling of the present invention or a pharmaceutically acceptable salt or solvate thereof.

[0225] Tumors suitable for the methods and uses of the present invention can include various solid tumors and hematologic tumors. Tumors suitable for the methods and uses of the present invention can be in the early, middle, or late stages, or can be metastatic cancers. In addition, tumors suitable for the methods and uses of the present invention can be tumors that have previously received treatment and have escaped immune toxicity. In some embodiments, the tumor is resistant to PD-1 or PD-L1 inhibitors.

[0226] In some embodiments, the tumor is a cold tumor with less T cell infiltration. Studies on PD-1 or PD-L1 inhibitor-resistant tumors (so-called "cold tumors") have found that the drug resistance of tumors is largely related to the quantity and quality of T cells infiltrating the tumor. Analysis of PD-L1 expression in some such tumors showed that although there was less immune cell infiltration in the tumor microenvironment, some tumor cells could show moderate levels of PD-L1 expression, and DCs and macrophages in the tumor microenvironment could show very high levels of PD-L1 expression. Therefore, it is expected that by activating DCs and / or macrophages in the tumor microenvironment, further stimulating T cell infiltration into the tumor and the anti-tumor function of T cells, it will be beneficial to overcome the drug resistance of such tumors. See, e.g., Rafael Winograd et al., Induction of T-cell immunity overcomes complete resistance to PD-1and CTLA-4 blockade and improves survival in pancreatic carcinoma, Cancer Immunol Res. 2015Apr; 3(4):399–411, doi:10.1158 / 2326-6066.CIR-14-0215. Haidong Tang et al., Facilitating T cell infiltration in tumor microenvironment overcomes resistance to PD-L1 blockade, Cancer Cell. 2016 March 14; 29(3):285–296. doi:10.1016 / j.ccell.2016.02.004.

[0227] In some embodiments, tumors treated according to the methods of the invention have at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, 100% PD-L1 positive cells, or a CPS score of 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, 100. PD-L1 expression levels can be assessed on tumor and tumor microenvironment biopsies by immunohistochemistry. The CPS score of a tumor can be determined with reference to the method described in Karina Kulangara et al. (Clinical Utility of the Combined Positive Score for Programmed Death Ligand-1 Expression and the Approval of Pembrolizumab for Treatment of Gastric Cancer, Arch Pathol Lab Med (2019) 143(3): 330–337, https: / / doi.org / 10.5858 / arpa.2018-0043-OA). In some embodiments, the application of the method of the present invention in the cancer induces tumor regression. In some embodiments, the application of the method of the present invention in the cancer results in tumor growth inhibition.

[0228] In any of the above embodiments of the methods of the present invention, the administration of an antibody or binding fragment thereof according to the present invention and an immunoconjugate or conjugate according to the present invention, or a pharmaceutically acceptable salt or solvate thereof, may comprise 1) a therapeutic measure that cures, slows down, alleviates the symptoms of, and / or stops the progression of, a diagnosed pathological condition or disorder; or 2) a preventative or prophylactic measure that prevents and / or slows the development of a pathological condition or disorder. Thus, in the methods of the present invention, the subject may be an individual already suffering from a disease, an individual susceptible to a disease, or an individual for whom a disease is to be prevented. The individual will benefit from the therapeutic or prophylactic measure and exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, disorder, condition, and / or symptom compared to an individual who has not received the treatment. In some embodiments, the present invention relates to the treatment of a disease or disorder; in other embodiments, the present invention relates to the prevention of a disease or disorder.

[0229] The antibodies or binding fragments thereof according to the present invention and the immunoconjugates or pharmaceutically acceptable salts or solvates thereof according to the present invention, and other therapeutic agents optionally used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.

[0230] For the prevention or treatment of disease, the appropriate dosage of the antibodies or binding fragments thereof according to the invention and the immunoconjugates or conjugates according to the invention, or pharmaceutically acceptable salts or solvates thereof, when used alone or in combination with one or more other therapeutic agents, will depend on the type of disease being treated, the specific type of drug used, the severity and course of the disease, whether the drug is being administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0231] In some embodiments, the present invention also provides the use of the immunoconjugates or conjugates of the present invention or their pharmaceutically acceptable salts or solvates and the antibodies or antibody fragments of the present invention as a medicament or for the preparation of a medicament. In some embodiments, the medicament is a medicament for use in the aforementioned treatment and prevention methods.

[0232] Any or all features described above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are for illustrative purposes and should not be construed as constituting any limitation.

[0233] Example

[0234] Example 1. Preparation and panning of PD-L1 phage display library

[0235] 1.1 Two healthy adult alpacas (Chengdu Apec) were selected and the human PD-L1 extracellular segment (UniProtKB accession number Q9NZQ7, Phe19-Arg238) recombinant protein (CST, product number: 71391S) was mixed with Freund's adjuvant in a 1:1 ratio. The alpacas were immunized by multiple subcutaneous injections for a total of five immunizations, with an interval of 2-3 weeks.

[0236] 1.2 Peripheral blood was collected from alpacas after the third, fourth, and fifth immunizations, lymphocytes were isolated, and Trizol reagent (Thermo Fisher Scientific) was added to extract total RNA. The extracted RNA was reverse transcribed using the PrimeScript Reverse Transcription Kit (Takara). PCR reactions were performed using the cDNA as templates to amplify antibody fragments. The phage display vector and PCR product were double-digested, and the digestion products were mixed in proportion and added to T4 ligase (Thermo Fisher Scientific) for ligation. The ligation products were electroporated into TG1 competent cells to construct a VHH antibody immune library.

[0237] The above library bacterial solution was inoculated and cultured to the logarithmic growth phase, and M13KO7 helper phage was added for infection to prepare a phage display library.

[0238] 1.3 Recombinant phage were subjected to two rounds of panning using the biotinylated antigen PD-L1-His (ACRO, Cat. No. PD1-H82E5-200UG). After each round of panning, the eluted recombinant phage were used to infect TG1 cells in the logarithmic growth phase to prepare a phage library for the next round of panning. Simultaneously, the bacterial solution after each round of infection was serially diluted and plated onto plates to form single colonies for binding ELISA analysis.

[0239] 1.4 Binding ELISA was used to detect binding activity.

[0240] Take the monoclonal plate from 1.3, select a single clone and induce expression with IPTG. Take the supernatant after induced expression for binding ELISA detection. Use Human PD-L1 antigen (ACRO, product number: PD1-H5258-1MG) to coat a 96-well ELISA plate. Add 80μL blocking agent and 20μL bacterial expression supernatant to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add Anti-Flag / HRP secondary antibody diluted in blocking agent, and incubate at room temperature for 1 hour. After washing with PBST, add TMB colorimetric solution and develop in the dark. After stopping the reaction with stop solution, read the plate on a microplate reader and measure the OD450nm absorbance value. Bacterial clones with a reading greater than 0.1 are positive clones and are screened for blocking function.

[0241] Example 2. Screening of PD-L1 VHH blocking antibodies

[0242] 2.1 Blocking ELISA screening of PD-L1 positive clones

[0243] Human PD-L1 FC (ACRO, Catalog No. PD1-H5258-1MG) antigen was diluted in PBS buffer and coated onto a 96-well ELISA plate. To the blocked 96-well plate, 80 μL of biotin-labeled PD-1 diluted in blocking agent and 20 μL of diluted bacterial expression supernatant were added and incubated at room temperature for 1 hour. The plate was washed three times with PBST, and HRP avidin secondary antibody (Thermo Fisher Scientific, Catalog No. 21126) diluted in blocking agent was added and incubated at room temperature for 1 hour. After washing with PBST, TMB colorimetric solution was added for color development in the dark, and the plate was stopped with stop solution. The plate was read using a microplate reader, and the absorbance at OD 450 nm was measured. Bacterial clones with a reading less than 0.15 were considered to have blocking function.

[0244] 2.2 Reporter assay screening of PD-L1 positive clones

[0245] A reporter molecule assay was performed using a similar method described in a previous literature (Liu, C. et al., Small molecule inhibitors against PD-1 / PD-L1 immune checkpoints and current methodologies for their development: a review. Cancer Cell Int 21, 239(2021). https: / / doi.org / 10.1186 / s12935-021-01946-4). Briefly, PD-1 / PD-L1 Blockade Bioassay Kit (Promega, J1252) was used to plate PD-L1α APC cells (artificial antigen-presenting cells that recombinantly express PD-L1) in 96-well culture plates according to the manufacturer's instructions. The cells were cultured overnight, and diluted bacterial supernatant and PD-1 effector cells (Jurkat cells stably expressing human PD-1 and NFAT-RE-luciferase reporter molecules) were added to the well plates. The cells were cultured for 6 h, and the colorimetric substrate was added to the well plates. The cells were incubated at room temperature, and the luminescence value was measured using a multifunctional microplate reader.

[0246] The bacterial clones with blocking function screened in the blocking ELISA and reporter assay were sent to the supplier for sequencing.

[0247] Example 3. Eukaryotic expression and purification of VHH candidate antibodies

[0248] Using molecular biology techniques, the anti-PD-L1 VHH antibody sequence with blocking function obtained in Example 2 was amplified by PCR and inserted into a eukaryotic expression vector, transfected into HEK-293, expressed and purified, and finally obtained the VHH-His recombinant antibody.

[0249] The purified antibodies were screened using the Blocking ELISA and reporter assay described in Example 2, and ultimately a lead molecule that could block the binding of PD-1 and PD-L1 was obtained.

[0250] For the full-length base sequence and amino acid sequence of the anti-PD-L1 VHH antibody (LA03C06) obtained in the present invention, as well as the sequence number, please refer to the attached sequence listing.

[0251] Example 4. Determination of the binding kinetics between the VHH antibody of the present invention and antigen using thin-layer interferometry

[0252] The equilibrium dissociation constant (KD) of the anti-PD-L1 VHH antibody of the present invention binding to human or monkey PD-L1 was determined using the thin-layer biofilm interferometry technique (ForteBio). The ForteBio affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8). Half an hour before the start of the experiment, according to the number of samples, an appropriate number of AHC (18-5060, Sartorius) sensors were taken and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%). 100 μl of SD buffer, antibody, and antigen were added to a 96-well black polystyrene half-volume microplate (Greiner, 675076). The plate was arranged according to the sample position and the sensor position was selected. The instrument was set up with the following parameters: run steps: Baseline 120 s, Loading ~1 nm, Baseline 120 s, Association 100 s, and Dissociation 120 s, speed 1000 rpm, temperature 30° C. KD values ​​were analyzed using ForteBio analysis software.

[0253] The affinity of the antibodies was determined as shown in Table 1:

[0254] Table 1. Affinity constants (equilibrium dissociation constants) of monovalent binding of PD-L1 antigen and antibody detected by ForteBio *The control molecule NB is from patent CN107686520A and has the VHH sequence of SEQ ID NO:31.

[0255] Example 5. PD-L1 VHH Antibody Humanization and Bispecific Antibody Protein Expression and Purification

[0256] 1. Humanization of PD-L1 VHH Antibody

[0257] Based on Discovery Studio and PyMOL software, the nanoantibody LA03C06 obtained by immunization of alpacas was humanized through the following steps:

[0258] (1) Determine the CDR region of the Nanobody;

[0259] (2) Screening the human germline sequence database for the closest homologous sequence to the V / J region;

[0260] (3) constructing the CDR regions of the Nanobody onto the human framework region;

[0261] (4) Based on sequence and structural characteristics, determine the amino acid positions in the framework region that play a role in maintaining CDR function, and perform back mutations at the sequence positions identified as important;

[0262] (5) After synthesizing the sequence and preparing the single-domain VHH-His antibody, the affinity of the humanized molecule to human PD-L1 was determined using biofilm thin layer interferometry (BLI) technology.

[0263] Based on the affinity data in Table 2, it can be concluded that the humanized hz03C06.g6 molecule maintains an affinity comparable to that of a camelid single-domain antibody. The amino acid sequences of the chimeric antibody and the humanized hz03C06.g6 molecule are shown in Table 2.

[0264] Table 2. Affinity constants (M) for monovalent antigen-antibody binding assays by ForteBio.

[0265] 2. Bispecific antibody protein preparation:

[0266] DNA encoding the bispecific antibody sequence was synthesized and inserted into the expression plasmid pcDNA3.1 to construct light chain plasmids and heavy chain plasmids expressing the 2+1 bispecific antibody Dace-hz03C06.g6-2+1 (SEQ ID NOs: 7-9) and the Dace-hz03C06.g6-2+2 bispecific antibody (SEQ ID NOs: 10 and 11), respectively. Light chain plasmids and heavy chain plasmids expressing the control anti-PD-L1 monoclonal antibody Atezolizumab (SEQ ID NOs: 12-13) and the anti-CD40 monoclonal antibody Selicrelumab (SEQ ID NOs: 14-15) were also constructed.

[0267] Expi293F cells (purchased from Gibco) were cultured with Expi293F medium (Gibco, REF#A14351-01). The cell density was checked one day before transfection (viability should be greater than 95%) and adjusted to 3 × 10 6 The cell density was adjusted to 3 × 10 cells / ml on the day of transfection. 6 cells / ml.

[0268] Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as the transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, where the light and heavy chain ratio of the 2+1 type antibody molecule is 2:1:1, and the light and heavy chain plasmid ratio of the remaining antibodies is 1:1, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, and then gently pour the mixture into the Expi293F cell suspension while shaking. The cells are cultured in a shaker under the conditions of 8% CO2, 36.5°C, and 120 rpm.

[0269] After 16-18 hours of culture, the cell suspension was supplemented with 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), a glucose solution to a final concentration of 5 g / L, and valproic acid sodium salt (Merk, Cat# P4543-100G) to a final concentration of 2.2 mM. The suspension was gently mixed and cultured for 7 days at 8% CO₂, 36.5°C, and 120 rpm before sampling. The cell suspension was then mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered using a 0.22 μm disposable vacuum filter.

[0270] Affinity chromatography to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1 M NaOH were passed through the tubing and affinity chromatography column, and then the tubing and column were washed with 10-20 column volumes of distilled water. The packed column was equilibrated with 5 column volumes of 1× PBS (Gibco); the filtered cell feed was passed through the column, and the packed column was washed with 10 column volumes of 1× PBS to remove non-specific binding proteins; the packed column was rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), the eluate was collected, the pH was adjusted to 6.0 with 2 M Tris, and the column was sterilized by filtration.

[0271] Ion exchange chromatography purification of bispecific antibodies: A Mono S 5 / 50GL (GE Healthcare) ion exchange chromatography column was used and placed in an AKTApure system (GE healthcare). The AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column was detoxified with 0.5 M NaOH for 2 hours, and then the system and column were rinsed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20 mM phosphate, pH 6.0) until the conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was re-equilibrated with 5 column volumes of loading buffer. A linear elution gradient of 0-40% elution buffer (20 mM phosphate, 1 M NaCl, pH 6.0) was performed over 30 column volumes, and samples were collected based on UV absorbance.

[0272] SDS-PAGE electrophoresis analysis of the collected samples. Take the protein sample after ion exchange, add reducing loading buffer (Yisheng Bio, catalog number: 20315ES05) or non-reducing loading buffer (Beijing Biolabs, catalog number: WE0289), heat at 70 degrees Celsius for 10 minutes, take an appropriate amount of sample and add precast gel 4–20% TGX TM Precast Gel (Bio-rad, catalog number: 4561095) was added, and a protein marker (Biorad, catalog number: 1610375) was added as a control. Electrophoresis was performed at 150 V for 50 minutes, and the gel was stained with a protein stainer (Gensher, L00760C). The gel was then photographed with a gel imager (Shanghai Tianneng, Tanon-1600).

[0273] The purity of the collected fractions was determined by size exclusion chromatography (SEC). Based on the results of SDS-PAGE and SEC, fractions with a purity greater than 95% were combined.

[0274] The protein sample that meets the purity standard is centrifuged in a 15ml ultrafiltration centrifuge tube at 4000 rpm for 10 minutes. The protein is diluted with PBS and centrifuged again at 4000 rpm for 10 minutes. This operation is repeated several times to replace the protein storage buffer. After the liquid is replaced, the sample is sterilized by filtration and the antibody concentration is tested. The composition and content of the bispecific antibody are further qualitatively and quantitatively analyzed by combining capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0275] Example 6. CD40 reporter assay for target molecules

[0276] 1. Detection method:

[0277] After 2-3 passages, the HEK-Blue CD40L cells (HEK293 cells that recombinantly express CD40 and the SEAP reporter gene under the control of the NF-κB response element, purchased from InvivoGen, product number hkb-cd40), MC38 cells, and MC38 PD-L1 cells (MC38 cells that recombinantly highly express PD-L1) were removed from the culture medium, washed with 10 ml PBS, digested with 2 ml trypsin, and terminated with culture medium. The cells were centrifuged at 400 x g for 3 minutes, the supernatant was discarded, the culture medium was resuspended, 20 μl of the cell suspension was transferred to a 1.5 ml EP tube, and the cells were counted. In a 15 ml centrifuge tube, HEK-Blue CD40L was prepared into a cell suspension, 3x10 4 / well, use a dispenser to press 50ul into each well of two 96-well plates (flat bottom); then press 0.5x10 4 Add 50ul MC38 and 50ul serially diluted bispecific antibody to be tested per well and mix well. 4 Add 50 μl of MC38 PD-L1 and 50 μl of serially diluted bispecific antibody to be tested to each well and mix thoroughly. Add 200 μl of PBS around the cells to prevent edge effects and place the cell plate in the incubator for an additional 20 hours.

[0278] Prepare QUANTI-Blue according to the manufacturer's instructions. TM(InvivoGen) solution. Briefly, take 10ml ddH2O in a 50ml centrifuge tube, add 100ul buffer and 100ul QUANTI-Blue (100x) in sequence, mix well, wrap with tin foil and store in a 4℃ refrigerator. Preheat for 30min before use, then pipette 180ul / well QUANTI-Blue into a 96-well plate (flat bottom) for later use. Prepare a new round-bottom 96-well plate, add 80ul culture medium, then add 20ul supernatant of HEK-Blue CD40L cells that have been co-cultured with the test drug and MC38 or MC38 PD-L1 for 20h, mix well (i.e., dilute 5 times), take 20ul to the cell plate with QUANTI-Blue, incubate at 37℃ for 1h, open the lid and place in a microplate reader, and read the plate at OD635nm.

[0279] 2CD40 reporter assay results:

[0280] HEK-Blue CD40L cells are stably transduced with CD40 and SEAP reporter gene constructs. Binding of CD40 ligand to cell surface CD40 triggers a signaling cascade, leading to NF-κB activation and subsequent SEAP secretion into the cell supernatant. QUANTI-Blue is an enzymatic colorimetric assay that can be used to measure SEAP levels in the cell supernatant, thereby characterizing CD40 ligand activity.

[0281] The results showed that the CD40 activation induced by Dace-hz03C06.g6-2+1 was about 91 times weaker than that of Selicrelumab in the absence of PD-L1 (as shown in Figure 1); while in the presence of PD-L1, it was significantly improved, about 12 times stronger than Selicrelumab (as shown in Figure 2). This result shows that the agonist activity of the Dace-hz03C06.g6-2+1 molecule on CD40 showed obvious PD-L1 dependence; through the design of the monovalent PD-L1 binding site, the ability of the antibody molecule to selectively activate the CD40 signaling pathway was significantly improved (compared to the absence of PD-L1, EC 50 About 8.6nM; in the presence of PD-L1, EC 50 about 0.009nM).

[0282] Example 7. DC activation experiment with dual anti-CD40 / PD-L1 molecules

[0283] 1. Detection method:

[0284] Isolation and induction of dendritic cells (DCs) from human PBMCs. Briefly, hPBMCs (50 million) were taken out of liquid nitrogen tank, thawed in a 37°C water bath, added with RPMI 1640 complete medium, centrifuged at 300 x g for 5 min, and then resuspended in 1 mL of culture medium to adjust the concentration to approximately 5 × 10 7 cells / mL, and transferred to a 5mL flow cytometry tube. Use a human monocyte enrichment kit (Stemcell, catalog number 19058) without removing CD16. According to the manufacturer's instructions, add 100uL enrichment cocktail, mix well, and let it stand at 4°C for 10 minutes; vortex the magnetic beads for 30 seconds, take 100uL magnetic beads and add them to the sample, mix well, and let it stand at 4°C for 5 minutes; add medium (RPMI 1640+10% FBS) to 2.5mL, and mix gently 2-3 times; place on a magnetic stand and let it stand at room temperature for 2.5 minutes, pour the supernatant into a new 5mL flow cytometry tube, count, and adjust the cell concentration to about 5E5 cells / mL; add GM-CSF and IL-4 with a final concentration of 40ng / mL, and place in a 37°C incubator for culture; on the third day, collect the cell suspension, centrifuge at 300x g for 5min, discard the supernatant, resuspend with culture medium, count, and adjust the cell density to 5E5 Cells were resuspended in culture medium and counted, and 50 μL of the culture medium was plated into each well of a 96-well round-bottom plate to a cell count of 1.2E5 cells / well.

[0285] Cultures of CHOK1 cells that do not express human PD-L1 (CHOK1 WT) and CHOKI cells that recombinantly express human PD-L1 (CHOKIPD-L1) were collected and the supernatant discarded. The cells were rinsed once with 10 mL of PBS and then digested with 4 mL of trypsin. After terminating the digestion reaction with culture medium, the cells were centrifuged at 400 x g for 3 minutes, the supernatant discarded, and the cells were resuspended in culture medium and counted. The resulting CHOK1 WT cells or CHOKIPD-L1 cells were plated into the wells of the aforementioned 96-well round-bottom plate, with 50 μL plated per well to a cell count of 0.8E5 cells / well.

[0286] The test antibody was serially diluted at a 1:4 ratio to obtain eight concentrations ranging from 0.009766 nM to 40 nM. Each concentration was added to the wells of the aforementioned 96-well round-bottom plate, with 50 μL of antibody added to each well. The plate was then incubated in an incubator for 48 hours. The 96-well plate was removed and centrifuged at 300 x g for 5 minutes. 20 μL of cell supernatant was added to 180 μL of culture medium and mixed thoroughly. The 10-fold diluted cell supernatant was analyzed for IL-12p40 levels using the Lianke Biotech EK1183-96 ELISA kit according to the manufacturer's instructions.

[0287] 2. DC activation experimental results:

[0288] CD40 on the surface of DCs can bind and interact with its ligand, activating various signaling pathways through signal transduction, promoting DC maturation and secreting the cytokine IL-12. Measuring the concentration of IL-12p40 in the cell supernatant using an ELISA kit can indicate the extent of DC activation by the antibody molecule.

[0289] As shown in Figure 3, consistent with previous reports that CD40 activation by Selicrelumab antibodies is independent of cross-linking, Selicrelumab has strong DC activation activity under conditions of both high and low PD-L1 expression. Both Dace-hz03C06.g6-2+1 and Dace-hz03C06.g6-2+2 showed dependence on PD-L1-mediated cross-linking, showing stronger DC activation when PD-L1 is highly expressed than when PD-L1 is low. In addition, as can be seen from Figure 3, under conditions of high PD-L1 expression, the activation of Dace-hz03C06.g6-2+1 is significantly stronger than that of Dace-hz03C06.g6-2+2, and exhibits a more significant selective DC activation effect. This result indicates that the 2+1 molecular configuration is more advantageous for the antibody of the present invention to fully utilize PD-L1 and selectively activate DC cells.

[0290] Example 8. Determination of the binding kinetics of Dace-hz03C06.g6-2+1 antibody and antigen using thin-layer biofilm interferometry

[0291] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human, monkey, rat, and mouse CD40 and PD-L1 was determined using the thin-layer biofilm interferometry technique (ForteBio). The ForteBio affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8). Half an hour before the start of the experiment, according to the number of samples, an appropriate number of AHC (18-5060, Sartorius) sensors were taken and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%). 100 μl of SD buffer, antibody, and antigen were added to a 96-well black polystyrene half-volume microplate (Greiner, 675076). The plate was arranged according to the sample position and the sensor position was selected. The instrument was set up with the following parameters: run steps: Baseline 120 s, Loading ~1 nm, Baseline 120 s, Association 100 s, and Dissociation 120 s, speed 1000 rpm, temperature 30° C. KD values ​​were analyzed using ForteBio analysis software.

[0292] The affinities of the antibodies determined are shown in Table 3:

[0293] Table 3. Affinity constants of monovalent binding of Dace-hz03C06.g6-2+1 antigen and antibody detected by ForteBio

[0294] NB means not bound

[0295] Example 9. Therapeutic Effect of Dace-hz03C06.g6-2+1 in the B16F10-hPD-L1 Model

[0296] In this experiment, B16F10-hPD-L1 cells were inoculated into C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice to determine the anti-tumor effect of the Dace-hz03C06.g6-2+1 antibody of the present invention.

[0297] C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice:

[0298] Female C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. They were SPF-grade and quality-inspected by Suzhou Xishan Biotechnology Co., Ltd., with a certificate number of 320726230100116536. Mice were acclimated for 3 days upon arrival before the study began.

[0299] cell:

[0300] B16F10-hPD-L1 melanoma cells were obtained from Innovent Biologics (Suzhou) Co., Ltd. (CAT#: N / A) and routinely subcultured according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in sterile PBS to adjust the cell density to 5 × 10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice to establish a B16F10-hPD-L1 tumor-bearing mouse model.

[0301] Dosage:

[0302] Seven days after tumor cell inoculation, the tumor volume of each mouse was measured. 3 Mice between the two groups were divided into groups according to tumor volume (7 mice per group). The dosage and method of administration are shown in Table 4, and h-IgG (purchased from EQUITECH-BIO) was used as a negative control. The drug was administered on the 7th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on the 18th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: A vernier caliper was used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0303] Table 4. Experimental design

[0304] The tumor inhibition results are shown in Figure 4 and Table 5. On day 18 after inoculation, the tumor inhibition rates in the Dace-hz03C06.g6-2+1, 0.3 mg / kg, Dace-hz03C06.g6-2+1, 1 mg / kg, and Dace-hz03C06.g6-2+1, 3 mg / kg groups were 27%, 63%, and 88%, respectively. Mouse body weights were also monitored, and as shown in Figure 5, no significant differences in mouse body weight were observed. Therefore, the Dace-hz03C06.g6-2+1 drug of the present invention exhibited a significant inhibitory effect on tumors, exhibiting a dose-response relationship.

[0305] Table 5. Tumor inhibition rate on day 18

[0306] Example 10. Therapeutic Effect of Dace-hz03C06.g6-2+1 in the B16F10-hPD-L1 Model

[0307] In this experiment, B16F10-hPD-L1 cells were inoculated into C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice to determine the anti-tumor effect of the Dace-hz03C06.G6-2+1 antibody of the present invention.

[0308] C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice:

[0309] Female C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. They were SPF-grade and quality-inspected by Suzhou Xishan Biotechnology Co., Ltd., with a certificate number of 320726230100038316. Mice were acclimated for 3 days upon arrival before the study began.

[0310] cell:

[0311] B16F10-hPD-L1 melanoma cells were purchased from Xinda Biopharmaceuticals (Suzhou) Co., Ltd. (CAT#: N / A) and routine subcultured in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in sterile PBS to adjust the cell density to 5×10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice to establish a B16F10-hPD-L1 tumor-bearing mouse model.

[0312] Dosage:

[0313] Six days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice with tumor volumes of approximately 22-116 mm were selected. 3 The mice were divided into groups according to the tumor volume (7 mice per group). The dosage and method of administration are shown in Table 6. h-IgG (purchased from EQUITECH-BIO, product number SLH56-0001) was used as a negative control and administered on the 6th day after inoculation. The tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration. The relative tumor inhibition rate (TGI%) was calculated on the 20th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: A vernier caliper was used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor. The tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0314] Table 6. Experimental design

[0315] The tumor inhibition rates are shown in Figure 6 and Table 7. On day 20 after inoculation, tumor inhibition rates were 4.2%, 97%, and 67% in the 10 mg / kg, 1.3 mg / kg, and 2.3 mg / kg groups, respectively. The 1.3 mg / kg and 2.3 mg / kg groups showed significantly greater efficacy than the 10 mg / kg PD-L1 atezolizumab group. Mouse body weights were also measured, and as shown in Figure 7, no significant differences were observed. Therefore, Dace-hz03C06.g6-2+1 and Dace-hz03C06.g6-2+2 have significant inhibitory effects on B16F10-hPD-L1 tumors, and the efficacy of Dace-hz03C06.g6-2+1 is significantly better than that of Dace-hz03C06.g6-2+2.

[0316] Table 7. Tumor inhibition rate on day 20

[0317] Example 11. Therapeutic Effect of Dace-hz03C06.g6-2+1 in the KPC-HPD-L1 Model

[0318] In this study, KPC-hPD-L1 cells were used to inoculate C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice to determine the anti-tumor effect of Dace-hz03C06.g6-2+1.

[0319] C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice:

[0320] Female C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. They were SPF-grade and quality-inspected by Suzhou Xishan Biotechnology Co., Ltd., with a certificate number of A202311090029. Mice were acclimated for 3 days upon arrival before the study began.

[0321] cell:

[0322] KPC-hPD-L1 pancreatic cancer cells were obtained from Innovent Biologics (Suzhou) Co., Ltd. (CAT#: N / A) and routinely subcultured according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in sterile PBS to adjust the cell density to 30 × 10 6 On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice to establish a KPC-hPD-L1 tumor-bearing mouse model.

[0323] Dosage:

[0324] Four days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice with tumor volumes of approximately 42-121 mm were selected. 3 The mice were divided into groups according to the tumor volume (6 mice per group). The dosage and method of administration are shown in Table 8, and h-IgG (purchased from EQUITECH-BIO) was used as a negative control. The drug was administered on the 4th day after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. The body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on the 29th day after inoculation. The calculation formula is as follows: TGI% = 100% * (tumor volume of the control group - tumor volume of the treatment group) / (tumor volume of the control group - tumor volume of the control group before administration). Tumor volume measurement: A vernier caliper was used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. Body weight was measured using an electronic balance.

[0325] Table 8. Experimental design

[0326] The tumor inhibition rate results are shown in Figure 8 and Table 9. On day 29 after inoculation, the Dace-hz03C06.g6-2+1 group achieved a 54% tumor inhibition rate. We also measured the mouse body weights, and as shown in Figure 9, no significant changes were observed. Therefore, the Dace-hz03C06.g6-2+1 group demonstrated a significant inhibitory effect on KPC-hPD-L1 tumors.

[0327] Table 9. Tumor inhibition rate on day 29

[0328] Example 12. Toxic Effects of Dace-hz03C06.g6-2+1 in C57 KI hPD-1 / hPD-L1 / hCD40 Mice

[0329] In this experiment, C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice were used. A single intraperitoneal injection of Dace-hz03C06.g6-2+1 was performed to observe the release of ALT, AST, and cytokines in the mouse serum to determine the toxicity of the drug.

[0330] C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice:

[0331] Female C57 KI hPD-1 / hPD-L1 / hCD40 transgenic mice were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. They were SPF-grade and quality-inspected by Suzhou Xishan Biotechnology Co., Ltd., with a certificate number of 320726230100133615. Mice were acclimated for 3 days upon arrival before the study began.

[0332] Dosage:

[0333] The mice were grouped and weighed according to the experimental design in Table 10. On day 0, h-IgG, Selicrelumab, and Dace-hz03C06.g6-2+1 drugs were injected intraperitoneally, and blood samples were collected on day 1.

[0334] Table 10. Experimental design

[0335] ALT and AST tests:

[0336] After sampling, blood samples were centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected and sent to Suzhou Xishan Zhongke Pharmaceutical Research and Development Co., Ltd. for testing of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The results, as shown in Figures 10 and 11, showed that the AST and ALT values ​​in the Dace-hz03C06.g6-2+1 group were much lower than those in the selicrelumab group and comparable to those in the IgG1 group. This indicates that compared with the reference drug selicrelumab, Dace-hz03C06.g6-2+1 caused less damage to the mouse liver.

[0337] Cytokine detection:

[0338] Serum cytokine levels were measured using the CBA (cytometric Beads Array) cytokine detection system using the Human Th1 / Th2 / Th17 CBA Kit (BD Biosciences, Cat. No. 560484). Th1 / Th2 / Th17 standards were resuspended in 2 ml of assay diluent and diluted 1:2 to obtain a gradient of standard solutions ranging from 5000 to 20 pg / ml. Next, the capture bead volume was determined based on the sample volume (10 μl of beads per sample). All beads were mixed and vortexed (microspheres tend to sediment easily, so thorough mixing was required before each aspiration). A 96-well plate was prepared and 50 μl of the following solutions were added: mixed capture beads, standards, serum sample, and PE detection reagent. Incubate at room temperature for 2 hours in the dark. After incubation, 200 μl of wash buffer was added, and the plates were centrifuged at 200 g for 5 minutes. The supernatant was removed and the beads were resuspended in 150 μl of wash buffer before analysis. The flow cytometry results were analyzed using FCAP Array software.

[0339] The CBA cytokine detection system captures single or multiple proteins through microspheres of known size and fluorescence intensity to achieve the purpose of soluble protein detection. In this detection system, each capture microsphere (capture beads) is coated with a specific antibody. When the capture beads, detection antibodies, and the sample to be tested are co-incubated, the three form a double antibody sandwich complex that can be detected by flow cytometry. The test results are shown in Figures 12 and 13. Compared with the CD40 agonist Selicrelumab, the TNF-α and IL-6 levels in the Dace-hz03C06.g6-2+1 group were significantly reduced and comparable to the IgG1 group. This suggests that Dace-03C06.g6-2+1 has a lower risk of inducing a cytokine storm.

[0340] The combined results of liver enzyme and cytokine tests showed that Dace-hz03C06.g6-2+1 exhibited better safety in mice compared with the CD40 agonist Selicrelumab.

[0341] Sequence Listing Overview

Claims

1. A bispecific antibody or antigen-binding fragment thereof that specifically binds to PD-L1 and CD40, wherein the antibody comprises at least one antigen-binding site that specifically binds to PD-L1 and at least one antigen-binding site that specifically binds to CD40.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein: - The PD-L1 antigen binding site binds to human PD-L1 with high affinity, preferably with a KD value of approximately 5x10 -9 M to 0.5x10 -9 M; - the CD40 antigen binding site selectively activates the CD40 signaling pathway in a PD-L1-dependent manner, preferably, the selectivity index defined as the ratio of the EC50 values ​​of the antibody in the absence and presence of PD-L1 expressing cells is greater than 100, for example, about 300-1000; and / or - The antibody is a trivalent bispecific antibody containing one PD-L1 antigen-binding site and two CD40 antigen-binding sites.

3. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the PD-L1 antigen-binding site comprises or consists of a VHH domain; and the CD40 antigen-binding site comprises or consists of a Fab or scFv domain.

4. The bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the PD-L1 antigen-binding site comprises the CDR1-3 sequence in the VHH domain of SEQ ID NO: 5 or 6; and wherein the CD40 antigen-binding site comprises the LCDR1-3 sequence in the light chain variable domain of SEQ ID NO: 16 and the HCDR1-3 sequence in the heavy chain variable domain of SEQ ID NO:

17.

5. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody further comprises an immunoglobulin Fc region, preferably, the Fc region is an IgG1 Fc region, and more preferably, the Fc region has a mutation that reduces or eliminates Fcγ receptor binding activity.

6. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody comprises or consists of an anti-CD40 full-length antibody and one or two (preferably one) anti-PD-L1 VHH domains conjugated thereto.

7. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The PD-L1 antigen binding site comprises a VHH domain, wherein the VHH domain comprises CDR1, CDR2 and CDR3 comprising or consisting of the amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, respectively. Preferably, the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 6, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 5 or 6, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 5 or 6, Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence shown in SEQ ID NO:

6.

8. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein: The CD40 antigen-binding site comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH domain comprises HCDR1, HCDR2 and HCDR3 comprising or consisting of the amino acid sequences shown in SEQ ID NOs: 21, 22 and 23, respectively, and the VL domain comprises LCDR1, LCDR2 and LCDR3 comprising or consisting of the amino acid sequences shown in SEQ ID NOs: 18, 19 and 20, respectively. Preferably, the VH domain comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 17, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 17; and The VL domain comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 16, or an amino acid sequence that has one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16; Most preferably, the VH domain comprises the amino acid sequence of SEQ ID NO: 17 and the VH domain comprises the amino acid sequence of SEQ ID NO:

16.

9. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein: The bispecific antibody comprises a connecting peptide having a length of 10-20 amino acids, preferably, comprising the amino acid sequence of SEQ ID NO:

30.

10. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the bispecific antibody comprises a first, a second and a third polypeptide chain, wherein: - the first polypeptide chain comprises, from N-terminus to C-terminus, a VH domain, a CH1 domain, an Fc region, a connecting peptide and a VHH domain; - the second polypeptide chain comprises a VH domain, a CH1 domain and an Fc region from N-terminus to C-terminus; - the third polypeptide chain comprises a VL domain and a CL domain from N-terminus to C-terminus; wherein the VHH domain specifically binds to PD-L1, and wherein the VH domain pairs with the VL domain and specifically binds to CD40, Preferably, the Fc regions of the first and second polypeptide chains respectively comprise a Knob mutation and a Hole mutation that promote heterodimerization of the first and second polypeptide chains, and optionally a cysteine ​​residue mutation for forming an interchain disulfide bond between the first and second polypeptide chains; Still more preferably, the Fc regions of the first and second polypeptide chains each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 25 or an amino acid sequence at least 95% identical thereto and SEQ ID NO: 26 or an amino acid sequence at least 95% identical thereto, Still more preferably, the bispecific antibody comprises a first, a second and a third polypeptide chain, wherein: The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; The second polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8; and The third polypeptide chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:

7.

11. The bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the bispecific antibody comprises a first and a second polypeptide chain, wherein: - the first polypeptide chain comprises, from N-terminus to C-terminus, a VH domain, a CH1 domain, an Fc region, a connecting peptide and a VHH domain; - the second polypeptide chain comprises a VL domain and a CL domain from N-terminus to C-terminus; wherein the VHH domain specifically binds to PD-L1, and wherein the VH domain pairs with the VL domain and specifically binds to CD40, Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; The second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

12. An anti-PD-L1 antibody or antigen-binding fragment thereof, comprising a VHH domain that specifically binds to PD-L1, wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences contained in SEQ ID NO: 5 or 6; Preferably, the CDR1, CDR2 and CDR3 sequences comprise or consist of the amino acid sequences of SEQ ID NOs: 2, 3 and 4, respectively; Still more preferably, the VHH domain comprises the amino acid sequence shown in SEQ ID NO: 5 or 6, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 5 or 6, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 5 or 6, Most preferably, the VHH domain comprises the amino acid sequence of SEQ ID NO: 6, or consists of the amino acid sequence shown in SEQ ID NO:

6.

13. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

14. A vector, preferably an expression vector, comprising the polynucleotide of claim 13.

15. A host cell comprising the polynucleotide of claim 13 or the vector of claim 14, optionally wherein the host cell is a mammalian cell.

16. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, comprising: Host cells containing a polynucleotide encoding the polypeptide chain of the antibody or antigen-binding fragment thereof are cultured under conditions suitable for expression of the polypeptide chain.

17. An immunoconjugate or immunofusion comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the immunoconjugate or immunofusion according to claim 17 and a pharmaceutically acceptable carrier.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the immunoconjugate or immunofusion according to claim 17 as a medicament or for the preparation of a medicament.

20. The use according to claim 19, wherein the medicament is for treating and / or preventing cancer in an individual, or as a diagnostic tool for a disease, preferably the individual is a mammal, more preferably a human.

21. A method for treating or preventing cancer, comprising administering to an individual in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the immunoconjugate or immunofusion according to claim 17.

22. The use or method of any one of claims 19-21, wherein the cancer is a solid tumor or a hematological tumor, preferably the tumor is a PD-L1 positive tumor.