WO2020151761A1

WO2020151761A1 - Bispecific antibody binding to pd-l1 and ox40

Info

Publication number: WO2020151761A1
Application number: PCT/CN2020/073959
Authority: WO
Inventors: 匡智慧; 刘心义; 陈炳良; 刘军建
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2019-01-25
Filing date: 2020-01-23
Publication date: 2020-07-30
Also published as: TWI793395B; CN113330036B; CN113330036A; TW202043278A

Abstract

An artificially designed antibody molecule, in particular an anti-PD-L1/OX40 bispecific antibody molecule capable of binding both PD-L1 and OX40.

Description

Bispecific antibodies that bind PD-L1 and OX40

Invention field

The present invention generally relates to the fields of immunology and antibody engineering. Specifically, the present invention relates to a novel artificially designed bispecific antibody molecule, particularly a bispecific antibody that simultaneously binds PD-L1 and OX40, a polynucleotide encoding the antibody molecule or each chain thereof, and The vector of the polynucleotide, the host cell containing the polynucleotide or the vector, the immunoconjugate and pharmaceutical composition containing the antibody molecule, and the antibody molecule in the immunotherapy, prevention and/or disease Diagnostic use.

Background of the invention

Antibody molecules can target and specifically bind to their corresponding antigens, and are increasingly becoming important therapeutic agents, preventive agents and preventive agents for various diseases (for example, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.) / Or diagnostic agent. However, monospecific antibodies directed against only one target have some limitations in clinical applications. Patients may develop resistance or non-response after receiving monospecific antibody treatment. With the research on cancer and other diseases, it has been recognized that there are often multiple signal transduction pathways involved in the occurrence and development of diseases, and single-target immunotherapy is usually not enough to play a therapeutic role in many diseases.

Since multispecific antibodies (for example, bispecific antibodies) can specifically bind to different antigens, they can be designed as signal transduction pathways that act on two or more different media at the same time. These advantages have opened up broad application prospects for multispecific antibodies (for example, bispecific antibodies).

A large number of imaginative multispecific antibody (e.g., bispecific antibody) formats have been developed through antibody engineering and their applicability in disease applications has been studied (Brinkmann U. and Kontermann RE, The making of bispecific antibodies, Mabs, 2017, 9(2):182-212).

Multispecific antibodies (for example, bispecific antibodies) can be divided into many types according to different components and construction methods. For example, multispecific antibodies can be divided into symmetrical structures and asymmetric structures according to the basic symmetry of the left and right structures of multispecific antibodies; according to whether the multispecific antibodies have IgG Fc regions, they can be divided into antibody styles with Fc regions and Fc regions without According to the number of antigen binding sites in a multispecific antibody, it can be divided into bivalent, trivalent, tetravalent or more valent antibodies.

The multispecific antibody format in the prior art has its own advantages and disadvantages in preparation and application. For example, although Blinatumomab can be produced by recombinant Chinese hamster ovary (CHO) cells for large-scale culture, it is easy to form aggregates and has a short half-life in vivo. In actual use, an additional continuous infusion device is required; Catumaxomab has a complicated production process and murine heterologous antibodies are more likely to cause immunogenicity problems in humans.

Therefore, there is still a need in the art for alternative multispecific antibodies with improved performance, especially bispecific antibodies. The present invention provides a new multispecific antibody format, which is easy to be effectively expressed in cultured cells in vitro and does not require complex production processes. At the same time, the bispecific antibody can simultaneously bind to different antigens, especially OX40 and PD-L1, and maintain the binding activity of each antigen binding site to the corresponding different epitopes, and other properties. Further, the bispecific antibody format of the present invention is physically stable and biologically stable, which allows the antibody to have better productivity and developability. Summary of the invention

This article discloses a new type of bispecific antibody molecule constructed by antibody engineering methods.

Therefore, in one aspect, the present invention provides bispecific antibody molecules having one or more of the following characteristics:

(a) Specific binding to one or two antigens with high affinity;

(b) It is easy to express in cultured cells in vitro, and the chains of antibody molecules can be correctly coupled or paired;

(c) It has good physical stability, in particular, good long-term thermal stability; and can maintain biological activity for a long time;

(d) After specifically binding to one or two antigens, it exerts biological functions by regulating (for example, inhibiting or activating) the signal transduction pathway in which each antigen participates;

(e) Play effector functions;

(f) It has better anti-tumor activity.

In one embodiment, the antibody molecule of the present invention comprises a whole antibody part and a single domain antibody part, the latter being connected to the C-terminus of the heavy chain constant region of the whole antibody part by a linker.

In one embodiment, the antibody molecule of the invention comprises or consists of the following parts:

The polypeptide chain of formula (I) (peptide chain #1):

VH-CH1-Fc-X-VHH; and

The polypeptide chain of formula (II) (peptide chain #2):

VL-CL;

among them:

VH represents the variable region of the heavy chain;

CH stands for heavy chain constant region domain;

Fc includes CH2, CH3, and optionally CH4;

CH1, CH2, CH3 and CH4 represent the

domains

1, 2, 3 and 4 of the heavy chain constant region, respectively,

X may not exist, or when it exists, it means a joint, such as a flexible joint;

VHH represents a single domain antigen binding site, such as a single domain antibody;

VL stands for the variable region of the light chain;

CL represents the constant region of the light chain;

Optionally, there is also a hinge region between CH1 and Fc.

In one embodiment, the antibody molecule of the present invention comprises at least one polypeptide chain of formula (I) and one polypeptide chain of formula (II). Preferably, the antibody molecule of the present invention comprises two (for example, the same) polypeptide chains of formula (I) and two (for example, the same) polypeptide chains of formula (II).

In one embodiment, the structure of the antibody molecule of the present invention is shown in Figure 1.

In one embodiment, the antibody molecule or fragment thereof of the present invention has 2 or 4 antigen binding sites, which bind to 2, 3 or 4 different antigens, or the same antigen. In one embodiment, the VH in formula (I) and the VL in formula (II) form an antigen binding site, and the VHH of formula (I) constitutes an antigen binding site (single domain antigen binding site).

In one embodiment, different antigen binding sites bind to the same epitope on the same antigen, or different epitopes.

In one embodiment, the linker X in formula (I) in the antibody molecule of the present invention is a flexible linker, such as a linker having glycine and/or serine residues alone or in combination. In one embodiment, the linker comprises an amino acid sequence (Gly ₄ Ser) n, where n is a positive integer equal to or greater than 1, for example, n is a positive integer from 1 to 7, for example, n is 2, 3, 4, 5, 6. In one embodiment, n is 1, 2, 3, or 4.

In one embodiment, the antigen binding site formed by the VH in formula (I) and the VL in formula (II) is human or humanized, or chimeric.

In one embodiment, the single domain antigen binding site (VHH) in the antibody molecule of the present invention is the heavy chain variable domain of an antibody that naturally lacks light chains (e.g., the heavy chain naturally occurring in Camelidae species). The heavy chain variable domain of a chain antibody) or a VH-like single domain in an immunoglobulin called a new antigen receptor (NAR) in fish (such as IgNAR that occurs naturally in shark serum), or Recombinant single domain antigen binding sites derived from them (e.g., camelized human VH domain or humanized camelid antibody heavy chain variable domain). In a preferred embodiment, the single domain antigen binding site in the antibody molecule of the present invention is selected from the heavy chain variable domain of a heavy chain antibody naturally occurring in camelid species, and the camelized human VH domain And humanized camelid antibody heavy chain variable domains.

In one embodiment, the VHH molecule in the peptide chain of formula (I) in the antibody molecule of the present invention may be derived from antibodies produced in camelid species such as camels, alpacas, dromedaries, llamas and guanacos. Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains, and the VHH of such heavy chain antibodies is also within the scope of the present invention.

In one embodiment, CH1 and Fc are derived from an antibody heavy chain, or a derivative thereof.

In one embodiment, "CH1-Fc" of formula (I) is in the form of IgG, such as IgG1, IgG2, or IgG4. In one embodiment, the heavy chain constant domain is derived from IgG2. It will be understood that the Fc in the constant domain can be mutated to achieve the effect of stabilizing the antibody or enhancing the effect of effector function. For example, in a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the amino acid mutation is present in the CH2 domain, for example, the antibody molecule is contained at positions 234 and 235 (EU numbering, according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) EU index is numbered). In a specific embodiment, the amino acid substitutions are L234A and L235A.

In one embodiment, there is a disulfide bond between CH1 and CL. In one embodiment, if two polypeptide chains of formula (I) are included, there are disulfide bonds between the hinge regions between CH1 and CH2 of the two polypeptide chains of formula (I), and the number of disulfide bonds depends on the antibody The form of the IgG from which the constant domains are derived can vary. In some embodiments, there are 2 or 4 disulfide bonds between the hinge regions.

In one embodiment, the light chain constant domain CL of formula (II) is derived from kappa or lambda.

In one embodiment, the binding site formed by the VH of formula (I) and the VL of formula (II) is specific to the first antigen. In one embodiment, the first antigen is OX40.

In one embodiment, the binding site formed by the VHH of formula (I) is specific for the second antigen. In one embodiment, the second antigen is PD-L1.

The type of antigen specifically bound by the antibody molecule of the present invention is not particularly limited, and the antigen may be, for example, cytokines, growth factors, hormones, signaling proteins, inflammatory mediators, ligands, cell surface receptors or fragments thereof. In one embodiment, the antigen to which the antibody molecule of the present invention specifically binds is selected from tumor-associated antigens, immune checkpoint molecules, angiogenesis-inducing factors, tumor necrosis factor receptor superfamily members, and costimulatory molecules in the immune system, and Ligands and/or receptors for these molecules, for example, OX40, CD47, PD1, PD-L1, PD-L2, LAG-3, 4-1BB (CD137), VEGF, and GITR.

In one embodiment, the VH-CH1-Fc of formula (I) constitutes the heavy chain of the whole antibody part, and the VL-CL of formula (II) constitutes the light chain of the whole antibody part.

In one embodiment, the VHH of formula (II) constitutes a single domain antibody.

In one embodiment, the antibody of the present invention also covers its antigen-binding fragments, such as Fab, Fab', Fab'-SH, Fv, single chain antibodies (e.g. scFv) or (Fab') ₂ , single domain antibodies, double Antibody (dAb) or linear antibody.

In one aspect, the present invention provides a nucleic acid encoding any one or more polypeptide chains in the antibody molecule of the present invention, a vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector.

In one aspect, the present invention provides a vector comprising a polynucleotide encoding any one or more polypeptide chains of the antibody molecule of the invention, preferably an expression vector, such as pXC vector or pTT5 vector, such as pXC17.4 or pXC18.4 . In one embodiment, the expression vector is constructed as a dual gene expression vector pXC vector.

In one aspect, the invention provides methods for producing antibody molecules of the invention or fragments thereof.

In some embodiments, the invention provides immunoconjugates, pharmaceutical compositions, kits, combination products or articles of manufacture comprising the antibodies of the invention.

In some embodiments, the antibody, pharmaceutical composition or immunoconjugate or combination product or kit of the present invention is used to prevent or treat diseases, such as autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunction Diseases etc. For example, the disease is tumor (e.g. cancer) or infection. In some embodiments, the tumor is tumor immune escape. Preferably, the tumor is, for example, colon cancer or colorectal cancer or rectal cancer or lung cancer. In another aspect, the present invention relates to a method of preventing or treating a disease in a subject or an individual, the method comprising administering to the subject an effective amount of any antibody or fragment thereof, pharmaceutical composition or immunization described herein Conjugate or combination product or kit. For example, the disease is tumor (e.g. cancer) or infection. In some embodiments, the tumor is tumor immune escape. In one embodiment, the tumor is, for example, colon cancer or colorectal cancer or rectal cancer or lung cancer. In another aspect, the present invention also relates to any of the antibodies or fragments or immunoconjugates described herein for the preparation of drugs or pharmaceutical compositions or kits or combinations for the treatment of tumors (such as cancer) or infections in a subject The purpose of the product. In some embodiments, the tumor is tumor immune escape. In one embodiment, the tumor is, for example, colon cancer or colorectal cancer or rectal cancer or lung cancer.

The invention also relates to methods for detecting antigens in samples.

The invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein or any combination thereof is applicable to any and all antibodies or fragments or immunoconjugates or pharmaceutical compositions or combination products or kits, methods and uses of the invention described herein.

Brief description of the drawings

When read together with the following drawings, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the invention, the figure shows a currently preferred embodiment. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figures 1A-1B illustrate the structure of the bispecific antibody of the present invention.

Figure 2 shows the purity of the anti-PD-L1/OX40 bispecific antibody prepared by the present invention detected by size exclusion chromatography (SEC).

Figure 3 shows the binding of the anti-PD-L1/OX40 bispecific antibody of the present invention and the anti-OX40 antibodies ADI-20057 and IgG2 as controls to CHO cells (CHO-OX40 cells) overexpressing human OX40. In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the average fluorescence intensity (MFI).

Figure 4 shows the anti-PD-L1/OX40 bispecific antibody of the present invention, as well as anti-PD-L1 humanized Nb-Fc and IgG2 as a control and CHO cells overexpressing human PD-L1 (CHO-PD-L1 Cell). In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the average fluorescence intensity (MFI).

Figure 5 shows the anti-PD-L1/OX40 bispecific antibody of the present invention, as well as other antibodies and controls (anti-PD-L1 humanized Nb-Fc, ADI-20057, anti-PD-L1 humanized Nb-Fc+ ADI-20057 and IgG2) simultaneously bind to CHO cells overexpressing OX40 (CHO-OX40) and CHO cells overexpressing PD-L1 (CHO-PD-L1).

Figure 6 shows the binding of the anti-PD-L1/OX40 bispecific antibody of the present invention and the anti-OX40 antibody ADI-20057 and IgG2 as a control to human T cells.

Fig. 7 shows the results of the anti-PD-L1/OX40 bispecific antibody of the present invention measured by the differential scanning fluorescence method (DSF).

Figure 8 shows that the anti-PD-L1/OX40 bispecific antibody of the present invention effectively relieves the blocking effect of the PD1/PD-L1 interaction on the NFAT signal pathway, and thus obtains the luminous signal. The effects of anti-PD-L1 humanized Nb-Fc and IgG2 as controls were also tested.

Figure 9 shows the blocking effect of the anti-PD-L1/OX40 bispecific antibody of the present invention and ADI-20057, Pogalizumab and IgG2 as controls on the binding of human OX40 ligand to OX40, demonstrating the bispecific antibody of the present invention There is no blocking effect on this binding.

Figure 10 shows the effects of the anti-PD-L1/OX40 bispecific antibody of the present invention and ADI-20057, Pogalizumab and IgG2 as controls on the binding of human OX40 ligand to OX40, proving that the bispecific antibody of the present invention does not hinder It breaks the binding of human OX40 ligand and OX40, and effectively enhances the activation of OX40 signal pathway mediated by OX40 ligand.

Figure 11 shows the activation of the anti-PD-L1/OX40 bispecific antibody of the present invention based on the luciferase reporter gene method and ADI-20057 and IgG2 as a control on OX40-mediated signal pathway activation.

Figure 12 shows the effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on PD-L1-dependent OX40-mediated signaling pathway. Among them, Raji cells that do not express PD-L1 are used. The effects of anti-PD-L1 humanized Nb-Fc, ADI-20057, anti-PD-L1 humanized Nb-Fc+ADI-20057, Pogalizumab and IgG2 were also tested.

Figure 13 shows the activation effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on PD-L1-dependent OX40-mediated signaling pathway. Among them, Raji cells expressing PD-L1 are used, which proves that the antibody of the present invention has a better OX40-mediated signal pathway activation effect in the presence of cells expressing PD-L1. The effects of anti-PD-L1 humanized Nb-Fc, ADI-20057, anti-PD-L1 humanized Nb-Fc+ADI-20057, Pogalizumab and IgG2 were also tested.

Figure 14 shows the activation effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on PD-L1-dependent OX40-mediated signaling pathway. The human lung cancer cell NCI-H292, which expresses PD-L1 on the surface, was used to prove that the antibody of the present invention has a better OX40-mediated signal pathway activation effect in the presence of tumor cells that naturally express PD-L1. The effects of anti-PD-L1 humanized Nb-Fc, ADI-20057, and anti-PD-L1 humanized Nb-Fc+ADI-20057 were also tested.

Figure 15 shows the activation effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on human T cells. The effects of anti-PD-L1 humanized Nb-Fc, ADI-20057, anti-PD-L1 humanized Nb-Fc+ADI-20057 and IgG2 were also tested.

Figure 16 shows the tumor suppressive effect of the anti-PD-L1/OX40 bispecific antibody of the present invention, other antibodies and controls on the LoVo cell tumor-bearing NPG mouse model.

Figure 17 shows the effects of the anti-PD-L1/OX40 bispecific antibody of the present invention, other antibodies and controls on the body weight of the LoVo cell tumor-bearing NPG mouse model after administration.

Figure 18 shows the tumor suppressive effect of the low-dose anti-PD-L1/OX40 bispecific antibody of the present invention and other antibodies and controls on the NOG mouse model bearing NCI-H292 cell tumor.

Figure 19 shows the tumor suppressive effects of the anti-PD-L1/OX40 bispecific antibody of the present invention and other antibodies and controls in the middle dose group on the NOG mouse model bearing NCI-H292 cell tumor.

Figure 20 shows the tumor suppressive effect of the high-dose anti-PD-L1/OX40 bispecific antibody of the present invention and other antibodies and controls on the NOG mouse model bearing NCI-H292 cell tumor.

Figure 21 shows the effect of the anti-PD-L1/OX40 bispecific antibody of the present invention, other antibodies and controls on the body weight of a NOG mouse model bearing NCI-H292 cell tumor.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. In addition, the materials, methods, and examples described herein are only illustrative and not intended to be limiting. Other features, objects, and advantages of the present invention will be apparent from this specification and drawings, and from the appended claims.

abbreviation

Unless otherwise specified, the abbreviations in this specification have the following meanings:

Use the following abbreviations:

ADCC antibody-dependent cell-mediated toxicity

CDC Complement-dependent cytotoxicity

CDR Complementarity determining region in immunoglobulin variable region

CHO Chinese Hamster Ovary

EC50 The concentration that results in 50% potency or binding

K _D equilibrium dissociation constant

ELISA Enzyme-linked immunosorbent assay

FACS flow cytometry

MOA mechanism of action, mechanism of action

MLR Mixed lymphocyte reaction

FR Antibody framework area

IC50 The concentration that produces 50% inhibition

Ig Immunoglobulin

Kabat passed the immunoglobulin comparison and numbering system established by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md.)

mAb or Mab or MAb monoclonal antibody

PCR polymerase chain reaction

IFN Interferon

VL Light chain variable region

VH Heavy chain variable region

LC Light chain

HC heavy chain

HCDR heavy chain complementarity determining region

LCDR Light chain complementary decision area

IL2 Interleukin-2

I. Definition

To interpret this specification, the following definitions will be used, and as long as appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

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

As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.

The term "antibody" is used in the broadest sense herein and refers to a protein containing an antigen-binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies ( For example, bispecific antibodies), single chain antibodies, whole antibodies, and antibody fragments.

The terms "full antibody", "full-length antibody", "full antibody" and "whole antibody" are used interchangeably herein to refer to a naturally occurring one comprising at least two heavy chains (H) interconnected by disulfide bonds. And two light chains (L) glycoproteins. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity determining regions (CDR)), with more conservative regions (framework regions (FR)) interposed between them. Each VH and VL consists of three CDRs and four The FR composition is arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits multiple effector functions.

"Antibody fragment" refers to a molecule that is different from an intact antibody, which contains a part of an intact antibody and binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibodies (such as scFv); single-domain antibodies; Specific antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antibody fragments.

As used herein, the term "epitope" refers to a portion of an antigen (for example, human OX40 or PD-L1) that specifically interacts with an antibody molecule.

An "antibody that binds to the same or overlapping epitope" as a reference antibody refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody in a competition assay. Antigen binding, on the contrary, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or more than 95% of the binding of the antibody to its antigen in a competition assay.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition Determination (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253).

An antibody that inhibits (for example, competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen . Conversely, the reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g. equilibrium dissociation constant). Methods for determining affinity are known in the art, such as ForteBio affinity determination.

An antibody that shows the same or similar binding affinity and/or specificity as the reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.

A "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point") area. CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (on the World Wide Web imgt.cines.fr/on), and based on the use of a large number of crystal structures of neighbor propagation clustering (affinity propagation clustering) North CDR definition.

For example, according to different CDR determination schemes, the residues of each CDR are as follows.

The CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).

Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

Unless otherwise specified, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it refers 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)).

In one embodiment, the CDR of the antibody of the present invention determines the boundary by the Kabat rule, or by the AbM rule, or by the combination thereof.

In one embodiment of the present invention, the HCDR1 of VH and VHH in formula (I) of the antibody of the present invention is determined by AbM rules, HCDR2 and HCDR3 are determined by Kabat rules, and VL CDR in formula (II) is determined by Kabat rules determine.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs are different from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-part of the CDR. As those skilled in the art know, the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced by conservative amino acid residues.

"Antibody in the form of IgG" refers to the form of IgG to which the constant region of the heavy chain of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in the form of IgG4 means that its heavy chain constant region is derived from IgG4.

The term "single domain antibody" as used herein generally refers to an antibody that consists of only one heavy chain variable region and has antigen binding activity, that is, contains only one chain from the C-terminus to the N-terminus: FR4-VHHCDR3 -FR3-VHHCDR2-FR2-VHHCDR1-FR1 antibody, which can be derived from the heavy chain variable domain of camelid heavy chain antibody, the VH-like single domain (v-NAR) of fish IgNAR), which can be naturally produced or by Gene engineering technology is produced. Examples of single-domain antibodies include single-domain antibodies (WO2005/035572) derived from camelids (llamas and camels) and cartilaginous fishes (such as nurse sharks).

The term "camelized human VH domain" refers to the transfer of key elements derived from camelid VHH to the human VH domain, so that the human VH domain no longer needs to be paired with the VL domain to recognize the target antigen. The human VH domain alone can confer antigen binding specificity.

As used herein, the term "binding site" or "antigen-binding site" refers to the area of an antibody molecule that actually binds to an antigen, including the antibody light chain variable domain (VL) and antibody heavy chain variable domain (VH ) Consisting of a VH/VL pair, a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) of an IgNAR from a shark, a camelized human VH domain, a human The variable domain of a camelid antibody heavy chain. In one embodiment of the present invention, the antibody molecule of the present invention contains at least four antigen binding sites, for example, two single domain antibody antigen binding sites (for example, VHH) and two VH/VL pairs are formed. Antigen binding site.

The term "single domain antigen binding site" refers to a region of an antibody molecule where a single variable domain (for example, a heavy chain variable domain (VH)) binds to an antigen. In one embodiment of the present invention, the single domain antigen binding site of the present invention may constitute a single domain antibody. In one embodiment, the antibody molecule of the present invention contains two single domain antigen binding sites, which respectively bind the same or different antigens. In another embodiment of the present invention, the antibody molecule of the present invention contains two single domain antigen binding sites, which respectively bind to the same or different epitopes.

As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen binding sites, each of the at least two antigen binding sites is different from a different epitope of the same antigen or is different from Different epitopes of the antigen bind. The antibodies provided herein are generally multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen.

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, IgG immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable domain (VH), also called a heavy chain variable domain, followed by three heavy chain constant domain domains (CH1, CH2 And CH3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). The heavy chains of immunoglobulins can belong to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into sub-classes. Classes, such as γ ₁ (IgG1), γ ₂ (IgG2), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). The light chains of immunoglobulins can be divided into one of two types based on the amino acid sequence of their constant domains, called kappa and lambda. An immunoglobulin basically consists of two Fab molecules and an Fc domain connected by the hinge region of an immunoglobulin.

The term "effector function" refers to those biological activities attributed to the immunoglobulin Fc region that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) , Cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (such as B cell receptors) and B cell activation.

The term "chimeric antibody" is an antibody molecule in which (a) the constant region or part thereof is changed, replaced or exchanged so that the antigen binding site is consistent with a different or changed category, effector function and/or species Region or completely different molecules (for example, enzymes, toxins, hormones, growth factors, drugs) that give chimeric antibody new properties; or (b) use variable regions or parts of them with different or changed antigen specificities The variable region is changed, replaced or exchanged. For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulin. Due to the replacement of the human constant region, the chimeric antibody can retain its specificity in recognizing antigens, while at the same time having reduced antigenicity in humans as compared with the original mouse antibody.

A "humanized" antibody is an antibody that retains the antigen-specific reactivity of a non-human antibody (such as a mouse monoclonal antibody), and at the same time has a lower immunogenicity when administered as a therapeutic drug to humans. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterpart (ie, the parts of the constant and variable regions that are not involved in binding are the corresponding parts of the human antibody). See, for example, Padlan, Anatomy of the antibody molecule, Mol. Immun., 1994, 31:169-217. Other examples of human antibody engineering technologies include but are not limited to the Xoma technology disclosed in US 5,766,886.

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

The term "...valent" antibody refers to the number of antigen binding sites present in an antibody molecule. "Bivalent", "trivalent" and "tetravalent" antibodies refer to the presence of 2 antigen binding sites, 3 antigen binding sites and 4 antigen binding sites in the antibody molecule, respectively. In one embodiment, the bispecific antibodies reported herein are "tetravalent".

The term "flexible linker" or "linker" refers to a connecting peptide composed of amino acids, such as glycine and/or serine residues used alone or in combination, to connect various variable domains in an antibody. In one embodiment, the flexible linker is a Gly/Ser linking peptide, including the amino acid sequence (Gly ₄ Ser) n, where n is a positive integer equal to or greater than 1, for example, n is a positive integer from 1 to 7, such as 2. , 3, or 4. In one embodiment, the flexible linker is (Gly ₄ Ser) ₂ (SEQ ID NO: 5). Also included within the scope of the present invention are the linkers described in WO2012/138475, which is incorporated herein by reference.

As used herein, the term "binding" or "specific binding" means that the binding 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.

The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled person will understand that any macromolecule, including substantially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. In some embodiments herein, the first antigen and the second antigen are two different antigens.

The terms "tumor-associated antigen" or "cancer antigen" interchangeably refer to molecules (usually proteins, carbohydrates or lipids) that are expressed completely or as fragments (e.g., MHC/peptides) on the surface of cancer cells compared to normal cells. Quality), and the molecule can be used in the preferential targeting of cancer cells by the agent. In some embodiments, the tumor-associated antigen is a cell surface molecule that is overexpressed in tumor cells compared to normal cells, for example, 1 fold overexpression, 2 fold overexpression, 3 fold overexpression, or more than normal cells Overexpression. In some embodiments, tumor-associated antigens are cell surface molecules that are inappropriately synthesized in tumor cells, such as molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, tumor-associated antigens are only expressed intact or expressed as fragments on the cell surface of tumor cells, and are not synthesized or expressed on the surface of normal cells.

The term "cytokine" is a generic term for proteins that are released by a cell population and act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monocytes, interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and Gamma interferon. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of natural sequence cytokines, including small molecular entities produced by artificial synthesis, and their pharmaceutically acceptable Derivatives and salts.

An "immunoconjugate" is an antibody conjugated to one or more other substances (including but not limited to cytotoxic agents or labels).

As used herein, the term "OX40" refers to any natural OX40 from any vertebrate source, including mammals such as primates (e.g. humans, monkeys, cynomolgus monkeys) and rodents (e.g. mice and rats), Unless otherwise indicated. The term encompasses "full length", unprocessed OX40 and any form of OX40 due to processing in the cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.

"OX40 activation" refers to the activation of the OX40 receptor. Generally, OX40 activation leads to signal transduction.

The terms "anti-OX40 antibody", "anti-OX40", "OX40 antibody" or "OX40-binding antibody" as used herein refer to antibodies that are capable of binding (human or monkey) OX40 protein or with sufficient affinity Its fragments are such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting (human or monkey) OX40. In one embodiment, the degree of binding of an anti-OX40 antibody to a non-(human or monkey) OX40 protein is less than about 10%, about 20%, about 30%, about about 10%, about 20%, about 30%, or about of the binding of the antibody to (human or cynomolgus) OX40 protein. 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or biofilm interferometry or MSD assay or SPR assay .

The terms "programmed cell death 1 ligand 1", "PD-L1", "programmed death ligand 1", "cluster of differentiation 274", "CD274" or "B7 homolog 1" as used herein refer to Any natural PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. You can find additional information about the human PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 29126. Additional information about mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found, for example, under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7, and an exemplary full-length mouse PD-L1 protein can be found, for example, under NCBI accession number NP_068693 or Uniprot accession number Q9EP73. L1 protein sequence.

The term "anti-PD-L1 antibody", "anti-PD-L1", "PD-L1 antibody" or "antibody that binds PD-L1" as used herein refers to an antibody that can bind to PD with sufficient affinity -L1 protein or fragments thereof. In one embodiment, the degree of binding of the anti-PD-L1 antibody to the non-PD-L1 protein is less than about 10%, about 20%, about 30%, about 40%, about 50% of the binding of the antibody to PD-L1 , About 60%, about 70%, about 80%, or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or biooptical interferometry or MSD assay.

The term "inhibitor" or "antagonist" includes substances that reduce certain parameters (eg, activity) of a given molecule. For example, this term includes substances that cause the given molecule to be inhibited by at least 5%, 10%, 20%, 30%, 40% or more of the activity (eg, PD-L1 activity). Therefore, the inhibitory effect need not be 100%.

The term "activator" includes substances that increase certain parameters (eg, activity) of a given molecule. For example, this term includes substances that increase the activity of a given molecule by at least 5%, 10%, 20%, 30%, 40%, or more (e.g., OX40 activity). Therefore, the activation effect does not have to be 100%.

The "functional Fc region" possesses the "effector function" of the native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.

"Effector functions" refer to those biological activities that can be attributed to the Fc region of an antibody and vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (such as B cell receptors) Body) down-regulation; and B cell activation.

"Human effector cells" refer to white blood cells that express one or more FcRs and perform effector functions. In certain embodiments, the cell at least expresses Fc to enable effector function and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from their natural sources, such as blood.

The term "effective amount" refers to the amount or dose of the antibody or fragment or conjugate or composition of the present invention that, after administration to the patient in single or multiple doses, produces the desired effect in the patient in need of treatment or prevention. The effective amount can be easily determined by the attending physician as a person skilled in the art by considering various factors such as the species of mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; The response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapy.

"Therapeutically effective amount" refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time. The therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition can vary according to various factors such as disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody fragment or its conjugate or composition are less than the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (such as tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%. % And still more preferably at least about 80%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system predicting efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the compound's ability to inhibit, said inhibition in vitro by assays known to the skilled artisan.

"Prophylactically effective amount" refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The "Fab" fragment includes the variable domain of the heavy chain and the variable domain of the light chain, and also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by adding some residues (including one or more cysteines from the hinge region of an antibody) to the carboxy terminus of the CH1 domain of the heavy chain. Fab'-SH is the name of Fab' in which the cysteine residue of the constant domain carries a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments, with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also called the EU index, such as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

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

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, regardless of the number of passages. The offspring may not be exactly the same as the parent cell in nucleic acid content, but may contain mutations. Included herein is the mutant progeny with the same function or biological activity screened or selected in the initially transformed cell. A host cell is any type of cell system that can be used to produce the antibody molecule of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, E. coli cells. Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cultured plant tissues or cells inside animal tissues.

The term "anti-tumor effect" refers to a biological effect that can be exhibited by a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is usually characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include lung cancer, colon cancer, rectal cancer, colorectal cancer, including metastatic forms of those cancers.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope label or fluorescent label) or, in the case of enzymatic labeling, can catalyze a chemical change of a detectable substrate compound or composition. The term is intended to cover the direct labeling of the probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody, and the indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labels include detection of primary antibodies using fluorescently labeled secondary antibodies and end labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.

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

An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to more than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) confirmed. For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule exists outside the chromosome or at a chromosomal location different from its natural chromosomal location.

The calculation of sequence identity between sequences is performed as follows.

In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences may be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity. In a preferred embodiment, the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. Available), using Blossum62 matrix or PAM250 matrix and

gap weight

16, 14, 12, 10, 8, 6 or 4 and

length weight

1, 2, 3, 4, 5 or 6, to determine the identity between two amino acid sequences Sex percentage. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 are used.

Length weights

1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

You can also use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using E. Meyers and W. Miller algorithms that have been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determine the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes hybridization and washing conditions. Instructions for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in the references and either method can be used. The specific hybridization conditions mentioned in this article are as follows: 1) Low stringency hybridization conditions are in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by at least 50°C (for low stringency conditions, you can Increase the washing temperature to 55°C) Wash twice in 0.2X SSC, 0.1% SDS; 2) Medium stringency hybridization conditions are about 45°C in 6X SSC, and then at 60°C in 0.2X SSC, 0.1% SDS Wash one or more times in medium; 3) High stringency hybridization conditions are in 6X SSC at about 45°C, and then wash one or more times in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) Very high The stringent hybridization conditions are washing one or more times in 0.5M sodium phosphate, 7% SDS at 65°C, and then in 0.2X SSC, 0.1% SDS at 65°C. The very high stringency condition (4) is the preferred condition and one that should be used unless otherwise specified.

The term "pharmaceutical composition" refers to a composition that exists in a form that allows the biological activity of the active ingredients contained therein to be effective, and does not contain an additional substance that has unacceptable toxicity to the subject to which the composition is administered. Ingredients.

The term "pharmaceutical excipients" refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), carriers, excipients or stabilizers, etc. administered together with the active substance.

As used herein, "treatment" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. The desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some embodiments, the antibody molecules of the present invention are used to delay or slow the progression of a disease.

As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or condition or symptoms of a specific disease or condition. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.

The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating diseases, such as tumors (such as cancer) and infections, including anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infections Active agents, small molecule drugs or immunomodulators.

"Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer, including but not limited to anti-tumor agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; anti-estrogens ; Anti-androgen; and non-steroidal anti-androgen.

The term "immunomodulator" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response can be a humoral response or a cellular response.

The term "small molecule drugs" refers to low molecular weight organic compounds capable of regulating biological processes.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.

The term "anti-infective agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, such as parasites, at the applied concentration and dosing interval, but is not lethal to the host. As used herein, the term anti-infective agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a specific aspect, the anti-infective active agent is non-toxic to the host at the applied concentration and dosing interval.

Antibacterial anti-infective actives or antibacterial agents can be broadly classified as bactericidal (ie, direct killing) or bacteriostatic (ie, preventing division). Antibacterial anti-infective active agents can be further classified as narrow-spectrum antibacterial agents (ie, affect only small bacterial subtypes, for example, Gram-negative, etc.) or broad-spectrum antibacterial agents (ie, affect a wide range of species).

The term "antiviral agent" includes any substance that inhibits or eliminates the growth, pathogenicity, and/or survival of a virus.

The term "antifungal agent" includes any substance that inhibits or eliminates the growth, pathogenicity, and/or survival of fungi.

The term "antiprotozoal agent" includes any substance that inhibits or eliminates the growth, morbidity, and/or survival of protozoan organisms, such as parasites.

The term "dysfunction" refers to a state of reduced immune responsiveness to antigenic stimuli in the context of immune dysfunction. As used herein, the term "dysfunction" also includes insensitivity or non-response to antigen recognition, in particular, conversion of antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (eg interferon gamma) And/or the ability to kill target cells is impaired.

"Activated T cells" means inducing, causing or stimulating effector or memory T cells to have the biological function of renewing, sustaining or amplifying. Examples of enhanced T cell function include: increased secretion of interferon gamma (such as IFNg) or interleukin (such as IL-2) from CD8 ⁺ effector T cells relative to such levels before intervention, and increased Secretion of gamma-interferon (eg IFNg) or interleukin (eg IL-2) from CD4 ⁺ memory and/or effector T cells, increased CD4 ⁺ effector and/or memory T cell proliferation, increased CD8 ⁺ Effector T cell proliferation, increased antigen responsiveness (e.g. clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 2 times, 3 times, 3 times, 4 times, 5 times , 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 Times, 140 times, 150 times, 160 times or higher. The way to measure this enhancement is known to those of ordinary skill in the art.

"Tumor immune escape" refers to the process of tumor evasion from immune recognition and clearance. In this way, as a treatment concept, tumor immunity is "treated" when such escape is weakened, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

"Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps clear tumor cells through an immune response.

As used herein, "agonist activity of an antibody" refers to the biological activity of an antibody that can activate the antigen to which it binds.

"Antiangiogenic agents" refer to compounds that block or interfere with the development of blood vessels to some extent. The anti-angiogenic agent may be, for example, a small molecule or antibody that binds to growth factors or growth factor receptors involved in promoting angiogenesis.

The term "combination product" refers to a fixed or non-fixed combination in the form of a dosage unit or a kit of parts for combined administration, in which two or more therapeutic agents can be independently administered at the same time or at a certain time. Separate administration within time intervals, especially when these time intervals allow the combination partner to demonstrate cooperation, for example, a synergistic effect. The term "fixed combination" means that the antibody of the present invention and the combination partner (eg, other therapeutic agent) are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the antibody of the present invention and the combination partner (such as other therapeutic agents) are administered to the patient simultaneously, concurrently or sequentially as separate entities, without a specific time limit, wherein such administration provides two treatments in the patient The therapeutically effective level of the agent. The latter also applies to cocktail therapy, such as the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents to treat cancer or infection as described in this disclosure. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration or separate administration or sequential administration for each active ingredient in multiple or in separate containers (eg, tablets, capsules, powders, and liquids). The powder and/or liquid can be reconstituted or diluted to the desired dosage before administration. In some embodiments, administration also includes the use of each type of therapeutic agent at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in the treatment of the conditions or conditions described herein.

The term "vector" when used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors can direct the expression of nucleic acids effectively linked to them. Such vectors are referred to herein as "expression vectors".

"Subject/patient sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from the subject's pregnancy or development at any time. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and so on. Examples of tumor samples include, but are not limited to, tumor biopsy, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulation Plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumors sample.

The term "package insert" is used to refer to the instructions usually included in the commercial packaging of therapeutic products, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings related to the application of such therapeutic products .

II. Antibody molecules of the invention

The present invention provides a new type of antibody molecule, which can be used for immunotherapy, prevention and/or diagnosis of various diseases. The antibody molecule of the present invention contains at least 2, 3 or 4 antigen binding sites, which can function as a monospecific antibody or a bispecific antibody or a multispecific antibody, preferably, it can function as a bispecific antibody Play a role.

In one embodiment, the single domain antigen binding site (VHH) of formula (I) in the antibody molecule of the present invention is a single heavy chain variable domain capable of specifically binding to a target antigen epitope with higher affinity, for example, derived From the heavy chain variable domain of camelid heavy chain antibody, from the v-NAR of shark IgNAR, camelized human VH domain, humanized camelid antibody heavy chain variable domain, and their Recombined single domain. In one embodiment, the single domain antigen binding site in the antibody molecule of the present invention is derived from the heavy chain variable domain of a camelid heavy chain antibody, camelized human VH domain and/or humanized camel Family antibody heavy chain variable domain. The size, structure, and antigenicity of antibody proteins obtained from camelid species (such as camels, alpacas, dromedaries, llamas, and guanacos) have been characterized in the prior art. In nature, some IgG antibodies from the camelid mammal family lack light chains, and therefore are structurally different from the common four-chain antibody structure with two heavy chains and two light chains from other animals. See PCT/EP93/02214 (WO94/04678 published on March 3, 1994).

The heavy chain variable domain VHH of camelid heavy chain antibody with high affinity to the target antigen can be obtained by genetic engineering method. See, for example, U.S. Patent No. 5,759,808, issued June 2, 1998. Like other non-human antibody fragments, the amino acid sequence of Camelidae VHH can be recombinantly changed to obtain a sequence that mimics the human sequence more realistically, that is, "humanization", thereby reducing the antigenicity of Camelidae VHH to humans. In addition, key elements derived from Camelidae VHH can also be transferred to the human VH domain to obtain a camelized human VH domain. The molecular weight of VHH is one-tenth that of a human IgG molecule, and has a physical diameter of only a few nanometers. VHH itself has extremely high thermal stability, stability to extreme pH and proteolytic digestion, and low antigenicity. Therefore, in an embodiment of the antibody molecule of the present invention, the VHH in formula (I) is used as a building block for the antibody molecule of the present invention. The stability and the low antigenicity of human subjects have contributed.

In one embodiment, the VHH in the antibody formula (I) of the present invention specifically binds PD-L1 (for example, human PD-L1).

In one embodiment, the VHH in the formula (I) of the antibody of the present invention that specifically binds to PD-L1 comprises

(i) The three complementarity determining regions (VHH CDR) contained in SEQ ID NO: 6, or

(ii) Relative to the sequence of (i), the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequences in the three CDR regions.

In a preferred embodiment, the VHH of the antibody formula (I) of the present invention that specifically binds to PD-L1 comprises:

Complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 10, or consists of the amino acid sequence, or VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 10 Compared with the amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions); VHH CDR2 includes the amino acid sequence of SEQ ID NO: 11, or consists of the amino acid sequence, or VHH CDR2 includes and SEQ ID The amino acid sequence of NO: 11 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequence; VHH CDR3 includes or consists of the amino acid sequence of SEQ ID NO: 12, or VHH CDR3 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 12.

In a preferred embodiment, the VHH that specifically binds PD-L1 in the formula (I) of the antibody of the present invention comprises or consists of:

(i) The sequence shown in SEQ ID NO: 6,

(ii) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, or

(iii) Compared with the amino acid sequence of SEQ ID NO: 6, there are one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, More preferably, the amino acid sequence is conservatively substituted), and preferably, the amino acid change does not occur in the CDR region.

In one embodiment, the antigen binding site formed by the VH of formula (I) and the VL of formula (II) specifically binds to OX40, such as human OX40.

In some specific embodiments, in the antibody molecule of the invention, the VH in formula (I) comprises

(i) The three complementarity determining regions HCDR of the heavy chain variable region VH shown in SEQ ID NO: 2, or

(ii) Relative to the sequence of (i), a sequence that includes at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the three CDR regions, and / or

VL in formula (II) contains

(i) The three complementarity determining regions LCDR of the light chain variable region VL shown in SEQ ID NO: 8, or

In some embodiments, in the antibody molecule of the present invention, the VH in formula (I) comprises

Complementarity determining regions (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence, or HCDR1 comprises the amino acid sequence of SEQ ID NO: 13. One or three changes (preferably amino acid substitutions, preferably conservative substitutions) of the amino acid sequence; HCDR2 includes the amino acid sequence of SEQ ID NO: 14, or consists of the amino acid sequence, or HCDR2 includes the amino acid sequence of SEQ ID NO: 14 Compared with the amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions); HCDR3 includes the amino acid sequence of SEQ ID NO: 15, or consists of the amino acid sequence, or HCDR3 includes and SEQ ID NO: The amino acid sequence of 15 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequence;

and / or

VL in formula (II) contains

Complementarity determining regions (CDR) LCDR1, LCDR2, and LCDR3, where LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 16, or LCDR1 includes one or two amino acid sequences of SEQ ID NO: 16 The amino acid sequence of one or three changes (preferably amino acid substitutions, conservative substitutions); LCDR2 includes the amino acid sequence of SEQ ID NO: 17, or consists of the amino acid sequence, or LCDR2 includes the amino acid sequence of SEQ ID NO: 17 Compared with the amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions); LCDR3 includes the amino acid sequence of SEQ ID NO: 18, or consists of the amino acid sequence, or LCDR3 includes and SEQ ID NO: Compared with the amino acid sequence of 18, the amino acid sequence has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions).

In some embodiments, in the antibody molecules of the invention,

(a) VH in formula (I)

(i) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 2, or Consist of; or

(ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 2; or

(iii) Comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 2 , More preferably the amino acid sequence of conservative substitution) or consisting of it, preferably, the amino acid change does not occur in the CDR region;

and / or

(b) VL in formula (II)

(i) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 8 or Consist of

(ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 8; or

(iii) Comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 8 It is more preferred that the amino acid sequence is conservatively substituted) or consists of it. Preferably, the amino acid change does not occur in the CDR region.

In some embodiments, the Fc of formula (I) of the antibody of the present invention is derived from IgG1, IgG2, or IgG4. In some embodiments, the Fc is derived from IgG2. In some embodiments, Fc

(i) Contains the amino acid sequence of SEQ ID NO: 4 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity Amino acid sequence or consist of it;

(ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 4; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 4 , More preferably amino acid sequence of conservative substitution) or composed of it.

In some embodiments, CH1 of formula (I) of the antibody of the present invention is derived from IgG1, IgG2 or IgG4. In some embodiments, CH1 is derived from IgG2. In some embodiments, CH1

(i) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 3 or Consist of

(ii) It comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 3 , More preferably amino acid sequence of conservative substitution) or composed of it.

In some embodiments, the CL of formula (I) of the antibody of the present invention

(i) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 9 or Consist of

(ii) It comprises or consists of the amino acid sequence of SEQ ID NO: 9; or

(iii) Comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 9 , More preferably amino acid sequence of conservative substitution) or composed of it.

In some embodiments, X of the antibody formula (I) of the present invention is a flexible linker, such as a linker with glycine and/or serine residues alone or in combination. In one embodiment, the linker comprises an amino acid sequence (Gly ₄ Ser) n, where n is a positive integer equal to or greater than 1, for example, n is a positive integer from 1 to 7, for example, n is 2, 3, 4, 5, 6. In one embodiment, n is 1, 2, 3, or 4. In one embodiment, X has the sequence shown in SEQ ID NO:5.

In some embodiments, in the antibody molecules of the invention,

(a) VH-CH1-Fc in formula (I)

(i) Comprising those having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 19 Amino acid sequence or consist of it;

(ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 19; or

(iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared with the amino acid sequence of SEQ ID NO: 19 ( Preferred amino acid substitutions, more preferred amino acid conservative substitutions) amino acid sequence, preferably, the amino acid changes do not occur in the CDR region of the heavy chain, more preferably, the amino acid changes do not occur in the heavy chain variable region;

and / or

(b) VL-CL of formula (II)

(i) Contains the amino acid sequence of SEQ ID NO: 7 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity Amino acid sequence or consist of it;

(ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7; or

(iii) Comprising one or more (preferably not more than 20 or 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes compared with the amino acid sequence of SEQ ID NO: 7 ( Preferred amino acid substitutions, more preferred amino acid conservative substitutions) amino acid sequence, preferably, the amino acid changes do not occur in the CDR region of the light chain, more preferably, the amino acid changes do not occur in the light chain variable region.

In some embodiments, the antibody molecule of the present invention further includes a signal peptide sequence at the N-terminus of the VH of formula (I) or VL of formula (II), for example, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 22).

In a preferred embodiment of the present invention, the present invention relates to an antibody molecule, wherein the polypeptide chain of formula (I) comprises the sequence shown in SEQ ID NO:1, or contains at least 85%, 90%, 91% thereof , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence; and/or wherein the polypeptide chain of formula (II) comprises SEQ ID NO: 7 Sequence, or comprise an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

In one embodiment, the antibody of the invention comprises a whole antibody portion as a part thereof. In some embodiments, the whole antibody portion of the present invention comprises VH-CH1-Fc of the chain of formula (I), and VL-CL of the chain of formula (II). VH constitutes the heavy chain variable region of the whole antibody part, CH1-Fc constitutes the heavy chain constant region of the whole antibody part, and the two together constitute the heavy chain of the whole antibody part; VL constitutes the light chain variable region of the whole antibody part, CL The light chain constant region constituting the whole antibody part, and the two together constitute the light chain of the whole antibody part.

In some embodiments, the whole antibody portion is a human antibody. In some embodiments, the whole antibody portion is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG4. In some embodiments, the whole antibody portion can independently constitute a monoclonal antibody. In some embodiments, the whole antibody portion is humanized. In some embodiments, the whole antibody portion is a chimeric antibody. In some embodiments, at least part of the framework sequence of the whole antibody portion is a human consensus framework sequence.

In a specific embodiment, the whole antibody portion is an anti-OX40 antibody.

In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDR (for example, in FR). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain.

In some embodiments, the substitutions are conservative substitutions. Conservative substitution refers to the replacement of an amino acid by another amino acid in the same category, for example, an acidic amino acid is replaced by another acidic amino acid, a basic amino acid is replaced by another basic amino acid, or a neutral amino acid is replaced by another neutral amino acid. Replacement. Exemplary substitutions are shown in Table A below:

Table A

原始残基Original residue	示例性置换Exemplary permutation	优选的置换Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Asp、Lys；ArgGln; His; Asp, Lys; Arg	GlnGln
Asp(D)Asp(D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys(C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln(Q)	Asn；GluAsn; Glu	AsnAsn
Glu(E)Glu(E)	Asp；GlnAsp; Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu，Val；Met；Ala；Phe；正亮氨酸Leu, Val; Met; Ala; Phe; Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val；SerVal; Ser	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine	LeuLeu

In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites.

For example, one or more amino acid substitutions can be performed to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody to the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861. Antibodies with altered types of glycosylation can be prepared, such as hypofucosylated antibodies with a reduced amount of fucosyl residues or antibodies with an increased aliquot GlcNac structure. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modification can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation system. Alternatively, a fucosidase can be used to cleave fucose residues of an antibody; for example, fucosidase α-L-fucosidase removes fucosyl residues from the antibody (Tarentino et al. 1975) Biochem. 14:5516-23).

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants, so as to enhance the effectiveness of the antibody in treating cancer or cell proliferative diseases, for example. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fc. The modification of Fc can also change the half-life of the antibody in the therapeutic antibody, thereby achieving a lower frequency of administration and thus increased convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731, pages 734-735.

In one embodiment, the number of cysteine residues of an antibody can be changed to modify antibody properties. For example, the hinge region of CH1 is modified to change (for example, increase or decrease) the number of cysteine residues in the hinge region. This approach is further elaborated in US Patent No. 5,677,425. The number of cysteine residues in the hinge region of CH1 can be changed, for example, to promote the assembly of light and heavy chains or to increase or decrease the stability of the antibody.

Optionally, the antibodies of the invention include post-translational modifications to the antibody chain. Exemplary post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulations, such as conjugation with labeled components.

In one embodiment of the invention, the antibodies or fragments of the invention are glycosylated with engineered yeast N-linked glycans or CHO N-linked glycans.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. The part suitable for antibody derivatization includes, but is not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof.

Another modification of the antibodies or fragments described herein covered by the present invention is pegylation. The antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy polyethylene glycol or poly Ethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention, for example, see EP0154316 and EP0401384.

In some embodiments, the antibody molecules of the invention are humanized. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are fully incorporated herein by reference (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13:1619-1633).

In some embodiments, the antibody molecules of the invention are human antibodies or humanized antibodies. Various techniques known in the art can be used to prepare human antibodies or humanized antibodies.

In some embodiments, the antibody molecules of the invention are chimeric antibodies.

In some embodiments, at least part of the framework sequences of the antibody molecules of the invention are human consensus framework sequences.

In one embodiment, the antibody molecule of the present invention also encompasses its antibody fragments, such as the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv) or (Fab') _2. Or linear antibody.

In some embodiments, the anti-PD-L1/OX40 bispecific antibody of the present invention has one or more of the following properties:

(1) The bispecific antibody or fragment thereof of the present invention simultaneously binds two human antigens with high affinity, for example, binds to human OX40 with the following equilibrium dissociation constant (K _D ), the K _{D is} less than or equal to about 150 nM, 140nM, 130nM, 120nM, 110nM, or 100nM, in some embodiments, the K _{D is} above about 90 nM or 95 nM; and at the same time, binds to human PD-L1 with the following equilibrium dissociation constant (K _D ), the K _{D is} less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, or 4 nM. In some embodiments, the K _{D is} above about 1 nM, 2 nM, 3 nM, or 3.5 nM; in some embodiments, the antibody binds Affinity is measured using SPR assay.

(2) The bispecific antibody or fragment thereof of the present invention simultaneously binds two monkey antigens with high affinity, for example, binds to monkey OX40 with the following equilibrium dissociation constant (K _D ), the K _{D being} less than or equal to about 50 nM, 40nM, 30nM, 25nM, 24nM, 23nM or 22nM, in some embodiments, the K _{D is} above about 10 nM or 15 nM or 20 nM; and at the same time, binds to monkey PD-L1 with the following equilibrium dissociation constant (K _D ) , The K _{D is} less than or equal to about 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 14 nM, or 13 nM. In some embodiments, the K _{D is} above about 10 nM, 11 nM, or 12 nM; in some embodiments, the antibody binds Affinity is measured using biofilm layer interference technique (for example, ForteBio affinity measurement).

(2) The antibody or fragment thereof of the present invention binds to cells expressing human PD-L1, for example, with an EC50 of less than or equal to about 10 nM, 9 nM, 8.9 nM or 8.8 nM (in some embodiments, the EC50 is about 7 nM , 8nM or 8.5nM or more), and at the same time, bind to cells expressing human OX40, for example, with an EC50 less than or equal to about 10nM, 9nM, 8.9nM, 8.8nM, 8.7nM, 8.6nM, or 8.5nM (in some embodiments , The EC50 is about 7 nM or more than 8 nM). In some embodiments, the binding is determined using flow cytometry (eg, FACS). In some embodiments, the cells expressing human OX40 are CHO cells expressing human OX40 and/or the cells expressing human PD-L1 are CHO cells expressing human PD-L1. In some embodiments, the antibodies or fragments thereof of the present invention induce cross-linking of cells expressing human OX40 with cells expressing human PD-L1.

(3) The antibody or fragment thereof of the present invention binds to human T cells with an EC50 of less than or equal to about 5nM, 4.5nM, 4.4nM, 4.3nM, 4.2nM or 4.1nM (in some embodiments, the EC50 is 3 or 3.5 or more than 4nM). In some embodiments, the binding is determined using flow cytometry (eg, FACS).

(4) The antibody or fragment thereof of the present invention has good thermal stability, such as long-term thermal stability. In some embodiments, for example in an accelerated stability test, for example, withstand at 40°C, for example for at least 30 days. In some embodiments, in an accelerated stability test, the antibody maintains at least 95%, 96%, 97%, 98%, or 99% of its monomer peak purity after being placed at 40°C for at least 10 days, 20 days, or 30 days. %. In some embodiments, the antibody or fragment thereof has a Tm greater than or equal to about 60°C, 61°C, 62°C, or 63°C as determined by differential scanning fluorescence.

(5) The antibody or fragment thereof of the present invention blocks the relevant activity of PD-L1 (for example, human PD-L1). In some embodiments, the relevant activity of PD-L1 is the binding of PD-L1 to PD-1 or the binding of PD-L2 to PD-1. In some embodiments, the antibody or fragment thereof of the present invention inhibits the binding of PD-L1 to PD-1 in a MOA (mechanisms of action) assay (functional biological activity detection system, for example from Promega). In some embodiments, in the luciferase reporter gene detection system, the antibody of the present invention relieves the inhibition of the PD-1/PD-L1 interaction on the NFAT signaling pathway, for example, at a rate of less than or equal to about 1 nM, 0.9 nM, 0.8 An EC50 of nM, 0.7nM, 0.6nM, or 0.5nM (in some embodiments, the EC50 is greater than or equal to about 0.3nM or 0.35nM or 0.4nM).

(6) The antibody or fragment thereof of the present invention does not block the binding of human OX40 ligand to OX40. In some embodiments, the antibody or fragment thereof has lower blocking than existing antibodies, such as pogalizumab. In some embodiments, the antibody or fragment thereof does not block the binding of human OX40 ligand to OX40 at all, for example, comparable to IgG.

(7) The antibody or fragment thereof of the present invention effectively activates the OX40 signaling pathway, such as the OX40 or OX40 ligand-mediated signaling pathway and/or its downstream signaling pathway (such as the NFkB signaling pathway).

(8) The antibody or fragment thereof of the present invention has PD-L1 (such as human PD-L1)-dependent ability to effectively activate the OX40 signaling pathway. In some embodiments, the antibody or fragment thereof of the present invention effectively activates the OX40 signaling pathway in the presence of cells (such as tumor cells) expressing (such as naturally expressed or engineered) PD-L1. In some embodiments, the The signal pathway includes, for example, a signal pathway mediated by OX40 or an OX40 ligand and/or its downstream signal pathway (for example, the NFkB signal pathway).

(9) The antibody or its fragment of the present invention effectively activates T cells (such as CD4+ T cells), for example, its activation effect is stronger than anti-PD-L1 antibody or anti-OX40 antibody or a combination of the two.

(10) The antibody of the present invention has a better tumor suppressing effect.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention has one or more of the following characteristics:

(i) Shows that the antibody of the present invention (e.g., comprising SEQ ID NO: 1 as the peptide chain of formula (I) and SEQ ID NO: 7 as the peptide chain of formula (II)) is identical or similar to OX40 and PD-L1 Binding affinity and/or specificity;

(ii) Inhibition (e.g., competitive inhibition) of the antibody of the present invention (e.g., comprising SEQ ID NO: 1 as the peptide chain of formula (I) and SEQ ID NO: 7 as the peptide chain of formula (II)) and OX40 and PD -L1 combination;

(iii) It binds the same or overlapping epitope with the antibody of the present invention (for example, comprising SEQ ID NO: 1 as the peptide chain of formula (I) and SEQ ID NO: 7 as the peptide chain of formula (II));

(iv) Compete with the antibody of the present invention (for example, comprising SEQ ID NO: 1 as the peptide chain of formula (I) and SEQ ID NO: 7 as the peptide chain of formula (II)) to compete for binding to OX40 and PD-L1;

(v) It has one or more biological properties of the antibody of the present invention (for example, comprising SEQ ID NO: 1 as the peptide chain of formula (I) and SEQ ID NO: 7 as the peptide chain of formula (II)).

III. Immunoconjugates

In some embodiments, the present invention also encompasses antibodies conjugated to other substances ("immunoconjugates"). In some embodiments, other substances such as therapeutic agents or markers, such as cytotoxic or immunosuppressive agents or chemotherapeutic agents. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art.

In addition, the antibody molecules of the present invention can be conjugated with tag sequences (such as peptides) to facilitate purification. In a preferred embodiment, the tag amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259Eton Avenue, Chatsworth, CA, 91311), and many of them are commercially available . As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine provides convenient purification of the fusion protein. Other peptide tags used for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and "flag" tags .

In other embodiments, the antibody molecule of the invention is conjugated to a diagnostic or detectable agent. Such antibodies can be used as part of clinical testing methods (such as determining the efficacy of a particular therapy) to monitor or predict the onset, formation, progression, and/or severity of diseases or disorders. This type of diagnosis and detection can be achieved by coupling antibodies to detectable substances, including but not limited to various enzymes; prosthetic groups; luminous substances; luminescent substances; radioactive substances; and various positrons Positron emitting metal and non-radioactive paramagnetic metal ions in emission imaging.

In addition, the antibody molecules of the present invention can be conjugated to therapeutic moieties or drug moieties that modulate a given biological response. The therapeutic part or the drug part shall not be interpreted as being restricted to classic chemotherapeutics. For example, the drug moiety can be a protein, peptide, or polypeptide possessing the desired biological activity. Such proteins may, for example, include toxins; proteins or biological response modifiers.

In addition, the antibody molecules of the present invention can be conjugated to therapeutic moieties such as radioactive metal ions or macrocyclic chelating agents that can be used to conjugate radioactive metal ions to polypeptides.

Techniques for conjugating therapeutic moieties to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", cited in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds), 243-256 pages (Alan R. Liss, Inc. 1985).

The antibody can also be attached to a solid support, which is particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

In some embodiments, the immunoconjugate is used to prevent or treat diseases, such as autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunction diseases and the like. For example, the disease is tumor (e.g. cancer) or infection. In some embodiments, the tumor is tumor immune escape. Preferably, the tumor is, for example, colon cancer or colorectal cancer or rectal cancer or lung cancer.

IV. The nucleic acid of the present invention and the host cell containing it

In one aspect, the invention provides a nucleic acid encoding any of the above antibodies or fragments or any chain thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (such as CHO cells or 293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

For example, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:1-9, or a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:1-9 A nucleic acid with an amino acid sequence of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

The present invention also covers nucleic acids that hybridize with the following nucleic acids under stringent conditions, or nucleic acids that have one or more substitutions (such as conservative substitutions), deletions or insertions with the following nucleic acids: comprising a code selected from SEQ ID NO:1- A nucleic acid comprising a nucleic acid sequence of the amino acid sequence shown in any one of 9; or a nucleic acid comprising a code selected from the amino acid sequence shown in any one of SEQ ID NO: 1-9 with at least 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the nucleic acid sequence.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC). For example, pXC vector or pTT5 vector, such as pXC17.4 or pXC18.4. In one embodiment, the expression vector is constructed as a dual gene expression vector.

Once the expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in a culture medium and screened for appropriate activity. The methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on the methods known in this specification and the prior art, depending on the specific expression vector and Changes or optimization of mammalian host cells.

In addition, it is possible to select cells that have stably incorporated DNA into their chromosomes by introducing one or more markers that allow the selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (for example, antibiotics), or heavy metal (for example, copper) resistance to the auxotrophic host. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements can include splicing signals, as well as transcription promoters, enhancers, and termination signals.

In one embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In some embodiments, a host cell comprising an expression vector of the invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

Suitable host cells include prokaryotic microorganisms such as E. coli. The host cell can also be a eukaryotic microorganism such as filamentous fungus or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for growth in suspension can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 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 (BRL3A), human lung cells (W138), human liver cells ( Hep G2), Chinese hamster ovary cells (CHO cells), CHOK1SV cells, CHOK1SV GS-KO cells, CHOS cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (Edited by B.K.C.Lo, Humana Press, Totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell, such as a CHOS cell, CHOK1SV cell or CHOK1SV and GS-KO, or the host cell is a 293 cell, such as a HEK293 cell.

V. Production and purification of antibody molecules of the invention

In one embodiment, the present invention provides a method for preparing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment), wherein the method includes a method suitable for expressing the antibody molecule of the present invention or a fragment thereof (preferred antigen-binding fragment). ) The host cell is cultured under the condition of nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated. In a certain embodiment, the method further comprises recovering the antibody molecule of the invention or a fragment thereof (preferably an antigen-binding fragment) from the host cell.

In one embodiment, there is provided a method of preparing the antibody molecule of the present invention, wherein the method comprises, under conditions suitable for expression of the antibody, culturing the antibody encoding the antibody (for example, any one polypeptide chain and/or multiple polypeptide chains) The nucleic acid or the host cell containing the expression vector of the nucleic acid, as provided above, and the antibody is optionally recovered from the host cell (or host cell culture medium).

In order to recombinantly produce the antibody molecule of the present invention, the nucleic acid encoding the antibody (such as the antibody described above, such as any one polypeptide chain and/or multiple polypeptide chains) is isolated, and inserted into one or more vectors for use in the host Further cloning and/or expression in the cell. Such nucleic acids are easy to isolate and sequence using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains).

The antibody molecules prepared as described herein can be purified by known existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography and the like. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be obvious to those skilled in the art. The purity of the antibody molecule 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, and the like.

VI. Assay

The antibody molecules provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities by various assays known in the art. On the one hand, the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot and the like. Methods known in the art can be used to determine binding to the bound antigen, and exemplary methods are disclosed herein, such as biofilm layer interference technology and SPR.

The present invention also provides an assay method for identifying antibodies with biological activity. Biological activities may include, for example, binding to antigens, binding to cell surface antigens, inhibition or activation of antigens, and the like. An antibody having such biological activity in vivo and/or in vitro is also provided.

In certain embodiments, the antibodies of the invention are tested for such biological activity.

The present invention also provides methods for identifying properties of antibodies, such as properties related to druggability. The drug-related properties include, for example, thermal stability, such as long-term thermal stability.

Cells for use in any of the above in vitro assays include cell lines that naturally express the antigen or are engineered to express the antigen. Such cells also include antigen-expressing cell lines and cell lines transfected with antigen-encoding DNA that does not normally express the antigen.

It is understood that the immunoconjugates of the invention can be used to replace or supplement the antibody molecules of the invention to perform any of the aforementioned assays.

It is understood that the antibody molecules of the present invention and other active agents can be used to perform any of the aforementioned assays.

In some embodiments, the antigen is PD-L1 (e.g., human PD-L1) and/or OX40 (e.g., human OX40 or monkey OX40).

VII. Pharmaceutical compositions and pharmaceutical preparations

In some embodiments, the present invention provides a composition comprising any antibody molecule described herein or a fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, the composition, for example, a pharmaceutical composition, comprises the antibody molecule of the present invention or a fragment or immunoconjugate thereof, and one or more other therapeutic agents (e.g., anti-angiogenic agents, chemotherapeutic agents, Cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators).

In some embodiments, the composition is used to prevent or treat diseases, such as autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunction diseases and the like. For example, the disease is tumor (e.g. cancer) or infection. In some embodiments, the tumor is tumor immune escape. Preferably, the tumor is, for example, colon cancer or colorectal cancer or rectal cancer or lung cancer.

The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) containing the antibodies of the present invention or immunoconjugates thereof and/or compositions (including pharmaceutical compositions or pharmaceutical preparations) containing polynucleotides encoding the antibodies of the present invention . In certain embodiments, the composition comprises one or more antibodies of the invention or fragments thereof or one or more polynucleotides encoding one or more antibodies of the invention or fragments thereof.

These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, etc. that are physiologically compatible. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. It is also possible to use saline solutions and aqueous dextrose and glycerol solutions as liquid carriers, especially for injectable solutions.

Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerin , Propylene, glycol, water, ethanol, etc. For the use and use of excipients, see also "Handbook of Pharmaceutical Excipients", fifth edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

The composition of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

It can be prepared by mixing the antibody of the present invention with the required purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). The pharmaceutical preparation of the antibody is preferably in the form of a lyophilized preparation or an aqueous solution.

An exemplary lyophilized antibody formulation is described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

The pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient that is required for the specific indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators. The active ingredients are appropriately combined in an amount effective for the intended use.

In some embodiments, the therapeutic agent is selected from one, two or all of the following categories (i)-(iii): (i) drugs that enhance antigen presentation (eg, tumor antigen presentation); (ii) enhance effects Drugs for cellular responses (eg, B cell and/or T cell activation and/or mobilization); or (iii) drugs that reduce immunosuppression.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules.

The pharmaceutical composition of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (for example, by injection or infusion).

The therapeutic composition should generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or lyophilized form. A sterile injectable solution can be prepared by adding the active compound (ie, antibody molecule) in the required amount to a suitable solvent, followed by filtration and sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients. Coating agents such as lecithin and the like can be used. In the case of a dispersion, the proper fluidity of the solution can be maintained by using a surfactant. Prolonged absorption of the injectable composition can be caused by including in the composition a substance that delays absorption such as monostearate and gelatin.

Kits containing the antibody molecules described herein are also within the scope of the present invention. The kit may contain one or more other elements, including, for example: a package insert; other reagents, such as a marker or reagent for coupling; a pharmaceutically acceptable carrier; and a device or other materials for administration to a subject.

VIII. Combination products or kits

In some embodiments, the present invention also provides a combination product, which comprises the antibody or antigen-binding fragment thereof of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (such as anti-angiogenic agents, chemotherapy Agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators, etc.).

In some embodiments, the combination product is used to prevent or treat diseases, such as autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunction diseases and the like. For example, the disease is tumor (e.g. cancer) or infection. In some embodiments, the tumor is tumor immune escape. Preferably, the tumor is, for example, colon cancer or colorectal cancer or rectal cancer or lung cancer.

In some scenarios, two or more components of the combination product may be administered to the subject in combination sequentially, separately, or simultaneously.

In some embodiments, the present invention also provides a kit comprising the antibody, pharmaceutical composition, immunoconjugate or combination product of the present invention, and optionally a package insert to guide administration.

In some embodiments, the present invention also provides a pharmaceutical product comprising the antibody, pharmaceutical composition, immunoconjugate, and combination product of the present invention. Optionally, the pharmaceutical product further includes a package insert to guide administration.

IX. Use of the antibody molecule of the present invention

In one aspect, the present invention relates to the use of the antibody molecule of the present invention in vivo to treat or prevent diseases that require modulation of immune response in a subject, thereby inhibiting or reducing related diseases such as cancerous tumors, autoimmune diseases, inflammatory diseases, The emergence or recurrence of infections, T cell dysfunction diseases. The antibody molecule of the present invention can be used alone. Alternatively, the antibody molecule can be administered in combination with other therapies (e.g., therapeutic/prophylactic/treatment modality). When the antibody molecule of the present invention is administered in combination with one or more other drugs, the combination can be administered sequentially, separately, or simultaneously in any order.

Therefore, in one embodiment, the present invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody molecule described herein. In another embodiment, the present invention provides a method of preventing the occurrence or recurrence of a disease in a subject, the method comprising administering to the subject a prophylactically effective amount of an antibody molecule described herein.

In some embodiments, the disease is a disease in which the patient has (e.g., elevated levels, such as nucleic acid or protein levels) PD-L1 or PD-1 or PD-L2. In some embodiments, the disease is a disease that has (e.g., elevated levels, e.g., nucleic acid or protein levels) PD-L1 or PD-1 or PD-L2 in the patient, such as cancer. In some embodiments, the disease is a disease with T cell dysfunction. In some embodiments, the disease is a disease with reduced levels (e.g., nucleic acid or protein levels) of OX40, such as a disease with reduced OX40 expression or activity.

In some embodiments, the disease is a disease that would benefit from PD-L1 or PD-1 or PD-L2 that inhibits nucleic acid or protein levels. In some embodiments, the disease benefits from blocking the binding of PD-1 to PD-L1, or the binding of PD-1 to PD-L2. In some embodiments, the disease benefits from activation of OX40 activity, such as activation of the OX40 signaling pathway, and or activation of T cells.

In some embodiments, the present invention relates to a method of inhibiting the action of an antigen and/or activating antigen activity or activating an antigen-mediated signaling pathway in an individual, the method comprising administering to the subject an effective amount of an antibody molecule disclosed herein (e.g., anti- PD-L1/OX40 antibody) or pharmaceutical composition or immunoconjugate or combination product or kit. In some embodiments, the antigen is OX40 (e.g., human OX40) and/or PD-L1 (e.g., human PD-L1). In some embodiments, inhibiting the antigen effect refers to blocking the binding of PD-1 and PD-L1, or blocking the binding of PD-1 and PD-L2. In some embodiments, activating antigen activity or activating an antigen-mediated signaling pathway refers to activating the OX40 signaling pathway.

In a specific embodiment, the anti-PD-L1/OX40 bispecific antibody of the present invention can activate T cells (such as CD4+ T cells), for example, enhance the immune stimulatory/effector function of T effector cells and/or make these cells Proliferate and/or down-regulate the immunosuppressive function of T regulatory cells. In some embodiments, the antibody is capable of causing antibody-dependent cell-mediated cytotoxicity (ADCC).

In a specific embodiment, the anti-PD-L1/OX40 bispecific antibody of the present invention enhances CD4+ effector T cell function, for example, by increasing the proliferation of CD4+ effector T cells and/or increasing the cytokine production of CD4+ effector T cells. In some embodiments, the cytokine is gamma-interferon, such as IFNg or interleukin, such as IL-2.

In some embodiments, the anti-PD-L1/OX40 bispecific antibody or fragment thereof of the present invention increases the number of (infiltrating) CD4+ effector T cells in a tumor (e.g., the total number of CD4+ effector T cells, or, for example, CD4+ in CD45+ cells). Percentage of cells). In some embodiments, the anti-PD-L1/OX40 bispecific antibodies or fragments thereof of the present invention increase the number of (infiltrating) CD4+ effector T cells in tumors expressing interferon-gamma (e.g., CD4+ interferon-expressing CD4+ The total number of effector T cells, or, for example, the percentage of CD4+ cells expressing gamma-interferon in the total CD4+ cells).

In some embodiments, the anti-PD-L1/OX40 bispecific antibody or fragment thereof of the present invention enhances memory T cell function, for example, by increasing memory T cell proliferation and/or increasing memory cell cytokine production. In some embodiments, the cytokine is gamma-interferon (e.g., IFNg) or interleukin (e.g., IL-2).

In another aspect, the present invention relates to a method of preventing or treating a tumor (e.g., cancer) in a subject, the method comprising administering to the subject an effective amount of the antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein. Compound or combination product or kit. In some embodiments, the tumor is tumor immune escape. In some embodiments, the tumor is cancer.

In another aspect, the present invention relates to a method of preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein Or combination products or kits.

In another aspect, the present invention relates to a method of preventing or treating autoimmune diseases and/or inflammatory diseases in a subject, the method comprising administering to the subject an effective amount of the antibody molecule or drug disclosed herein Composition or immunoconjugate or combination product or kit.

The subject may be a mammal, for example, a primate, preferably, a higher primate, for example, a human (for example, a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject has or is at risk of suffering from a disease described herein (eg, a tumor or infection or an autoimmune disease as described herein). In certain embodiments, the subject has received or has received other treatments, such as chemotherapy treatments and/or radiation therapy. Alternatively or in combination, the subject is immunocompromised due to infection or is at risk of being immunocompromised due to infection.

In some embodiments, cancers treated and/or prevented with antibody molecules include, but are not limited to, solid tumors, hematological cancers, and metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors. The cancer can be early, middle or late or metastatic cancer.

In some embodiments of any of the methods of the invention, the cancers described herein display human effector cells (eg, are infiltrated by human effector cells). Methods used to detect human effector cells are well known in the art and include, for example, by IHC. In some embodiments, the cancer displays high levels of human effector cells. In some embodiments, the human effector cells are one or more of NK cells, macrophages, and monocytes. In some embodiments of any of the methods of the invention, the cancer described herein displays FcR-expressing cells (e.g., is infiltrated by FcR-expressing cells). Methods for detecting FcR are well known in the art and include, for example, by IHC. In some embodiments, the cancer displays high levels of FcR-expressing cells. In some embodiments, FcR is FcyR. In some embodiments, FcR is an activating FcγR.

The methods and compositions disclosed herein can be used to treat metastatic lesions associated with the aforementioned cancers.

In some embodiments, the tumor is a tumor that requires activation of T cells, such as cancer, such as a tumor or cancer with T cell dysfunction. In some embodiments, the tumor is a tumor that expresses (e.g., elevated levels) PD-L1, such as a cancer. In some embodiments, the tumor is a tumor in which the expression or activity of OX40 is reduced. In some embodiments, the tumor is a tumor that benefits from activation of the OX40 signaling pathway, such as cancer.

In some embodiments, the infectious diseases treated and/or prevented with antibody molecules include pathogens for which no effective vaccine currently exists or pathogens for which conventional vaccines are not fully effective. The blocking effect of the antibody molecules exemplified in the present invention on PD-L1 is particularly useful for combating infections established by pathogens whose variant antigens appear as the infection process progresses. These variant antigens can be regarded as foreign antigens when the antibody of the present invention is administered. Therefore, the antibody molecules exemplified in the present invention can stimulate a strong T cell response that is not inhibited by negative signals through PD-L1.

In some embodiments, the antibody molecules of the present invention are used to treat and/or prevent inflammatory and autoimmune diseases and graft-versus-host disease (GvHD) to down-regulate the immune system.

In other aspects, the present invention provides the use of antibody molecules or fragments or immunoconjugates or compositions or combination products or kits in the production or preparation of drugs for the treatment of related diseases or disorders mentioned herein .

In some embodiments, the antibody or antibody fragment or immunoconjugate or composition or combination product or kit of the present invention will delay the onset of the disorder and/or symptoms related to the disorder.

In some embodiments, the antibody or pharmaceutical composition or immunoconjugate or combination product or kit of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for use herein Said prevention and/or treatment.

In some embodiments, the treatment modality includes surgery; radiotherapy, localized irradiation or focused irradiation, and the like.

In some embodiments, the therapeutic agent is selected from anti-angiogenic agents, chemotherapeutics, cytotoxic agents, vaccines, other antibodies, anti-infective agents, or immunomodulators.

Exemplary vaccines include but are not limited to cancer vaccines. The vaccine can be a DNA-based vaccine, an RNA-based vaccine, or a virus-transduction-based vaccine. Cancer vaccines can be prophylactic or therapeutic.

Exemplary anti-infective agents include, but are not limited to, antiviral agents, antifungal agents, antiprotozoal agents, and antibacterial agents.

In some further embodiments, the antibodies or fragments thereof of the present invention can also be used in combination with tyrosine kinase inhibitors.

In some embodiments of any of the methods of the invention, the administration of an antibody or fragment thereof of the invention is combined with the administration of a tumor antigen. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises nucleic acid. In some embodiments, the tumor antigen is a tumor cell.

In some embodiments, the antibodies of the present invention or fragments thereof may be administered in combination with anti-tumor agents.

In some embodiments, the antibodies of the invention or fragments thereof can be administered in combination with cytokines. The cytokine can be administered as a fusion molecule with the antibody molecule of the present invention, or as a separate composition. In one embodiment, the antibodies of the invention are administered in combination with one, two, three or more cytokines (e.g., as a fusion molecule or as a separate composition).

In some embodiments, the antibodies or fragments thereof of the present invention can be combined with conventional cancer therapies in the art. Conventional cancer therapies include but are not limited to: (i) radiation therapy or ionizing radiation to kill cancer cells and shrink tumors. Radiotherapy can be administered via external radiotherapy (EBRT) or via internal brachytherapy; (ii) chemotherapy, or application of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or specific effects Agents that deregulate cancer cell proteins; (iv) immunotherapy, or enhance the host immune response (e.g., vaccine); (v) hormone therapy, or block hormones (e.g., when the tumor is hormone sensitive), (vi) Angiogenesis inhibitors, or block blood vessel formation and growth, and (vii) palliative care, or such treatments, which involve improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding.

In some embodiments, the antibodies or fragments thereof of the present invention can be combined with conventional methods for enhancing the immune function of the host.

The various combination therapies described above can be further combined for treatment.

Such combination therapies encompass combined administration (in which two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case, other therapies, such as treatment modality and The administration of the antibody of the invention occurs before, at the same time, and/or after the therapeutic agent. Antibody molecules and/or other therapies, such as therapeutic agents or treatment modalities, can be administered during active disease or during remission or less active disease. The antibody molecule can be administered before other treatments, at the same time as other treatments, after treatment, or during disease remission.

In one embodiment, the administration of the antibody of the present invention and the administration of another therapy (such as a treatment modality or therapeutic agent) are within about one month of each other, or within about one, two, or three weeks, or about 1, 2, 3 , 4, 5, or 6 days.

It is understood that the immunoconjugate or composition or combination product or kit of the present invention can be used to replace or supplement the antibody of the present invention for any treatment.

The antibodies of the present invention (and pharmaceutical compositions or immunoconjugates containing them, and any additional therapeutic agents) can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, And, if local treatment is needed, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. To a certain extent, it depends on whether the medication is short-term or long-term, and it can be administered by any suitable route, such as injection, such as intravenous or subcutaneous injection. Various medication schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

In order to prevent or treat disease, the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progress of the disease , Whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient in one treatment or over a series of treatments.

The dosage and treatment regimen of the antibody molecule of the present invention can be determined by the skilled person. In some embodiments, the dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus injection can be administered, several divided doses can be administered over time, or the dose can be reduced or increased proportionally as shown by the criticality of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of active compound, which is calculated when combined with the required pharmaceutical carrier Produce the desired therapeutic effect. The specifications used in the dosage unit form of the present invention are directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) mixing this active compound for use in the field of sensitive therapy in individuals. limit.

X. Methods and compositions for diagnosis and detection

In certain embodiments, any antibody or antigen-binding fragment thereof provided herein can be used to detect the presence of the bound antigen in a biological sample. When the term "detection" is used herein, it includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assay Method, PCR-technology (for example, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

In one aspect, the present invention provides in vitro or in vivo diagnostic methods for detecting the presence of related antigens in biological samples, such as serum, semen or urine or tissue biopsy samples (e.g., from hyperproliferative or cancerous lesions). The diagnostic method includes: (i) contacting a sample (and optionally a control sample) with an antibody molecule as described herein or administering the antibody molecule to a subject under conditions that allow interaction to occur, and (ii) The formation of a complex between the antibody molecule and the sample (and optionally the control sample) is detected. The formation of the complex indicates the presence of the relevant antigen, and may indicate the applicability or demand for the treatment and/or prevention described herein.

In one embodiment, the antibodies of the present invention can be used to diagnose diseases, for example, to evaluate (eg, monitor) the treatment or progression, diagnosis, and/or staging of diseases (eg, tumors or infections) described herein in a subject. In certain embodiments, labeled antibodies of the invention are provided. Labels include, but are not limited to, labels or parts that are directly detected (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), and parts that are indirectly detected, such as enzymes or ligands, such as , Through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125I, 3H and 131I, fluorescing groups such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Umbelliferone, luceriferase, for example, firefly luceriferase and bacterial luceriferase (U.S. Patent No. 4,737,456), umbelliferone, 2,3-dihydrophthalazinedione, Horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lyase, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6 -Phosphate dehydrogenase, heterocyclic oxidase such as uricase and xanthine oxidase, and enzymes that use hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, Biotin/avidin, spin label, phage label, stable free radical, etc.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the antibody of the invention. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, the relevant antigen is detected before treatment, for example, before starting treatment or before some treatment after the treatment interval.

In some embodiments, the level and/or distribution of the relevant antigen is determined in vivo, for example, in a non-invasive manner (e.g., by using a suitable imaging technique (e.g., positron emission tomography (PET) scan) to detect the detectable In one embodiment, for example, by detecting the antibody molecule of the present invention that is detectably labeled with a PET reagent (for example, ¹⁸ F-fluorodeoxyglucose (FDG)), the in vivo measurement of the relevant antigen Level and/or distribution.

In one embodiment, the invention provides a diagnostic kit comprising the antibody molecule described herein and instructions for use.

In some embodiments, a method of treating a disease is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a sample of a subject containing cancer cells) for the presence of a relevant antigen, thereby determining its Value, compare the value with a control value (for example, the value of a relevant antigen in a healthy individual), and if it is greater than the control value, then administer a therapeutically effective amount of the subject, optionally combined with one or more other therapies, to the subject Invented antibodies, thus treating diseases.

In some embodiments, the antigen is PD-L1 (e.g., human PD-L1) and/or OX40 (e.g., human OX40).

It can be understood that the various embodiments described in each part of the present invention, such as diseases, therapeutic agents, treatment methods, and administration, are also applicable to the embodiments of other parts of the present invention, or can be combined with the embodiments of other parts. The embodiments described in each part of the present invention that are applicable to the properties, uses, and methods of antibody molecules are also applicable to compositions, conjugates, combination products and kits containing antibodies.

The following examples are described to assist the understanding of the present invention. It is not intended and should not be interpreted in any way to limit the scope of protection of the present invention.

Example 1. Construction of anti-PD-L1/OX40 bispecific antibody

In this example, molecular cloning technology was used to construct an anti-PD-L1/OX40 bispecific antibody. This bispecific antibody format contains four polypeptide chains and can bind to two antigens, antigen A is OX40, and antigen B is PD-L1. The parent antibody used to construct the bispecific antibody is anti-OX40 monoclonal antibody (ADI-20057 in the form of IgG2, Chinese invention patent application number: 201710185399.9; anti-PD-L1 nanobody humanized Nb-Fc (Chinese invention patent application number) : PCT/CN2017/095884). The construction method is as follows: the structure of the antibody is shown in Figure 1A, consisting of four symmetrical polypeptide chains, the left half is composed of peptide chain #1 and peptide chain #2, and the right half is the same It is composed of peptide chain #1 and peptide chain #2. The structures of peptide chain #1 and peptide chain #2 are shown in Figure 1B. The anti-PD-L1/OX40 bispecific antibody is described below. Anti-PD- The amino acid sequence of the anti-PD-L1/OX40 bispecific antibody peptide chain #1 shown in SEQ ID NO: 1 of the left half of the L1/OX40 bispecific antibody includes the amino acid sequence shown in SEQ ID NO: 2 from the N-terminus to the C-terminus Anti-OX40 antibody ADI-20057VH amino acid sequence, CH1 amino acid sequence shown in SEQ ID NO: 3, Fc amino acid sequence shown in SEQ ID NO: 4, (G4S) ₂ flexible connecting peptide chain amino acid shown in SEQ ID NO: 5 Sequence and the amino acid sequence of the anti-PD-L1 single domain antibody shown in SEQ ID NO: 6. The anti-PD-L1/OX40 shown in SEQ ID NO: 7 of the left half of the anti-PD-L1/OX40 bispecific antibody The amino acid sequence of the bispecific antibody peptide chain #2 includes the amino acid sequence of the anti-OX40 antibody ADI-20057VL shown in SEQ ID NO: 8 and the CL amino acid sequence shown in SEQ ID NO: 9 from N to C.

SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6 are the CDR regions of the anti-PD-L1/OX40 bispecific antibody of the present invention (wherein the HCDR1 in the VHH and VH regions is used AbM rule definition; HCDR2&3 use Kabat rule definition; LCDR: use Kabat rule definition). See the sequence table for specific sequence information.

Example 2. Expression and purification of anti-PD-L1/OX40 bispecific antibody protein

In this example, the nucleotide sequences encoding the peptide chain #1 and peptide chain #2 of the anti-PD-L1/OX40 bispecific antibody constructed in Example 1 were respectively linked to a commercially available genuine product via multiple cloning sites. The nuclear expression vector pXC vector was expressed and purified in eukaryotic cells to obtain the anti-PD-L1/OX40 bispecific antibody. The specific operation is as follows.

In this example, Suzhou Genewiz was commissioned to synthesize the nucleotide sequences encoding the peptide chains of the anti-PD-L1/OX40 bispecific antibody and the IgG2 control antibody. Use appropriate restriction endonucleases and ligases to ligate the synthesized nucleotide sequences encoding the peptide chains into the vector respectively, wherein the two peptide chains encoding DNA sequences of the anti-PD-L1/OX40 bispecific antibody are separately The eukaryotic expression vector Double-GeneVectors (pXC17.4, pXC18.4) (purchased from Lonza) was constructed and sequenced to verify the correctness of the sequence, and recombinant vectors containing the nucleotide sequence encoding the peptide chain were obtained. Peptides #1 and #2 were transferred into CHOK1SV GS-KO Cell line (purchased from Lonza) by electrotransfection method to construct a protein expression cell line for protein expression. The protein expression process is as follows:

1) Take the required CHOK1SV GS-KO Cell line (Lonza) protein expression cell line, subculture it on CD CHO Medium (GIBCO, 10743-029), and culture it to a cell density of 0.8×10 ⁶ cells/ml;

2) After overnight incubation, supplement with 200g/L FEED (Sigma, H6784-100G) with a culture volume of 5/100 every other day, and supplement with glucose (D(+)-Glucose anhydrous, Merck, 1.37048.5000) to the final concentration 5g/L;

3) When the culture is continuously cultured to the 14th day or when the cell viability is ≤60%, the culture is collected, centrifuged at 7500 rpm for 30 minutes, and the cell supernatant is taken for purification.

The cell culture supernatant is purified by affinity chromatography to purify the target bispecific antibody protein. The specific process is as follows: 1) Affinity purification: Use MabSelect SuRe (GE Healthcare, catalog number: 17-5438-03) affinity chromatography column and place it in the AKTA pure system (GE Healthcare). Use 0.1M NaOH to detoxify the AKTA system (overnight). On the day of sample collection, the cells were centrifuged at 7500 rpm/min for 30 minutes and filtered using SARTOPORE (Sartorius, 5441307H4). Before purification, use 5 column volumes of Binding Buffer (Tris20mM, NaCl150mM, pH7.2) to clean the system and equilibrate the column. Pass the filtered supernatant to be purified through the column. Use 5-10 times the column volume of the Binding Buffer to rebalance, and use the UV detection device equipped with the AKTApure system to monitor until the UV is flat. The antibody was eluted with Elution Buffer (citric acid + sodium citrate 100 mM, pH 3.5), and samples were collected according to the UV absorption value. Add 80μl of Neutralizing Buffer (Tris-HCl2M) to each 1ml of the collection solution for neutralization;

2) Replacing the buffer: Detect the purity of the collected samples in each sub-tube according to size exclusion chromatography (SEC), and combine the samples with a purity greater than 95%. Centrifuge the combined solution using a 15ml ultrafiltration centrifuge tube, 4500rpm, 30min. Use PBS to dilute the protein and continue centrifugation, 4500rpm, 30min, repeat 2 times to replace the buffer. Combine the antibodies after changing the buffer and measure the antibody concentration.

The purified protein was tested for purity by SEC, and the result is shown in Figure 2. After two-step purification, the anti-PD-L1/OX40 bispecific antibody of the present invention has a relatively high purity, and its monomer main peak purity is 100.00%, which is suitable for later development.

In this example, Pogalizumab is a human IgG1OX40 antibody for protein expression in HEK293 cells, which uses the INN: list 114 from the suggestion (see http://www.who.int/medicines/publications/druginformation/innlists/PL114.pdf ) Of the heavy and light chain sequences. Entrusted Suzhou Genewiz Biotechnology Co., Ltd. (Genewiz) to synthesize the nucleotide sequences encoding the above-mentioned peptide chains of the antibody. Using appropriate restriction enzymes and ligases, the synthesized nucleotide sequences encoding the peptide chains were respectively ligated into the vector pTT5, and recombinant vectors containing the nucleotide sequences encoding the peptide chains were obtained.

Polyethyleneimine (PEI) was used to transfect into HEK293 cells for protein expression. The transfection process is as follows:

1) Subculture HEK293 cells (purchased from Invitrogen) in Expi293 cell culture medium (purchased from Invitrogen) according to the required transfection volume. The cell culture was centrifuged the day before transfection to obtain a cell pellet, and the cells were suspended in a fresh Expi293 cell culture medium, and the cell density was adjusted to 1×10 ⁶ cells/ml. Continue to culture HEK293 cells so that the cell density in the culture on the day of transfection is about 2×10 ⁶ cells/ml;

2) F17 medium (purchased from Gibco, catalog number A13835-01) of 1/10 of the final volume of the HEK293 cell suspension was used as the transfection buffer. Add 200 μg of the recombinant plasmids containing the nucleotide sequences of each strand in a molar ratio of 1:1 to each milliliter of transfection buffer, and mix them;

3) Add 30 μg of PEI (Polysciences, 23966) to the plasmid, mix and incubate at room temperature for 10 minutes.

Pour the mixture gently into the HEK293 cell suspension. Gently mix and incubate overnight at 8% CO ₂ at 36.5°C;

4) After overnight culture, add FEED (Sigma, catalog number: H6784-100G) with a concentration of 200g/L at a concentration of 1/50 of the transfection culture volume and 1/50 of the transfection culture volume to the culture flask. Glucose solution with a concentration of 200 g/L, mix gently, and place it in 8% CO ₂ at 36.5°C to continue the culture. After 20 hours, add VPA (Gibco, catalog number: 11140-050) to a final concentration of 2mM/L;

5) When the culture is continuously cultured to the 7th day or when the cell viability is ≤60%, collect the culture, centrifuge at 7500 rpm for 30 minutes, and take the cell supernatant for purification;

The cell culture supernatant is purified by affinity chromatography to purify the target bispecific antibody protein. The specific process is as follows:

1) Affinity purification: Use MabSelect SuRe (GE Healthcare, catalog number: 17-5438-03) affinity chromatography column and place it in the AKTApure system (GE Healthcare). Use 0.1M NaOH to detoxify the AKTA system (GE Healthcare) (overnight). On the day of sample collection, the cells were centrifuged at 7500 rpm/min for 30 minutes and filtered using SARTOPORE (Sartorius, 5441307H4). Before purification, use 5 column volumes of Binding Buffer (Tris20mM, NaCl150mM, pH7.2) to clean the system and equilibrate the column. Pass the filtered supernatant to be purified through the column. Use 5-10 times the column volume of the Binding Buffer to rebalance, and use the UV detection device equipped with the AKTApure system to monitor until the UV is flat. The antibody was eluted with Elution Buffer (citric acid + sodium citrate 100 mM, pH 3.5), and samples were collected according to the UV absorption value. Add 80μl of Neutralizing Buffer (Tris-HCl2M) to each 1ml of the collection solution for neutralization;

The anti-OX40 monoclonal antibody (ADI-20057 in the form of IgG2) was prepared using known methods, see Chinese invention patent application number: 201710185399.9; anti-PD-L1 nanobody humanized Nb-Fc (see Chinese invention patent application number: PCT/ CN2017/095884), and the same purification was performed to obtain ADI-20057 and anti-PD-L1 humanized Nb-Fc, which were used in subsequent experiments.

Example 3. Detection of binding activity of anti-PD-L1/OX40 bispecific antibody and antigen

Example 3.1. SPR assay to determine the affinity of anti-PD-L1/OX40 bispecific antibodies

The surface plasmon resonance method (SPR) is used to determine the equilibrium dissociation constant (K _D ) of the antibody of the present invention binding to human PD-L1 or human OX40. Based on the SPR principle, when a beam of polarized light is incident on the end face of the prism at a certain angle, a surface plasmon wave will be generated at the interface between the prism and the gold film, causing free electrons in the metal film to resonate, that is, surface plasmon resonance. When analyzing, first fix a layer of biomolecular recognition film on the surface of the sensor chip, and then flow the sample to be tested across the chip surface. If there are molecules in the sample that can interact with the biomolecular recognition film on the chip surface, it will cause the gold film surface The change of refractive index eventually leads to the change of SPR angle. By detecting the change of SPR angle, information such as the affinity and kinetic constant of the analyte can be obtained.

In this example, the K _D of the anti-PD-L1/OX40 bispecific antibody was determined by Biacore (GE Healthcare, T200). The specific method is as follows: After the antibody is captured by the anti-human Fc antibody on the chip, the detection of the antigen and the captured antibody Binding and dissociation between the two to obtain affinity and kinetic constants. The method includes chip preparation and affinity detection. In the measurement process, 10×HBS-EP+(BR-1006-69, GE Healthcare) diluted by 10 times was used as the experimental buffer. The chip preparation process uses the amino coupling kit (BR-1006-33, GE Healthcare), and the anti-human Fc antibody is coupled to the surface of the CM5 chip (29-1496-03, GE Healthcare). The specific process is as follows: 50mM N-hydroxysuccinimide (NHS) and 200mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) are freshly mixed and injected into the dual channel of the chip to activate 7 minute. Then the anti-human Fc antibody was diluted in 10mM Acetate (pH5.0) and injected into the CM5 chip dual channel to make the protein covalently coupled to the surface of the chip channel with a coupling height of about 6000RU. Finally, 1M ethanolamine was injected, and the remaining activated sites were blocked for 7 minutes. Each cycle of affinity detection includes capturing antibody, binding a concentration of antigen and chip regeneration:

Capture antibody: First, dilute the antibody prepared and purified as in Example 2 to 0.5 μg/mL, and capture it in the second channel of the CM5 chip at a flow rate of 10 μL/min for 30 seconds.

Binding antigen: According to the optimal concentration range of SPR, use experimental buffer to dilute the antigen (between 0.15nM-20nM) (human PD-L1 (ACRO, PD1-H5229), human OX40 (ACRO, OXO-H5224)), from low concentration to high concentration, inject into the dual channel of the CM5 chip, the binding time is 180s and the dissociation time is 600s.

Chip regeneration: Use 10mM Glycine pH1.5 (BR-1003-54, GE Healthcare) to regenerate the chip before the next antibody determination.

The data results use 1:1 combination model for dynamic analysis.

The test results are shown in Table 1 and Table 2. The anti-PD-L1/OX40 bispecific antibody of the present invention can bind to human PD-L1 and human OX40 protein, and maintains the parent antibody ADI-20057 and anti-PD-L1 human The affinity constant of the sourced Nb-Fc and each corresponding antigen.

Table 1. Affinity of anti-PD-L1/OX40 bispecific antibodies to human OX40 determined by SPR assay

Table 2. Affinity of anti-PD-L1/OX40 bispecific antibodies to human PD-L1 determined by SPR assay

Example 3.2. Determination of the affinity of anti-PD-L1/OX40 bispecific antibodies with monkey antigen ForteBio assay

In this example, the Octet Red96 system (manufactured by ForteBio) was used to determine the equilibrium dissociation constant (K _D) of the exemplary anti-PD-L1/OX40 bispecific antibody of the present invention that binds to OX40 and PD-L1 through a kinetic binding assay. ). The ForteBio affinity determination was performed according to the method reported in the literature (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5(2): p.270-278). The experiment process is as follows:

1) Prepare the sensor: half an hour before the start of the experiment, according to the number of samples, take an appropriate number of AHQ sensors (Pall, 1506091) and soak them in SD buffer (PBS1×, BSA 0.1%, Tween-20 0.05%), pre-wet 20 minute;

2) Experimental process: Add 100μl of SD buffer as a blank control (for background deduction) and 100μl of 100nM purified anti-PD-L1/OX40 double into the wells of 96-well black polystyrene half-volume microplate (Greiner). Specific antibody and anti-PD-L1 humanized Nb-Fc antibody as a control, anti-OX40 antibody ADI-20057, 100μl of monkey PD-L1 (ACRO, PD1-C52H4) as an antigen diluted in SD buffer, monkey OX40 (ACRO, OX0-C5220) solution. The anti-human IgG Fc biosensor AHQ was immersed in the wells respectively containing the antibody solution, and the sample was immersed for 600 seconds at room temperature. The sensor was then washed in SD buffer until it reached the baseline, and then immersed in a well containing 100ul of antigen solution to monitor the binding of antibody to antigen. Then transfer the sensor to a well containing 100ul SD buffer to monitor the antibody dissociation (set the running steps and time: Baseline, Loading ~ 1nm, Baseline, Association and Dissociation time depends on the sample binding and dissociation speed). The speed is 400 rpm and the temperature is 30°C. The background-corrected binding and dissociation curves were fitted by Octet analysis software (ForteBio) to generate the binding (K _on ) and dissociation (k _dis ) rate constants, which were then used to calculate the equilibrium dissociation constant (K _D ).

The test results are shown in Table 3 and Table 4. The anti-PD-L1/OX40 bispecific antibody of the present invention can bind to monkey PD-L1 and monkey OX40, and maintains the parental antibody ADI-20057 and anti-PD-L1 humanization The affinity constant of Nb-Fc and each corresponding antigen.

Table 3. Binding affinity of anti-PD-L1/OX40 bispecific antibodies to monkey OX40 determined by ForteBio kinetic binding assay

Table 4. Binding affinity of anti-PD-L1/OX40 bispecific antibodies to monkey PD-L1 determined by ForteBio kinetic binding assay

Example 3.3. Binding analysis of the anti-PD-L1/OX40 bispecific antibody of the present invention and CHO cells overexpressing human OX40 or human PD-L1

In order to verify whether the anti-PD-L1/OX40 bispecific antibody of the present invention can bind to the antigen expressed on the cell surface, this example uses flow cytometry to detect the anti-PD-L1/OX40 bispecific antibody and overexpressing human OX40 or human PD-L1 binding to CHO cells, the experimental process is as follows:

1) Cell preparation: Transfect the pCHO1.0 vector (Invitrogen) carrying the human OX40 cDNA (SinoBiological Inc) cloned into the multi-cloning site MCS into Chinese hamster ovarian cancer cells (CHO) (Invitrogen), and obtain stable expression after pressure screening CHO cells of human OX40 (CHO-OX40 cells), the pCHO1.0 vector (Invitrogen) carrying human PD-L1 cDNA (SinoBiological Inc) cloned into the multi-cloning site MCS was transfected into Chinese hamster ovarian cancer cells (CHO) (Invitrogen) ), CHO cells (CHO-PD-L1 cells) stably expressing human PD-L1 were obtained through pressure screening, and CHO-PD-L1/CHO-OX40 cells were counted and diluted to 2×10 ⁶ cells/ml. Add 100μl/well to the U-shaped bottom 96-well plate;

2) Detection steps: 400g, 5min, centrifuge, remove the cell culture medium. Add serially diluted samples (bispecific antibodies, anti-PD-L1 antibodies, and anti-OX40 antibodies) to the U-shaped plate and resuspend the cells, add 100 μl to each well, and let stand on ice for 30 minutes. 400g, 5min, remove the supernatant, wash the cells with PBS once to remove unbound antibody. 400g, 5min remove PBS, add 100μl 1:200 diluted PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) to each well. Incubate for 30min on ice, protected from light. 400g, 5min, remove the supernatant, wash the cells with PBS once to remove unconjugated antibody. Resuspend the cells with 100μl PBS, flow cytometer (BD, ACCURIC6) to detect the binding of antibody to cells.

The test results are shown in Figure 3. The anti-PD-L1/OX40 bispecific antibody of the present invention can bind to human OX40 expressed on the cell surface, with a binding EC50 of 8.463 nM, and the binding ability of the parent anti-OX40 antibody ADI-20057 ( EC50 is 4.710nM) similar. As shown in Figure 4, the anti-PD-L1/OX40 bispecific antibody of the present invention can bind to human PD-L1 expressed on the cell surface, with a binding EC50 of 8.732 nM, and the parent antibody anti-PD-L1 humanized Nb- The binding capacity of Fc (EC50 is 9.651 nM) is similar.

Example 3.4. Analysis of the anti-PD-L1/OX40 bispecific antibody of the present invention simultaneously binding to CHO cells overexpressing human OX40 and CHO cells overexpressing human PD-L1

In order to verify whether the anti-PD-L1/OX40 bispecific antibody of the present invention can simultaneously bind to target antigens from different cells, this example uses flow cytometry to detect the cross-linking of different cells induced by the bispecific antibody . The specific experiment process is as follows:

1) The CHO-PD-L1 and CHO-OX40 cells obtained as described in Example 3.3 were centrifuged at 400g for 5 minutes to remove the medium, washed with PBS, and then resuspended in PBS. After counting, adjust the cell density to 2× 10 ⁶ / ml; the CHO-PD-L1 and CHO-OX40 cells were 1: 5000 was added ^{CellTracker TM Deep Red (Thermo, C34565} ) , and cell Trace CFSE (Invitrogen, C34554) dye, placed in 37 ℃ 30 minutes. After centrifugation at 400g for 5 minutes, remove the supernatant and wash the cells once with PBS;

2) Add serially diluted samples (bispecific antibody, anti-PD-L1 antibody, anti-OX40 antibody and IgG2 control (see sequence table)) respectively into U-shaped bottom 96-well plates and stained CHO-PD-L1 cells , Mix (the final cell density is 1.5×10 ⁶ cells/ml). Place the U-shaped bottom 96-well plate at 4°C for 30 minutes and then take it out, 400g, centrifuge for 5 minutes, wash four times with PBS, and resuspend the cells with PBS;

3) Add the CHO-OX40 cells stained in 1) above (the final cell density is 1×10 ⁶ cells/ml) to the above cells in 2), and place them at room temperature for 1 hour before performing flow cytometry (BD, ACCURIC6) detection. The ratio of double-positive cells in channel 2 and channel 4 can reflect the cell cross-linking caused by the anti-PD-L1/OX40 bispecific antibody.

The FACS detection result is shown in Figure 5. The anti-PD-L1/OX40 bispecific antibody of the present invention can induce the cross-linking of CHO-PD-L1 cells and CHO-OX40 cells, thus indicating that the bispecific antibody of the present invention can Simultaneously bind target antigens from different cell surfaces.

Example 4 Human T cell binding detection of anti-PD-L1/OX40 bispecific antibody of the present invention

In order to verify whether the anti-PD-L1/OX40 bispecific antibody of the present invention can bind to human T cells, this example uses flow cytometry to detect the binding of the anti-PD-L1/OX40 bispecific antibody to human T cells Ability, the experiment process is as follows:

1) Cell preparation: Resuscitate human PBMC cells (ALLCELLS, PB005F), use Human T cell enrichment kit (STEMCELL, 19051) to isolate CD4+ cells, and add Dynabeads Human T-Activator according to CD4+: anti-CD3/CD28Beads=1:1 CD3/CD28 (Gibco, 11131D), stimulate for 3 days;

2) Detection steps: Centrifuge the cell culture in 1) at 400g for 5min, remove the cell culture medium, resuspend the cells with PBS, and after counting, adjust the cell density to 2×10 ⁶ cells/ml, toward the U-shaped bottom Add 100μl/well to 96-well plate. Add the serially diluted samples (anti-PD-L1/OX40 bispecific antibody, ADI-20057 and IgG2 control) to the U-shaped plate and mix, add 100 μl to each well, and let it stand on ice for 30 minutes. Centrifuge at 400g for 5min to remove the supernatant, and wash the cells with PBS once. Centrifuge at 400g for 5min to remove PBS, and add 100μl of 1:200 diluted PE-anti-human Fc antibody (SOUTHERNBIOTECH, 2040-09) to each well. Incubate for 30min on ice, protected from light. Centrifuge at 400g for 5min to remove the supernatant, and wash the cells with PBS once. Resuspend the cells with 100μl PBS and detect by flow cytometry (BD, ACCURIC6).

The test results are shown in Figure 6. The anti-PD-L1/OX40 bispecific antibody of the present invention can bind to human T cells, with a binding EC50 of 4.062 nM, and binds to the parental anti-OX40 antibody ADI-20057

The capacity (EC50 is 2.571 nM) is similar.

Example 5 Accelerated stability detection of the anti-PD-L1/OX40 bispecific antibody of the present invention

In order to confirm the stability of the bispecific antibody, this example evaluated the long-term changes in the purity of a batch of antibodies prepared at 40°C for 0, 1, 3, 7, 10, 20, and 30 days. Thermal stability. The experimental method is as follows:

1. Concentrate the antibody sample obtained above to 10 mg/ml (dissolved in PBS), and distribute it in EP tubes, 200 μl/tube, and store at 40°C in the dark.

2. On the 0th, 1, 3, 7, 10, 20, and 30 days, take one tube and use size exclusion chromatography (SEC) to detect the purity of the main monomer peak.

The experimental results are shown in Table 5. When the anti-PD-L1/OX40 bispecific antibody of the present invention is placed at 40°C for 30 days, the reduction in the ratio of the monomer main peak is only 1.28%. The results show that the anti-PD-L1/OX40 bispecific antibody of the present invention has good stability and is suitable for later development.

Table 5. Changes in the ratio of the main monomer peaks of the bispecific antibody cultured at 40°C

放置于40℃(天)Placed at 40℃ (days)	抗PD-L1/OX40双特异性抗体Anti-PD-L1/OX40 bispecific antibody
00	100.00％100.00%
11	100.00％100.00%
33	99.46％99.46%
77	99.32％99.32%
1010	99.09％99.09%
2020	98.95％98.95%
3030	98.72％98.72%

Example 6 Detection of T _m of the anti-PD-L1/OX40 bispecific antibody of the present invention

Differential scanning fluorescence (DSF) can provide information about structural stability according to the fluorescence change process in the map, and detect the configuration change of the protein. The temperature corresponding to the maximum absolute value of the fluorescence curve is the Tm of the protein. In this example, the DSF method was used to determine the T _m value of the anti-PD-L1/OX40 bispecific antibody of the present invention. The experimental process is as follows:

1) Dilute the anti-PD-L1/OX40 bispecific antibody sample prepared above to 1 mg/ml with PBS. Dilute SYPRO Orange protein gel staining (GIBCO, S6650) by 50 times with PBS, that is, 4μl SYPRO Orange protein gel staining mother liquor plus 196μl PBS;

2) Add sample to 96-well PCR plate: 50μl diluted antibody sample + 10μl SYPRO Orange protein gel staining diluent (obtained in step 1) + 40μl water. Put it in 7500 real-time PCR system (Applied Biosystems, AB/7500) for testing.

The experimental results are shown in Table 6 and Figure 7. The anti-PD-L1/OX40 bispecific antibody of the present invention has a Tm greater than 60°C and is suitable for later development.

Table 6. Tm measurement results of bispecific antibodies

抗体的名称The name of the antibody	T _m1 T _m 1	T _m2 T _m 2	T _m3 T _m 3
抗PD-L1/OX40双特异性抗体Anti-PD-L1/OX40 bispecific antibody	63.5963.59	71.2071.20	74.8174.81

Example 7 Detection of anti-PD-L1 activity of anti-PD-L1/OX40 bispecific antibody based on luciferase reporter gene

In order to determine whether the anti-PD-L1/OX40 bispecific antibody can relieve the inhibitory effect of the PD-1/PD-L1 interaction on the NFAT signaling pathway, this example uses the luciferase reporter gene to detect the cell line (Promega, CS187109 ), by detecting the expression of luciferase, the ability of the bispecific antibody to inhibit the PD-1/PD-L1 interaction is reflected. The detailed experimental process is as follows:

Considering that the exploration of antibodies should be based on the understanding of their mechanism of action (MOA) and biological activity, this example uses PD-1/PD-L1 Blockade Bioassay, Cell Propagation Model (Promega Company) ), studied the anti-PD-L1 biological activity of the bispecific antibody of the present invention.

Promega's PD-1/PD-L1 Blockade Bioassay is a biologically relevant MOA-based assay used to determine the efficacy and stability of antibodies that can block the PD-1/PD-L1 interaction. The assay consists of two genetically engineered cell lines:

PD-1 effector cells: stably expressing human PD-1 and Jurkat T cells that express luciferase induced by nuclear factor of activated T cells (NFAT).

PD-L1 aAPC/CHO-K1 cells: CHO-K1 cells that stably express human PD-L1 and cell surface proteins that activate the corresponding TCR in an antigen-independent manner.

The combination of PD-1 and PD-L1 can block the downstream signal transduction of NFAT, thereby inhibiting the expression of luciferase. When PD-1 antibody or PD-L1 antibody is added, this blocking effect is reversed. Luciferase is expressed, thereby detecting the luminous signal. The detection method has good sensitivity, specificity, accuracy, and stability.

Test according to the manufacturer's product specification.

1) Pave PD-L1 aAPC/CHO-K1 cells the day before the activity test: discard the culture supernatant, wash once with PBS, add appropriate amount of pancreatin (Gibco, 25200072), incubate at 37°C, 5% CO ₂ for 3-5 min, Double the volume of RPMI1640 (Gibco, 22400-071) medium containing 10% FBS (HyClone, SH30084.03) to terminate the digestion, collect the cells, take a small amount of cell mixture to determine the cell concentration, take the required volume of cells, 400g, and centrifuge for 10 minutes , Discard the supernatant, and resuspend the cells in RPMI1640 (Gibco, 22400-071) medium containing 10% FBS (HyClone, SH30084.03) as the assay buffer to make the cell density 4×10 ⁵ cells/ml. The cells were added to a 96-well white cell culture plate (Nunclon, 136101) 100 μL/well, and PBS was added to the side holes, 200 μl/well. Cells were cultured overnight in a carbon dioxide incubator at 37°C and a 5% CO ₂ incubator;

2) Take a sterile 96-well plate (Nunclon, 442404), and dilute the sample to be tested (anti-PD-L1/OX40 bispecific antibody, anti-PD-L1 humanized Nb-Fc and IgG2 control) to 400nM as the starting concentration , The second concentration point 2 to the 10th concentration point 3 times serial dilution, a total of 12 concentration points;

3) Take PD-1 effector cells, count, 400g, centrifuge for 5 minutes, resuspend the cells in assay buffer to make the cell concentration 1.25×10 ⁶ cells/ml, take 96 white cell culture plates (NUNC, 136101), each well Add 100μl of cells;

4) Take out the white cell culture plate in step 1) from the incubator, discard 95μl/well, and add 40μl of antibody diluted in step 2) and Jurkat/PD-1 cells in step 3), 40μl per well;

5) The culture plate obtained in step 4) is cultured in a carbon dioxide incubator at 37°C and 5% CO ₂ for 6 hours;

6) Take out the white cell culture plate in step 5) and let it stand at room temperature for 5-10 minutes;

7) Melt Bio-Glo ^TM buffer (Promega, G7940), add Bio-Glo ^TM substrate (Promega, G7940), and mix well. The obtained Bio-Glo ^TM reagent was added at 80 μl/well to the well of the test plate (obtained in step 6) after 6 hours of incubation. Leave at room temperature for 5 to 10 minutes;

8) Use Spectra Max I3 microplate reader (Thermo, Maxi3) to collect all-wavelength chemiluminescence, and the collection time per well is 1000ms.

The experimental results are shown in Figure 8. The anti-PD-L1/OX40 bispecific antibody of the present invention can effectively relieve the blocking effect of PD1/PD-L1 interaction on the NFAT signaling pathway, and its activity is comparable to that of anti-PD-L1 humanization Nb-Fc is similar (the EC50 of anti-PD-L1/OX40 bispecific antibody is 0.4085, and the EC50 of anti-PD-L1 humanized Nb-Fc is 0.4271).

Example 8 The anti-PD-L1/OX40 bispecific antibody of the present invention does not block the interaction between human OX40 ligand and human OX40 on CHO cells

In order to verify whether the bispecific antibody of the present invention blocks the binding of human OX40 ligand to human OX40, this example uses flow cytometry to detect that the bispecific antibody does not block the binding of human OX40 ligand to CHO cells expressing human OX40. The experiment process is as follows:

1) The biotin labeling of Human OX40 Ligand Fc Tag (ACRO, OXL-H526X-1MG) is operated in accordance with the EZ-Link Sulfo-NHS-LC-BiotinNoWeighFormat (PIERCE, 21327) instruction manual, and the desalting is performed in accordance with 2ml Zeba ^TM centrifugal desalting Column (PIERCE, 89890) manual operation;

2) Cell preparation: Pipette the CHO-OX40 cells obtained in Example 3.3 into a 50ml centrifuge tube, count on a hemocytometer, take 2.4×10 ⁷ cells/ml in a new 50ml centrifuge tube, and centrifuge at 400g for 5min. Remove the supernatant, resuspend the cells in 20ml PBS, centrifuge again for 5 minutes, remove the supernatant, and resuspend the cells in 5ml PBS;

3) Add 50μl of CHO-OX40 cells processed in 2) to each well of 96-well U-bottom hemagglutination plate for use;

4) Gradient concentration sample solution preparation: Add 3.2ml PBS to 96μl of Biotin-labeled Human OX40 Lig and Fc Tag (Biotin-Human OX40 Lig and Fc Tag), mix well, and configure into Biotin-Human OX40 Lig and Fc Tag PBS mixture. Dilute the antibody sample (anti-PD-L1/OX40 bispecific antibody, ADI-20057, Pogalizumab and IgG2 control) with Biotin-Human OX40 Ligand Fc Tag PBS mixture, with 2000 nM as the starting concentration, and the next 11 concentration points 3 Double serial dilution, a total of 12 concentration points;

5) Add 50μl of the prepared gradient concentration sample to the 96-well U-bottom hemagglutination plate obtained in 3), mix well, and incubate at 4°C for 30 minutes;

6) Centrifuge the cell culture obtained in 5) at 400 g for 5 min to remove the supernatant, add 150 μl PBS to each well, and then centrifuge at 400 g for 5 min to remove the supernatant, repeat three times;

7) Add 100μl of Streptavidin-R-phycoerythrin (SAPE) (THERMO, S21388) diluted 1:200 to each well of the 96-well U-bottom hemagglutination plate obtained in 6), 4°C, 30min; 8) to 7) Add 150μl PBS, 400g, 5min to each well of 96-well U-bottom hemagglutination plate, centrifuge, remove the supernatant, repeat twice;

9) The cells in the 96-well U-bottom hemagglutination plate obtained in 8) were resuspended in 100 μl PBS, and detected by a flow cytometer (BD, ACCURIC6).

The test results are shown in Figure 9. In the experiment, Pogalizumab easily blocked the binding of human OX40 ligand to OX40 at a concentration greater than about 1 nM or higher. However, we found that the anti-PD-L1/ The OX40 bispecific antibody did not show a blocking effect, similar to the IgG control.

Example 9 OX40 blocking assay for detection of anti-PD-L1/OX40 bispecific antibodies based on luciferase reporter gene

The non-blocking activity of the anti-PD-L1/OX40 bispecific antibody of the present invention can be evaluated by measuring the performance of the antibody to block OX40 activation mediated by OX40 ligand.

NFkB-mediated transcriptional activation is measured to evaluate the activator activity of the anti-OX40 antibody of the anti-PD-L1/OX40 bispecific antibody of the present invention. With Anti-Human CD3 (BD, 555329), Anti-Human CD28 (BD, 555725) plus the antibody in the solution activated overexpression human OX40 (purchased from Sino) and NFkB-luciferase construct (NFkB promoter- Luc, Promega) Jurkat cells (ATCC, USA) (Jurkat-OX40-NFkB-Luc-Rep), and then add Bio-Glo ^TM reagent for color development. The specific experiment process is as follows:

Solution preparation: (1) Jurkat-OX40-NFkB-Luc-Rep cell complete medium: RPIM-1640 (90%) (Gibco, 22400-071), FBS (10%) (HyClone, SH30084.03), Hygromycin B (200μg/ml) (INVITROGEN, 10687010), Puromycin (2μg/ml) (GBICO, A11138-02);

(2) Analysis buffer A: RPIM-1640 (90%), FBS (10%), Anti-Human CD3 (4μg/ml), Anti-Human CD28 (16μg/ml), 20μg/ml Human OX40 Ligand Fc Tag , Ready to use;

(3) Analysis buffer B: RPIM-1640 (90%), FBS (10%), Anti-Human CD3 (4μg/ml), Anti-Human CD28 (16μg/ml), ready for use.

Experimental steps:

1) Jurkat-OX40-NFkB-Luc-Rep cell treatment: Take the Jurkat-OX40-NFkB-Luc-Rep cell count in the logarithmic growth phase, 400g, centrifuge for 5min, and regenerate it with complete medium

Suspend the cells and adjust the cell density to 8×10 ⁶ cells/ml for later use;

2) Preparation of gradient concentration sample solution: Dilute the test antibody sample and control to 200nM as the starting concentration with the processed analysis buffer A, and the second concentration point 2 to the 10th concentration point 3 times serial dilution, a total of 9 Concentration point. Use the processed analysis buffer B as a control group;

3) Add samples: Add 50μl of the cell suspension prepared in 1) and 50μl of the diluted antibody sample and 50μl of the control sample in 1) to each well of the white opaque cell culture plate, and place the cell plate in a carbon dioxide incubator 37 Cultivate for 12 hours under 5% CO ₂ conditions;

4) Color development: Melt Bio-Glo ^TM buffer (Promega, G7940), add Bio-Glo ^TM substrate (Promega, G7940), and mix well to obtain Bio-Glo ^TM detection reagent. The obtained Bio-GloTM detection reagent was added at 80 μl/well to the well of the detection plate after culturing for 12 hours in the above 3). Leave it at room temperature for 5 to 10 minutes, and read the luminous signal value.

5) Read the plate: Use Spectra Max I3 microplate reader (Thermo, Max i3) to collect full-wavelength chemiluminescence, and the collection time per well is 1000ms.

The experimental results are shown in Figure 10. Pogalizumab easily blocks the activation of the OX40 signaling pathway mediated by OX40 ligand at a concentration greater than about 0.4 nM or higher. However, we were surprised to find that the anti-PD-L1/OX40 bispecific antibody of the present invention and its parental anti-OX40 antibody increased the level of luciferase, indicating that it has non-OX40 ligand blocking properties and aggregates OX40 receptors. Contribute.

The anti-PD-L1/OX40 bispecific antibody of the present invention effectively enhances the activation of the OX40 signal pathway mediated by the OX40 ligand, with EC50=1.993 nM, and the activation effect of the parent anti-OX40 antibody is similar (EC50=2.326 nM).

Example 10 Detection of the activation of the anti-PD-L1/OX40 bispecific antibody on the OX40-mediated signal pathway based on the luciferase reporter gene method

In order to detect the in vitro biological activity of the bispecific antibody, this example uses the Jurkat-OX40-NFkB-Luc-Rep stable cell line from Cinda Biopharmaceutical (Suzhou) Co., Ltd. (see Example 9 for the construction process). Cells (ATCC, CCL-86TM) enhance the activation of OX40-specific antibodies, and at the same time add Anti-Human CD3 (BD, 555329) and Anti-Human CD28 (BD, 555725) to the cell reaction system, thereby activating downstream NFκB lining After the expression of luciferase reporter gene, the substrate of luciferase is added to lyse the cells and produce luminescence. The intensity of luminescence reflects the biological activity of anti-OX40 antibody.

The detailed experiment process is as follows:

(2) Analysis buffer: RPIM-1640 (90%), FBS (10%), Anti-Human CD3 (10μg/ml) (BD, 555329), Anti-Human CD28 (10μg/ml) (BD, 555725) , Ready to use;

(3) Raji complete medium: RPIM-1640 (90%) (Gibco, 22400-071), FBS (10%) (HyClone, SH30084.03).

Experimental steps:

1) Raji cell processing: Take Raji cells to count, 400g, centrifuge for 5min, resuspend the cells with analysis buffer and adjust the cell density to 2.0×10 ⁶ cells/ml for later use;

2) Add the Raji cells obtained in 1) to the white opaque cell culture plate according to the experimental layout, and then add the processed Jurkat-OX40-NFkB-Luc-Rep cells (the steps are the same as the experimental step 1 of Example 9) ) Add 25μl/well to the above white opaque cell culture plate according to the experimental layout, gently shake it horizontally, and place it in a 37°C, 5% CO ₂ incubator for use;

3) Preparation of gradient concentration sample solution: Dilute the antibody sample (anti-PD-L1/OX40 bispecific antibody, ADI-20057 and IgG2 control) with analysis buffer, with 800nM as the starting concentration, the second concentration point 2 to 3 times serial dilution for the 12th concentration point, a total of 12 concentration points;

4) Adding samples: Set up 3 multiple wells in the sample group, add each concentration sample (50μl) to the white opaque cell culture plate obtained in 2) (each concentration sample is added in triplicate), and place the cell plate Incubate in a carbon dioxide incubator at 37°C and 5% CO ₂ for 16 hours;

5) Color development: Melt Bio-Glo ^TM buffer (Promega, G7940), add Bio-Glo ^TM substrate (Promega, G7940), and mix well to obtain Bio-Glo ^TM detection reagent. The obtained Bio-Glo ^TM detection reagent was added at 80 μl/well to the well of the detection plate obtained in 4) after 16 hours of incubation. Leave it at room temperature for 5 to 10 minutes, and read the luminous signal value.

6) Reading plate: Use Spectra Max I3 microplate reader (Thermo, Max i3) to collect full-wavelength chemiluminescence, and the collection time per well is 1000ms.

The results are shown in Figure 11, the anti-PD-L1/OX40 bispecific antibody of the present invention can activate the OX40 signaling pathway with an activation EC50 = 0.6712 nM, and the activation effect is similar to that of the parent (EC50 = 0.1455 nM).

Example 11 The anti-PD-L1/OX40 bispecific antibody of the present invention mediates the detection of PD-L1-dependent activation of OX40-mediated signal pathway

Example 11.1 The anti-PD-L1/OX40 bispecific antibody of the present invention activates the biological activity of OX40-mediated signaling pathway in the presence of highly expressing PD-L1 Raji cells

In order to detect that the anti-PD-L1/OX40 bispecific antibody of the present invention activates the biological activity of the OX40-mediated signaling pathway in the presence of highly expressing PD-L1 Raji cells. The pCHO1.0 vector (Invitrogen) carrying the human PD-L1 cDNA (Sino Biological) cloned into the multi-cloning site MCS was transfected into Raji (ATCC, CCL-86TM) host cells, and the stable expression of human PD-L1 was obtained by pressure screening Raji cells (Raji-PD-L1 cells). This example uses the cell line to detect the specific activation of the anti-PD-L1/OX40 bispecific antibody on the NFkB signaling pathway downstream of OX40 in the presence of Raji-PD-L1.

Solution preparation: (1) Jurkat-OX40-NFkB-Luc-Rep cell complete medium: RPIM-1640 (90%),

FBS (10%), Hygromycin B (200μg/ml), Puromycin (2μg/ml).

(2) Analysis buffer: RPIM-1640 (90%), FBS (10%), ready for use.

(3) Raji complete medium: RPIM-1640 (90%), FBS (10%).

Experimental steps:

1) Take a small amount of cell suspension, measure the cell density with a cell counting plate, perform 400g centrifugation on the cell culture, centrifuge for 10 minutes, remove the supernatant, add the analysis buffer to gently resuspend the cells, and mix Jurkat-OX40-NFkB-Luc-Rep Adjust the cell density to 4×10 ⁵ cells/ml; adjust the Raji cell density to 1.2×10 ⁵ cells/ml; adjust the Raji-PD-L1 cell density to 1.2×10 ⁵ cells/ml;

2) Transfer the cell suspension obtained in 1) into the sample tank, and take a 96-well white cell culture plate. Add 66.5μL of Jurkat-OX40-NFkB-Luc-Rep (see Example 9 for the construction process) and Raji cell suspension (or Raji-PD-L1 cell suspension) processed in 1) in the first well. Add 50 μL of the Jurkat-OX40-NFkB-Luc-Rep cell and Raji cell suspension (or Raji-PD-L1) processed in 1) to the twelfth well;

3) Adding samples: add the antibody to be tested in the first well, dilute to 25nM as the starting concentration, and dilute the second concentration point 2 to the 12th concentration point by 4 times, a total of 12 concentration points;

4) Place the cell plate in a carbon dioxide incubator at 37°C and 5% CO ₂ for 16 hours;

5) Melt Bio-Glo ^TM buffer (Promega, G7940), add Bio-Glo ^TM detection substrate (Promega, G7940), and mix to obtain. The obtained Bio-Glo ^TM detection reagent was added at 80 μl/well to the wells of the detection plate after 16 hours of incubation. Leave it at room temperature for 5 to 10 minutes, and use Spectra Max I3 microplate reader (Thermo, Max i3) to collect all-wavelength chemiluminescence, and the collection time per well is 1000 ms.

The experimental results are shown in Figures 12 and 13. As shown in Figure 12, in the cell reaction system without PD-L1 expression, the anti-PD-L1/OX40 bispecific antibody of the present invention and the anti-OX40 antibody (ADI-20057) are used alone, and the anti-PD-L1 antibody (anti PD-L1 humanized Nb-Fc) alone, anti-OX40 antibody (ADI-20057) and anti-PD-L1 antibody (anti-PD-L1 humanized Nb-Fc) combined use, none of them can activate the NFkB signal downstream of OX40 path.

However, we were surprised to find that in a cell system with PD-L1 expression (experimental results are shown in Figure 13), the anti-PD-L1/OX40 bispecific antibody of the present invention has better performance than anti-OX40 antibody and anti-PD-L1 antibody. Used alone, anti-OX40 antibody and anti-PD-L1 antibody combined use, Pogalizumab has a more pronounced NFkB signaling pathway activation effect. The anti-PD-L1/OX40 bispecific antibody of the present invention shows that in the presence of cells expressing PD-L1, it can better activate the NFkB signaling pathway downstream of OX40.

Example 11.2 The anti-PD-L1/OX40 bispecific antibody of the present invention specifically activates the NFkB signaling pathway downstream of OX40 in the presence of tumor cells

The PD-L1 protein is expressed on the surface of tumor cells in the human body, and most anti-PD-L1 monoclonal antibodies will bind to the tumor cells. In this example, the Jurkat-OX40-NFkB-Luc-Rep cell line of Cinda Biopharmaceutical (Suzhou) Co., Ltd. (see Example 9 for the construction process) and the tumor cell NCI-H292 human lung cancer cell (ATCC, CRL-1848) After incubation, it was tested whether the anti-PD-L1/OX40 bispecific antibody of the present invention has a specific activation effect on the NFkB signaling pathway downstream of OX40 in the presence of tumor cells. The specific experiment process is as follows:

Solution preparation: (1) Jurkat-OX40-NF _κ B-luc cell complete medium: RPIM-1640 (90%), FBS (10%), Hygromycin B (200 μg/ml), Puromycin (2 μg/ml).

(2) Analysis buffer: RPIM-1640 (90%), FBS (10%), ready for use.

(3) NCI-H292 human lung cancer cell complete medium: RPIM-1640 (90%), FBS (10%).

Experimental steps:

1) NCI-H292 human lung cancer cell treatment: Take NCI-H292 cell count, centrifuge at 1000 rpm/min for 5 minutes, resuspend the cells in analysis buffer and adjust the cell density to 1.6×10 ⁶ cells/ml for use;

2) Jurkat-OX40-NFκB-luc cell processing: Take the Jurkat-OX40-NFκB-luc cell count in the logarithmic growth phase, 400g, centrifuge for 5min, resuspend the cells with analysis buffer and adjust the cell density to 0.8×10 ⁶ cells /ml spare;

3) Add the treated NCI-H292 human lung cancer cells to the white opaque cell culture plate according to the experimental layout 25μl/well, and then add the treated Jurkat-OX40-NFκB-luc cells according to the experimental layout 25μl/well to the white opaque cells In the culture plate, gently shake it horizontally and place it in a 37°C, 5% CO ₂ incubator for later use;

4) Preparation of gradient concentration sample solution: The antibody samples (anti-PD-L1/OX40 bispecific antibody prepared as described above, ADI-20057, anti-PD-L1 humanized Nb-Fc, ADI- 20057+anti-PD-L1 humanized Nb-Fc) diluted to 20nM as the starting concentration, the second concentration point 2 to the 12th concentration point 3-fold serial dilution, a total of 12 concentration points;

5) Adding samples: Set up 3 multiple wells in the sample group, add each concentration sample (50μl) to 3) to obtain a white opaque cell culture plate (each concentration sample well is in triplicate), and place the cell plate in carbon dioxide Incubate in an incubator at 37°C and 5% CO ₂ for 16 hours;

6) Color development: Melt Bio-Glo ^TM buffer (Promega, G7940), add Bio-Glo ^TM substrate (Promega, G7940), and mix well to obtain Bio-Glo ^TM detection reagent. The obtained Bio-Glo ^TM detection reagent was added at 80 μl/well to the wells of the detection plate after 16 hours of incubation. Leave it at room temperature for 5 to 10 minutes, and use Spectra Max I3 microplate reader (Thermo, Max i3) to collect all-wavelength chemiluminescence, and the collection time per well is 1000 ms.

The experimental results are shown in Figure 14. In the cell reaction system of NCI-H292 human lung cancer cells, anti-OX40 antibody (ADI-20057) was used alone, and anti-PD-L1 antibody (anti-PD-L1 humanized Nb-Fc) was used alone. Use, anti-OX40 antibody and anti-PD-L1 antibody combined (ADI-20057+anti-PD-L1 humanized Nb-Fc) can not activate the NFkB signaling pathway downstream of OX40. However, we were surprised to find that the anti-PD-L1/OX40 bispecific antibody of the present invention has a more significant NFkB signaling pathway than anti-OX40 antibody, anti-PD-L1 antibody alone, anti-OX40 antibody and anti-PD-L1 antibody combined use The activation effect. Bispecific antibodies can better activate the NFkB signaling pathway downstream of OX40 in the presence of tumor cells that naturally express PD-L1.

Example 12 Detection of the activation effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on human T cells

In order to detect the in vitro biological activity of the bispecific antibody, this example tested the activation effect of the bispecific antibody on human T cells in vitro. The detailed experimental process is as follows:

Resuscitate human PBMC cells (ALLCELLS, PB005F), stand for 3 hours and then become monocytes, add 10ml AIM

Medium CTS (GIBCO, A3021002) medium, add IL4 (20ng/ml) (R&D, 204-IL), GM-CSF (10ng/ml) (R&D, 215-GM) to induce monocytes to differentiate into DC cells, culture To the 5th day, add TNFα (1000U/ml, 10ng/ml) (R&D, 210-TA), RhIL-1β (5ng/ml) (R&D, 201-LB) that induce DC maturation into the cell culture. , RhIL-6 (10ng/ml) (R&D, 206-IL), 1μM PGE (Tocris, 2296), in a carbon dioxide incubator at 37℃, 5% CO ₂ culture conditions for 2 days, as a lymphocyte mixed reaction ( MLR) mature DC cells (moDC);

Human PBMC cells (ALLCELLS, PB005F) were resuscitated, and CD4+ cell isolation was performed according to the instructions of the Human CD4+ T cell enrichment kit (STEMCELL, 19052). In short, the above-mentioned suspended cell liquid drawn from the PBMC static culture for 2 hours is placed in a 20ml centrifuge tube, centrifuged at 300g for 10 minutes, and 500μl of the separation solution provided in the kit and 100μl of the kit are added to the cell pellet Incubate the equipped purified antibody for 20 minutes at 4°C, wash once with the separating solution, add 500μl of the bead buffer in the kit and incubate for 15 minutes, remove the beads by magnetic field, and use AIM

Medium CTS (GIBCO, A3021002) medium wash once, use 8ml AIM

CD4+ cells were cultured in Medium CTS (GIBCO, A3021002) medium at 37°C and 5% CO ₂ . According to CD4+: anti-CD3/CD28Beads=1:1, add Dynabeads Human T-Activator CD3/CD28 (INVITROGEN, 11131D), incubate in a carbon dioxide incubator at 37℃, 5% CO ₂ for 3 days, and perform beads on CD4+ cells stimulate;

Mix the above isolated mature DC cells with bead-stimulated CD4+ cells, each well with a volume of 200μl, 12,000 DC cells, 120,000 CD4+ cells, add Staphylococcal entererotoxin E superantigen (Toxin technology, ET404), 1ng/ml, add antibody Sample (anti-PD-L1/OX40 bispecific antibody, ADI-20057, anti-PD-L1 humanized Nb-Fc, ADI-20057+anti-PD-L1 humanized Nb-Fc and IgG2 prepared as described above Control), 100 nM is the starting concentration, 3 times dilution, a total of ten concentration points. Mixed culture for 3 days, using the cisbio IL2 detection kit (CISBIO, 62HIL02PEG) to detect the expression of IL2 in each sample. The expression of IL2 of different antibodies reflects the ability to activate T cells.

The results are shown in Figure 15. The anti-PD-L1/OX40 bispecific antibody of the present invention can activate T cells in vitro, and its activation effect is better than that of anti-OX40 antibody (ADI-20057) and anti-PD-L1 antibody (anti-PD-L1 Humanized Nb-Fc) alone, anti-OX40 antibody and anti-PD-L1 antibody combined (ADI-20057+anti-PD-L1 humanized Nb-Fc) are more powerful.

Example 13 Anti-tumor effect in vivo of the anti-PD-L1/OX40 bispecific antibody of the present invention

Example 13.1. Anti-tumor effect of the anti-PD-L1/OX40 bispecific antibody of the present invention in a LoVo cell tumor-bearing NPG mouse model

In this example, NPG mice were mixed inoculated with LoVo (ATCC, CAT#CCL-229TM) cells and PBMC (All Cells, PB005F) cells to generate tumor-bearing mice, and the anti-OX40/PD of the present invention was tested. -The anti-tumor effect of L1 bispecific antibody.

NPG mice:

Female NPG mice (18g/35 days old) were purchased from Beijing Weitongda Experimental Animal Technology Co., Ltd. The grade is SPF, and the quantity is 75. The quality inspection unit is Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the certificate number is NO.11806300011459. The mice were acclimatized and quarantined for 7 days after arrival, and then began research.

Culture cells and inoculate mice:

Cell: LoVo: ATCC CAT#CCL-229TM Lot#60380843

PBMC: All Cells Lot#3000417

The LoVo cells were routinely subcultured for subsequent in vivo experiments. The PBMC cells were recovered one day in advance, and the PBMC cell suspension was collected by centrifugation the next day. LoVo cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation, and LoVo cells were dispersed with PBS (1×). The LoVo cells and PBMC cells were mixed 4:1 and dispersed with PBS (1×), that is, the density of LoVo cells was 12.5×10 ⁶ cells/ml and the density of PBMC cells was 3.125×10 ⁶ cells/ml. The right back of the mouse was shaved, and 0.2ml of the mixed cell suspension was subcutaneously inoculated into the right abdominal area of the NPG mouse on day 0 to establish a humanized LoVo tumor-bearing mouse model.

Administration: 3 days after tumor cell inoculation, random groups (5-7 mice per group), the dosage and manner of administration are shown in Table 7, and h-IgG (purchased from Equitech-Bio) was used as a negative control. They were administered on the 3rd, 7th, 10th, and 14th days after vaccination, and the tumor volume and body weight of the mice were monitored twice a week. The monitoring ended after 31 days. The average tumor volume of the h-IgG control group before administration was 46 mm ³ . Calculate the relative tumor inhibition rate (TGI%) on the 28th day after vaccination. The calculation formula is as follows: TGI%=100%×(h-IgG control group tumor volume – treatment group tumor volume)/(h-IgG control group tumor volume – h -IgG control group initial tumor volume).

Tumor volume measurement: the largest long axis (L) and largest wide axis (W) of the tumor are measured with a vernier caliper, and the tumor volume is calculated according to the following formula: V=L×W ² /2. An electronic balance is used to determine body weight. Throughout the study, when the tumors reached the endpoint (tumor volume> 3000mm ³⁾ or when the mice with> 20% body weight loss, the mice were euthanized.

Table 7. Experimental design table

*Dose every 3-4 days, 4 times in total

The experimental results are shown in Figure 16 and Figure 17 and Table 8. It can be seen that the anti-PD-L1/OX40 bispecific antibody of the present invention has a significantly better tumor inhibitory effect than monoclonal antibodies (anti-PD-L1 humanized Nb-Fc, ADI-20057) And the combination of anti-PD-L1 humanized Nb-Fc and ADI-20057.

Table 8. Tumor suppression rate on day 28

Example 13.2. The anti-tumor effect of the anti-PD-L1/OX40 bispecific antibody of the present invention in a NOG mouse model bearing NCI-H292 cell tumor

In this example, after inoculation with PBMC cells, NOG mice were inoculated with NCI-H292 cells (ATCC, CRL-1848) to determine the anti-tumor effect of the anti-PD-L1/OX40 bispecific antibody of the present invention.

NOG mice: Female NOG mice (15-18g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The grade is SPF, and the number is 110. The quality inspection unit is Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the certificate number is NO.11400700339672. The mice were domesticated for 7 days after arrival, and then the study began.

The NCI-H292 cells were routinely subcultured for subsequent in vivo experiments. Resuscitate PBMC cells (All Cells, PB005F), collect the PBMC cell suspension by centrifugation, and disperse the PBMC cells with PBS (1×) to prepare a cell suspension with a cell concentration of 12.5×10 ⁶ cells/ml. On day 0, 0.2ml of cell suspension was subcutaneously inoculated into the orbital vein of NOG mice. On the 5th day, the NCI-H292 cells were collected by centrifugation, and the NCI-H292 cells were dispersed with PBS (1×) to prepare a cell suspension with a cell concentration of 25×10 ⁶ cells/ml. 0.2ml of cell suspension was subcutaneously inoculated into the right abdominal region of NOG mice to establish a NCI-H292 tumor-bearing mouse model.

Administration:

The tumor cells were randomly divided into groups (5-9 mice per group) 1 day after inoculation. The dosage and method of administration are shown in Table 9. h-IgG (purchased from Equitech-Bio) was used as a negative control, and they were divided into groups on the first day after inoculation. , 4, 8, and 11 days, and monitor the tumor volume and body weight of mice twice a week. The monitoring ended after 26 days. Calculate the relative tumor inhibition rate (TGI%) on the 25th day after vaccination. The calculation formula is as follows: TGI%=100%×(h-IgG control group tumor volume – treatment group tumor volume)/(h-IgG control group tumor volume – h -IgG control group initial tumor volume). The average tumor volume of the h-IgG control group before administration was 78 mm ³ . Tumor volume measurement: the largest long axis (L) and largest wide axis (W) of the tumor are measured with a vernier caliper, and the tumor volume is calculated according to the following formula: V=L×W ² /2. Throughout the study, when the tumors reached the endpoint (tumor volume> 3000mm ³⁾ or when the mice with> 20% body weight loss, the mice were euthanized.

Table 9. Experimental design table

The results of tumor inhibition rate are shown in Figure 18-20 and Table 10. As shown in Figure 18, on the 25th day after vaccination, the low-dose group was compared with h-IgG control, anti-PD-L1 humanized Nb-Fc 0.01 mg The inhibitory rates of ADI-200570.02 mg/kg and ADI-2005 were 20% and 37%, respectively. The combination of anti-PD-L1 humanized Nb-Fc and ADI-200570.01+0.02mg/kg has no obvious tumor suppression; anti-PD-L1/OX40 bispecific antibody 0.023mg/kg tumor suppression rate is 62%. The anti-PD-L1/OX40 bispecific antibody 0.023mg/kg has a better anti-tumor effect.

As shown in Figure 19 and Table 10, compared with the h-IgG control in the middle dose group, the anti-PD-L1 humanized Nb-Fc 0.1 mg/kg and ADI-20057 0.2 mg/kg single-drug inhibition rates were 48% and 38%. Anti-PD-L1 humanized Nb-Fc combined with ADI-20057 0.1+0.2mg/kg has a tumor suppression rate of 45%; anti-PD-L1/OX40 bispecific antibody 0.23mg/kg has a tumor suppression rate of 90% . The anti-PD-L1/OX40 bispecific antibody 0.23mg/kg has stronger anti-tumor effect than single drug and combination drug.

As shown in Figure 20 and Table 10, compared with hIgG in the high-dose group, the anti-PD-L1 humanized Nb-Fc1mg/kg and ADI-200572mg/kg single-drug inhibition rates were 51% and 47%, respectively. Anti-PD-L1 humanized Nb-Fc combined with ADI-200571+2mg/kg has a tumor inhibition rate of 59%; anti-PD-L1/OX40 bispecific antibody 2.3mg/kg has a tumor inhibition rate of 94%. The anti-PD-L1/OX40 bispecific antibody 2.3mg/kg has the best tumor suppressing effect, and the tumor suppressing effect is stronger than single drug and combination drug. The high-dose group has better anti-tumor effect than the middle-dose and low-dose groups, and has a dose-dependent effect.

At the same time, we tested the weight of the mice, and the results are shown in Figure 21. There is a slight difference in the weight of the mice in the later stage.

Table 10. Tumor Inhibition Rate on Day 25

Table 11. Sequence information of exemplary anti-PD-L1/OX40 bispecific antibodies and controls

Table 12 Sequence:

Claims

A bispecific antibody molecule or antigen-binding fragment thereof that binds OX40 and PD-L1, which comprises or consists of the following peptide chains:

(i) The polypeptide chain of formula (I):

VH-CH1-Fc-X-VHH; and

(ii) The polypeptide chain of formula (II):

VL-CL;

among them:

VH represents the variable region of the heavy chain;

CH stands for the constant region of the heavy chain;

Fc includes CH2, CH3, and optionally CH4;

CH1, CH2, CH3 and CH4 represent the domains 1, 2, 3 and 4 of the heavy chain constant region, respectively;

X may not exist, or when it exists, it means a joint;

VHH stands for single domain antigen binding site;

VL stands for the variable region of the light chain;

CL represents the constant region of the light chain;

Optionally, there is a hinge region between CH1 and Fc;

The antigen binding site formed by VH and VL is specific to OX40, and the antigen binding site formed by VHH is specific to PD-L1.
The antibody molecule or antigen-binding fragment thereof according to claim 1, which comprises or consists of one or two polypeptide chains of formula (I) and one or two polypeptide chains of formula (II).
The antibody molecule or antigen-binding fragment thereof of claim 1 or 2, wherein the linker is a flexible linker, preferably, the linker comprises an amino acid sequence (Gly 4 Ser) n, wherein n is a positive integer equal to or greater than 1, for example , N is a positive integer from 1-7, for example, n is 1, 2, 3, 4, 5 or 6.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or fragment thereof is a human antibody or a humanized antibody, or a chimeric antibody.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the single domain antigen binding site (VHH) is the heavy chain variable domain of an antibody that naturally lacks light chains, for example, Camelidae (Camelidae) the heavy chain variable domain of the naturally occurring heavy chain antibody in the species or the VH-like single domain in the immunoglobulin called the novel antigen receptor in fish (such as the naturally occurring IgNAR in shark serum); Or a recombinant single domain antigen binding site derived from them (for example, a camelized human VH domain or a humanized camelid antibody heavy chain variable domain); preferably, the single domain antigen The binding site is selected from the heavy chain variable domain of a heavy chain antibody naturally occurring in camelid species, a camelized human VH domain and a humanized camelid antibody heavy chain variable domain.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein "CH1-Fc" of formula (I) is in the form of IgG, such as IgG1, IgG2 or IgG4, and/or formula (II) The CL comes from κ or λ.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein OX40 is human OX40 or monkey OX40; and/or wherein PD-L1 is human PD-L1.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1-7, wherein the VHH in formula (I) comprises

(i) The three complementarity determining regions (VHH CDR) contained in SEQ ID NO: 6, or

(ii) Complementarity determining region (CDR) VHH CDR1, VHH CDR2 and VHH CDR3, wherein VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 10 or consists of the amino acid sequence; VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 11 , Or consisting of the amino acid sequence; VHH CDR3 includes or consists of the amino acid sequence of SEQ ID NO: 12; or

(iii) The sequence shown in SEQ ID NO: 6 or consists of it, or

(iv) An amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 6 or is determined by Its composition.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1-8, wherein

VH in formula (I) contains

(i) The three complementarity determining regions HCDRs of the heavy chain variable region VH shown in SEQ ID NO: 2; or

(ii) Complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence; HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, or is composed of Amino acid sequence composition; HCDR3 comprises the amino acid sequence of SEQ ID NO: 15 or consists of the amino acid sequence; or

(iii) The amino acid sequence shown in SEQ ID NO: 2 or consists of it; or

(iv) An amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 2 or is determined by Its composition

and / or

VL contains

(i) The three complementarity determining regions LCDR of the heavy chain variable region VL shown in SEQ ID NO: 8; or

(ii) Complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, where LCDR1 includes the amino acid sequence of SEQ ID NO: 16, or consists of the amino acid sequence; LCDR2 includes the amino acid sequence of SEQ ID NO: 17, or is composed of Amino acid sequence composition; LCDR3 includes the amino acid sequence of SEQ ID NO: 18, or consists of the amino acid sequence; or

(iii) The amino acid sequence shown in SEQ ID NO: 8 or consists of it; or

(iv) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8 or is determined by Its composition.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1-9, wherein the Fc of formula (I) is derived from IgG1, IgG2 or IgG4, preferably, the Fc is derived from IgG2, and more preferably, the Fc

(i) Contains the amino acid sequence of SEQ ID NO: 4 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity Amino acid sequence or consist of it;

(ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 4; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 4 , More preferably amino acid sequence of conservative substitution) or composed of it.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1-10, wherein CH1 of (I) is derived from IgG1, IgG2 or IgG4, preferably CH1 is derived from IgG2, preferably CH1

(i) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 3 or Consist of

(ii) It comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

(iii) Comprising one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared with the amino acid sequence of SEQ ID NO: 3 , More preferably amino acid sequence of conservative substitution) or composed of it.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1-11, wherein

(a) VH-CH1-Fc in formula (I)

(i) Comprising those having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 19 The amino acid sequence or consists of it; or

(ii) It comprises or consists of the amino acid sequence of SEQ ID NO: 19;

and / or

(b) VL-CL of formula (II)

(i) Contains the amino acid sequence of SEQ ID NO: 7 having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity The amino acid sequence or consists of it; or

(ii) It comprises or consists of the amino acid sequence of SEQ ID NO: 7.
The antibody molecule or antigen-binding fragment thereof according to any one of claims 1-12, wherein the polypeptide chain of formula (I) comprises or consists of the sequence shown in SEQ ID NO:1, or contains at least 85%, 90% of the sequence shown in SEQ ID NO:1. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequence or composed thereof; and/or wherein the polypeptide chain of formula (II) comprises The sequence shown in SEQ ID NO: 7 is composed of, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % Identity or consist of amino acid sequence.
An isolated nucleic acid encoding any one or more polypeptide chains of the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 13.
A vector comprising the nucleic acid of claim 14, preferably the vector is an expression vector, such as a pXC vector or a pTT5 vector.
A host cell comprising the nucleic acid of claim 14 or the vector of claim 15, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from E. coli cells, yeast cells, mammalian cells or suitable for preparation Other cells of antibodies or antigen-binding fragments thereof, most preferably, the host cells are 293 cells or CHO cells.
A method for preparing the antibody molecule or antigen-binding fragment thereof according to any one of claims 1 to 13, said method comprising culturing the host cell of claim 16 under conditions suitable for expressing the nucleic acid of claim 14, optionally Separating the antibody or antigen-binding fragment thereof, optionally the method further comprises recovering the antibody or antigen-binding fragment thereof from the host cell.
An immunoconjugate comprising the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 13 and other substances, such as therapeutic agents or labels, such as cytotoxic agents or anti-angiogenic agents.
A pharmaceutical composition comprising the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 13 or the immunoconjugate of claim 18, and optionally pharmaceutical excipients.
A pharmaceutical composition comprising the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 13 or the immunoconjugate of claim 17, and other therapeutic agents, and optionally pharmaceutical adjuvants; preferably, the The other therapeutic agents are selected from anti-angiogenesis agents, chemotherapeutics, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators.
A combination product comprising the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 13 or the immunoconjugate of claim 18, and one or more other therapeutic agents, such as anti-angiogenic agents, chemotherapeutic agents , Cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators.
A method for preventing or treating a disease in a subject, which comprises administering to the subject an effective amount of the antibody molecule of any one of claims 1 to 13, or the immunoconjugate of claim 18, Or the pharmaceutical composition of claim 19 or 20 or the combination product of claim 21, wherein the disease such as autoimmune disease, inflammatory disease, infection, tumor, T cell dysfunction disease, for example, the tumor is cancer, For example, a cancer having an elevated expression level of PD-1, PD-L1 or PD-L2 and/or a reduced expression level or activity of OX40, such as colon cancer or rectal cancer or colorectal cancer or lung cancer.
The method of claim 22, further comprising administering one or more other therapies to the subject in combination, the therapies including, for example, a treatment modality and/or other therapeutic agents, preferably, the treatment modality includes surgery Treatment and/or radiotherapy, or the therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators.
A method for detecting the antigen OX40 and/or PD-L1 in a sample, the method comprising

(a) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1-13; and

(b) Detect the formation of a complex between the antibody or its antigen-binding fragment and OX40 and/or PD-L1, optionally, the antibody is detectably labeled.