TWI793395B - Bispecific antibodies that bind to pd-l1 and ox40 - Google Patents

Abstract

The present invention provides a novel, artificially designed antibody molecule, in particular an anti-PD-L1/OX 40 bispecific antibody molecule, which is capable of binding both PD-L1 and OX 40.

Description

Bispecific antibodies that bind PD-L1 and OX40

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

Antibody molecules can specifically bind to their corresponding antigens, and are increasingly becoming important therapeutic, preventive and/or therapeutic agents for various diseases (such as cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.) or diagnostic agents. However, monospecific antibodies against only one target have some limitations in clinical application. Patients may develop drug resistance or non-response after receiving monospecific antibody therapy. With the research on cancer and many other diseases, it has been realized that multiple signal transduction pathways are often 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 (eg, bispecific antibodies) can specifically bind different antigens, they can be designed to simultaneously act on two or more signal transduction pathways of different mediators. These advantageous properties open up broad application prospects for multispecific antibodies (eg, bispecific antibodies).

A large number of imaginative multispecific antibody (eg, bispecific antibody) formats have been developed by antibody engineering and their applicability to disease applications has been investigated (Brinkmann U. and Kontermann R.E., The making of bispecific antibodies, Mabs, 2017, 9(2): 182-212).

Multispecific antibodies (eg, bispecific antibodies) can be divided into many types according to different components and construction methods. For example, according to the left-right symmetry of the multispecific antibody structure, it can be divided into symmetrical structure and asymmetric structure; according to whether the multispecific antibody has an IgG Fc region, it can be divided into antibodies with an Fc region and those without an Fc region. Antibody format; according to the number of antigen-binding sites in the multispecific antibody, it can be divided into bivalent, trivalent, tetravalent or more valent antibodies, etc.

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

Therefore, there remains a need in the art for alternative multispecific antibodies, particularly bispecific antibodies, with improved properties. The present invention provides a new multispecific antibody format, which can be easily and effectively expressed in cultured cells in vitro without complex production process. At the same time, the bispecific antibody can simultaneously bind to different antigens, especially OX40 and PD-L1, maintain the binding activity of each antigen-binding site to the corresponding different epitopes, and other properties. Further, the double special of the present invention The heterologous antibody format is physically stable and biologically stable, which allows for better production and developability of the antibody.

This paper discloses a novel bispecific antibody molecule constructed by antibody engineering method.

Accordingly, in one aspect, the invention provides bispecific antibody molecules having one or more of the following properties:

(a) specifically binds to one or two antigens with high affinity;

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

(c) have good physical stability, in particular, have 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) exert effector function;

(f) have better antitumor activity.

In one embodiment, an antibody molecule of the invention comprises a full antibody portion and a single domain antibody portion, the latter linked by a linker to the C-terminus of the heavy chain constant region of the full antibody portion.

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

Polypeptide chain of formula (I) (peptide chain #1):

VH-CH1-Fc-X-VHH; and

Polypeptide chain of formula (II) (peptide chain #2):

VL-CL;

in:

VH means heavy chain variable region;

CH represents the heavy chain constant region domain;

Fc comprises CH2, CH3, and optionally CH4;

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

X may be absent or, when present, represent a linker, such as a flexible linker;

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

VL represents the light chain variable region;

CL represents the light chain constant region;

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

In one embodiment, the antibody molecule of the invention comprises at least one polypeptide chain of formula (I) and one polypeptide chain of formula (II). Preferably, the antibody molecule of the invention comprises two (eg identical) polypeptide chains of formula (I) and two (eg identical) 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 invention has 2 or 4 antigen binding sites which bind 2, 3 or 4 different antigens, or the same antigen. In one embodiment, VH in formula (I) and VL in formula (II) form one antigen binding site, and VHH of formula (I) constitutes one antigen binding site (single domain antigen binding site).

In one embodiment, different antigen binding sites bind 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 invention is a flexible linker, for example 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, wherein 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 VH in formula (I) and VL in formula (II) is human or humanized, or chimeric.

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

In one embodiment, the VHH molecule in the peptide chain of formula (I) in the antibody molecule of the present invention can be derived from antibodies produced in Camelidae species such as camelids, alpacas, dromedaries, alpacas and guanacos. Species other than Camelidae can also produce heavy chain antibodies that naturally lack light chains, and the VHH of such heavy chain antibodies are also within the scope of the invention.

In one embodiment, CH1 and Fc are from antibody heavy chains, or derivatives 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 from IgG2. It will be appreciated that mutations may be made to the Fc in the constant domains to achieve the effect of stabilizing the antibody, or to enhance 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 comprises positions 234 and 235 (EU numbering, according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Amino acid substitutions at Public Health Service, National Institutes of Health, Bethesda, MD (1991) EU index numbering). In a specific embodiment, said 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), the number of disulfide bonds depends on the antibody The IgG form from which the constant domains are derived can vary, and 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 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 for a 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 a 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 specifically bound by the antibody molecule of the present invention is selected from Tumor-associated antigens, immune checkpoint molecules, angiogenesis-inducing factors, tumor necrosis factor receptor superfamily members, and co-stimulatory molecules in the immune system, as well as ligands and/or receptors for these molecules, e.g., OX40, CD47, PD1 , PD-LI, PD-L2, LAG-3, 4-1BB (CD137), VEGF, and GITR.

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

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

In one embodiment, the antibodies of the invention also encompass antigen-binding fragments thereof, such as Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv) or (Fab') ₂ , single domain antibody, diabody 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 comprising the nucleic acid, and a host cell comprising 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 in the antibody molecule of the present invention, preferably an expression vector, such as a pXC vector or a pTT5 vector, such as pXC17.4 or pXC18. 4. In one embodiment, the expression vector is constructed as a double gene expression vector pXC vector.

In one aspect, the invention provides methods for producing an antibody molecule of the invention or a fragment thereof.

In some embodiments, the invention provides immunoconjugates, pharmaceutical compositions, kits, combinations or articles of manufacture comprising an antibody 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 disease etc. For example, the disease is a tumor (eg cancer) or an infection. in some real In an embodiment, the tumor is an immune escape tumor. Preferably, the tumor is eg colon or colorectal or rectal or lung cancer. In another aspect, the present invention relates to a method of preventing or treating a disease in a subject or individual, said method comprising administering to said subject an effective amount of any antibody or fragment thereof, pharmaceutical composition or immunological composition described herein. Conjugate or combination products or kits. For example, the disease is a tumor (eg cancer) or an infection. In some embodiments, the tumor is an immune escape tumor. In one embodiment, the tumor is eg colon or colorectal or rectal or lung cancer. In another aspect, the present invention also relates to any antibody or fragment thereof or immunoconjugate described herein for the preparation of a medicament or pharmaceutical composition or kit or combination for use in the treatment of a tumor (e.g. cancer) or infection in a subject The purpose of the product. In some embodiments, the tumor is an immune escape tumor. In one embodiment, the tumor is eg colon or colorectal or rectal or lung cancer.

The invention also relates to methods for detecting antigens in a sample.

The present invention also covers any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof applies to any and all antibodies or fragments thereof or immunoconjugates or pharmaceutical compositions or combinations or kits, methods and uses of the invention described herein.

The preferred embodiments of the invention described in detail below will be better understood when read in conjunction with the following drawings. For purposes of illustrating the invention, the drawings show a presently preferred embodiment. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1A and Figure 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, as well as the anti-OX40 antibody ADI-20057 and IgG2 as a control, to CHO cells overexpressing human OX40 (CHO-OX40 cells). In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the mean 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- combination of L1 cells). In the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the mean 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 OX40-overexpressing CHO cells (CHO-OX40) and PD-L1-overexpressing CHO cells (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.

Figure 7 shows the results of the anti-PD-L1/OX40 bispecific antibody of the present invention measured by differential scanning fluorescence (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 signaling pathway, and then obtains a fluorescent signal. The effect of anti-PD-L1 humanized Nb-Fc and IgG2 as controls was also examined.

Figure 9 shows the anti-PD-L1/OX40 bispecific antibody of the present invention, as well as the blocking effect of ADI-20057, Pogalizumab (Pogalizumab) and IgG2 as a control on the binding of human OX40 ligand to OX40 , proving that the bispecific antibody of the present invention has no blocking effect on this binding.

Figure 10 shows the anti-PD-L1/OX40 bispecific antibody of the present invention, as well as ADI-20057, Bojializumab (Pogalizumab) and IgG2 as a control, on the binding of human OX40 ligand to OX40 It proves that the bispecific antibody of the present invention does not block the binding of the human OX40 ligand to OX40, and effectively enhances the activation of the OX40 signaling pathway mediated by the OX40 ligand.

Figure 11 shows the activation of the anti-PD-L1/OX40 bispecific antibody of the present invention and ADI-20057 and IgG2 as controls on the OX40-mediated signaling pathway detected by the luciferase reporter gene method.

Figure 12 shows the effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on the PD-L1-dependent OX40-mediated signaling pathway. Raji cells that do not express PD-L1 were 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 examined.

Figure 13 shows the activation of the PD-L1-dependent OX40-mediated signaling pathway by the anti-PD-L1/OX40 bispecific antibody of the present invention. Among them, the use of Raji cells with high expression of PD-L1 proves that the antibody of the present invention has better OX40-mediated signaling pathway activation 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 examined.

Figure 14 shows the activation of the PD-L1-dependent OX40-mediated signaling pathway by the anti-PD-L1/OX40 bispecific antibody of the present invention. Among them, the human lung cancer cell NCI-H292 expressing PD-L1 on the surface was used to prove that the antibody of the present invention has a better OX40-mediated signaling pathway activation in the presence of tumor cells naturally expressing 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 of human T cells by the anti-PD-L1/OX40 bispecific antibody of the present invention. The effects of anti-PD-L1 humanized Nb-Fc, ADI-20057, anti-PD-L1 humanized NNb-Fc+ADI-20057 and IgG2 were also examined.

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

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

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

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

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

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

Detailed description of the invention

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

abbreviation

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

Use the following abbreviations:

I‧Definition

In order to explain this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

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

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

Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.

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

The terms "whole antibody", "full-length antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to a naturally occurring antibody comprising at least two heavy chains (H) and two Glycoproteins with light chains (L). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Heavy The chain constant region consists of three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interspersed with more conservative regions (framework regions (FRs)). 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 carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.

"Antibody fragment" refers to a molecule that is distinct from an intact antibody, comprises a portion of an intact antibody and binds the same 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 (e.g. scFv); Specific antibodies or fragments thereof; camelid antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

As used herein, the term "epitope" refers to a portion of an antigen (eg, 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% or more of said reference antibody and its Antigen binding, conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of that 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 more than 50%, 60%, 70%, 80%, 90%, or 95% of the binding of said reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of that 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 radioimmunoassays (RIA), solid-phase Phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see eg Stahli et al., 1983, Methods in Enzymology 9:242-253).

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

An antibody exhibiting the same or similar binding affinity and/or specificity as a reference antibody is an antibody capable of binding at least 50%, 60%, 70%, 80%, 90% or more than 95% 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-contacting residues ( "antigen contact point"). The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as 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 combination of a number of well-known antibody CDR assignment systems, which Assignment systems include, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the 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, U.S. Department 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 at imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

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

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

Unless otherwise stated, in the present invention, the term "'CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways.

Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it is meant according to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

In one embodiment, the CDRs of an antibody of the invention are bounded by Kabat rules, or by AbM rules, or by a combination thereof.

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

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the region of minimal overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. A minimal binding unit may be a subsection of a CDR. As will be apparent to those skilled in the art, the residues of the remainder of the CDR sequence can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.

"An antibody in IgG form" refers to the IgG form to which the heavy chain constant region 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 IgG4 form means that the heavy chain constant region is from IgG4.

The term "single domain antibody" as used herein generally refers to an antibody consisting of only one heavy chain variable region with antigen-binding activity, that is, comprising only one chain from the C-terminus to the N-terminus: FR4-VHHCDR3 -Antibody against FR3-VHHCDR2-FR2-VHHCDR1-FR1, which can be derived from camelid heavy chain anti heavy chain variable domain of body, VH-like single domain (v-NAR) of fish IgNAR), which can be produced naturally or by genetic engineering techniques. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fishes (eg nurse sharks) (WO 2005/035572).

The term "camelized human VH domain" refers to the transfer of key elements derived from camelid VHH to the human VH domain, resulting in the human VH domain no longer needing to be paired with the VL domain to recognize the target antigen, camelized Human VH domains alone confer antigen-binding specificity.

As used herein, the term "binding site" or "antigen-binding site" refers to the region of an antibody molecule that actually binds to an antigen, comprising the antibody light chain variable domain (VL) and the antibody heavy chain variable domain (VH ), a VH/VL pair composed of Camelidae heavy chain antibodies, a VH-like single domain (v-NAR) from an IgNAR from a shark, a camelized human VH domain, a human A heavy chain variable domain of a camelid antibody. In one embodiment of the invention, an antibody molecule of the invention comprises at least four antigen binding sites, e.g., two single domain antibody antigen binding sites (e.g., VHH) and two VH/VL pairs forming Antigen binding site.

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

As used herein, the term "multispecific" antibody refers to an antibody that has at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope. Antigen binding to different epitopes. Antibodies provided herein are typically multispecific antibodies, e.g. such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different antigenic 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 class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains that are disulfide bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three domains of the heavy chain constant region (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 be assigned to one of five classes, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subclasses 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 assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked by an immunoglobulin hinge region.

The term "effector functions" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: 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, downregulation of cell surface receptors (eg, 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 altered, replaced or exchanged such that the antigen binding site is of a different or altered class, effector function and/or species constant regions or entirely different molecules that confer novel properties on chimeric antibodies (e.g., enzymes, toxins, hormones, growth factors, drugs) etc.; or (b) altering, replacing or exchanging the variable region or part thereof with a variable region having a different or altered antigen specificity. For example, mouse antibodies can be modified by exchanging their constant regions with those from human immunoglobulins. Due to the exchange of human constant regions, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original mouse antibody.

A "humanized" antibody is one that retains the antigen-specific reactivity of a non-human antibody (eg, a mouse monoclonal antibody) while being less immunogenic when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding sites and replacing the remainder of the antibodies with their human counterparts (ie, the constant regions and the parts of the variable domains that do not participate in binding are those of human antibodies). See, eg, 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 corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using Human antibody libraries or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

The term "...valent" antibody refers to the number of antigen-binding sites present in the antibody molecule. "Bivalent", "trivalent" and "tetravalent" antibodies refer to the existence 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 consisting of amino acids, such as glycine and/or serine residues, alone or in combination, to link the various variable domains in an antibody. In one embodiment, the flexible linker is a Gly/Ser linking peptide comprising the 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 to 7, For example 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 "bind" or "specifically bind" 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 a particular 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 antibody production or activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially any protein or peptide, can be used as an antigen. Furthermore, 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 (typically proteins, carbohydrates or lipids) expressed entirely or as fragments (e.g., MHC/peptides) on the surface of cancer cells, preferably as compared to normal cells ), and the molecule can be used in the preferential targeting of cancer cells by agents. In some embodiments, the tumor-associated antigen is a cell surface molecule that is overexpressed in tumor cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more than normal cells Overexpression. In some embodiments, tumor-associated antigens are cell surface molecules that are inappropriately synthesized in tumor cells, eg, molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, tumor-associated antigens are expressed intact or as fragments only on the cell surface of tumor cells, and are not synthesized or expressed on the surface of normal cells.

The term "cytokine" is a general term for proteins released by one cell population to act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, 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 factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL ) and γ-interferon. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines, including small molecular entities produced artificially, and pharmaceutically available Derivatives and salts accepted.

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 native 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 as well as any form of OX40 that results from processing in the cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.

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

As used herein, the term "anti-OX40 antibody", "anti-OX40", "OX40 antibody" or "OX40-binding antibody" refers to an antibody that is capable of binding with sufficient affinity to a (human or monkey) OX40 protein or Fragments thereof such that the antibody can be used as a diagnostic and/or therapeutic agent targeting (human or monkey) OX40. In one embodiment, the anti-OX40 antibody binds to a non-(human or monkey) OX40 protein to a degree less than about 10%, about 20%, about 30%, about 30% less than the binding of said antibody to (human or cynomolgus monkey) OX40. 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 native PD-L1 of any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats). The term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 that results from 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 four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. Additional information on the human PD-L1 gene, including the genomic DNA sequence, can be found under NCBI Gene ID No. 29126. Additional information on the mouse PD-L1 gene, including the 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 No. NP_001254653 or UniProt Accession No. Q9NZQ7, while an exemplary full-length mouse PD-L1 can be found, for example, under NCBI Accession No. NP_068693 or Uniprot Accession No. Q9EP73. L1 protein sequence.

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

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

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

A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; Such effector functions generally require the association of an Fc region with a binding domain (eg, an antibody variable domain), and can be assessed using a variety of assays, such as those disclosed herein.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; body) downregulation; and B cell activation.

"Human effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cell expresses at least Fc 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 source, such as blood.

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

A "therapeutically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result. A therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof may vary depending on factors such as the disease state, 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 one in which any toxic or detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g. tumor growth rate) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60% relative to an untreated subject. % or 70% and still more preferably at least about 80%. Compounds can be evaluated for their ability to inhibit a measurable parameter (eg, cancer) in animal model systems predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by examining the ability of the compound to inhibit, in vitro, by assays known to the skilled artisan.

A "prophylactically effective amount" refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.

A "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 the addition of some residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for a Fab' in which the cysteine residue of the constant domain bears a free thiol group. F(ab') ₂ antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. 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 region comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, 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 domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, eg, Kindt et al. Kuby Immunology, 6 ^th ed., WH Freeman and Co. p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind a particular antigen can be used to isolate antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

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 the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Included herein are those with the specificity of screening or selection in initially transformed cells. Mutant progeny with the same function or biological activity. A host cell is any type of cellular system that can be used to produce an antibody molecule of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells. Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

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

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

The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is often 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 to all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to herein.

As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused. The label may itself be detectable (eg, radioisotopic or fluorescent label) or, in the case of an enzymatic label, may catalyze a detectable chemical alteration of a substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled . indirect marking Examples 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, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, antibodies are purified to greater 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 determined by HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.

Calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., the first and second amino acid sequences can be separated for optimal alignment) or introduce gaps in one or both of the nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the aligned 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 at corresponding amino acid positions or nucleotide positions are then compared or Nucleotides. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http://www.gcg. com), determine two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 The percent identity between. In yet another preferred embodiment, using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weight 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

It is also possible to use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4: 11- 17) Determining the percent identity between two amino acid sequences or nucleotide sequences.

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

As used herein, the term "hybridizes under conditions of low stringency, medium stringency, high stringency, or very high stringency" describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), incorporated by reference Found in 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in the reference and either method can be used. The specific hybridization conditions mentioned herein 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) twice in 0.2X SSC, 0.1% SDS; 2) medium stringency hybridization conditions are about 45°C in 6X SSC, followed by 60°C in 0.2X SSC, 0.1% Wash one or more times in SDS; 3) high stringency hybridization condition is at about 45 ℃ in 6 X SSC, then wash one or more times in 0.2X SSC, 0.1% SDS at 65 ℃; And preferably 4 ) Very high stringency hybridization conditions are one or more washes in 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the one that should be used unless otherwise stated.

The term "pharmaceutical composition" refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.

The term "pharmaceutical excipient" refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), carrier, excipient or stabilizer, etc., which are administered together with the active substance.

As used herein, "treating" means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibody molecules of the invention are used to delay the development of a disease or to slow the progression of a disease.

As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder. 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 a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

The term "therapeutic agent" as used herein encompasses any substance that is effective in the prevention or treatment of diseases such as tumors (e.g. cancer) and infections, including anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs or immunomodulators.

"Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer, including but not limited to antineoplastic agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; antiestrogens ; antiandrogens; and nonsteroidal antiandrogens.

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

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

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

The term "anti-infective active agent" includes any molecule that, at the concentration and dosing interval administered, specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi or protozoa, eg parasites, but is not lethal to the host. As used herein, the term anti-infective active agent includes antibiotics, antibacterials, antivirals, antifungals and antiprotozoals. In a specific aspect, the anti-infective agent is nontoxic to the host at the concentration and dosing interval administered.

Antibacterial anti-infective actives or antibacterial agents can be broadly classified as bactericidal (ie, kill directly) or bacteriostatic (ie, prevent division). Antibacterial anti-infective active agents can be further sub-categorized as narrow-spectrum antibacterials (ie, affect only a small bacterial subtype, eg, Gram-negative, etc.) or broad-spectrum antibacterials (ie, affect a wide range of species).

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

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

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

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

"Activating T cells" means inducing, causing or stimulating effector or memory T cells to have renewed, sustained or amplified biological functions. Examples of enhanced T cell function include: increased secretion of gamma-interferon (eg, IFNg) or interleukin (eg, IL-2) from CD8 ⁺ effector T cells relative to such levels before intervention, increased Gamma-interferon (eg, IFNg) or interleukin (eg, IL-2) secretion from CD4 ⁺ memory and/or effector T cells, elevated CD4 ⁺ effector and/or memory T cell proliferation, elevated CD8 ⁺ Proliferation of effector T cells, increased antigen responsiveness (eg clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 2-fold, 3-fold, 3-fold, 4-fold, 5-fold , 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 times, 140 times, 150 times, 160 times or higher. Means of measuring this enhancement are known to those of ordinary skill in the art.

"Tumor immune evasion" refers to the process by which tumors evade immune recognition and elimination. Thus, as a therapeutic concept, tumor immunity is "cured" when such evasions are 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 facilitates clearance of tumor cells by immune responses.

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

"Anti-angiogenic agent" refers to a compound that blocks or interferes in some way with the development of blood vessels. Anti-angiogenic agents can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis.

The term "combination product" refers to a fixed or non-fixed combination in dosage unit form or a kit of parts for combined administration in which two or more therapeutic agents can be administered independently at the same time or at the same time The administration is separated at intervals, especially when these intervals allow the combination partners to exhibit synergy, eg, a synergistic effect. The term "fixed combination" refers to the simultaneous administration of an antibody of the invention and a combination partner (eg, other therapeutic agent) to a patient as a single entity or dosage. The term "non-fixed combination" means that an antibody of the invention and a combination partner (e.g., other therapeutic agent) are administered to a patient as separate entities simultaneously, concurrently or sequentially, with no specific time limit, wherein such administration provides both treatments in the patient therapeutically effective levels of the agent. The latter also applies to cocktail therapy, eg 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 a cancer or an infection as described in this disclosure. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule with fixed ratios of the active ingredients. Alternatively, such administration includes co-administration or separate administration or sequential administration for each active ingredient in multiples or in separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration. In some embodiments, administering also includes using each type of therapeutic agent at about the same time, or in a sequential fashion at different times. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.

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

"Subject/patient sample" refers to a collection of cells or fluid obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject. Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimen, circulating tumor cells, serum, plasma, circulating Plasma proteins in 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 commonly included in commercial packages of therapeutic products that contain information on the indications, usage, dosage, administration, combination therapies, contraindications and/or warnings concerning the use of such therapeutic products. information.

II. Antibody Molecules of the Invention

The present invention provides a novel antibody molecule, which can be used for immunotherapy, prevention and/or diagnosis of various diseases. The antibody molecule of the present invention comprises at least 2, 3 or 4 antigen binding sites, it can function as a monospecific antibody or a bispecific antibody or a multispecific antibody, preferably, it can function as a bispecific antibody Antibodies do their job.

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, e.g., derived Heavy chain variable domains from camelid heavy chain antibodies, v-NAR from IgNARs from sharks, camelized human VH domains, humanized camelid antibody heavy chain variable domains, and their Recombined single domain. In one embodiment, the single domain antigen binding site in an antibody molecule of the invention is a heavy chain variable domain derived from a camelid heavy chain antibody, a camelized human VH domain and/or a humanized camelid Antibody heavy chain variable domains. The size, structure and antigenicity of antibody proteins obtained from species of Camelidae (eg, llama, alpaca, dromedary, llama and guanaco) have been characterized in the prior art for their antigenicity against human subjects. Certain IgG antibodies from the Camelidae mammalian family lack light chains in nature, and thus differ structurally from the common four-chain antibody structure with two heavy and two light chains from other animals. See PCT/EP 93/02214 (WO 94/04678 published March 3, 1994).

The heavy chain variable domain VHH of the camelid heavy chain antibody with high affinity to the target antigen can be obtained by genetic engineering. See e.g. U.S. Patent No. issued June 2, 1998 5,759,808. Like other non-human antibody fragments, the amino acid sequences of Camelid VHHs can be recombinantly altered to obtain sequences that more closely mimic human sequences, i.e., "humanized", thereby reducing the antigenicity of Camelid VHHs to humans . In addition, key elements derived from camelid VHH can also be transferred to human VH domain to obtain camelized human VH domain. The molecular weight of a 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 one 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. Contributed to the stability and low antigenicity of human subjects.

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

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

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

(ii) relative to the sequence of (i), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

In a preferred embodiment, the VHH specifically binding to PD-L1 in the antibody molecular formula (I) of the present invention 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 said amino acid sequence, or VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 10 VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 11, or Consists of said amino acid sequence, or VHH CDR2 comprises the same sequence as SEQ ID NO: 11 VHH CDR3 comprises the amino acid sequence of SEQ ID NO: 12 or is composed of The amino acid sequence composition, or the VHH CDR3 comprises amino groups with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared with the amino acid sequence of SEQ ID NO: 12 acid sequence.

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

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

(ii) an amino group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:6 acid sequence, or

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

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

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

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

(ii) relative to the sequence of (i), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), and/or

VL in formula (II) contains

(i) the 3 complementarity determining regions LCDR of the light chain variable region VL as shown in SEQ ID NO: 8, or

(ii) relative to the sequence of (i), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

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

Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13, or HCDR1 comprises the amino acid sequence of SEQ ID NO: 13 Compared with an amino acid sequence with one, two or three changes (preferably amino acid substitution, preferably conservative substitution); HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, or consists of said amino acid sequence Sequence composition, or HCDR2 comprises the amino acid sequence that has one, two or three changes (preferred amino acid substitution, preferred conservative substitution) compared with the amino acid sequence of SEQ ID NO: 14; HCDR3 comprises the amino acid sequence of SEQ ID NO:14 The amino acid sequence of ID NO: 15, or consists of said amino acid sequence, or HCDR3 comprises one, two or three changes compared with the amino acid sequence of SEQ ID NO: 15 (preferably amino acid sequence Acid substitution, preferably conservative substitution) amino acid sequence;

and / or

VL in formula (II) contains

Complementarity Determining Regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, or LCDR1 comprises the amino acid sequence of SEQ ID NO: 16 Compared with an amino acid sequence with one, two or three changes (preferably amino acid substitution, preferably conservative substitution); LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, or is composed of said amino acid Sequence composition, or LCDR2 comprises the amino acid sequence that has one, two or three changes (preferred amino acid substitution, preferred conservative substitution) compared with the amino acid sequence of SEQ ID NO: 17; LCDR3 comprises the amino acid sequence of SEQ ID NO:17 The amino acid sequence of ID NO: 18, or consists of said amino acid sequence, or LCDR3 contains and The amino acid sequence of SEQ ID NO: 18 has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence.

In some embodiments, in an antibody molecule of the invention,

(a) VH in formula (I)

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

(ii) comprises or consists 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 acids compared with the amino acid sequence of SEQ ID NO: 2 Altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of it, preferably, the amino acid change does not occur in the CDR region;

and / or

(b) VL in formula (II)

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

(ii) comprises or consists 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 acids compared with the amino acid sequence of SEQ ID NO: 8 Altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists thereof, preferably, said amino acid alteration does not occur in the CDR region.

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

(i) comprising an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 or consisting of a specific amino acid sequence;

(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence of SEQ ID NO: 4 Altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of it.

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

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

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

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence of SEQ ID NO: 3 Altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of it.

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

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

(ii) 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 acids compared with the amino acid sequence of SEQ ID NO: 9 Altered (preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or consists of it.

In some embodiments, X of formula (I) of an antibody of the invention is a flexible linker, eg, 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, wherein 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 an antibody molecule of the invention,

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

(i) comprising an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19 or consisting of a specific amino acid sequence;

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

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

and / or

(b) VL-CL of formula (II)

(i) comprising an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7 or consisting of a specific amino acid sequence;

(ii) comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) compared with the amino acid sequence of SEQ ID NO: 7 Amino acid changes (preferably amino acid substitutions, preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region of the light chain, more preferably, the amino acid change does not occur in the light chain variable region middle.

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

In a preferred embodiment of the present invention, the present invention relates to such antibody molecules, wherein the polypeptide chain of formula (I) comprises the sequence shown in SEQ ID NO: 1, or comprises at least 85%, 90%, 91 %, 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 The sequence shown, or comprising an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

In one embodiment, an antibody of the invention comprises, as part of it, a whole antibody portion. In some embodiments, a whole antibody portion of the invention comprises VH-CH1-Fc of the chain of formula (I), and VL-CL of the chain of formula (II). Among them, 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 part of the whole antibody part; VL constitutes the light chain variable region of the whole antibody part, and CL The light chain constant regions that make up the whole antibody portion, together make up the light chains of the whole antibody portion.

In some embodiments, the whole antibody portion is a human antibody. In some embodiments, the whole antibody portion is an IgG1 form of the antibody or an IgG2 form of the antibody or an IgG4 form of the antibody. In some embodiments, portions of whole antibodies may independently constitute monoclonal antibodies. In some embodiments, whole antibody portions are humanized. In some embodiments, the whole antibody portion is a chimeric antibody. In some embodiments, at least part of the framework sequences of the whole antibody portion are human consensus framework sequences.

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 preferred embodiments, the amino acid changes described in the present invention occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitutions are conservative substitutions. A conservative substitution is when one amino acid is replaced by another within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid , or the replacement of one neutral amino acid by another neutral amino acid. Exemplary permutations are shown in Table A below:

Table A

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

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

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to enhance, for example, the effectiveness of the antibody in treating cancer or cell proliferative diseases . Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fcs. Modifications to the Fc can also alter the half-life of the antibody in the therapeutic antibody, enabling less frequent dosing and thus increased convenience and reduced material usage. See Presta (2005) J. Allergy Clin. Immunol. 116:731, pp. 734-735.

In one embodiment, the number of cysteine residues in an antibody can be altered to modify antibody properties. For example, a modification is made to the hinge region of CH1 such that the number of cysteine residues in the hinge region is altered (eg, increased or decreased). This approach is further described in US Patent No. 5,677,425. Can The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease antibody stability.

Antibodies of the invention optionally include post-translational modifications to the antibody chains. Exemplary post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

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-proteinaceous moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are 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 copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n - vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol, and mixtures thereof.

Another modification of the antibodies or fragments thereof described herein contemplated by the present invention is pegylation. Antibodies can be PEGylated, for example, to increase the biological (eg, 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 PEGylation of proteins are known in the art and can be applied to the antibodies of the invention, see eg EP 0154316 and EP 0401384.

In some embodiments, antibody molecules of the invention are humanized. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the content of which is hereby incorporated by reference in its entirety (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. Human or humanized antibodies can be prepared using various techniques known in the art.

In some embodiments, 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 molecules of the invention of the invention also encompass antibody fragments thereof, such as antibody fragments of: 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 ), said K _D being less than or equal to approximately 150 nM, 140 nM , 130nM, 120nM, 110nM or 100nM, in some embodiments, the K _D is above about 90nM or 95nM; 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 is above about 1 nM, 2 nM, 3 nM, or 3.5 nM; in some embodiments, the antibody binding affinity is Determined using SPR assay.

(2) The bispecific antibody or fragment thereof of the present invention simultaneously binds to two monkey antigens with high affinity, for example, binds to monkey OX40 with the following equilibrium dissociation constant (K _D ), said K _D is less than or equal to about 50 nM, 40 nM , 30nM, 25nM, 24nM, 23nM or 22nM, in some embodiments, the K _D is about 10nM or 15nM or 20nM or more; and at the same time, it binds to monkey PD-L1 with the following equilibrium dissociation constant (K _D ), so The _K is less than or equal to about 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 14 nM, or 13 nM, and in some embodiments, the _K is above about 10 nM, 11 nM, or 12 nM; in some embodiments, the antibody binding affinity is Assayed using biofilm layer interferometry (eg ForteBio affinity assay).

(2) The antibodies of the invention or fragments thereof bind 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 at about 7 nM , 8 nM or more than 8.5 nM), and at the same time, bind to cells expressing human OX40, for example, with an EC50 of less than or equal to about 10 nM, 9 nM, 8.9 nM, 8.8 nM, 8.7 nM, 8.6 nM or 8.5 nM (in some embodiments in which the EC50 is above about 7 nM or 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, an antibody or fragment thereof of the invention induces cross-linking of cells expressing human OX40 with cells expressing human PD-L1.

(3) an antibody or fragment thereof of the invention binds to human T cells with an EC50 of less than or equal to about 5 nM, 4.5 nM, 4.4 nM, 4.3 nM, 4.2 nM, or 4.1 nM (in some embodiments, the EC50 is between 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, at 40° C., for example, for at least 30 sky. In some embodiments, the antibody retains at least 95%, 96%, 97%, 98%, or 99% of its monomeric main peak purity in an accelerated stability test, e.g., at 40°C for at least 10 days, 20 days, or 30 days. %. In some embodiments, the antibody or fragment thereof has a Tm, as determined by differential scanning fluorometry, greater than or equal to about 60°C, 61°C, 62°C, or 63°C.

(5) The antibody or fragment thereof of the present invention blocks the related activities of PD-L1 (such as 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, such as from Promega). In some embodiments, in the luciferase reporter gene detection system, the antibody of the present invention releases the inhibition of PD-1/PD-L1 interaction on the NFAT signaling pathway, for example, at 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, an EC50 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 antibodies or fragments thereof block less 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, eg, comparable to IgG.

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

(8) The antibody or fragment thereof of the present invention has the ability to effectively activate the OX40 signaling pathway dependent on PD-L1 (such as human PD-L1). In some embodiments, the antibodies or fragments thereof of the present invention effectively activate OX40 signaling in the presence of cells (e.g., tumor cells) expressing (e.g., naturally expressing or engineered to express) PD-L1 In some embodiments, the signaling pathway includes, for example, OX40 or OX40 ligand-mediated signaling pathway and/or its downstream signaling pathway (eg, NFkB signaling pathway).

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

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

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

(i) show the same or similar to OX40 and PD-L1 as the antibody of the present invention (for example, comprising SEQ ID NO: 1 as the peptide chain of formula (I) and comprising SEQ ID NO: 7 as the peptide chain of formula (II)) binding affinity and/or specificity;

(ii) inhibit (for example, competitively inhibit) the antibody of the present invention (for example comprising SEQ ID NO: 1 as the peptide chain of formula (I) and comprise SEQ ID NO: 7 as the peptide chain of formula (II)) and OX40 and PD - binding of L1;

(iii) binds to 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 comprising SEQ ID NO: 7 as the peptide chain of formula (II);

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

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

III‧Immunoconjugate

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

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

In other embodiments, antibody molecules of the invention are conjugated to diagnostic or detectable agents. Such antibodies may be used as part of clinical assays (eg, to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression and/or severity of a disease or condition. Such diagnostics and detection can be accomplished by conjugating antibodies to detectable substances including, but not limited to, various enzymes; prosthetic groups; fluorescent substances; luminescent substances; radioactive substances; Positron-emitting metal and nonradioactive paramagnetic metal ions in imaging.

Additionally, antibody molecules of the invention may be conjugated to therapeutic or drug moieties that modulate a given biological response. The therapeutic moiety or drug moiety should not be construed as limited to classic chemotherapeutic drugs. For example, the drug moiety can be a protein, peptide or polypeptide possessing the desired biological activity. Such proteins may include, for example, toxins; proteins or biological response modifiers.

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

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

Antibodies can also be attached to solid supports, which are 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 dysfunctional diseases, and the like. For example, the disease is a tumor (eg cancer) or an infection. In some embodiments, the tumor is an immune escape tumor. Preferably, the tumor is eg colon or colorectal or rectal or lung cancer.

IV. Nucleic acids of the invention and host cells comprising them

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

For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-9, or encoding an amine sequence selected from any one of SEQ ID NOs: 1-9. A nucleic acid having an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence.

The present invention also encompasses nucleic acids that hybridize under stringent conditions or that have one or more substitutions (e.g., conservative substitutions), deletions or insertions with the following nucleic acids: Nucleic acid of the nucleic acid sequence of the amino acid sequence shown in any one of 9; or comprise the nucleic acid of coding and be selected from the amino acid sequence shown in any one of SEQ ID NO:1-9 have at least 85%, 90%, 91 A nucleic acid that is a nucleic acid sequence of an amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In one embodiment, one or more vectors comprising said 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 (YACs). For example pXC vector or pTT5 vector, eg pXC17.4 or pXC18.4. In one embodiment, the expression vector is constructed as a double 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. Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the particular expression vector and mammalian species used. Animal host cell alteration or optimization.

Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. A marker can, for example, confer prototrophy, biocidal (eg, antibiotic) or heavy metal (eg, copper) resistance, etc. to an 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 middle. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splicing signals, as well as transcriptional promoters, enhancers and termination signals.

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

Suitable host cells include prokaryotic microorganisms such as E. coli. The host cells can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as insect cells and the like. Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted 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 (HEK 293 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 (BRL 3A), human lung cells (W138), human liver cells 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, NSO, P3X63 and Sp2/0, etc. For a review of mammalian host cell lines suitable for protein production see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell, such as a CHOS cell, CHOK1SV cell or CHOK1SV 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 an antibody molecule of the present invention or a fragment thereof (preferably an antigen-binding fragment), wherein said method comprises a method suitable for expressing an antibody molecule or fragment thereof (preferably an antigen-binding fragment) encoding the present invention. The host cell is cultured under conditions that bind the nucleic acid of the fragment), and optionally the antibody or fragment thereof (preferably an antigen-binding fragment) is 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 for preparing an antibody molecule of the present invention, wherein the method comprises, under conditions suitable for antibody expression, culturing a protein comprising an antibody encoding the antibody (eg, any polypeptide chain and/or multiple polypeptide chains) A host cell of the nucleic acid or an expression vector comprising the nucleic acid, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium).

In order to recombinantly produce the antibody molecule of the present invention, nucleic acid encoding an antibody (such as an antibody described above, such as any polypeptide chain and/or multiple polypeptide chains) is isolated and inserted into one or more vectors for use in a host Further cloning and/or expression in cells. Such nucleic acids are readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the antibody heavy and light chains).

Antibody molecules prepared as described herein can be purified by known art 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 will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. The purity of antibody molecules of the 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 for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the antibodies of the present invention are tested for their antigen-binding activity, for example, by known methods such as ELISA, Western blotting and the like. Binding to bound antigen can be determined using methods known in the art, exemplary methods are disclosed herein, such as biofilm layer interferometry and SPR.

The invention also provides assays for identifying biologically active antibodies. Biological activities may include, for example, binding to antigens, binding to cell surface antigens, inhibition or activation of antigens, and the like. Also provided are antibodies having such biological activity in vivo and/or in vitro.

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

The invention also provides methods for identifying properties of antibodies, such as druggability-related properties. Such druggability-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 either naturally express the antigen or have been engineered to express the antigen. Such cells also include cell lines that express the antigen and cell lines transfected with DNA encoding the antigen that do not normally express the antigen.

It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to the antibody molecules of the invention.

It will be appreciated that any of the above assays can be performed using the antibody molecules of the invention and additional active agents.

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

VII. Pharmaceutical compositions and pharmaceutical preparations

In some embodiments, the present invention provides a composition, preferably a pharmaceutical composition, comprising any antibody molecule or fragment thereof (preferably an antigen-binding fragment thereof) described herein or an immunoconjugate thereof. In one embodiment, the composition further includes pharmaceutical excipients. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises an antibody molecule of the invention, or a fragment thereof, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., anti-angiogenic, chemotherapeutic, 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 a tumor (eg cancer) or an infection. In some embodiments, the tumor is an immune escape tumor. Preferably, the tumor is eg colon or colorectal or rectal or lung cancer.

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

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

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in 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. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

Suitable excipients include starch, dextrose, 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 of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

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

The compositions of the invention can be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, 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 (eg, 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.

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

Exemplary lyophilized antibody formulations are 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 a histidine-acetate buffer.

The pharmaceutical compositions or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be 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, among others. The active ingredients are suitably present in combination in amounts effective for the intended use.

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

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

The pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).

Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes or in lyophilized form. Sterile injectable solutions can be prepared by incorporating the active compound (ie, antibody molecule) in the required amount in an appropriate solvent, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which 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, proper fluidity of the solution can be maintained by using a surfactant. can be obtained by using the Prolonged absorption of the injectable compositions can be brought about by including agents which delay absorption such as monostearate salts and gelatin in the compositions.

Kits comprising the antibody molecules described herein are also within the scope of the invention. A kit may comprise one or more additional elements including, for example: a package insert; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject .

VIII. Combination Products or Kits

In some embodiments, the invention also provides a combination product comprising an antibody of the invention, or an antigen-binding fragment thereof, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., anti-angiogenic agents, chemotherapeutic agents, Other antibodies, cytotoxic agents, vaccines, anti-infective active agents, small molecule drugs or immunomodulators, etc.).

In some embodiments, the combination product is used for preventing or treating diseases, such as autoimmune diseases, inflammatory diseases, infections, tumors, T cell dysfunction diseases and the like. For example, the disease is a tumor (eg cancer) or an infection. In some embodiments, the tumor is an immune escape tumor. Preferably, the tumor is eg colon or colorectal or rectal or lung cancer.

In some aspects, two or more components of the combination may be administered to the subject sequentially, separately, or in combination at the same time.

In some embodiments, the invention also provides kits comprising the antibodies, pharmaceutical compositions, immunoconjugates or combinations of the invention, and optionally package inserts directing administration.

In some embodiments, the present invention also provides pharmaceutical preparations comprising the antibodies, pharmaceutical compositions, immunoconjugates, and combination products of the present invention. Optionally, the pharmaceutical preparations further include a package insert to guide administration.

IX. Uses of the Antibody Molecules of the Invention

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

Accordingly, in one embodiment, the 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 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 (eg, elevated levels, eg, at the nucleic acid or protein level) of PD-L1 or PD-1 or PD-L2. In some embodiments, the disease is a disease in which PD-L1 or PD-1 or PD-L2 is present (eg, elevated levels, eg, at the nucleic acid or protein level) in a patient, eg, cancer. In some embodiments, the disease is a disease with T cell dysfunction. In some embodiments, the disease is a disease with reduced levels (eg, nucleic acid or protein level) of OX40, eg, a disease with reduced expression or activity of OX40.

In some embodiments, the disease is one that would benefit from inhibition of PD-L1 or PD-1 or PD-L2 at the nucleic acid or protein level. 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, eg, 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 Antibody molecules (eg, anti-PD-L1/OX40 antibody) or pharmaceutical compositions or immunoconjugates or combination products or kits disclosed herein. In some embodiments, the antigen is OX40 (eg, human OX40) and/or PD-L1 (eg, human PD-L1). In some embodiments, inhibiting the effect of an antigen refers to blocking the binding of PD-1 to PD-L1, or blocking the binding of PD-1 to PD-L2. In some embodiments, activating antigen activity or activating antigen-mediated signaling pathway refers to activating 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), such as enhancing the immune stimulation/effector function of T effector cells and/or making these cells Proliferate and/or downregulate 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 the function of CD4+ effector T cells, 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 an interleukin, such as IL-2.

In some embodiments, the anti-PD-L1/OX40 bispecific antibody or fragment thereof of the invention increases the number of (infiltrating) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or, e.g., CD4+ among CD45+ cells) in a tumor. percentage of cells). In some embodiments, the anti-PD-L1/OX40 bispecific antibody or fragment thereof of the invention increases the number of intratumoral (infiltrating) CD4+ effector T cells expressing γ-interferon (e.g. CD4+ γ-expressing interferon The total number of effector T cells, or eg the percentage of CD4+ cells expressing interferon-gamma among 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 (eg, IFNg) or interleukin (eg, IL-2).

In another aspect, the present invention relates to a method of preventing or treating a tumor (eg, cancer) in a subject, said method comprising administering to said subject an effective amount of an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein. compounds or combination products or kits. In some embodiments, the tumor is an immune escape tumor. 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, said method comprising administering to said subject an effective amount of an 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 an autoimmune disease and/or an inflammatory disease in a subject, said method comprising administering to said subject an effective amount of an antibody molecule or a medicament disclosed herein Compositions or immunoconjugates or combination products or kits.

The subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, a patient suffering from or at risk of having a disease described herein). In one embodiment, the subject has or is at risk of having a disease described herein (eg, a tumor or an infection or an autoimmune disease as described herein). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy. Alternatively or in combination, the subject is or is at risk of being immunocompromised by 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. Cancer can be early, middle or advanced or metastatic.

In some embodiments of any of the methods of the invention, the cancer described herein exhibits (eg, is infiltrated with) human effector cells. Methods for detecting human effector cells are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cells are one or more of NK cells, macrophages, monocytes. In some embodiments of any of the methods of the invention, the cancer described herein displays FcR-expressing cells (eg, is infiltrated by FcR-expressing cells). Methods for detecting FcRs are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of FcR-expressing cells. In some embodiments, the FcR is an FcγR. In some embodiments, the 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, eg, cancer, eg, a tumor or cancer with T cell dysfunction. In some embodiments, the tumor is a tumor expressing (eg, elevated levels of) PD-L1, eg, cancer. In some embodiments, the tumor is a tumor in which the expression or activity of OX40 has been reduced. In some embodiments, the tumor is a tumor that benefits from activation of the OX40 signaling pathway, eg, cancer.

In some embodiments, the infectious diseases to be treated and/or prevented with antibody molecules include pathogens for which there is currently no effective vaccine or 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 that present mutated antigens as the infection progresses. These variant antigens can be regarded as foreign antigens when the antibodies of the present invention are administered, thus, the exemplified antibody molecules of the present invention can stimulate strong T cell responses that are not inhibited by negative signals through PD-L1.

In some embodiments, the antibody molecules of the 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 thereof or immunoconjugates or compositions or combination products or kits in the production or preparation of medicaments for the treatment of related diseases or conditions mentioned herein .

In some embodiments, an antibody or antibody fragment or immunoconjugate or composition or combination or kit of the invention delays the onset of a disorder and/or symptoms associated with a disorder.

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

In some embodiments, treatment modalities include surgery; radiation therapy, localized or focused irradiation, and the like.

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

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

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

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

In some embodiments of any of the methods of the invention, administration of an antibody or fragment thereof of the invention is combined with administration of a tumor antigen. An antigen may eg be 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, tumor antigens comprise nucleic acids. In some embodiments, the tumor antigen is a tumor cell.

In some embodiments, an antibody or fragment thereof of the invention may be administered in combination with an antineoplastic agent.

In some embodiments, antibodies or fragments thereof of the invention may be administered in conjunction with cytokines. Cytokines can be administered as fusion molecules with antibody molecules of the invention, or as separate compositions. In one embodiment, an antibody of the invention is administered in combination with one, two, three or more cytokines (eg, as a fusion molecule or as a separate composition).

In some embodiments, the antibodies or fragments thereof of the invention may be combined with conventional cancer therapies in the art, including but not limited to: (i) radiation therapy or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered via external beam radiation therapy (EBRT) or via internal brachytherapy; (ii) chemotherapy, or application of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or specifically affect Agents that deregulate cancer cell proteins; (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines); (v) hormone therapy, or blocking hormones (e.g., when the tumor is hormone-sensitive), (vi) Angiogenesis inhibitors, or blocking blood vessel formation and growth, and (vii) palliative care, or treatments that involve improving 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 therapy encompasses combined administration (where two or more therapeutic agents are contained in the same formulation or in separate formulations), and separate administration, in which case it may be administered in combination with other therapies, such as treatment modalities and Administration of the antibody of the invention occurs before, simultaneously with, and/or after/or the therapeutic agent. Antibody molecules and/or other therapies, eg, therapeutic agents or treatment modalities, may be administered during active disease or during remission or less active disease. Antibody molecules can be administered before, concurrently with, after, or during remission of other treatments.

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

It is understood that any therapy can be performed using the immunoconjugate or composition or combination product or kit of the invention in place of or in addition to the antibody of the invention.

Antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising same, and any additional therapeutic agents) may be administered by any suitable method, including parenteral, intrapulmonary, and intranasal , and, if required for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, eg, by injection, eg intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prophylaxis or treatment of disease, the appropriate dose of the antibody of the 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 course of the disease, the Whether the antibody is administered prophylactically or therapeutically, previous treatment therapy, the patient's clinical history and response to the antibodies, and the judgment of the attending physician. The antibody is suitably administered to the patient in one treatment or over a series of treatments.

Dosages and treatment regimens for the antibody molecules of the invention can be determined by the skilled artisan. In some embodiments, dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus injection can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially 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 suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to be in association with the required pharmaceutical carrier. produce the desired therapeutic effect. The specifications for the dosage unit forms of the invention are directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the art in which such active compound is to be combined for the treatment of sensitivity in an individual. limit.

X. Methods and Compositions for Diagnosis and Detection

In certain embodiments, any antibody provided herein, or antigen-binding fragment thereof, can be used to detect the presence of an antigen to which it binds in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, a biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

In one aspect, the invention provides in vitro or in vivo methods for detecting the presence of relevant antigens in biological samples such as serum, semen or urine or tissue biopsy samples (for example, from hyperproliferative or cancerous lesions). diagnosis method. The diagnostic method comprises: (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 the interaction to occur and (ii) Complex formation between the antibody molecule and the sample (and optionally, a control sample) is detected. Formation of the complex indicates the presence of the relevant antigen and may indicate suitability or need for treatment and/or prophylaxis as described herein.

In one embodiment, antibodies of the invention can be used to diagnose a disease, eg, evaluate (eg, monitor) treatment or progression, diagnosis and/or staging of a disease described herein (eg, tumor or infection) in a subject. In certain embodiments, labeled antibodies of the invention are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are indirectly detected, such as enzymes or ligands, such as , by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioactive isotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, eg, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, Horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, eg, glucose oxidase, galactose oxidase, and glucose-6- Phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / Avidin, spin tags, phage tags, stable free radicals and more.

In some embodiments of any of the inventions provided herein, a sample is obtained prior to treatment with an 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 implementations In protocols, the sample is a biopsy (eg, a core biopsy), a surgical specimen (eg, a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, the relevant antigen is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

In some embodiments, the level and/or distribution of the relevant antigen is determined in vivo, e.g., non-invasively (e.g., by scanning using a suitable imaging technique, e.g., positron emission tomography (PET)). Detector-labeled antibody molecule of the invention. In one embodiment, an in vivo assay correlates, for example, by detecting an antibody molecule of the invention detectably labeled with a PET reagent (eg, ¹⁸ F-fluorodeoxyglucose (FDG)). Levels and/or distribution of antigens.

In one embodiment, the invention provides diagnostic kits comprising the antibody molecules 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 from a subject comprising cancer cells) for the presence of an antigen of interest, thereby determining its value , comparing this value to a control value (e.g., the value of a relevant antigen in a healthy individual), and if greater than the control value, administering to the subject a therapeutically effective amount of an antibody of the invention optionally in combination with one or more other therapies , thus treating the disease.

In some embodiments, the antigen is PD-L1 (eg, human PD-L1) and/or OX40 (eg, 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, etc., are also applicable to or can be combined with the embodiments of other parts of the present invention. Properties that are applicable to antibody molecules described in the various sections of the present invention Embodiments such as properties, uses, and methods are also applicable to compositions, conjugates, combination products, kits, etc. comprising antibodies.

The following examples are described to aid in the understanding of the present invention. The examples are not intended and should not be construed in any way as limiting the scope of the invention.

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

In this example, an anti-PD-L1/OX40 bispecific antibody was constructed using molecular cloning technology. The bispecific antibody format contains four polypeptide chains and can bind to two antigens, antigen A is OX40 and antigen B is PD-L1. The parental antibody used to construct the bispecific antibody is an 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, which is composed of four symmetrical polypeptide chains, the left half of which is composed of peptide chain #1 and peptide chain #2, and the right The half part is also 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 The amino acid sequence of anti-PD-L1/OX40 bispecific antibody peptide chain #1 shown in SEQ ID NO: 1 of the left half of the anti-PD-L1/OX40 bispecific antibody includes SEQ ID from the N-terminus to the C-terminus NO: Anti-OX40 antibody ADI-20057 VH amino acid sequence shown in 2, CH1 amino acid sequence shown in SEQ ID NO: 3, Fc amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: The (G4S) ₂ flexible linker peptide chain amino acid sequence shown in 5, and the anti-PD-L1 single domain antibody amino acid sequence shown in SEQ ID NO: 6. The left side of the anti-PD-L1/OX40 bispecific antibody Half of the peptide chain #2 amino acid sequence of the anti-PD-L1/OX40 bispecific antibody shown in SEQ ID NO: 7 contains the anti-OX40 antibody ADI-20057 shown in SEQ ID NO: 8 from the N-terminal to the C-terminal VL amino acid sequence and CL amino acid sequence shown in SEQ ID NO:9.

SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 6 in bold italics are the CDR regions of the anti-PD-L1/OX40 bispecific antibody of the present invention (the HCDR1 in the VHH and VH regions Defined using AbM rules; HCDR2 & 3 defined using Kabat rules; LCDR: defined using Kabat rules). See the sequence listing for specific sequence information.

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

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

In this example, Suzhou Genewiz Biotechnology Co., Ltd. (Genewiz) was commissioned to synthesize the coding nucleotide sequences of the peptide chains of the anti-PD-L1/OX40 bispecific antibody and the IgG2 control antibody. Using appropriate restriction enzymes and ligases, the synthesized nucleotide sequences encoding the peptide chains were respectively ligated into vectors, wherein the encoding DNA sequences of the two peptide chains of the anti-PD-L1/OX40 bispecific antibody were respectively The eukaryotic expression vectors Double-GeneVectors (pXC17.4, pXC18.4) (purchased from Lonza Company) were constructed and the correctness of the sequence was verified by sequencing, and the recombinant vectors respectively containing the nucleotide sequences encoding the peptide chains were obtained. Peptide chains #1 and #2 were transferred into CHOK1SV GS-KO Cell line (purchased from Lonza) cells by electrotransfection to construct protein expression cell lines for protein expression. The protein expression process was as follows:

1) Take the desired CHOK1SVGS-KO Cell line (Lonza) protein expression cell line, subculture it in CD CHO Medium (GIBCO, 10743-029), and culture it until the cell density is 0.8×10 ⁶ cells/ml;

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

60%時，收集培養物，以7500轉/分鐘離心30分鐘，取細胞上清，用以純化。 3) Continuous culture until the 14th day or cell viability

At 60%, the culture was collected, centrifuged at 7500 rpm for 30 minutes, and the cell supernatant was taken for purification.

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

1) Affinity purification: MabSelect SuRe (GE Healthcare, catalog number: 17-5438-03) affinity chromatography column was selected and placed in the AKTApure system (GE Healthcare). The AKTA system was detoxified (overnight) with 0.1 M NaOH. On the day of sample collection, the cells were centrifuged at 7500 rpm/min for 30 min and filtered using SARTOPORE (Sartorius, 5441307H4). Before purification, wash the system and equilibrate the column with Binding Buffer (Tris 20mM, NaCl 150mM, pH 7.2) with 5 column volumes. Pass the above-mentioned filtered supernatant that needs to be purified through the column. Use 5-10 times the column volume Binding Buffer to rebalance, and use the UV detection device equipped with the AKTApure system to monitor until the UV levels out. Antibody was eluted with Elution Buffer (citric acid + sodium citrate 100mM, pH 3.5), and samples were collected according to the UV absorption value. Add 80 μl of Neutralizing Buffer (Tris-HCl 2M) to each 1ml of collected solution for neutralization;

2) Replacing the buffer: According to size exclusion chromatography (size exclusion chromatography; SEC), the purity of the collected samples in the separate tubes was detected, and the samples with a purity greater than 95% were combined. The combined solution was centrifuged in a 15ml ultrafiltration centrifuge tube at 4500rpm for 30min. Dilute the protein with PBS and continue to centrifuge at 4500rpm for 30min, repeating twice to replace the buffer. Combine the antibodies after buffer replacement and measure the antibody concentration.

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

In this example, Pogalizumab is a human IgG1 OX40 antibody expressed in HEK293 cells using the proposed INN: List 114 (see http://www.who.int/medicines/publications/druginformation/innlists/PL114. pdf) of the heavy and light chain sequences. Suzhou Jinweizhi Biotechnology Co., Ltd. (Genewiz) was entrusted to synthesize the coding nucleotide sequences of the above-mentioned peptide chains of the antibody. The synthesized nucleotide sequences encoding peptide chains were respectively ligated into vector pTT5 by using appropriate restriction enzymes and ligases to obtain recombinant vectors respectively containing the nucleotide sequences encoding peptide chains.

Transfect HEK293 cells with Polyethyleneimine (PEI) 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. Centrifuge the cell culture one day before transfection to obtain the cell pellet, suspend the cells with fresh Expi293 cell culture medium, and adjust the cell density 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) with a final volume of 1/10 of the HEK293 cell suspension was used as a transfection buffer. Add 200 μg of the 1:1 molar ratio of the above-mentioned recombinant plasmids respectively containing the nucleotide sequences of each chain to each milliliter of transfection buffer, and mix well;

3) Add 30 μg of PEI (Polysciences, 23966) to the plasmid, mix well and incubate at room temperature for 10 min. Gently pour the mixture into the HEK293 cell suspension. Gently mix, place in 8% CO ₂ , 36.5°C overnight culture;

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

60%時，收集培養物，以7500轉/分鐘離心30分鐘，取細胞上清，用以純化； 5) Continuous culture until the 7th day or cell viability

At 60%, the culture was collected, centrifuged at 7500 rpm for 30 minutes, and the cell supernatant was 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: MabSelect SuRe (GE Healthcare, catalog number: 17-5438-03) affinity chromatography column was selected and placed in the AKTApure system (GE Healthcare). The AKTA system (GE Healthcare) was detoxified (overnight) with 0.1 M NaOH. On the day of sample collection, the cells were centrifuged at 7500 rpm/min for 30 min and filtered using SARTOPORE (Sartorius, 5441307H4). Before purification, wash the system and equilibrate the column with Binding Buffer (Tris 20 mM, NaCl 150 mM, pH 7.2) with 5 column volumes. Pass the above-mentioned filtered supernatant that needs to be purified through the column. Use 5-10 times the column volume Binding Buffer to rebalance, and use the UV detection device equipped with the AKTApure system to monitor until the UV levels out. Antibody was eluted with Elution Buffer (citric acid + sodium citrate 100mM, pH 3.5), and samples were collected according to the UV absorption value. Add 80 μl of Neutralizing Buffer (Tris-HCl 2M) to each 1ml of collected solution for neutralization;

2) Replacing the buffer: According to size exclusion chromatography (size exclusion chromatography; SEC), the purity of the collected samples in the separate tubes was detected, and the samples with a purity greater than 95% were combined. The combined solution was centrifuged in a 15ml ultrafiltration centrifuge tube at 4500rpm for 30min. Dilute the protein with PBS and continue to centrifuge at 4500rpm for 30min, repeating twice to replace the buffer. Combine the antibodies after buffer replacement and measure the antibody concentration.

Anti-OX40 monoclonal antibody (ADI-20057 in IgG2 form, see Chinese invention patent application number: 201710185399.9; anti-PD-L1 nanobody humanized Nb-Fc (see See Chinese Invention Patent Application No.: PCT/CN2017/095884), and perform the above purification in the same way to obtain ADI-20057 and anti-PD-L1 humanized Nb-Fc for subsequent experiments.

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

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

The equilibrium dissociation constant (K _D ) of the antibody of the present invention binding to human PD-L1 or human OX40 is determined by surface plasmon resonance (SPR). Based on the SPR principle, when a beam of polarized light is incident on the end face of the prism at a certain angle, surface plasmon waves 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. During the analysis, a layer of biomolecular recognition film is first fixed on the surface of the sensor chip, and then the sample to be tested flows over the surface of the chip. If there are molecules in the sample that can interact with the biomolecular recognition film on the surface of the chip, it will cause refraction on the surface of the gold film. The change of the rate will eventually lead to the change of the SPR angle. By detecting the change of the SPR angle, information such as the affinity and kinetic constant of the analyte can be obtained.

In this example, Biacore (GE Healthcare, T200) was used to measure the K _D of the anti-PD-L1/OX40 bispecific antibody. Binding and dissociation between antibodies to obtain affinity and kinetic constants. The method includes chip preparation and affinity detection. The assay process used 10×HBS-EP+ (BR-1006-69, GE Healthcare) diluted 10 times as the experimental buffer. The chip preparation process used the amine coupling kit (BR-1006-33, GE Healthcare), and the anti-human Fc antibody was coupled to the surface of the CM5 chip (29-1496-03, GE Healthcare). The specific process was as follows: first 50mM N-hydroxysuccinimide (NHS) and 200mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were freshly mixed and injected into the dual channel of the chip, Activate for 7 minutes. Then the anti-human Fc antibody was diluted in 10mM Acetate (pH 5.0), and injected into the dual channels of the CM5 chip, so that the protein was covalently coupled on the surface of the chip channel, and the coupling height was about 6000 RU. Finally, 1M ethanolamine was injected to block the remaining activated sites for 7 minutes. Each cycle of affinity detection includes capturing antibody, binding a concentration of antigen, and chip regeneration:

Capture antibody: Firstly, the antibody prepared and purified as in Example 2 was diluted to 0.5 μg/mL, captured on the second channel of the CM5 chip at a flow rate of 10 μL/min, and the capture time was 30 s.

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

Chip regeneration: Before the next antibody assay, the chip was regenerated using 10 mM Glycine pH 1.5 (BR-1003-54, GE Healthcare).

The data results were analyzed kinetically using a 1:1 binding model.

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 proteins, and maintain the parental antibody ADI-20057 and anti-PD-L1 Affinity constants of humanized Nb-Fc with 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 antibody to monkey antigen ForteBio assay

In this example, the Octet Red96 system (produced by ForteBio) was used to determine the equilibrium dissociation constant ( _KD ). 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 experimental 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 (PBS 1×, BSA 0.1%, Tween-20 0.05%) and pre-wet them for 20 minutes ;

2) Experimental process: Add 100 μl of SD buffer as a blank control (for background subtraction) and 100 μl of 100 nM purified anti-PD-L1/OX40 double wells into the wells of a 96-well black polystyrene half volume microplate (Greiner). Specific antibody and anti-PD-L1 humanized Nb-Fc antibody as control, anti-OX40 antibody antibody ADI-20057, 100 μl diluted in SD buffer as antigen monkey PD-L1 (ACRO, PD1-C52H4), monkey Solution of OX40 (ACRO, OXO-C5220). The anti-human IgG Fc biosensor AHQ was immersed in the wells respectively containing the antibody solution, and sampled at room temperature for 600 seconds. The sensor was then washed in SD buffer to baseline and then submerged in wells containing 100ul of antigen solution to monitor the binding of antibody to antigen. Then transfer the sensor to the well containing 100ul SD buffer to monitor the dissociation of the antibody (set the running steps and time: Baseline, Loading~1nm, Baseline, Association and Dissociation time depend on the sample binding and dissociation speed). The rotation speed is 400 rpm, and the temperature is 30°C. Background corrected binding and dissociation curves were fitted by Octet analysis software (ForteBio) to generate association (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 maintain the parental antibody ADI-20057 and anti-PD-L1 human Affinity constants of FeNb-Fc with 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. Analysis of the binding of the anti-PD-L1/OX40 bispecific antibody of the present invention to 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 overexpressed human The binding of OX40 or human PD-L1 to CHO cells, the experimental procedure is as follows:

1) Cell preparation: Transfect the pCHO1.0 vector (Invitrogen) carrying the human OX40 cDNA (SinoBiological Inc) from the pure strain to the multi-selection site MCS into Chinese hamster ovarian cancer cells (CHO) (Invitrogen), and obtain by pressure screening CHO cells (CHO-OX40 cells) stably expressing human OX40 were transfected with pCHO1.0 vector (Invitrogen) carrying human PD-L1 cDNA (SinoBiological Inc) from a pure strain to multi-selective colony site MCS into Chinese hamster ovarian cancer cells (CHO) (Invitrogen), CHO cells stably expressing human PD-L1 (CHO-PD-L1 cells) were obtained by pressure screening, and the CHO-PD-L1/CHO-OX40 cells were counted and diluted to 2×10 ⁶ Cells/ml, add 100 μl/well to U-bottom 96-well plate;

2) Detection steps: centrifuge at 400 g for 5 min, and remove the cell culture medium. Add the serially diluted samples (bispecific antibody, anti-PD-L1 antibody and anti-OX40 antibody) 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 to remove the supernatant, wash the cells with PBS once to remove unbound antibodies. 400g, 5min to remove PBS, add 100μl 1:200 diluted PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) to each well. Incubate on ice for 30 min in the dark. 400g, 5min to remove the supernatant, wash the cells once with PBS, and remove unconjugated antibodies. The cells were resuspended in 100 μl of PBS, and the binding of the antibody to the cells was detected by flow cytometry (BD, ACCURIC6).

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 8.463nM, and the binding to the parental anti-OX40 antibody ADI-20057 Potency (EC50 of 4.710 nM) was 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 8.732nM, and the anti-PD-L1 humanization of the parental antibody The binding ability of Nb-Fc (EC50 of 9.651 nM) was similar.

Example 3.4. Analysis of the simultaneous binding of the anti-PD-L1/OX40 bispecific antibody of the present invention 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 experimental process is as follows:

1) The CHO-PD-L1 and CHO-OX40 cells obtained as described in Example 3.3 were subjected to 400 g, centrifuged for 5 minutes to remove the medium, washed once with PBS, and the cells were resuspended in PBS. After counting, the cell density was adjusted to 2×10 ⁶ cells/ml; CHO-PD-L1 and CHO-OX40 cells were added CellTraclker ^TM Deep Red (Thermo, C34565) and Cell Trace CFSE (Invitrogen, C34554) dyes at a ratio of 1:5000, respectively, and placed at 37°C for 30 minutes. After centrifugation at 400g for 5min, remove the supernatant and wash the cells once with PBS;

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

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

The FACS test results are 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 Capable of simultaneously binding target antigens from different cell surfaces.

Example 4 Human T cell binding detection of the 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 anti-PD-L1/OX40 bispecific antibodies to human T cells ability, the experimental process is as follows:

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

2) Detection steps: centrifuge the cell culture in 1) at 400 g for 5 min, remove the cell culture medium, resuspend the cells with PBS, and after counting, adjust the cell density to 2×10 ⁶ cells/ml, transfer to the U-shaped bottom 96 Add 100 μl/well to the well plate. Add the serially diluted samples (anti-PD-L1/OX40 bispecific antibody, ADI-20057 and IgG2 control) to the U-shaped plate, mix well, add 100 μl to each well, and let stand on ice for 30 minutes. Centrifuge at 400g for 5 minutes to remove the supernatant, and wash the cells once with PBS. Centrifuge at 400 g for 5 min to remove PBS, and add 100 μl of 1:200 diluted PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) to each well. Incubate on ice for 30 min in the dark. Centrifuge at 400g for 5 minutes to remove the supernatant, and wash the cells once with PBS. The cells were resuspended in 100 μl PBS, and detected 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, and the binding EC50 is 4.062nM, and the binding ability of the parental anti-OX40 antibody ADI-20057 (EC50 is 2.571nM) are 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 bispecific antibodies, this example evaluates the purity of a batch of antibodies prepared by detecting the changes in the purity of the prepared antibodies after 0, 1, 3, 7, 10, 20, and 30 days at 40°C. Long term thermal stability. The experimental method is as follows:

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

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

The experimental results are shown in Table 5. The anti-PD-L1/OX40 bispecific antibody of the present invention was placed at 40°C for 30 days, and the proportion of the main monomer peak decreased by 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 proportion of the main peak of the monomer when the bispecific antibody was incubated at 40°C

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

Differential Scanning Fluorescence (DSF) can provide information about structural stability based on the fluorescence change process in the map, and detect the conformational changes of proteins. The temperature corresponding to the maximum absolute value of the fluorescence curve is the Tm of the protein. In this example, the _Tm value of the anti-PD-L1/OX40 bispecific antibody of the present invention was determined by using the DSF method. The experimental process is as follows:

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

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

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

Table 6. Determination results of bispecific antibody Tm value

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 a luciferase reporter gene to detect cell lines (Promega, CS187109 ), by detecting the expression of luciferase to reflect the ability of the bispecific antibody to inhibit the interaction of PD-1/PD-L1, the detailed experimental process is as follows:

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

Promega's PD-1/PD-L1 Blockade Bioassay is a biologically relevant MOA-based assay for the determination of the potency and stability of antibodies capable of blocking the PD-1/PD-L1 interaction. The assay consists of two genetically engineered cell lines:

˙PD-1 effector cells: Jurkat T cells that stably express human PD-1 and induce the expression of luciferase by nuclear factor of activated T cells (NFAT).

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

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

Test according to the manufacturer's product instructions.

1) Plate PD-L1 aAPC/CHO-K1 cells one day before activity detection: Discard the culture supernatant, wash once with PBS, add an appropriate amount of trypsin (Gibco, 25200072), incubate at 37°C, 5% CO ₂ for 3-5min, wash with four Double the volume of RPMI1640 (Gibco, 22400-071) medium containing 10% FBS (HyClone, SH30084.03) to stop the digestion, collect the cells, take a small amount of cell mixture to measure the cell concentration, take the required volume of cells, 400g, centrifuge for 10min, The supernatant was discarded, and the RPMI1640 (Gibco, 22400-071) medium containing 10% FBS (HyClone, SH30084.03) was used as the assay buffer to resuspend the cells so that the cell density was 4×10 ⁵ cells/ml. The cells were added to a 96-well white cell culture plate (Nunclon, 136101) at 100 μL/well, and PBS was added to the side wells at 200 μl/well. Cells were cultured overnight in a carbon dioxide incubator at 37°C in 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 initial concentration, 3-fold serial dilution from the second concentration point to the tenth concentration point, a total of 12 concentration points;

3) Take PD-1 effector cells, count, 400g, centrifuge for 5min, resuspend the cells with assay buffer, so that the cell concentration is 1.25× ¹⁰⁶ cells/ml, take 96 white cell culture plates (NUNC, 136101), each well Add 100 μl cells;

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

5) Cultivate the culture plate obtained in step 4) 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 5-10min;

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

8) Use a Spectra Max I3 microplate reader (Thermo, Maxi3) to collect full-wavelength chemiluminescence, and the collection time for each 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 similar to that of anti-PD-L1 human Nb-Fc was similar (EC50 for anti-PD-L1/OX40 bispecific antibody was 0.4085 and EC50 for anti-PD-L1 humanized Nb-Fc was 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 ability, the experimental process is as follows:

1) Biotin labeling of Human OX40 Ligand Fc Tag (ACRO, OXL-H526X-1MG) was performed according to the instruction manual of EZ-Link Sulfo-NHS-LC-BiotinNoWeighFormat (PIERCE, 21327), and desalting was performed according to 2ml Zeba ^TM centrifugal desalting Column (PIERCE, 89890) instruction manual operation;

2) Cell preparation: draw the CHO-OX40 cells obtained in Example 3.3 into a 50ml centrifuge tube, count on a hemocytometer, take 2.4× ¹⁰⁷ cells/ml in a new 50ml centrifuge tube, centrifuge at 400g for 5min, Remove the supernatant, resuspend the cells with 20ml PBS, centrifuge again for 5 minutes, remove the supernatant, and resuspend the cells with 5ml PBS;

3) Add 50 μl per well of CHO-OX40 cells treated in 2) to a 96-well U-bottom hemagglutination plate for later use;

4) Preparation of gradient concentration sample solution: add 3.2ml PBS to 96 μl of biotin-labeled Human OX40 Ligand Fc Tag (Biotin-Human OX40 Ligand Fc Tag), mix well, and prepare Biotin-Human OX40 Ligand Fc Tag PBS mixture. Antibody samples (anti-PD-L1/OX40 bispecific antibody, ADI-20057, Pogalizumab and IgG2 control) were diluted with Biotin-Human OX40 Ligand Fc Tag PBS mixture, starting at 2000nM, followed by 11 concentration points 3 1-fold serial dilution, a total of 12 concentration points;

5) Add 50 μl of prepared gradient concentration samples 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, remove the supernatant, add 150 μl of PBS to each well, then centrifuge at 400 g for 5 min, remove the supernatant, and repeat three times;

7) Add 100 μl of 1:200 diluted Streptavidin-R-phycoerythrin (SAPE) (THERMO, S21388) to each well of the 96-well U-bottom hemagglutination plate obtained in 6), 4°C, 30min;

8) Add 150 μl of PBS to each well of the 96-well U-bottom hemagglutination plate obtained in 7), centrifuge at 400 g for 5 min, remove the supernatant, and repeat twice;

9) Resuspend the cells in the 96-well U-bottom hemagglutination plate obtained in 8) with 100 μl of PBS, and detect with a flow cytometer (BD, ACCURIC6).

The detection 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 of the present invention 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 Antibody 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 ability of the antibody to block OX40 ligand-mediated activation of OX40.

The activator activity of the anti-OX40 antibody of the anti-PD-L1/OX40 bispecific antibody of the present invention was evaluated by measuring NFkB-mediated transcriptional activation. Anti-HumanCD3 (BD, 555329), Anti-Human CD28 (BD, 555725) plus antibody activation in solution overexpressed human OX40 (purchased from Sino) and NFkB-luciferase construct (NFkB promoter-luc , Promega) Jurkat cells (ATCC, USA) (Jurkat-OX40-NFkB-Luc-Rep), and then added Bio-Glo ^TM reagent for color development. The specific experimental 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) Assay 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 (4 μg/ml), Anti-Human CD28 (16 μg/ml), ready to use.

Experimental steps:

1) Jurkat-OX40-NFkB-Luc-Rep cell treatment: count the Jurkat-OX40-NFkB-Luc-Rep cells in the logarithmic growth phase, 400g, centrifuge for 5min, resuspend the cells with complete medium and adjust the cell density to 8×10 ⁶ pieces/ml spare;

2) Gradient concentration sample solution preparation: use the processed analysis buffer A to dilute the antibody sample to be tested and the control to 200nM as the initial concentration, and serially dilute the second concentration point 2 to the tenth concentration point 3 times, a total of 9 concentration point. Use the processed analysis buffer B as the control group;

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

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. Add the obtained Bio-Glo™ detection reagent to the wells of the detection plate after incubation in the above 3) for 12 hours at 80 μl/well. Leave it at room temperature for 5 to 10 minutes, and read the fluorescent signal value.

5) Plate reading: use a Spectra Max I3 microplate reader (Thermo, Max i3) to collect full-wavelength chemiluminescence, and the collection time for each well is 1000ms.

The experimental results are shown in Figure 10, Pogalizumab easily blocks the activation of the OX40 signaling pathway mediated by the OX40 ligand at a concentration greater than about 0.4nM or higher. However we are surprised The anti-PD-L1/OX40 bispecific antibody of the invention and its parental anti-OX40 antibody were found to increase luciferase levels, suggesting that they have non-OX40 ligand blocking properties and contribute to OX40 receptor aggregation.

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

Example 10 Detection of Activation of OX40-Mediated Signaling Pathway by Anti-PD-L1/OX40 Bispecific Antibody Based on Luciferase Reporter Gene Method

In order to detect the in vitro biological activity of bispecific antibodies, this example uses the Jurkat-OX40-NFkB-Luc-Rep stable cell line of Innovent Biopharmaceutical (Suzhou) Co., Ltd. (see Example 9 for the construction process), by adding Raji 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 the downstream NFκB fluorescent The luciferase reporter gene is expressed, and then the luciferase substrate is added to lyse the cells and generate fluorescence, and the biological activity of the anti-OX40 antibody is reflected by the intensity of the fluorescence.

The detailed experimental 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) Assay buffer: RPIM-1640 (90%), FBS (10%), Anti-Human CD3 (10μg/ml) (BD, 555329), Anti-Human CD28 (10μg/ml) (BD, 555725) , ready-to-use;

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

Experimental steps:

1) Raji cell treatment: count Raji cells, 400g, centrifuge for 5min, resuspend the cells with assay buffer and adjust the cell density to 2.0× ¹⁰⁶ cells/ml for later use;

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

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

4) Adding samples: set 3 duplicate wells in the sample group, add samples (50 μl) of each concentration to the white opaque cell culture plate obtained in 2) (each concentration sample is added in triplicate), and place the cell plate in Cultivate 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. 80 μl/well of the obtained Bio-Glo ^™ detection reagent was added to the wells of the detection plate obtained in 4) after culturing for 16 hours above. Leave it at room temperature for 5 to 10 minutes, and read the fluorescent signal value.

6) Plate reading: use a Spectra Max I3 microplate reader (Thermo, Max i3) to collect full-wavelength chemiluminescence, and the collection time for each 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=0.6712nM, which is similar to the activation effect of the parent (EC50=0.1455nM).

Example 11 Detection of the PD-L1-dependent activation of the OX40-mediated signaling pathway mediated by the anti-PD-L1/OX40 bispecific antibody of the present invention

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 Raji cells highly expressing PD-L1

In order to detect the biological activity of the OX40-mediated signaling pathway activated by the anti-PD-L1/OX40 bispecific antibody of the present invention in the presence of high-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 selection Raji cells (Raji-PD-L1 cells). In this example, the cell line was used 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 (200 μg/ml), Puromycin (2 μg/ml).

(2) Analysis buffer: RPIM-1640 (90%), FBS (10%), ready to 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, centrifuge the cell culture at 400g for 10min, remove the supernatant, add the assay 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 66.5 μL of Jurkat-OX40-NFkB-Luc-Rep (see Example 9 for the construction process) and Raji cell suspension (or Raji-PD-L1 cell suspension) treated in 1) to the first well, and the second well Add 50 μL of Jurkat-OX40-NFkB-Luc-Rep cells and Raji cell suspension (or Raji-PD-L1) treated in 1) to the twelfth well;

3) Adding samples: add the antibody to be tested in the first well, dilute to 25nM as the initial concentration, and serially dilute the second concentration point 2 to the 12th concentration point 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 well. 80 μl/well of the obtained Bio-Glo ^™ detection reagent was added to the wells of the above-mentioned detection plate after culturing for 16 hours. Leave it at room temperature for 5 to 10 minutes, and collect full-wavelength chemiluminescence with a Spectra Max I3 microplate reader (Thermo, Max i3). The collection time for each well is 1000ms.

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) were used alone, and the anti-PD-L1 antibody ( Neither anti-PD-L1 humanized Nb-Fc) alone nor anti-OX40 antibody (ADI-20057) combined with anti-PD-L1 antibody (anti-PD-L1 humanized Nb-Fc) could activate NFkB downstream of OX40 signal path.

However, we were surprised to find that in the cell system with PD-L1 expression (the experimental results are shown in Figure 13), the anti-PD-L1/OX40 bispecific antibody of the present invention has a higher activity than the anti-OX40 antibody and anti-PD-L1 Antibody used alone, combined use of anti-OX40 antibody and anti-PD-L1 antibody, and Pogalizumab had more significant activation of NFkB signaling pathway. Anti-PD-L1/OX40 bispecificity of the present invention The antibody showed better activation of the NFkB signaling pathway downstream of OX40 in the presence of PD-L1 expressing cells.

Example 11.2 The specific activation effect of the anti-PD-L1/OX40 bispecific antibody of the present invention on 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 tumor cells. In this example, the Jurkat-OX40-NFkB-Luc-Rep cell line of Innovent Biopharmaceutical (Suzhou) Co., Ltd. (see Example 9 for the construction process) and tumor cells NCI-H292 human lung cancer cells (ATCC, CRL-1848) were co-incubated , to detect 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 experimental process is as follows:

Solution preparation: (1) Jurkat-OX40-NFκB-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 to 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: count NCI-H292 cells, centrifuge at 1000rpm/min for 5min, resuspend the cells in assay buffer and adjust the cell density to 1.6× ¹⁰⁶ cells/ml for later use;

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

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

4) Preparation of gradient concentration sample solution: 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 initial 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 duplicate wells for the sample group, add samples (50 μl) of each concentration to the white opaque cell culture plate obtained in 3) respectively (each concentration sample well is in triplicate), and place the cell plate in carbon dioxide Cultivate 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. 80 μl/well of the obtained Bio-Glo ^™ detection reagent was added to the wells of the above-mentioned detection plate after culturing for 16 hours. Leave it at room temperature for 5 to 10 minutes, and collect full-wavelength chemiluminescence with a Spectra Max I3 microplate reader (Thermo, Max i3). The collection time for each well is 1000ms.

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, anti-PD-L1 antibody (anti-PD-L1 humanized Nb-Fc) Neither alone nor in combination with anti-OX40 antibody and anti-PD-L1 antibody (ADI-20057+anti-PD-L1 humanized Nb-Fc) could 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 activation. In the presence of tumor cells that naturally express PD-L1, the bispecific antibody can better activate the NFkB signaling pathway downstream of OX40.

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 biological activity of bispecific antibodies in vitro, this example detected the activation of bispecific antibodies on human T cells in vitro. The detailed experimental process is as follows:

Human PBMC cells (ALLCELLS, PB005F) were resuscitated, and after standing for 3 hours, they became monocytes, added 10ml AIM V® Medium CTS (GIBCO, A3021002) medium, added IL4 (20ng/ml) (R&D, 204- IL), GM-CSF (10ng/ml) (R&D, 215-GM) induced monocytes to differentiate into DC cells, cultured to the 5th day, and added the cytokine TNFα (1000U/ml , 10ng/ml) (R&D, 210-TA), RhIL-1β (5ng/ml) (R&D, 201-LB), RhIL-6 (10ng/ml) (R&D, 206-IL), 1μM PGE (Tocris, 2296), continued to culture for 2 days in a carbon dioxide incubator at 37°C and 5% CO ₂ as mature DC cells (moDC) of mixed lymphocyte reaction (MLR);

Human PBMC cells (ALLCELLS, PB005F) were revived, and CD4+ cells were isolated according to the instructions of the Human CD4+ T cell enrichment kit (STEMCELL, 19052). In short, the above-mentioned PBMCs were cultured for 2 hours and then the suspended cell liquid drawn up was placed in a 20ml centrifuge tube, centrifuged at 300g for 10 minutes, and 500μl of the separation medium provided in the kit and 100μl of the kit were added to the cell pellet. Prepare the purified antibody, incubate at 4°C for 20 minutes, wash once with the separation medium, add 500 μl of the bead buffer provided in the kit and incubate for 15 minutes, remove the beads with a magnetic field, and wash once with AIM V® Medium CTS (GIBCO, A3021002) medium , using 8 ml of AIM V® Medium CTS (GIBCO, A3021002) medium to culture the obtained CD4+ cells at 37°C and 5% CO ₂ . Add Dynabeads Human T-Activator CD3/CD28 (INVITROGEN, 11131D) according to CD4+:anti-CD3/CD28 Beads=1:1, culture in a carbon dioxide incubator at 37°C and 5% CO ₂ for 3 days, and implement the CD4+ cells bead stimulation;

Mix the mature DC cells separated above with the CD4+ cells stimulated by beads, the volume of each well is 200 μl, 12000 DC cells, 120000 CD4+ cells, add Staphylococcal enterotoxin E superantigen (Toxin technology, ET404), 1ng/ml, add antibody Samples (anti-PD-L1/OX40 bispecific antibody prepared as previously described, ADI-20057, anti-PD-L1 humanized Nb-Fc, ADI-20057+anti-PD-L1 humanized Nb-Fc and IgG2 Control), 100nM is the initial concentration, 3-fold dilution, a total of ten concentration points. After mixed culture for 3 days, the expression level of IL2 in each sample was detected using a cisbio IL2 detection kit (CISBIO, 62HIL02PEG). The expression level of IL2 of different antibodies reflects the activation ability of 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 anti-OX40 antibody (ADI-20057), anti-PD-L1 antibody (anti-PD- L1 humanized Nb-Fc) alone, anti-OX40 antibody and anti-PD-L1 antibody combination (ADI-20057+anti-PD-L1 humanized Nb-Fc) is stronger.

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

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

In this example, tumor-bearing mice were generated by using LoVo (ATCC, CAT# CCL-229TM) cells mixed with PBMC (All Cells, PB005F) cells to inoculate NPG mice, and the anti-OX40/ Antitumor effect of PD-L1 bispecific antibody.

NPG mice:

Female NPG mice (18 g/35 days old) were purchased from Beijing Weitongda Experimental Animal Technology Co., Ltd. The grade is SPF grade, and the quantity is 75. The quality inspection unit is Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and the certificate number is NO.11806300011459. The mice were acclimated to quarantine for 7 days upon arrival prior to the start of the study.

To grow cells and inoculate mice:

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

PBMC: All Cells Lot# 3000417

LoVo cells were routinely subcultured for subsequent in vivo experiments. The PBMC cells were revived 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 in 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 LoVo tumor-bearing humanized mouse model.

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

Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V=L×W ² /2. Body weight was measured using an electronic balance. Mice were euthanized when tumors reached endpoint (tumor volume >3000 mm ³ ) or when mice had >20% body weight loss throughout the study period.

Table 7. Experimental design table

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 inhibition rate on day 28

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

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

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

NCI-H292 cells were routinely subcultured for subsequent in vivo experiments. PBMC cells (All Cells, PB005F) were revived, and the PBMC cell suspension was collected by centrifugation, and the PBMC cells were dispersed 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. 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of NOG mice to establish the NCI-H292 tumor-bearing mouse model.

Administration:

One day after tumor cell inoculation, the mice were randomly divided into groups (5-9 mice in each group). Administer on days 1, 4, 8, and 11, and monitor the tumor volume and body weight of mice twice a week. Monitoring ended after 26 days. The relative tumor inhibition rate (TGI%) was calculated on the 25th day after inoculation, and the calculation formula was as follows: TGI%=100%×(tumor volume of h-IgG control group-tumor volume of treatment group)/(tumor volume of h-IgG control group-h - IgG control group initial tumor volume). The average tumor volume in the h-IgG control group before administration was 78mm ³ . Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V=L×W ² /2. Mice were euthanized when tumors reached endpoint (tumor volume >3000 mm ³ ) or when mice had >20% body weight loss throughout the study period.

Table 9. Experimental design table

The results of tumor inhibition rate are shown in Figure 18 to Figure 20 and Table 10: As shown in Figure 18, on day 25 after inoculation, the low-dose group was compared with the h-IgG control, and the anti-PD-L1 humanized Nb -Fc 0.01mg/kg and ADI-20057 0.02mg/kg single drug inhibition rate were 20% and 37%. Anti-PD-L1 humanized Nb-Fc combined with ADI-20057 0.01+0.02mg/kg had no obvious tumor inhibition; anti-PD- The tumor inhibition rate of 0.023mg/kg of L1/OX40 bispecific antibody was 62%. Anti-PD-L1/OX40 bispecific antibody 0.023mg/kg has better tumor inhibitory effect.

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

As shown in Figure 20 and Table 10, compared with hIgG in the high-dose group, the single-drug inhibition rates of anti-PD-L1 humanized Nb-Fc 1mg/kg and ADI-20057 2mg/kg were 51% and 47%, respectively. The tumor inhibition rate of anti-PD-L1 humanized Nb-Fc combined with ADI-20057 1+2mg/kg was 59%; the tumor inhibition rate of anti-PD-L1/OX40 bispecific antibody 2.3mg/kg was 94%. The anti-PD-L1/OX40 bispecific antibody 2.3mg/kg has the best tumor inhibitory effect, and the tumor inhibitory effect is stronger than single drug and combined drug. Compared with the middle dose and low dose groups, the high-dose group has better antitumor effect, which has a dose-dependent effect.

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

Table 10. Tumor inhibition rate on day 25

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

Table 12 sequence:

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<120> Bispecific antibody binding to PD-L1 and OX40

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<400> 3

<210> 4

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Claims (22)

A bispecific antibody molecule or antigen-binding fragment thereof that binds to OX40 and PD-L1, comprising or consisting of the following peptide chains: (i) a polypeptide chain of formula (I): VH-CH1-Fc-X-VHH; and (ii) a polypeptide chain of formula (II): VL-CL; wherein: VH represents a heavy chain variable region comprising the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3; CH represents a heavy chain constant region; Fc comprises CH2 , CH3, and CH4; CH1, CH2, CH3, and CH4 denote domains 1, 2, 3, and 4 of the heavy chain constant region, respectively; X may be absent or, when present, denote a linker; VHH denotes a single domain antigen-binding site Point, it comprises 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 is made up of said amino acid sequence; VHH CDR2 comprises SEQ ID NO : the amino acid sequence of 11, or consists of said amino acid sequence; VHH CDR3 comprises the amino acid sequence of SEQ ID NO: 12 or consists of said amino acid sequence; VL represents the light chain variable region, which Contains complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3; CL indicates the light chain constant region; there is a hinge region between CH1 and Fc; Wherein, the VH-CH1-Fc in the formula (I) comprises the amino acid sequence of SEQ ID NO: 19 or consists of it; and the VL-CL of the formula (II) comprises the amino acid sequence of SEQ ID NO: 7 or Consists of which; wherein the antigen binding site formed by VH and VL is specific for OX40, and the antigen binding site formed by VHH is specific for PD-L1. The antibody molecule or antigen-binding fragment thereof as described in item 1 of the scope of the patent application, which comprises 1 or 2 polypeptide chains of formula (I) and 1 or 2 polypeptide chains of formula (II), or consists of Polypeptide chain composition. The antibody molecule or antigen-binding fragment thereof as described in claim 1 or 2 of the patent claims, 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 as described in any one of the first or second claims of the patent application, wherein the "CH1-Fc" of formula (I) is in the form of IgG and/or the CL of formula (II) is derived from κ or lambda. The antibody molecule or antigen-binding fragment thereof as described in claim 4 of the patent application, wherein the form of IgG is IgG1, IgG2 or IgG4. The antibody molecule or antigen-binding fragment thereof as described in item 1 or 2 of the patent claims, 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 as described in claim 1 or 2, wherein the VHH in formula (I) comprises or consists of the sequence shown in SEQ ID NO:6. The antibody molecule or antigen-binding fragment thereof as described in item 1 or 2 of the scope of patent application, wherein the polypeptide chain of formula (I) comprises or consists of the sequence shown in SEQ ID NO: 1; and wherein the polypeptide chain of formula (II) The polypeptide chain comprises or consists of the sequence shown in SEQ ID NO:7. An isolated nucleic acid encoding the antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 8 of the claims. An expression vector comprising the nucleic acid described in item 9 of the patent application. The expression vector as described in item 10 of the patent application, wherein the expression vector is a pXC vector or a pTT5 vector. A host cell comprising the nucleic acid described in claim 9 of the patent application or the expression vector described in claim 10 or 11 of the patent application, the host cell is a 293 cell or a CHO cell. An immunoconjugate comprising the antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 8 of the patent application and other substances, wherein the other substances are therapeutic agents or labels. The immunoconjugate as described in claim 13, wherein the therapeutic agent is a cytotoxic agent or an anti-angiogenic agent. A pharmaceutical composition, which comprises the antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 8 of the patent application, or the immunoconjugate as described in claim 13 or 14 of the patent application, and the drug Use accessories. A combination product comprising the antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 8 or the immunoconjugate as described in claim 13 or 14, and one or Various other therapeutic agents. The combination product as described in item 16 of the patent application, wherein the therapeutic agent is an anti-angiogenic agent, a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, a small molecule drug or an immunomodulator. A use of the antibody molecule or antigen-binding fragment thereof as described in any one of the claims 1 to 8 or the immunoconjugate as described in the claims 13 or 14 in the preparation of medicines, wherein The medicament is for preventing or treating a disease in a subject, wherein the disease is an autoimmune disease, an inflammatory disease, an infection, cancer, or a T cell dysfunctional disease. The use as described in claim 18 of the patent application, wherein the cancer is a cancer with increased expression level of PD-1, PD-L1 or PD-L2 and/or decreased expression level or activity of OX40. The use as described in item 18 of the patent application, wherein the cancer is colon cancer or rectal cancer or colorectal cancer or lung cancer. The use as described in any one of claims 18 to 20, wherein the medicament is combined with one or more other therapies including treatment modalities and/or other therapeutic agents. Use as described in item 21 of the scope of patent application, wherein the treatment includes surgical 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.

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