TW202305011A - Specific binding protein targeting pd-1 and ox40 - Google Patents

Abstract

Disclosed are an antibody targeting PD1, or an antigen-binding fragment thereof, and a bispecific antibody targeting PD1 and OX40, or a fragment thereof. Further disclosed are a nucleic acid molecule encoding the antibody or the antigen-binding fragment thereof, the antibody targeting PD1, or the antigen-binding fragment thereof, the bispecific antibody targeting PD1 and OX40, or the fragment thereof, and the use of the encoding nucleic acid molecule in the treatment, prevention and/or diagnosis of a disease. The disease may be, for example, an immune disease, an acute inflammatory disease, a chronic inflammatory disease or a tumor disease.

Description

Specific binding proteins targeting PD-1 and/or OX40

The present invention relates to the field of biomedicine, more specifically to the field of antibody therapy.

The mammalian immune system is a finely balanced system, but it can sometimes be disrupted by diseases such as cancer. Immune checkpoint receptors play an important role in the immune system's response to disease by exerting co-stimulatory or co-inhibitory roles, and the delicate balance of the two determines the efficacy of the immune response. Co-inhibitors inhibit T cell proliferation and induce anti-inflammatory cytokine release. They reduce inflammation and avoid organ/tissue damage from an overactive immune response. On the other hand, costimulators promote the development of a protective immune response by promoting T cell line expansion (clonal expansion), effector differentiation and survival.

A well-established cancer immunotherapy approach could target these checkpoint receptors with antibodies that either block co-inhibitory receptor function or induce co-stimulatory receptor activity, triggering the immune system to recognize and kill tumor cells (Pardoll, 2012) . Antibodies that block the activity of co-inhibitory receptors have shown promising clinical activity and are currently approved for the treatment of cancer (Larkin et al., 2015). Antibodies that induce co-stimulatory receptor activity have shown great potential in preclinical model systems (Moran et al., 2013; Schaer et al., 2014), and several agents are currently in clinical trials (Mayes et al., 2018; Melero et al., 2013 ). These antibodies are designed to mimic the ligands of these co-stimulatory receptors and are therefore also known as agonist antibodies.

OX40 (also known as CD134, ACT45, TNFRSF4) is a member of the tumor necrosis factor receptor (TNFR) superfamily and is mainly expressed on activated T cells, including CD4+ T cells, CD8+ T cells, type 1 and type 2 T helper cells (Th1 and Th2) and regulatory T (Treg) cells, and is expressed on activated natural killer (NK) cells. The interaction of OX40 expressed on antigen-presenting cells (APCs) and its ligand OX40 ligand (OX40L) increases the lineage expansion, differentiation and survival of T cells and enhances the generation of memory T cells (Croft et al., 2009). OX40 stimulation can have direct effects on T cells, promoting their proliferation and survival, or indirect effects by enhancing the production of inflammatory cytokines such as IL2 and IFNγ. OX40 signaling can also regulate Treg cell function, although OX40 signaling on Treg cells can abolish the suppressive activity of Treg cells (Takeda et al., 2004). OX40 has been found to be expressed in tumor-infiltrating T cells from patients with head and neck cancer, melanoma, and colorectal cancer, however, high levels of OX40-positive lymphocytes are associated with better patient survival (Petty et al., 2002; Vetto et al. , 1997). Agonist antibodies to OX40 are currently in clinical trials for cancer, most showing good safety and limited clinical activity. The effect of targeting OX40 as a monotherapy is not ideal, and has not always achieved the expected results.

PD-1 (programmed death-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immune suppression. PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA. Two cell surface glycoprotein ligands of PD-1 have been identified, programmed cell death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), They are expressed on antigen-presenting cells as well as many types of human cancer cells, and they have been shown to downregulate T cell activation and cytokine secretion by binding to PD-1. The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancer cells.

Although the anti-cancer principle of using agonists to stimulate co-stimulatory receptors or checkpoint receptor inhibitors is clear: CTLA4, PD1/PDL1 and other checkpoint receptor inhibitors enhance the anti-cancer effect of T cells, while co-stimulatory receptors The body agonists CD28, 4-1BB, OX40, GITR, CD27 and ICOS promote the anti-tumor immunity of T cells. However, co-administration of co-stimulatory receptor agonists and checkpoint receptor inhibitors has not achieved good clinical results. In theory, stimulation of these co-stimulatory receptors with agonists should enhance antitumor immunity in the highly immunosuppressive tumor microenvironment. A large number of mouse data also confirmed the therapeutic potential of this class of drugs. For example, Genentech combined an OX40 agonist antibody with a PD-L1 antibody, and in a mouse model, 90% of colorectal cancer tumors achieved complete remission. However, while the data from animal studies is promising, the data from clinical trials is not ideal. In Genentech's phase Ib trial of the OX40 agonist MOXR0916 combined with the PD-L1 antibody atezolizumab, only 4% (2 of 51) of patients achieved a partial response, so Genentech shut down its OX40 program. In most cases, the combination of chemotherapy drugs may have a synergistic effect, so that the tumor treatment effect is better, but the combination of immunotherapy is not simply 1+1>2, it may be far from the expected results , or quite the opposite. Data from two sets of mouse models published in 2017 showed that when OX40 agonists and PD-1 inhibitors were administered simultaneously, the effects of the two antibodies counteracted each other due to T cell exhaustion or programmed cell death. However, sequential administration enhanced the antitumor activity of these drugs. One hypothesis is that co-stimulation must first promote tumor-specific T cells to the extent that checkpoint-suppressed immune responses exist before checkpoint inhibitors can be effective.

At present, there is no method that can use antibodies targeting PD1 and OX40 together to achieve effective anti-cancer. Therefore, it is of great clinical significance to discover methods that can enable both functions to function simultaneously, such as preparing bispecific antibodies.

The present invention provides a specific binding protein capable of binding to one or more antigens with high affinity and high specificity, the antigen being PD-1 or a fragment thereof, and/or OX40 or a fragment thereof. The present invention also provides nucleic acid molecules encoding the specific binding protein, expression vectors for producing the specific binding protein, host cells and methods for preparing the specific binding protein. The invention also relates to the use of said specific binding protein in the treatment, prevention and/or diagnosis of diseases, such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

In a first aspect, the present invention provides a specific antibody or an antigen-binding fragment thereof, which binds to PD-1 or a fragment thereof, comprising a light chain variable region VL and a heavy chain variable region VH, wherein , the VL comprises CDR1, CDR2 and CDR3, and its amino acid sequences are respectively shown in SEQ ID NO: 39, 46 and 54, and the VH comprises CDR1, CDR2 and CDR3, and its amino acid sequences are respectively as shown in SEQ ID NO : 8, 18 and 28, or respectively as shown in SEQ ID NO: 81, 86 and 91. In some embodiments, 1 to 3 amino acid substitutions may be included in these CDRs.

In some embodiments, the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54, respectively, and the VH comprises CDR1, CDR1, The amino acid sequences of CDR2 and CDR3 are shown in SEQ ID NO: 8, 18 and 28, respectively. In some embodiments, the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54, respectively, and the VH comprises CDR1, CDR2 and CDR3. The amino acid sequences of CDR3 are shown in SEQ ID NO: 81, 86 and 91, respectively. In some embodiments, these CDRs may contain amino acid mutations, and can maintain the function of the antibody specifically binding to PD-1. Preferably, the amino acid mutation is amino acid substitution, and the number of amino acid substitutions can be 1-3.

In some embodiments, the VL of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, Amino acid sequences that are 95%, 97%, 98%, 99% or 100% identical. In a specific embodiment, the VL of the above-mentioned specific binding protein comprises the amino acid sequence shown in SEQ ID NO: 67.

In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 62, or has at least 80%, 85%, 88%, 90%, 92%, Amino acid sequences that are 95%, 97%, 98%, 99% or 100% identical. In a specific embodiment, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 102, or has at least 80%, 85%, 88%, 90%, 92%, Amino acid sequences that are 95%, 97%, 98%, 99% or 100% identical. In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 102.

In some embodiments, the above-mentioned specific antibody or antigen-binding fragment thereof is selected from IgG, Fab, Fab', F(ab') ₂ , Fv or scFv. In some embodiments, the above-mentioned specific antibody or antigen-binding fragment thereof is IgG. In some embodiments, the specific antibody or antigen-binding fragment thereof described above includes a heavy chain constant region and/or a light chain constant region. In some embodiments, the heavy chain constant region described above may be derived from human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the light chain constant region may be selected from a kappa chain or a lambda chain. In some embodiments, the above-mentioned specific antibodies or antigen-binding fragments thereof may be prepared polyclonal antibodies or monoclonal antibodies.

In some embodiments, an amino acid substitution occurs at position 234 and/or position 235 of the Fc of the specific antibody or antigen-binding fragment thereof. In some embodiments, the above-mentioned specific antibodies or antigen-binding fragments thereof comprise L234A and/or L235A mutations. In some embodiments, the above-mentioned specific binding protein comprises two mutations, L234A and L235A.

In some embodiments, the Fc of the above-mentioned specific antibody is the Fc of human IgG1. In some embodiments, the Fc of the above-mentioned specific antibody is the Fc of human IgG4. In some embodiments, the Fc comprises the L234A and L235A mutations.

In some embodiments, the aforementioned specific antibodies or antigen-binding fragments thereof include heavy chains and light chains. In some embodiments, the light chain comprises or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 77 %, 99% or 100% identical amino acid sequence; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 72 or has at least 80%, 85%, 88%, 90%, 92% thereof %, 95%, 97%, 98%, 99% or 100% identical amino acid sequences. In some embodiments, the light chain comprises or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 77 %, 99% or 100% identical amino acid sequence; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 106 or has at least 80%, 85%, 88%, 90%, 92 %, 95%, 97%, 98%, 99% or 100% identical amino acid sequences.

In a second aspect, the present invention provides a specific binding protein comprising at least two structural domains capable of binding PD-1 or a fragment thereof, and/or OX40 or a fragment thereof.

In some embodiments, the specific binding protein is a bispecific antibody or an antigen-binding antibody thereof comprising a first domain and a second domain, the first domain binds PD-1 or a fragment thereof, and The second domain binds OX40 or a fragment thereof. In some embodiments, the second domain is a VH containing only the heavy chain and no light chain.

In some embodiments, the first domain is a PD-1 antibody or an antigen-binding fragment thereof.

In some embodiments, the second domain is an OX40 antibody or antigen-binding fragment thereof.

In some embodiments, the structure of the first domain and/or the second domain is selected from one of IgG, Fab, Fab', F(ab') ₂ , Fv, scFv, VH, or HCAb, Preferably, the number of Fab, Fab', F(ab') ₂ , Fv, scFv, VH is one or more.

In some embodiments, the first domain is in the form of an IgG. Preferably, the IgG heavy chain constant region is a human heavy chain constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region; preferably, the IgG in the first domain The Fc is the Fc of human IgG1 or IgG4.

In some embodiments, the human IgG preferably comprises one, two or three mutations among L234A, L235A and P329G, more preferably comprises two mutations of L234A and L235A or comprises three mutations of L234A, L235A and P329G;

Preferably, the Fc of IgG in the first domain is the Fc of human IgG1, more preferably, the Fc includes L234A, L235A and P329G mutations;

Preferably, the IgG Fc in the first domain is the Fc of human IgG4, and more preferably, the Fc includes L234A, L235A and P329G mutations.

In some embodiments, the human IgG preferably comprises one, two or three mutations among L234A, L235A and G237A, more preferably comprises two mutations of L234A and L235A or comprises three mutations of L234A, L235A and G237A;

Preferably, the Fc of IgG in the first domain is the Fc of human IgG1, more preferably, the Fc includes L234A, L235A and G237A mutations;

Preferably, the Fc of IgG in the first domain is the Fc of human IgG4, and more preferably, the Fc comprises L234A, L235A and G237A mutations.

Preferably, the first domain is a tetravalent structure.

Preferably, the second domain is a VH structure, and the number of VHs is preferably 1, 2, 3 or 4; more preferably, the number of VHs is 2.

In some embodiments, the first domain and the second domain are linked directly or via a linker peptide L to form a bispecific binding protein.

In some embodiments, the specific binding protein is a bispecific antibody with IgG_HC-VH tetravalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N'-VL ₁ -CL ₁ -C'; polypeptide chain 1, from the amino terminus to the carboxyl terminus, which comprises N'-VH ₁ -CH ₁ -h-CH2-CH3-L-VH ₂ -C'; wherein, the VL ₁ and VH ₁ are the VL and VH of the first domain, the VH ₂ is the VH of the second domain, the h is the hinge region of the IgG antibody, the CL ₁ is the CL of the first domain, the CH ₁ is CH1 of the first structural domain, the L is a connecting peptide, and CH3 of polypeptide chain 1 is connected to VH ₂ via L.

In some embodiments, the specific binding protein is a bispecific antibody of VH-IgG_HC tetravalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N'-VL ₁ -CL ₁ -C'; polypeptide chain 1, from the amino terminus to the carboxyl terminus, which comprises N'-VH ₂ -L-VH ₁ -CH ₁ -h-CH2-CH3-C'; wherein, the VL ₁ and VH ₁ are the VL and VH of the first domain, the VH ₂ is the VH of the second domain, the h is the hinge region, the CL ₁ is the CL of the first domain, and the CH1 is the second CH ₁ of a structural domain, said L is a connecting peptide, and VH ₂ of polypeptide chain 1 is connected to VH ₁ via connecting peptide L.

In some embodiments, the specific binding protein is a bispecific antibody with a tetravalent symmetrical structure of Fab(CL)-VH-Fc, which comprises two polypeptide chains, polypeptide chain 2, from the amino terminal to the carboxyl terminal, which comprises N'-VH'-CH1'-C'; polypeptide chain 1, from amino-terminus to carboxy-terminus, comprising N'-VL'-CL'-L- _VH2 -h- _CH2 - _CH3 -C' . Wherein, said VL' and VH' are VL and VH of the first structural domain respectively, said _VH2 is VH of the second structural domain, said h is a hinge region, said L is a connecting peptide, and said CL ' is the CL of the first structural domain, the CH1' is the CH1 of the first structural domain, and the CL' of the polypeptide chain 1 is directly fused with VH ₂ , that is, the length of L is 0.

In some embodiments, the specific binding protein is a bispecific antibody with IgG_HC-VH-VH hexavalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N'- VL ₁ -CL ₁ -C'; polypeptide chain 1, from amino terminus to carboxyl terminus, comprising N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L ₁ -VH ₂ -L2-VH ₂ -C'. Wherein, said VL ₁ and VH ₁ are respectively VL and VH of the first structural domain, said VH ₂ is VH of the second structural domain, said h is a hinge region, and said L1 and L2 are connecting peptides, so The CL ₁ is the CL of the first domain, the CH ₁ is the CH1 of the first domain, the CH3 of the polypeptide chain 1 is connected to the VH ₂ through L1, and the two VH ₂ are connected through L2.

In some embodiments, the specific binding protein is a bispecific antibody of IgG_HC-VH-VH-VH octavalent symmetrical structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminal to the carboxyl terminal, which contains N '-VL1-CL1-C'; polypeptide chain 1, from amino-terminus to carboxy-terminus, comprising N'-VH1-CH1-h-CH2-CH3-L1-VH2-L2-VH2-L3-VH2-C' . Wherein, said VL1 and VH1 are respectively the VL and VH of the first structural domain, said VH2 is the VH of the second structural domain, said h is a hinge region, said L1, L2 and L3 are connecting peptides, said CL1 is the CL of the first structural domain, the CH1 is the CH1 of the first structural domain, CH3 in the polypeptide chain 1 is connected to the first VH2 from the N-terminal to the C-terminal of the polypeptide chain 1 via the connecting peptide L1, the first The VH2 is connected to the second VH2 through the connecting peptide L2, and the second VH2 is connected to the third VH2 through the connecting peptide L3.

Wherein, the hinge region h is a common hinge region in the immunoglobulin field, usually contains a large amount of proline, is flexible, and forms 2-5 disulfide bonds.

Preferably, said L is a peptide with a length of 0-30 amino acids, and its amino acid sequence is shown in any one of SEQ ID NO: 116-140, see Table 1.

In some embodiments, CH3 of the second polypeptide chain is directly linked to _VH2 , ie the length of L is zero.

Table 1. Linker peptide sequences SEQ ID NO: linker peptide name length sequence SEQ ID NO: 116 H1 15 EPKSSDKTHTPPPPP SEQ ID NO: 117 (G2S)2 6 GGSGGS SEQ ID NO: 118 (G4S)3 15 GGGGSGGGGSGGGGS SEQ ID NO: 119 (G4S)2 10 GGGGSGGGGS SEQ ID NO: 120 GS_2 2 GS SEQ ID NO: 121 GS_4 4 GSGS SEQ ID NO: 122 GS_5 5 GGGGS SEQ ID NO: 123 GS_7 7 GGGGSGS SEQ ID NO: 124 GS_20 20 GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 125 GS_25 25 GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 126 LH1 10 DKTHTCPPCP SEQ ID NO: 127 G5-LH 15 GGGGGDKTHTCPPCP SEQ ID NO: 128 H1_15-RT 17 EPKSSDKTHTPPPPPRT SEQ ID NO: 129 L-GS_15-RT 18 LGGGGSGGGGSGGGGSRT SEQ ID NO: 130 L-H1_15-RT 18 LEPKSSDKTHTPPPPPRT SEQ ID NO: 131 KL-H1_15-RT 19 KLEPKSSDKTHTPPPPPRT SEQ ID NO: 132 KL-H1_15-AS 19 KLEPKSSDKTHTPPPPPAS SEQ ID NO: 133 RT-GS_5-KL 9 RTGGGGSKL SEQ ID NO: 134 RT-GS_15-KL 19 RTGGGGSGGGGSGGGGSKL SEQ ID NO: 135 RT-GS_25-KL 29 RTGGGGSGGGGSGGGGSGGGGSGGGGSKL SEQ ID NO: 136 human IgG1 hinge 15 EPKSCDKTHTCPPCP SEQ ID NO: 137 Human IgG1 Hinge (C220S) 15 EPKSSDKTHTCPPCP SEQ ID NO: 138 human IgG2 hinge 12 ERKCCVECPPCP SEQ ID NO: 139 human IgG4 hinge 12 ESKYGPPCPSCP SEQ ID NO: 140 Human IgG4 hinge (S228P) 12 ESKYGPPCPPCP

In some embodiments, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), and the VL comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID As shown in NO: 39, 46 and 54, the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 8, 18 and 28 or respectively in SEQ ID NO: 81, 86 and 91 shown.

In some embodiments, the first domain comprises VL and VH. In some embodiments, the amino acid sequence of the VL is as shown in SEQ ID NO: 67 or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% consistency. In a specific embodiment, the amino acid sequence of the VL is shown in SEQ ID NO: 67. In some embodiments, the VH has an amino acid sequence such as SEQ ID NO: 62 or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% thereof Or 100% consistency. In a specific embodiment, the amino acid sequence of the VH is as SEQ ID NO: 62. In some embodiments, the amino acid sequence of the VH is as shown in SEQ ID NO: 102, or at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% thereof , 99% or 100% consistency. In a specific embodiment, the amino acid sequence of the VH is shown in SEQ ID NO: 102.

In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid set forth in SEQ ID NO: 77 , a light chain of 99% or 100% identity, and at least 80%, 85%, 88%, 90% of the amino acid shown in SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO: 106 , 92%, 95%, 97%, 98%, 99% or 100% identical heavy chains.

In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid set forth in SEQ ID NO: 77 , a light chain of 99% or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid shown in SEQ ID NO: 71 , 99% or 100% identical heavy chains. In specific embodiments, the first domain comprises a light chain as shown in SEQ ID NO: 77, and a heavy chain as shown in SEQ ID NO: 71.

In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid set forth in SEQ ID NO: 77 , a light chain of 99% or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid shown in SEQ ID NO: 72 , 99% or 100% identical heavy chains. In specific embodiments, said first domain comprises a light chain as set forth in SEQ ID NO: 77 and a heavy chain as set forth in SEQ ID NO: 72.

In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid set forth in SEQ ID NO: 77 , a light chain of 99% or 100% identity, and at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid shown in SEQ ID NO: 106 , 99% or 100% identical heavy chains. In specific embodiments, said first domain comprises a light chain as set forth in SEQ ID NO: 77 and a heavy chain as set forth in SEQ ID NO: 106.

In some embodiments, the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively . In some embodiments, the second domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 85 and 90, respectively .

In some embodiments, the second domain comprises a VH having an amino acid sequence as shown in SEQ ID NO: 63, or at least 80%, 85%, 88%, 90%, 92% thereof , 95%, 97%, 98%, 99% or 100% consistency. In a specific embodiment, the amino acid sequence of the VH of the second domain is shown in SEQ ID NO: 63. In some embodiments, the second domain comprises a VH having an amino acid sequence as shown in SEQ ID NO: 101, or at least 80%, 85%, 88%, 90%, 92% thereof , 95%, 97%, 98%, 99% or 100% consistency. In a specific embodiment, the amino acid sequence of the VH of the second domain is shown in SEQ ID NO: 101.

In some embodiments, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 73 %, 99% or 100% identical heavy chains. In specific embodiments, said second domain comprises a heavy chain as set forth in SEQ ID NO:73. In some embodiments, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% of the amino acid sequence shown in SEQ ID NO: 105 %, 99% or 100% identical heavy chains. In specific embodiments, said second domain comprises a heavy chain as set forth in SEQ ID NO: 105.

In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the Said VL comprises CDR1, CDR2 and CDR3, and its amino acid sequences are respectively shown in SEQ ID NO: 39, 46 and 54, and said VH comprises CDR1, CDR2 and CDR3, and its amino acid sequences are respectively shown in SEQ ID NO: 8 , 18 and 28; and, the second structural domain comprises a heavy chain variable region (VH), and the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29.

In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises VL and VH, the VL comprises CDR1, CDR2 and CDR3, the amino acid sequence of which is As shown in SEQ ID NO: 39, 46 and 54 respectively, the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 81, 86 and 91 respectively; and, the second structure The domain comprises VH comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 19 and 29, respectively.

In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises VL and VH, the VL comprises CDR1, CDR2 and CDR3, the amino acid sequence of which is As shown in SEQ ID NO: 39, 46 and 54 respectively, the VH comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 81, 86 and 91 respectively; and, the second structure The domain comprises a VH comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 9, 85 and 90, respectively.

In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the The amino acid sequences of the VL and VH are shown in SEQ ID NO: 67 and SEQ ID NO: 62 respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO: 63 shown.

In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the The amino acid sequences of the VL and VH are shown in SEQ ID NO: 67 and SEQ ID NO: 102, respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO: 63 shown.

In a specific embodiment, the bispecific antibody comprises a first domain and a second domain, the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the The amino acid sequences of the VL and VH are shown in SEQ ID NO: 67 and SEQ ID NO: 102, respectively; and, the second domain comprises VH, and the amino acid sequence of the VH is shown in SEQ ID NO: 101 shown.

In some embodiments, the bispecific binding protein comprises polypeptide chain 1 and polypeptide chain 2, wherein polypeptide chain 1 is a long chain and polypeptide chain 2 is a short chain.

In some embodiments, the polypeptide chain 1 has SEQ ID NO:79, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:114, SEQ ID The amino acid sequence shown in any one of NO:115, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity; The polypeptide chain 2 has the amino acid sequence shown in any one of SEQ ID NO:77, SEQ ID NO:113, or has at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 79; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 The amino acid sequence shown.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 109; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 The amino acid sequence shown.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 110; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 The amino acid sequence shown.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 111; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 The amino acid sequence shown.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 112; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 The amino acid sequence shown.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 114; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 113 The amino acid sequence shown.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein, polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 115; polypeptide chain 2 comprises the amino acid sequence shown in SEQ ID NO: 77 The amino acid sequence shown.

In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof in the first aspect of the present invention, the specific binding protein or fragment thereof in the second aspect.

In some embodiments, the nucleic acid molecule is an mRNA molecule.

In a fourth aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule described in the third aspect of the present invention.

The expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a retrovirus vector, a lentivirus vector, a phage vector, an adenovirus vector, an adeno-associated vector or a herpes simplex vector.

In some embodiments, the expression vector is present in nanoparticles, liposomes, exosomes, microvesicles, or gene guns.

In a fifth aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the third aspect, or the expression vector of the fourth aspect.

The host cell is a conventional host cell in the art, as long as the expression vector of the fourth aspect can stably express the carried nucleic acid molecule as the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody described in the second aspect. Sexual antibodies. Preferably, the host cells are prokaryotic cells and/or eukaryotic cells, the prokaryotic cells are preferably E. coli cells such as TG1, BL21 (expressing single-chain antibodies or Fab antibodies), and the eukaryotic cells are preferably HEK293 cells or CHO cells (expressing full-length IgG antibodies). The host cell of the present invention can be obtained by transforming the expression vector of the fourth aspect into a host cell. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

In a sixth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect.

In some embodiments, the antibody or antigen-binding fragment thereof of the first aspect or the bispecific antibody of the second aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization technology.

In some embodiments, the method of preparation includes the step of culturing the host cell of the fourth aspect.

In some embodiments, Harbor HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the second domain of the bispecific antibody of the second aspect.

The HCAb transgenic mouse is a transgenic mouse carrying the immune repertoire of human immunoglobulins, capable of producing novel "heavy chain only" antibodies that are half the size of traditional IgG antibodies. It produces antibodies with only human antibody "heavy chain" variable domains and mouse Fc constant domains.

Preferably, the method for preparing the second domain of the bispecific antibody of the second aspect using HCAb transgenic mice comprises the following steps: (a) using human OX40 to immunize HCAb transgenic mice, preferably, the human OX40 antigen is recombinant human OX40-ECD-Fc, specifically, the antigen is a recombinant fusion protein composed of the extracellular region of OX40 linked to Fc; (b) Utilize the cDNA after the RNA reverse transcription of the splenic B cells of the immunized HCAb transgenic mice, and specific primers to amplify the human VH gene; (c) constructing the amplified VH gene into a mammalian cell expression vector encoding the human IgG antibody heavy chain Fc domain sequence; and (d) The fully human OX40 monoclonal antibody obtained in the purification step (c). Preferably, the IgG antibody is an IgG1 antibody or an IgG4 antibody.

In some embodiments, Harbor H2L2 transgenic mice (hereinafter referred to as H2L2 transgenic mice) are used to prepare the specific antibody or antigen-binding fragment thereof of the first aspect or the first domain of the bispecific antibody of the second aspect.

The H2L2 transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune library, and the antibody produced by it has a complete human antibody variable domain and a rat constant domain.

Preferably, the method of using H2L2 transgenic mice to prepare the specific antibody or antigen-binding fragment thereof of the first aspect or the first domain of the bispecific antibody of the second aspect comprises the following steps: (a) Using human PD-1 to immunize H2L2 transgenic mice, more preferably, the human PD-1 antigen is soluble recombinant human PD-1-hFc, specifically, the antigen is composed of PD-1 linked Fc The recombinant fusion protein; (b) The splenocytes of immunized H2L2 transgenic mice were fused with myeloma cell lines to obtain hybridoma cells, and the isolated hybridomas expressed heavy and light chain antibodies with complete human variable domains and rat constant domains molecular; (c) The antibody light chain variable domain sequence (VL) obtained in step (b) is gene-synthesized and cloned into a mammalian cell expression vector encoding the human antibody kappa light chain constant domain sequence to encode the antibody produced the full-length light chain of (d) The antibody heavy chain variable domain sequence (VH) obtained in step (b) is gene-synthesized and colonized into a mammalian cell expression vector encoding the heavy chain constant domain sequence of a human IgG antibody, so as to encode and produce IgG the full-length heavy chain of the antibody; (e) Simultaneously transfect the expression vectors of steps (c) and (d) into mammalian host cells, and use conventional recombinant protein expression and purification techniques to obtain recombinant antibodies with correct pairing and assembly of light and heavy chains.

Preferably, the antibody heavy chain variable domain sequence (VH) obtained in step (b) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human IgG4 antibody heavy chain constant domain sequence to encode the The full-length heavy chain of an IgG4 antibody.

Optionally, the antibody heavy chain variable domain sequence (VH) obtained in step (b) is gene-synthesized and cloned into a mammalian cell expression vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the The full-length heavy chain of an IgG1 antibody.

In some embodiments, the prepared second domain and the prepared first domain are combined into a specific binding protein. The specific binding protein can simultaneously bind two targets, wherein the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, and the second domain can bind OX40 molecules on T cells. After the protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.

In some embodiments, the prepared second domain and the prepared first domain are constructed into a bispecific binding protein. The bispecific binding protein can simultaneously bind two targets, wherein the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, and the second domain can bind OX40 molecules on T cells. After the binding protein binds to the surface of tumor cells, it can recruit and activate T cells in the vicinity of tumor cells, thereby killing tumor cells.

Preferably, the bispecific binding protein has a bivalent structure or a tetravalent symmetrical structure. More preferably, the bispecific binding protein has a tetravalent symmetrical structure.

Preferably, the bispecific binding protein has the structure and sequence described in the second aspect.

In the seventh aspect, the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof in the first aspect or the bispecific antibody in the second aspect, and a pharmaceutically acceptable carrier .

In some embodiments, the pharmaceutical composition further includes other ingredients as active ingredients, such as other small molecule drugs or antibodies or polypeptides as active ingredients.

The pharmaceutically acceptable carrier can be a conventional carrier in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, preferably including pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the specific binding protein and/or other small molecule drugs or antibodies or polypeptides, and 0.01-99.99% of the pharmaceutical carrier, the percentage being The mass percentage of the pharmaceutical composition.

The administration route of the pharmaceutical composition can be parenteral, injection or oral administration. The pharmaceutical composition can be prepared in a form suitable for administration, such as solid, semi-solid or liquid form, which can be aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion form. Administration can be intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation, or intrathoracic instillation. The pharmaceutical composition can also be administered in the form of aerosol or spray, such as nasal administration; or, intrathecal, intramedullary or intraventricular administration, and can also be transdermal, transdermal, topical, enteral, intravaginal , sublingual or rectal administration. The pharmaceutical composition can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dose beneficial to the patient.

In some embodiments, the specific binding protein and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.

In some embodiments, the specific binding protein is a bispecific binding protein.

In the eighth aspect, the present invention provides the antibody or antigen-binding fragment thereof in the first aspect, the bispecific antibody in the second aspect, the isolated nucleic acid molecule in the third aspect, and the pharmaceutical composition in the seventh aspect Application in preparation of medicines for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

The tumor can be breast cancer, renal cell carcinoma, melanoma, colon cancer, and B-cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin's lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignancy, etc.

The inflammatory disease may be atopic dermatitis, ulcerative colitis and the like.

The immune disease may be graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma and the like.

In the ninth aspect, the present invention provides a method for detecting OX40 and PD-1 in a sample, the method comprising using the antibody or antigen-binding fragment described in the first aspect or the bispecific antibody in the second aspect to detect OX40 and PD-1 in the sample OX40 and PD-1 steps.

The sample may be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.

In some embodiments, the method of detecting OX40 and PD-1 in a sample is for non-diagnostic purposes.

In the tenth aspect, the present invention also provides a kit, which includes one or more kits, comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the bismuth as described in the second aspect. Specific antibody or pharmaceutical composition as described in the seventh aspect of the present invention.

In some embodiments, the set of kits comprises a first kit comprising the antibody or antigen-binding fragment thereof of the first aspect, or the first domain and the second structure of the second aspect Domain-composed bispecific antibodies.

In some embodiments, the kit of parts may further comprise a second kit comprising other therapeutic agents including, but not limited to, chemotherapeutics, radiotherapeutics, immunosuppressive agents and cytotoxic drugs.

In some embodiments, the above-mentioned first medicine box and the second medicine box can be used at the same time, or the above-mentioned first medicine box can be used first and then the above-mentioned second medicine box, or the above-mentioned second medicine box can be used first and then the above-mentioned first medicine box can be used. A medicine box can be determined according to the actual needs of specific applications.

In a tenth aspect, the present invention provides methods for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antibody of the first aspect of the present invention or its The antigen-binding fragment or the bispecific antibody of the second aspect or the pharmaceutical composition of the seventh aspect.

The specific binding protein of the present invention has the following technical effects: 1. The PD-1-specific antibody of the present invention can specifically bind to cells expressing human PD-1 and cells expressing cynomolgus monkey PD-1; 2. The first domain of the PD1xOX40 bispecific antibody of the present invention can specifically bind to cells expressing human PD-1 and cells expressing cynomolgus PD-1; the second domain can bind to human OX40 protein and cynomolgus monkey OX40 protein; the second domain showed concentration-dependent enhancement of NF-κb signaling pathway under CHO-K1/CD32b cross-linking conditions; 3. The PD1xOX40 bispecific antibody of the present invention is PD-1 cross-linking dependent; under the assistance of CHO-K1/PD-1 cell cross-linking, it can specifically induce concentration-dependent enhancement of NF-κB signaling pathway. It can specifically induce the promotion of PD-1 cross-linking-dependent OX40-mediated NF-κB signaling pathway, and the signal strength it causes increases in a positive correlation with its concentration; 4. The PD-1xOX40 bispecific antibody of the present invention can inhibit the PD-1 signaling pathway; can enhance the secretion of TNFα and IFNγ; can inhibit the secretion of IL-10 by Treg cells; can promote the cytokine Granzyme B in T cells secretion. 5. The PD-1xOX40 bispecific antibody of the present invention can not only enhance the function and survival of effector T cells, but also inhibit the inhibitory function of Treg; The combination of 1 mAb and OX40 mAb showed significant synergistic activity.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa.

"About" and "approximately" generally mean an acceptable range of error for the measured value given the nature or precision of the measurement. Typically the margin of error is within 20%, typically within 10%, and even more typically within 5% of a given value or range of values.

The term "antigen binding molecule" or "specific binding protein" refers broadly to a molecule that specifically binds an antigenic determinant. Antigen binding molecules include, for example, antibodies, antibody fragments, and backbone antigen binding proteins.

The term "antibody" of the present invention encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single chain molecules and Antibody fragments may be used as long as they exhibit the desired antigen-binding activity.

The term "monoclonal antibody" in the present invention refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, except for possible traces of variant antibodies (such as those containing naturally occurring mutations or produced during the production of monoclonal antibody preparations). , usually present in small amounts), the individual antibodies comprised by the population of antibodies are identical and/or bind the same epitope. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

The term "multispecific antibody" of the present invention is used in its broadest sense to encompass antibodies with polyepitopic specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; Antibodies with VL and VH domains, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable domains, each single variable domain binds to Different targets or different epitope binding of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently, etc. .

The term "bispecific binding protein" or "bispecific antibody" of the present invention refers to the ability to specifically bind at least two different antigenic determinants, for example each composed of an antibody heavy chain variable domain (VH) and antibody light The two binding sites formed by the chain variable domain (VL) bind different antigens or different epitopes on the same antigen. Bispecific antibodies can be in a 1+1 format, a 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (comprising a second antigen or epitope). two binding sites for one antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies comprise two antigen-binding sites, each specific for a different antigenic determinant.

The term "valence" in the present invention means that an antigen-binding molecule has a specified number of binding domains present. Thus, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two binding domains, four binding domains and six binding domains, respectively, in the antigen-binding molecule. The bispecific antibody is at least "bivalent", and may be "trivalent", "tetravalent" or "more valent"). In some instances, the antibodies have two or more binding sites and are bispecific. That is, antibodies can be bispecific even where there are more than two binding sites (ie, the antibody is trivalent or multivalent).

The terms "full-length antibody" and "intact antibody" of the present invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody. "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, antibodies of the native IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) ( Also known as the heavy chain constant region). From N-terminus to C-terminus, each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain domain). chain constant region). The heavy chain of an antibody can be of one of five types, alpha (IgA), delta (IgD), ɛ (IgE), gamma (IgG), or mu (IgM), and can be further divided into subclasses Types such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of an antibody, based on the amino acid sequence of their constant domains, can be of one of two types, kappa and lambda light chains.

Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids . Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

"Antibody fragments" comprise a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') _2, and Fv; diabodies, triabodies, tetrabodies, crossed Fab fragments; linear antibodies; single chain antibody molecules (e.g. scFv); Multispecific antibodies formed from antibody fragments and single domain antibodies.

The term "antigen-binding domain" or "antigen-binding site" in the present invention refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a part of an antibody comprising a region that specifically binds and is complementary to a part or all of an antigen. In cases where the antigen molecule is large, the antigen-binding molecule can bind only a specific part of the antigen, called an epitope. An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, an antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

The term "antigenic determinant" in the present invention is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (for example, a stretch of continuous amino acids or a group consisting of different regions of non-contiguous amino acids) conformational configuration) to which the antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. The antigenic determinant may be present, for example, on the surface of tumor cells, microbially infected cells, other diseased cells, immune cells, free matter in serum and/or in the extracellular matrix (ECM). Unless otherwise stated, the proteins used as antigens in the present invention may be proteins in any native form from any vertebrate source, including, for example, primates (e.g., humans) and rodents (e.g., mice and rats) and other mammals. The antigen can also be a human protein, or the antigen can be "full length", unprocessed protein, any form of protein resulting from intracellular processing, or a naturally occurring protein variant, such as a splice variant or an allelic variant. body.

"Specifically binds" means that binding is selective for an antigen and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques as well as traditional binding assays. In one embodiment In certain embodiments, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, as measured, for example, by SPR. In certain embodiments, dissociation of the molecule bound to the antigen The constant (Kd) is ≤1μM, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM or ≤0.001nM (eg 10 ^-7 M or lower, eg 10 ^-7 M to 10 ^-13 M, eg ^10-9M to ^10-13M ).

"Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Binding affinity can generally be expressed in terms of a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (k _off and k _on , respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

The term "high affinity" in the present invention means that the Kd of the antibody for the target antigen is 10 ^-9 M or lower, even 10 ^-10 M or lower. The term "low affinity" in the present invention means that the Kd of the antibody is 10 ^-8 M or higher.

An "affinity matured" antibody is one that has one or more modifications in one or more hypervariable regions (HVRs) that result in the antibody being more sensitive to Improvement of antigen affinity.

The terms "single domain antibody" and "nanobody" in the present invention have the same meaning, referring to only having the variable region of the heavy chain of an antibody, and constructing a single domain antibody composed of only one heavy chain variable region, which has a complete Functional minimal antigen-binding fragment.

The term "heavy chain antibody" in the present invention is also called HCAb antibody, which refers to an antibody that lacks the light chain of the antibody and only contains the heavy chain, compared to the double-chain antibody (immunoglobulin), specifically contains the variable structure of the heavy chain domain and Fc constant domain.

The term "bispecific antibody comprising a first antigen-binding domain specifically binding to PD1 and a second antigen-binding domain specifically binding to OX40", "bispecific antibody specifically binding to PD1 and OX40", "specific to PD1 and OX40 bispecific antigen-binding molecule" or "anti-PD1/anti-OX40 antibody" or "PD-1xOX40 bispecific antibody" are used interchangeably herein and refer to the ability to bind PD1 and OX40 with sufficient affinity such that the Antibodies can be used as bispecific antibodies targeting PD1 and OX40 as diagnostic and/or therapeutic agents.

The term "T effector cells" in the present invention refers to T cells with cytolytic activity (for example, CD4+ and CD8+ T cells) and T helper (Th) cells, T effector cells secrete cytokines, and activate and guide other immune cells, Regulatory T cells (Treg cells) are not included. The anti-OX40 antibody of the present invention can activate T effector cells, such as CD4+ and CD8+ T effector cells.

The term "regulatory T cell" or "Treg cell" in the present invention refers to a special type of CD4+ T cell that can block the response of other T cells. Treg cells are characterized by the expression of CD4, the alpha subunit of the IL-2 receptor (CD25) and the transcription factor FOXP3, and play a crucial role in the induction and maintenance of peripheral self-tolerance against tumor-expressed antigen.

The term "PD1" of the present invention, also known as programmed cell death protein 1, is a type I membrane protein composed of 288 amino acids and belongs to the immunoglobulin superfamily, which was first disclosed in 1992 (Ishida et al., EMBO J., 11(1992), 3887-3895). PD-1 is a member of the extended CD28/CTLA-4 family of T cell regulators and has at least two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein structure consists of an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif, suggesting that PD-1 negatively regulates TCR signaling in T cell receptors. This is consistent with the binding of SHP-1 phosphatase and SHP-2 phosphatase to the cytoplasmic tail of PD-1 after ligand binding. Although PD-1 is not expressed on naive T cells, it is upregulated after T cell receptor (TCR)-mediated priming and is observed on both primed and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately. Although PD-1 has a relatively broad expression profile, its most important role may be as a co-inhibitory receptor on T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, current therapeutic approaches mainly block the interaction of PD-1 with its ligands to enhance T cell responses. The terms "programmed death 1" "programmed cell death 1" "protein PD-1" "PD-1" "PD1" "PDCD1" "hPD-1" and "hPD-1" are used interchangeably and include human PD -1 variants, isoforms, species homologues, and analogs that share at least one common epitope with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) accession number Q15116.

The term "PD1 antibody" of the present invention is capable of binding to PD1, especially a PD1 polypeptide expressed on the cell surface, with sufficient affinity so that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD1. Typically, PD1 antibodies bind less to irrelevant, non-PD1 proteins than do the antibodies, as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system. About 10% of the binding capacity of PD-1. In certain embodiments, the KD value of the antigen binding protein that binds to human PD1 is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (for example, 10 ⁻⁸ M or lower, eg 10 ^-13 M to 10 ^-8 M, eg 10 ^-13 M to 10 ^-9 M). In some embodiments, in a surface plasmon resonance assay, human PD1 (PD1-ECD) is used to determine the corresponding KD value of the binding affinity to obtain the PD-1 binding affinity. Therapeutic strategies with PD-1/PD-L1 antibodies are a standard treatment strategy in several metastatic tumors and have shown their role in early disease stages and adjuvant therapy, especially in melanoma and non-small cell lung cancer.

The term "OX40" in the present invention is also called CD134, which is expressed in activated CD4+T cells, CD8+T cells, dendritic cells, neutrophils and Treg cells 1-3 days after activation. The ligand OX40L of OX40 is expressed on the surface of antigen-presenting cells (APC) such as dendritic cells and B cells. The interaction of OX40/OX40L can recruit TRAF molecules in the intracellular region of OX40, and can also initiate the canonical NF-κB1 pathway or non-canonical NF-κB2 pathway, PI3k/PKB and NFAT pathways, thereby regulating T cell division and survival The genes that promote the transcription of cytokine genes and the expression of cytokine receptors can also promote the differentiation of B cells into plasma cells and the production of antibodies, etc. In other words, it can enhance the activity of effector T cells, NK, and NK-T cells , Relieving the immunosuppressive effect of Treg can not only enhance the specific immune response, but also enhance the innate immune response, thereby enhancing anti-tumor immunity.

The term "OX40 antibody" of the present invention is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting OX40. Typically, an OX40 antibody binds to an unrelated OX40 protein less than the antibody binds to OX40 as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system. about 10% of its binding capacity. In certain embodiments, the antibody that binds OX40 has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, such as 10 ⁻¹³ M to 10 ^-8 M, eg, 10 ^-13 M to 10 ^-9 M) dissociation constant (KD).

The term "H2L2 transgenic mouse" or "Harbour H2L2 mouse" in the present invention is a transgenic mouse carrying an immune repertoire of human immunoglobulins, which produces a fully human variable region composed of two heavy chains and Traditional tetrameric antibody composed of two light chains (H2L2) with fully human antibody variable domains and rat constant domains. The antibodies produced by the transgenic mice are affinity matured, fully humanized variable regions, and have excellent solubility.

The term "HarbourHCAb mouse" (WO2002/085945A2) in the present invention is a transgenic mouse carrying a human immunoglobulin immune repertoire capable of producing a novel "heavy chain only" antibody half the size of a traditional IgG antibody . It produces antibodies with only human antibody "heavy chain" variable domains and mouse Fc constant domains. Due to the absence of light chains, this "heavy chain only" antibody almost solves the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with traditional antibody platforms.

The term "variable region" or "variable domain" of the present invention refers to the domains of antibody heavy or light chains that participate in the binding of an antigen-binding molecule to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "variable" in the present invention means that certain segments of the variable domains generally differ in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β-sheet configuration, connected by three HVRs, which form loops connecting and in some cases forming part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th edition, National Institute of Health, Bethesda , MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.

The term "hypervariable region" or "HVR" according to the present invention refers to the region in the variable domain region of an antibody that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, native four-chain antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)" which have the highest sequence variability and/or Involved in antigen recognition. Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1) , 53-55(H2) and 96-101(H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 ) occurs at amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89-97 (L3), amino acid residues 31-35 (H1) , amino acid residues 50-65 (H2), and amino acid residues 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health , Bethesda, MD (1991). As a comparison, the corresponding amino acid residues of the CDRs defined in the references cited above are listed in Table 2. In the present invention, the amino acid residues of the CDRs listed above The acid sequences are all shown according to the Chothia definition rules (the claims of the present invention are also sequences shown according to the Chothia definition rules). However, it is well known to those skilled in the art that the antibodies can be defined in various ways in the art CDR, such as the Kabat definition rule based on sequence variability and the Chothia definition rule based on the position of the structural loop region (seeing J Mol Biol 273:927-48, 1997).In the present invention, it is also possible to use the Kabat definition and the Chothia definition rule Combined definition rule determines the amino acid residue in the variable domain sequence.Wherein Combined definition rule is exactly the scope that Kabat definition and Chothia definition are combined, based on this, got a bigger scope. Those skilled in the art should understand Note that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g. a variable region) are to be understood as covering any of the above known schemes as described by the present invention Defined complementarity determining region. Although the scope of protection claimed in the present invention is based on the sequence shown by the Chothia definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.

Table 2. Amino acid numbering system for each CDR region (see http://bioinf.org.uk/abs/) CDR\numbering system Kabat Chothia Combined LCDR1 24-34 26-32 24-34 LCDR2 50-56 50-52 50-56 LCDR3 89-97 91-96 89-97 HCDR1 31-35 26-32 26-35 HCDR2 50-65 52-58 50-65 HCR3 95-102 95-102 95-102

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The "class" of an antibody refers to the type of constant domain or constant region that the heavy chain of the antibody has. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ɛ, γ, and μ, respectively.

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

A "human antibody" has 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 repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

The term "Fc domain" or "Fc region" of the present invention is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The CH3 domain herein may be a native sequence CH3 domain or a variant CH3 domain. The CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fcy receptors (eg, FcyRI, FcyRIIa, FcyRIIb, FcyRIII) and/or complement. It is hypothesized that reducing or eliminating binding to Fc receptor gamma will reduce or eliminate ADCC mediated by antibody molecules. Similarly, reducing or eliminating binding to complement is expected to reduce or eliminate CDC mediated by antibody molecules. Mutations that reduce or eliminate binding of the CH2 domain to one or more Fcγ receptors and/or complement are known in the art (Wang et al., 2018). These mutations include the so-called "LALA mutation", which involves the replacement of the leucine residues at positions 1.3 and 1.2 of the IMGT position of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, by replacing the CH2 structure The asparagine (N) at position 84.4 of the IMGT position in the domain is mutated to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q), thereby converting the conserved N-chain sugar It is also known to reduce IgG1 effector function by mutating sylation sites to produce a-glycosyl antibodies (Wang et al., 2018). Alternatively, it is known that complement initiation (C1q binding) and ADCC can be achieved by incorporating the CH2 domain Mutation of proline at position 114 of the IMGT to alanine or glycine (P114A or P114G) reduces (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to produce Antibody molecules with ADCC or CDC activity.

An "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind a given antigen (eg, PD-1 or OX4). The antigen binding function of an antibody can be performed by fragments of an intact antibody. Examples of the term antigen-binding portion of an antibody or antigen-binding fragment include, but are not limited to, a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; F(ab) ₂ fragment, a Fab fragment comprising two A bivalent fragment of a bivalent fragment, the two Fab fragments are connected by a disulfide bridge at the hinge region; an Fd fragment, which consists of VH and CH1 domains; an Fv fragment, which consists of VL and VH domains of a single arm of an antibody; A single domain antibody (dAb) fragment consisting of a VH domain or a VL domain; and isolated complementarity determining regions (CDRs).

"A region equivalent to the Fc region of an immunoglobulin" includes naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as variants having substitutions, additions or deletions that do not substantially reduce immunoglobulin-mediated effector functions ( Modified variants with capabilities such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see eg, Bowie, J.U. et al., Science 247:1306-10 (1990)).

The term "effector function" of the present invention is attributable to the Fc region of an antibody and is a biological activity that varies with the antibody isotype. Examples of antibody 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 priming, among others.

The term "peptide linker" or "connecting peptide" of the present invention refers to a peptide comprising one or more amino acids, usually about 2 to 20 amino acids. The connecting peptide is a connecting peptide known in the art or described herein.

The terms "fused to", "fusion-linked" or "linked to" of the present invention refer to segments (eg antigen binding domain and FC domain) linked by peptide bonds either directly or via one or more linking peptides.

The invention also relates to amino acid sequence variants of the bispecific antibodies of the invention. Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at a final construct having the desired properties, such as antigen binding activity. Sites for substitution typically include HVRs and frameworks (FRs). See Table 3 for possible substitutions of amino acids.

Table 3. Conservative Amino Acid Substitutions amino acid residue conservative substitution Preferred conservative substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser; Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Nle Leu Leu(L) Nle; Ile; Val; Met; Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Trp(W) Tyr; Tyr Tyr(Y) Trp; Phe; Thr; Phe Val(V) Ile; Leu; Met; Phe; Leu

The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" or "nucleic acid molecule" in the present invention refers to an isolated nucleic acid molecule or construct such as messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA ). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA segments, present in a polynucleotide.

The term "isolated" nucleic acid molecule or polynucleotide according to the present invention refers to a nucleic acid molecule, DNA or RNA, which has been separated from its natural environment. In the present invention, the recombinant polynucleotide encoding the polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. An isolated polynucleotide includes a polynucleotide molecule normally contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, in plus- and minus-strand form, as well as in double-stranded form. Isolated polynucleotides or nucleic acids of the invention further include such molecules produced synthetically. Additionally, a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, ribosomal binding site, or transcription terminator.

The term "expression cassette" according to the present invention refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow the transcription of a particular nucleic acid in a target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.

The term "vector" or "expression vector" and "expression construct" in the present invention can be used interchangeably, and a specific gene operably linked to it is introduced into a target cell and directs the expression of a DNA molecule. The vector includes a vector that is a self-replicating nucleic acid structure as well as a vector that is incorporated into the genome of a host cell into which it has been introduced. The expression vectors of the present invention comprise expression cassettes. Expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants/transformants" and "transformed cells", including the primary transformed cell and progeny derived therefrom. The nucleic acid of the progeny may not be identical to that of the parental cell and may contain mutations. A host cell is any type of cell that can be used to produce a bispecific antigen binding molecule of the invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or hybridoma cells, yeast cells, insect cells and plant cells, also cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.

An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which it is administered. An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to permit or facilitate diagnosis. Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dosage of administration and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

The bispecific antibodies according to the invention have a synergistic effect. "Synergistic effect" means that the combined effect of two drugs is greater than the sum of their individual effects and statistically different from the control and single drugs. The additive effect of the present invention means that the combined effect of the two drugs is the sum of their individual effects and is statistically different from the control and/or single drug.

A "therapeutically effective amount" of a drug (eg, a pharmaceutical composition) refers to that amount, in dosage and administration interval and time, effective to achieve the desired therapeutic or prophylactic effect. For example, a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes or prevents the adverse effects of a disease.

"Individual" or "subject" refers to a mammal. 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 particular, an individual or subject is a human.

"Pharmaceutical composition" means a mixture comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure and other chemical components such as physiologically/pharmaceutically acceptable carriers or excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.

"Pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition other than the active ingredient that is nontoxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, diluents, stabilizers and/or preservatives.

The term "cancer" is meant to describe diseases in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal carcinoma, hepatocellular carcinoma, Gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, Endometrial or cervical cancer, salivary gland cancer, kidney or ureter cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, bile duct cancer, central nervous system (CNS) tumor , spinal axis tumors, brainstem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, myeloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, and Ewing superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute Lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplantation lymphoproliferative disorder (PTLD), as well as phakomatoses, edema (such as that associated with brain tumors), and Abnormal vascular proliferation associated with Meigs syndrome, brain tumors and cancers, as well as head or neck cancer and associated metastases.

"Treatment" refers to administering to a patient an internal or external therapeutic agent, such as a composition comprising any specific binding protein of the present disclosure, a bispecific antibody, an antibody or an antigen-binding fragment thereof or an encoding specific binding protein, bispecific Antibodies, nucleic acid molecules of antibodies or antigen-binding fragments thereof, said patient has one or more diseases or conditions for which said therapeutic agent has a therapeutic effect. Generally, a therapeutic agent is administered in a patient or population to be treated in an amount effective to ameliorate one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.

The term "preventing cancer" means delaying, inhibiting or preventing the onset of cancer in a mammal in which the onset of carcinogenesis or tumorigenesis has not been proven, but has been identified, for example, by genetic screening or other methods have a susceptibility to cancer. The term also includes treating a mammal with a premalignant condition to halt the progression of the premalignant condition to a malignancy or to cause its regression.

The term "liposome" is meant to include a variety of unilamellar and multilamellar lipid carriers formed by the generation of closed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.

The term "administration" refers to providing the specific binding protein, isolated nucleic acid molecule, expression vector, host cell or pharmaceutical composition of the present invention to a subject in need, for example, by oral administration, injection or local administration. who apply.

See Table 4 for the sequence numbers (SEQ ID NO:) of the light chain, heavy chain and variable region of the antibody of the present invention.

Table 4. Sequence numbers (SEQ ID NO:) of the light chain, heavy chain and variable region of the antibody of the present invention Antibody number light chain heavy chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 PR000674 77 71 67 62 39 46 54 8 18 28 PR001985 77 72 67 62 39 46 54 8 18 28 PR006429 77 106 67 102 39 46 54 81 86 91 PR002063 105 101 9 85 90 PR000150 75 69 65 60 37 44 52 6 16 26 PR000324 76 70 66 61 38 45 53 7 17 27 PR002067 73 63 9 19 29 PR003475 78 74 68 64 40 47 55 10 20 30 PR200526 108 107 104 103 95 97 99 82 87 92

See Table 5 for the sequence number (SEQ ID NO:) of the framework region of the antibody of the present invention.

Table 5. Sequence numbering (SEQ ID NO:) of the framework region of the antibody of the present invention Antibody number VH FWR1 VH FWR2 VH FWR3 VH FWR4 VLFWR1 VLFWR2 VLFWR3 VLFWR4 PR000674 3 13 twenty three 33 34 41 50 58 PR001985 3 13 twenty three 33 34 41 50 58 PR000150 1 11 twenty one 31 34 41 48 56 PR000324 2 12 twenty two 32 35 42 49 57 PR002067 4 14 twenty four 33 PR003475 5 15 25 33 36 43 51 59 PR006429 3 13 twenty three 33 34 41 50 58 PR002063 4 83 88 93 PR200526 80 84 89 33 94 96 98 100

The examples shown below are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of this specification or the scope of claims in any way. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, to insert genes encoding proteins into such vectors and plasmids, or to introduce plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E. F． and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press. Example 1 The acquisition of anti-OX40 fully human HCAb antibody

The Harbor HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying an immune repertoire of human immunoglobulins capable of producing novel "heavy chain only" antibodies half the size of traditional IgG antibodies . It produces antibodies with only human antibody "heavy chain" variable domains and mouse Fc constant domains. Due to the fact that it does not contain light chains, the antibody almost solves the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with traditional antibody platforms.

1.1 Immunization of HCAb mice

The above-mentioned Harbor HCAb human antibody transgenic mice aged 6 to 8 weeks were immunized with Harbor HCAb mice for multiple rounds using two immunization schemes. Specifically: immunization scheme 1, immunization with recombinant human OX40-ECD-Fc (ChemPartner, #21127-022) antigen protein. The total injected dose per mouse received by subcutaneous inguinal injection or by intraperitoneal injection was 100 [mu]L per immunization. In the first round of immunization, each mouse was immunized with an immunogen reagent prepared by mixing 50 μg of antigenic protein with complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of booster immunization, each mouse was immunized with 25 μg of antigenic protein mixed with Ribi adjuvant (Sigma Adjuvant System, Sigma, #S6322). Immunization scheme 2, immunization with HEK293/OX40 (ChemPartner, Shanghai) stable cell line overexpressing human OX40. Each mouse was intraperitoneally injected with 2×10 ⁶ cell suspension each time it was immunized. The interval between each booster round is at least two weeks and usually no more than five booster rounds. The immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected. Five days before HCAb mouse splenic B cell isolation, a final booster immunization was performed at a dose of 25 μg OX40-ECD-Fc (ChemPartner, #21127-022) antigenic protein per mouse.

Collect mouse blood, dilute the blood 10 times, take 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), and put it on the ELISA plate coated with human OX40-ECD-Fc ELISA was performed to determine the titer of anti-human OX40 in mouse blood, and two concentrations of mouse blood (1:100, 1:1000) were detected by flow cytometry against CHO-K1/hOX40 cells with high expression of OX40 (Chempartner, Shanghai) and atopic reactivity of CHO-K1 blasts. The blank control group (PB) was the serum of mice before immunization.

1.2 Obtain the anti-OX40 HCAb antibody sequence

When the OX40-specific antibody titer in the serum of the above-mentioned mice reaches a certain level, the splenocytes of the mice are taken out to separate B cells, and CD138-positive cells are sorted with a BD flow cytometry sorter (BD Biosciences, FACS AriaII Cell Sorter) Plasma cells and human OX40 antigen positive B cell population. Extract B cell RNA, reverse transcribe cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, #18091200), and then amplify human VH gene by PCR with specific primers. PCR forward primer 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3' (SEQ ID NO: 141), PCR reverse primer 5'- AATCCCTGGGCACTGAAGAGACGGTGACC-3' (SEQ ID NO: 142). The amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the heavy chain Fc domain sequence of human IgG1 antibody.

The constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain HCAb antibodies. To detect the binding of the HCAb-expressing supernatant to the stable cell line CHO-K1/OX40 (CHO-K1/hu OX40, Genscript, #M00561) overexpressing human OX40, while using a positive antibody (porcizumab) as a positive control , for mirrorball® fluorescent cytometry (SPT Labtech Ltd.) screening. The specific steps are: wash CHO-K1/OX40 cells with serum-free F12K medium (Thermo, #21127022), and resuspend them with serum-free medium to 1×10 ⁶ /mL. Add Draq5 fluorescent probe (Cell Signaling Technology, #4048L) (1 μL Draq5 to 1 mL CHO-K1/OX40 cells, 1:1000 dilution), and incubate for 30 minutes in a dark place. After centrifuging the cells, wash the cells with culture medium and adjust the cell density to 1×10 ⁵ cells/mL. Then add 1:1000 diluted Alexa Fluor® 488, AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., #109-545-098), and add 30 μL of the mixture per well to a 384-well plate (Greiner Bio One, #781091). Add 10 μL positive control or supernatant expressing HCAB to the 384-well plate and incubate for 2 hours. Fluorescence values were read on a mirrorball fluorescent flow instrument. The positive clone antibody was further tested by ELISA with human OX40 protein (Acrobiosystem, #OX0-H5224) and cynomolgus monkey OX40 protein (Novoprotein, #CB17) to verify the cross-binding activity. At the same time, the binding activity to CHO-K1/hu OX40# cells was further detected by FACS. The nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence of the pure antibody are obtained by conventional sequencing means. After removing the repetitive sequences, the remaining sequenced pure antibody plasmids were transfected into HEK293 cells for expression, and the obtained supernatant was tested for NF-kb function again, thus obtaining 64 proteins that simultaneously bind to CHO-K1/hu OX40 and cynomolgus monkey OX40 A functional fully human OX40 monoclonal antibody with a unique sequence. According to the results of human-monkey binding ability and NF-Kb function test, select the antibody with the highest comprehensive ranking for recombinant expression.

1.3 Preparation of anti-OX40 fully human recombinant antibody

The plasmid encoding the HCAb antibody obtained above was transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and purified anti-OX40 recombinant heavy chain antibody was obtained by using conventional recombinant protein expression and purification techniques. Specifically, HEK293 cells were expanded in FreeStyle™ F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, adjust the cell concentration to 6×10 ⁵ cells/mL, and culture in a shaker at 37°C with 8% CO ₂ for 24 hours, with the cell concentration at 1.2×10 ⁶ cells/mL. To prepare 30 mL of cultured cells, dissolve 30 µg of the above-mentioned plasmid encoding HCAb heavy chain in 1.5 mL of Opti-MEM serum-free medium (Thermo, #31985088), and then dissolve 1.5 mL of Opti-MEM into 1 mg/mL PEI (Polysciences , Inc, # 23966-2) 120 µL, let stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop into the plasmid PEI mixed solution while shaking the culture bottle, and incubate for 5 days in a shaker at 37°C with 8% CO ₂ . After 5 days, the cell viability was observed. The culture was collected, centrifuged at 3300G for 10 minutes and the supernatant was taken; then the supernatant was centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect™ (GE Healthcare Life Science, #71-5020-91 AE) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. The target protein was then eluted with 0.1M glycine at pH 3.5, adjusted to neutrality with Tris-HCl at pH 8.0, and finally concentrated and exchanged with ultrafiltration tubes (Millipore, #UFC901024) to PBS buffer to obtain purification Anti-human OX40 HCAb monoclonal antibody solution. Antibody concentration was determined by NanoDrop detection of 280nm absorbance, and the purity of antibody was determined by SEC-HPLC and SDS-PAGE.

The OX40 antibodies PR002067 and PR002063 were obtained, and the corresponding heavy chain amino acid sequences are shown in SEQ ID NO: 73 and SEQ ID NO: 105. At the same time, the present invention produced and prepared the anti-OX40 positive control antibody Pogalizumab (pogalizumab) analog (PR003475), its amino acid sequence is derived from IMGT data, and the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 74, For the amino acid sequence of the light chain of the antibody, refer to SEQ ID NO:78.

1.4 Analysis of protein purity and polymers by HPLC-SEC

The antibody protein samples obtained above were analyzed for purity and aggregated form using analytical size exclusion chromatography (SEC). The analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 µm, 7.8 mm × 30 cm) was connected to a high-pressure liquid chromatography (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II), with PBS buffer at room temperature Equilibrate for at least 1 hour. An appropriate amount of protein sample (at least 10 µg, the sample concentration adjusted to 1 mg/mL) was filtered with a 0.22 µm filter membrane and injected into the system, and the HPLC program was set: the sample was flowed through the chromatogram at a flow rate of 1.0 mL/min with pH 7.4 PBS buffer Column, the maximum time is 20 minutes; the detection wavelength is 280 nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample.

1.5 Analysis of protein purity and hydrophobicity by HPLC-HIC

The purity and hydrophobicity of the antibody protein samples obtained above were analyzed using analytical hydrophobic interaction chromatography (HIC). The analytical chromatographic column TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6 mm × 3.5 cm) was connected to a high pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), with PBS buffer at room temperature Equilibrate for at least 1 hour. The set method consisted of a linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) in 16 minutes at a flow rate of 0.7 mL/min, protein sample concentration 1 mg/mL, injection volume 20 µL, detection wavelength 280 nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample.

1.6 Using DSF to measure the thermal stability of protein molecules

Differential Scanning Fluorimetry (DSF) is a commonly used high-throughput method for measuring protein thermal stability. It uses a real-time fluorescent quantitative PCR instrument to reflect the denaturation process of the protein by monitoring the change of the fluorescence intensity of the dye combined with the unfolded protein molecule, thereby reflecting the thermal stability of the protein molecule. In this example, the DSF method is used to determine the thermal denaturation temperature (Tm) of protein molecules. 10 µg of protein was added to a 96-well PCR plate (Thermo, #AB-0700/W), followed by 2 µL of 100× diluted dye SYPROTM (Invitrogen, #2008138), followed by buffer to a final volume of 40 µL per well. Seal the PCR plate, place it in a real-time fluorescent quantitative PCR instrument (Bio-Rad, model CFX96 PCR System), incubate at 25°C for 5 minutes, and then gradually increase the temperature from 25°C to 95°C with a gradient of 0.2°C/0.2 minutes. The temperature was lowered to 25°C at the end of the test. Use FRET scan mode and use Bio-Rad CFX Maestro software for data analysis and calculate the Tm of the sample.

Table 6 shows the detection results of the physical and chemical properties of the OX40 antibodies PR002067 and PR002063.

Table 6. Physicochemical Properties of OX40 Antibody recombinant antibody Expression system and volume Yield after the first step of purification (mg/L) HPLC-SEC purity (%) Concentration (mg/mL) Endotoxin level (EU/mg) Total (mg) HPLC-HIC retention time (min) DSF TM1 (°C) PR002067 HEK293 (40mL) 87.5 99.29 3.5 <1 3.5 17.469 45.4 PR002063 HEK293 (40ml) 137.5 99.3 5.5 <1 5.5 18.109 51.8

1.7 ELISA detection of binding ability of HCAb monoclonal antibody protein level of OX40

This example is to study the activity of the prepared anti-OX40 HCAb monobody in vitro binding to human and cynomolgus monkey OX40 proteins. Human OX40 protein (Acro biosystem, #OX0-H5224) and cynomolgus monkey OX40 protein (Novoprotein, #CB17) were used to conduct antibody binding experiments at the protein level. Briefly, each well of a 384-well plate (PerkinElmer, #6007509) was coated with 20 μL of 1 ug/mL human OX40 protein and cynomolgus monkey OX40 protein dissolved in PBS, overnight at 4°C. The next day, wash the 384-well plate three times with PBS containing 0.05% Tween (MEDICAGO, #09-9410-100), and block with PBS containing 2% milk (Bio-Rad, #170-6404) at 37°C for 1 hour . The initial concentration of the OX40 antibody to be tested and the positive antibody (Pogalizumab) is 10nM, and a 4-fold serial dilution is made. The blocked 384-well plate was washed three times with PBST, and 10 μL of PBS or 10 μL of 4-fold serially diluted antibody and positive control (Pogalizumab) were added to the plate, and incubated at room temperature for 1 hour. After washing three times, 20 μL of goat anti-human Fc horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc., #109-035-098) was added to each well, and incubated at 37°C for 40 minutes. After washing three times, add 20 μL TMB (Sera Care, #5120-0077) to each well and incubate at room temperature for 5-15 minutes. Add 20 μL of stop solution (BBI life sciences, #E661006-0200) to each well, and use a plate reader (Molecular Devices, model SpectraMax Plus) to read the OD _450-650 value. The value was analyzed and graphed with Graphad 8.0.

The OX40 PR002067 antibody and PR002063 antibody in this example can bind to human OX40 and cynomolgus monkey (cyno) OX40 proteins, and the detected antibody binding ability increases in a positive correlation with the antibody concentration. Compared with the reference antibody Tab (Pogalizumab (Pogalizumab, Roche), the EC50 of PR002067 and PR002063 binding to human OX40 protein and cynomolgus monkey OX40 protein was equivalent to that of Tab (Pogalizumab), indicating that the antibody can be more effective at a lower concentration. Sensitively binds human OX40.

The binding activity of OX40 antibody to human OX40 protein is shown in Table 7.

Table 7. Binding activity of OX40 antibodies to human OX40 protein Antibody EC50 (pM) ODmax _ PR002067 80.6 2.378 PR002063 100.9 2.281 Pogalizumab 82.59 2.134

The binding activity of OX40 antibody to cynomolgus monkey OX40 protein is shown in Table 8.

Table 8. Binding activity of OX40 antibody to cynomolgus monkey OX40 protein Antibody EC50 (pM) ODmax _ PR002067 232 2.163 PR002063 275.8 2.318 Pogalizumab 194.3 2.226

Example 2 Preparation of anti-PD-1 H2L2 antibody

In order to prepare antibody molecules specifically binding to PD-1, experimental animals can usually be immunized with PD-1 antigen, and the experimental animals can be mice, rats, rabbits, sheep, camels, etc. Typically, the resulting antibody molecules are non-human. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability. However, the humanization process of antibodies has its own technical complexity, and molecules after humanization often reduce the affinity for antigens. On the other hand, advances in transgenic technology have made it possible to create genetically engineered mice that carry the human immunoglobulin immune repertoire and have their endogenous murine immune repertoire deleted. The antibody produced by this transgenic mouse has a fully human sequence, so there is no need for further humanization, which greatly improves the efficiency of therapeutic antibody development.

The Harbor H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune repertoire that produces antibodies with fully human antibody variable domains and rat constant domains.

2.1 PD-1 immune H2L2 mice

Harbor H2L2 mice were immunized for multiple rounds with soluble recombinant human PD-1-hFc fusion protein (Shanghai ChemPartner). The antigenic protein is mixed with an immune adjuvant to form an immunogenic reagent, which is then injected subcutaneously through the groin or intraperitoneally. In each round of immunization, each mouse received a total injection dose of 100 μL. In the first round of immunization, each mouse was immunized with 50 μg of antigen protein (human PD-1-hFc) and complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of booster immunization, each mouse was immunized with 25 μg of antigenic protein mixed with Ribi adjuvant (Sigma Adjuvant System, #S6322). The interval between each booster round is at least two weeks and usually no more than five booster rounds. The immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected. Three days before cell fusion, the last booster immunization was performed at a dose of 25 μg antigenic protein per mouse.

Collect mouse blood, dilute the blood 10 times, take 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), in the human PD-1-His (Shanghai ChemPartner ) ELISA plate for ELISA detection to determine the titer of anti-human PD-1 in mouse blood, and the anti-PD-1 titer of two concentrations of mouse blood (1:100, 1:1000) was detected by flow cytometry Specific reactivity of expressed CHO-K1/hPD-1 cells (Shanghai ChemPartner) and CHO-K1 blasts. The blank control group (PB) was the serum of mice before immunization.

2.2 Obtain hybridoma single strain and antibody sequence

When the PD-1-specific antibody titer in the serum of H2L2 mice reaches a certain level, the splenocytes of the mice are taken out and fused with myeloma cell lines to obtain hybridoma cells; the hybridoma cells have undergone multiple rounds of screening and selection. After breeding, at least 8 hybridomas expressing anti-PD-1 monoclonal antibody molecules were isolated. The isolated hybridoma cells and their expressed monoclonal antibodies are indicated by the corresponding colony numbers, for example: 4004_10H9A12, 4004_12H9C1 and so on. The isolated hybridomas express antibody molecules with intact human variable domains and heavy and light chains of rat constant domains. The above monoclonal antibodies were further identified, and several hybridoma clones were selected according to their binding ability to human PD-1, binding ability to cynomolgus monkey PD-1, and ability to inhibit the binding of PD-1 and PD-L1. Perform sequencing. The nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence were obtained by conventional hybridoma sequencing means. In this example, the sequence of the variable domain of the anti-PD-1 monoclonal antibody molecule obtained from the immunized Harbor H2L2 mouse is a human antibody sequence. In the present invention, the CDR sequences are divided by Chothia definition rules.

The heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic high-frequency mutation of the germline gene V, D, and J gene fragments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline gene V and J gene fragments of the light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is high. Use algorithms such as IMGT/DomainGapAlign (http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) or NCBI/IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/) The possible germline gene fragments when gene rearrangement occurs can be deduced from the variable domain sequences of antibodies.

The germline gene analysis of the antibodies obtained above is shown in Table 9, and the sequence numbers of the antibodies are shown in Table 10.

At the same time, the anti-PD-1 positive control antibody Pembrolizumab (Keytruda) analog (PR000150) of the present invention, its corresponding amino acid sequence is derived from the IMGT database, the heavy chain sequence of the antibody is shown in SEQ ID NO: 69, the antibody The light chain sequence is shown in SEQ ID NO:75.

Table 9. Anti-PD-1 H2L2 sequence germline gene analysis clone number recombinant antibody Molecular Structure IgG subtype VH germline V gene VL germline V gene 4004_12H9C1 PR000674 H2L2 Human IgG4 IGHV3-33 IGKV3-11

Table 10. Sequence numbers (SEQ ID NO:) of anti-PD-1 H2L2 antibodies Antibody number light chain heavy chain VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 Pembrolizumab 75 69 65 60 37 44 52 6 16 26 PR000674 77 71 67 62 39 46 54 8 18 28 PR001985 77 72 67 62 39 46 54 8 18 28 PR006429 77 106 67 102 39 46 54 81 86 91

2.3 Preparation of recombinant fully human antibody

After obtaining the light chain and heavy chain variable domain sequences encoding antibody molecules in the above-mentioned Examples 2.1 and 2.2, conventional recombinant DNA technology can be used to combine the light chain and heavy chain variable domain sequences with the corresponding human antibody light Chain and heavy chain constant domain sequences are fused and expressed to obtain recombinant antibody molecules.

In this example, the antibody light chain variable domain sequence (VL) obtained from Harbor H2L2 mice was synthesized by gene and cloned into a mammalian cell expression plasmid vector encoding the human antibody κ light chain constant domain sequence to encode The full length light chain of the antibody is produced. Antibody heavy chain variable domain sequence (VH) was synthesized by gene and cloned into mammalian cell expression plasmid vector encoding human IgG4 antibody heavy chain constant domain sequence (SEQ ID NO: 143) to encode the IgG4 antibody full length heavy chain.

The plasmid encoding the heavy chain of the Harbor H2L2 antibody and the plasmid encoding the light chain of the antibody are simultaneously transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and using conventional recombinant protein expression and purification techniques, the correct paired assembly of the light and heavy chains can be obtained. Purified recombinant antibodies.

Specifically, HEK293 cells were expanded in FreeStyle™ F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, adjust the cell concentration to (6~8)×10 ⁵ cells/mL, culture in a shaker at 37°C 8% CO ₂ for 24 hours, and the cell concentration is 1.2×10 ⁶ cells/mL. Prepare 30 mL of cultured cells. The above-mentioned heavy chain plasmid and light chain plasmid encoding the H2L2 antibody were mixed and dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088) at a ratio of 2:3, and sterilized by filtration with a 0.22 μm filter membrane. Take 1.5 mL of Opti-MEM and dissolve it into 120 µL of 1 mg/mL PEI (Polysciences Inc, #23966-2), and let stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop into the plasmid PEI mixed solution while shaking the culture bottle, and incubate in a 37°C 8% _CO2 shaker for 5 days. After 5 days, the cell viability was observed. The cultures were collected, centrifuged at 3300 g for 10 minutes, and the supernatant was taken; then the supernatant was centrifuged at high speed to remove impurities. A gravity column (Bio-Rad, #7311550) containing MabSelect™ (GE Healthcare Life Science, #71-5020-91 AE) was equilibrated with PBS (pH 7.4) and washed with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. The target protein was then eluted with 0.1M glycine at pH 3.5, then adjusted to neutrality with Tris-HCl at pH 8.0, and finally concentrated and exchanged into PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain purified antibody solution. Then use NanoDrop (Thermo Scientific™ NanoDrop™ One) to measure the concentration, aliquot and store for future use.

2.4 Analysis of protein purity and multimeric form by HPLC-SEC

Protein samples were analyzed for purity and aggregated form using analytical size-exclusion chromatography (SEC). The analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 µm, 7.8 mm x 30 cm) was connected to a high-pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), with a PBS buffer chamber Equilibrate at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 µg, the sample concentration was adjusted to 1 mg/mL) was filtered with a 0.22 µm filter membrane and injected into the system, and the HPLC program was set: the sample was mixed with PBS (pH 7.4) buffer at a flow rate of 1.0 mL/min Flow through the chromatographic column, the maximum time is 25 minutes; the detection wavelength is 280 nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention time of components of different molecular sizes in the sample. The yield and purity analysis results of PD-1 antibody are shown in Table 11.

Table 11. Yield and purity analysis of PD-1 antibody recombinant antibody molecular type Expression system and volume Yield after the first step of purification (mg/L) HPLC-SEC Purity (%) PR000674 H2L2 HEK293-F (30mL) 119.0 99.53

2.5 Changing the Fc sequence of H2L2 antibody

Transform the Fc of PR000674, switch the antibody type from IgG4 to IgG1 and introduce LALA (L234A and L235A mutations, according to EU index numbering) mutations to obtain PR001985, and the variable light and heavy chains of the PR001985 antibody are listed in Table 10 Domain amino acid sequence, light chain full-length amino acid sequence, heavy chain (human IgG1) full-length amino acid sequence, and amino acid sequences of CDRs defined according to Chothia definition rules.

2.6 Sequence optimization of antibodies

In this example, the potential PTM site of the PR001985 antibody was mutated to obtain a new antibody molecule PR006429 (called a PTM removal variant). Table 10 lists the amino acid sequences of the light and heavy chain variable domains of the PR006429 antibody, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG1), and the CDRs defined according to the Chothia definition rules amino acid sequence. All designed PTM-removal variants PR006429 were purified recombinant antibodies according to the method described in Example 2.3, and further verified in subsequent functional experiments.

2.7 Binding of antigen-binding proteins to cells overexpressing human/cynomolgus PD-1 (FACS)

In order to study the activity of PD-1 antigen binding protein binding to human/cynomolgus monkey PD-1 in vitro. Cell-level binding experiments were performed using CHO-K1 cell lines (CHO-K1-hPD-1, CHO-K1-cPD-1, derived from GenScript) overexpressing human or cynomolgus monkey PD-1. Briefly, digest CHO-K1-hPD-1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x ¹⁰⁶ cells/mL. Inoculate 100 µL cells/well in 96-well V-bottom plate (Corning, Cat#3894), then add 100 µL/well, 5-fold serial dilution of the antigen-binding protein to be tested, which is 2 times the final concentration, mix well, and the antigen The highest final concentration of binding protein was 100 nM or 300 nM, a total of 8 to 11 concentrations, and hIgG was used as a control. Cells were incubated at 4°C for 1 hour in the dark. Then, add 100 µL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Add 100 µL/well fluorescent secondary antibody (goat anti-human IgG (H+L) secondary antibody (Alexa Fluor® 488 conjugate, Invitrogen, Cat #A11013, 1:1000)) and incubate at 4°C for 30 minutes in the dark. Wash the cells twice with 200 µL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 µL/well pre-cooled PBS, and use BD FACS CANTOII to read the fluorescence signal value.

The results show that the PD-1 antigen binding protein PR000674 of the present invention can bind to CHO-K1 cells overexpressing human PD-1 and CHO-K1 cells overexpressing cynomolgus monkey PD-1.

The binding activity of PD-1 antigen binding protein to CHO-K1 cells overexpressing human PD-1 is shown in Table 12.

Table 12. Binding activity of PD-1 antigen binding proteins to CHO-K1 cells overexpressing human PD-1 Antibody EC50 (nM) MFImaximum PR000674 1.939 2347018 Pembrolizumab 1.634 2382166

The binding activity of PD-1 antigen binding protein to CHO-K1 cells overexpressing cynomolgus monkey PD-1 is shown in Table 13.

Table 13. Binding activity of PD-1 antigen binding proteins to CHO-K1 cells overexpressing cynomolgus monkey PD-1 Antibody EC50 (nM) MFImaximum PR000674 2.349 41162 Pembrolizumab 0.998 19091

2.8 Antigen-binding proteins block the binding of human PD-L1 to CHO-K1 cells overexpressing human PD-1

In order to study the activity of human PD-1 binding protein in blocking the binding of human PD-1 and human PD-L1 in vitro, the CHO-K1 cell line (CHO-K1-hPD-1) overexpressing human PD-1 was used to carry out the cell level detection. Human PD-1/human PD-L1 binding blocking experiment. Briefly, digest CHO-K1-hPD-1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x ¹⁰⁶ cells/mL. Inoculate 100 μL cells/well in 96-well V-bottom plate (Corning, Cat#3894), then add 100 μL/well, 2 times the final concentration of 3 times or 5 times the concentration of the antigen-binding protein to be tested, and mix evenly. The highest final concentration of antigen-binding protein was 100nM or 300nM, a total of 8 concentrations, and hIgG was used as a control. Cells were incubated at 4°C for 1 hour in the dark. Afterwards, centrifuge at 4°C for 5 minutes, discard the supernatant, then add 50 μL/well of biotin-labeled human PD-L1 protein (AcroBiosystems, PD1-H82F2) at a concentration of 1 μg/mL, and incubate at 4°C for 30 minutes in the dark. Add 100 μL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant. Add 100 μL/well fluorescent secondary antibody (PE Streptavidin, BD, Cat#554061, 1:200), and incubate at 4°C for 30 minutes in the dark. Wash the cells twice with 200 μL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well pre-cooled PBS, use BD FACS CANTOII to read the fluorescent signal value, and calculate IC50.

The results are shown in Figure 6 and Table 14. Figure 6 and Table 14 show that both antibodies PR001985 and PR006429 can block the binding of human PD-L1 to human PD-1 on the cell surface, and the inhibitory ability is equivalent to that of the positive control Keytruda (pembrolizumab).

Table 14 PD-1 antigen binding proteins block the binding of PD-1 and PD-L1 Abs Keytruda PR001985 PR006429 Maximum RUL 4831 4785 4944 EC50 2.267 2.04 2.216

Example 3 Preparation of anti-PD-1 H2L2 × OX40 bispecific antibody

Combining the anti-OX40 antibody prepared in Example 1 and the anti-PD-1 antibody prepared in Example 2 is used to prepare a bispecific antibody, which can bind to two targets at the same time, and one end (the first domain) can recognize the surface of tumor cells Specifically expressed PD-1, while the other end (second domain) can bind to OX40 molecules on T cells. When the PD-1×OX40 double antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.

The PD-1×OX40 bispecific antibody prepared in this example is IgG1, PR003787 has Fc L234A, L235A and P329G mutations (according to EU index numbering), R200538, PR200539, PR200531, PR200536, PR200528 and PR200600 have Fc L234A, L235A and G237A), the molecular structure is shown in Figure 1-5.

3.1 Construction of a bispecific antibody with IgG_HC-VH tetravalent symmetrical structure (structure 1)

Bispecific antibodies PR003787, PR200531 and PR200528 with IgG_HC-VH tetravalent symmetrical structure were constructed by using anti-PD-1 H2L2 antibody and anti-OX40 HCAb antibody. The binding protein of IgG_HC-VH tetravalent symmetrical structure (see Figure 1) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL ₁ -CL ₁ -C'; polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L-VH ₂ -C'. Wherein, said VL ₁ and VH ₁ are respectively the VL and VH of the first structural domain, said VH ₂ is the VH of the second structural domain, said h is the hinge region of an IgG antibody, and said CL ₁ is the first structural domain The CL of the domain, the CH ₁ is the CH1 of the first domain, the L is the connecting peptide, and the CH3 of the polypeptide chain 1 is connected to the VH ₂ via L. The connecting peptide L in the bispecific antibody PR003787 is H1, and its amino acid sequence is shown in SEQ ID NO: 116 in Table 15. The connecting peptide L in the bispecific antibodies PR200531 and PR200528 is (G2S) ₂ , which The sequence is shown as SEQ ID NO: 117 in Table 15.

3.2 Construction of bispecific antibody with VH-IgG_HC tetravalent symmetrical structure (structure 2)

The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200538 with VH-IgG_HC tetravalent symmetrical structure. The binding protein of the VH-IgG_HC tetravalent symmetrical structure (see Figure 2) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL ₁ -CL ₁ - C'; Polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which comprises N'-VH ₂ -L-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -C'. . Wherein, said VL ₁ and VH ₁ are respectively VL and VH of the first structural domain, said VH ₂ is the VH of the second structural domain, said h is the hinge region, and said CL ₁ is the CL of the first structural domain , the CH ₁ is the CH1 of the first structural domain, and the VH ₂ of the polypeptide chain 1 is connected to VH1 via the linker peptide L, and the L is the linker peptide (G4S) ₃ , its sequence is as shown in SEQ ID NO: 118 in Table 15 shown

3.3 Construction of bispecific antibody with Fab(CL)-VH-Fc tetravalent symmetrical structure (structure 3)

The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200539 with a tetravalent symmetrical structure of Fab(CL)-VH-Fc. The binding protein of Fab(CL)-VH-Fc tetravalent symmetrical structure (see Figure 3) contains two polypeptide chains: Polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'- VH'-CH1'-C'; Polypeptide chain 1, also known as long chain, from the amino terminus to the carboxyl terminus, which contains N'-VL'-CL'-L-VH ₂ -h-CH2-CH3-C' . Wherein, said VL' and VH' are VL and VH of the first structural domain respectively, said _VH2 is VH of the second structural domain, said h is a hinge region, said L is a connecting peptide, and said CL ' is the CL of the first structural domain, the CH1' is the CH1 of the first structural domain, and the CL' of the polypeptide chain 1 in PR200539 is directly fused with VH2, that is, the length of L is 0.

3.4 Construction of bispecific antibody with IgG_HC-VH-VH hexavalent symmetrical structure (structure 4)

The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002607 were used to construct the bispecific antibody PR200536 with IgG_HC-VH-VH hexavalent symmetrical structure. The binding protein of the IgG_HC-VH-VH hexavalent symmetrical structure (see Figure 4) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL ₁ - CL ₁ -C'; Polypeptide chain 1, also called long chain, from amino terminus to carboxyl terminus, which contains N'-VH1-CH ₁ -h-CH2-CH3-L1-VH ₂ -L2-VH ₂ -C '. Wherein, said VL ₁ and VH ₁ are respectively VL and VH of the first structural domain, said VH ₂ is VH of the second structural domain, said h is a hinge region, and said L1 and L2 are connecting peptides, so The CL1 is the CL of the first domain, and the _CH1 is the CH1 of the first domain. CH3 of polypeptide chain 1 is connected to VH ₂ through L1, and the two VH ₂ are connected through L2. The sequences of L1 and L2 are shown in SEQ ID NO: 117 and 118 in Table 15, respectively.

3.5 Construction of bispecific antibody with IgG_HC-VH-VH-VH octavalent symmetrical structure (structure 5)

The anti-PD-1 H2L2 antibody PR006429 and the anti-OX40 HCAb antibody PR002603 were used to construct the bispecific antibody PR200600 with an octavalent symmetrical structure of IgG_HC-VH-VH-VH. The binding protein of the IgG_HC-VH-VH-VH octavalent symmetrical structure (see Figure 5) contains two polypeptide chains: polypeptide chain 2, also known as the short chain, from the amino terminal to the carboxyl terminal, which contains N'-VL ₁ -CL ₁ -C'; Polypeptide chain 1, also called long chain, from the amino terminus to the carboxyl terminus, which contains N'-VH1-CH ₁ -h-CH ₂ -CH3-L1-VH ₂ -L ₂ - _VH2 -L3- _VH2 -C'. Wherein, said VL ₁ and VH ₁ are VL and VH of the first structural domain respectively, said VH ₂ is VH of the second structural domain, said h is a hinge region, and said L1, _L2 and _L3 are A connecting peptide, the CL ₁ is the CL of the first domain, the CH ₁ is the CH ₁ of the first domain. CH3 in polypeptide chain 1 is connected to the first VH ₂ via connecting peptide L1 (ordered from the N-terminal to the C-terminal of polypeptide chain-1), the first VH 2 is connected to the second VH ₂ through connecting peptide L2, and the first VH ₂ is connected to the second VH 2 through the connecting peptide L2. The two VH ₂ and the third VH ₂ are linked by linking peptide L3, wherein L1 is (G2S) 2 , L2 is (G4S) 3 , and L3 is (G4S) ₂ , the sequences of L1, L2 and L3 are shown in Table 15 Shown in SEQ117, 118, 119.

Table 15. List of connecting peptide sequences linker peptide name linker peptide length connecting peptide sequence Sequence number SEQ ID NO: H1 15 EPKSSDKTHTPPPPP 116 (G2S) ₂ 6 GGSGGS 117 (G4S) ₃ 15 GGGGSGGGGSGGGGS 118 (G4S) ₂ 10 GGGGSGGGGS 119

The amino acid sequence of the polypeptide chain of the double antibody molecule obtained in the present invention is shown in Table 16 in the numbering in the sequence listing.

Table 16. Sequence number (SEQ ID NO:) of the double antibody molecule obtained in the present invention Antibody number polypeptide chain 1 polypeptide chain 2 PR003787 79 77 PR200528 109 77 PR200531 110 77 PR200536 111 77 PR200538 112 77 PR200539 114 113 PR200600 115 77

The CDR sequence numbers of the antigenic domains of the PD-1×OX40 bispecific antibody are shown in Table 17. In Table 17, the 1# sequence number is the antigenic domain that binds to PD-1, and the 2# sequence number is the antigenic domain that binds to OX40.

Table 17. CDR sequence numbers (SEQ ID NO:) of the antigen-binding domain of the bis-antibody structure number Antibody number Antigen binding domain number LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 1 PR003787 #1 39 46 54 8 18 28 #2 — — — 9 19 29 2 PR200531 #1 39 46 54 81 86 91 #2 — — — 9 19 29 3 PR200528 #1 39 46 54 81 86 91 #2 — — — 9 85 90 4 PR200538 #1 39 46 54 81 86 91 #2 — — — 9 19 29 5 PR200539 #1 39 46 54 81 86 91 #2 — — — 9 19 29 6 PR200536 #1 39 46 54 81 86 91 #2 — — — 9 19 29 7 PR200600 #1 39 46 54 81 86 91 #2 — — — 9 85 90

The molecular structure information of the bispecific antibody of the present invention is shown in Table 18.

Table 18. PD-1×OX40 double antibody molecule with IgG-VH tetravalent symmetrical structure Double Antibody Molecule PD-1 antibody (IgG) OX40 Antibody (VH ₂ ) VH ₂ position relative to IgG Linker peptide ( between VH ₂ and IgG ) Fc type (mutation) Linker peptide length ( between VH2 and IgG ) PR003787 PR001985 PR002067 heavy chain C-terminal EPKSSDKTHTPPPPP ( SEQ ID NO: 116 ) Human IgG1 (L234A, L235A, P329G) 15 PR200531 PR006429 PR002067 heavy chain C-terminal GGSGGS (SEQ ID NO: 117) Human IgG1 (L234A, L235A, G237A) 6 PR200528 PR006429 PR002063 heavy chain C-terminal GGSGGS9 (SEQ ID NO: 117) Human IgG1 (L234A, L235A, G237A) 6 PR200538 PR006429 PR002067 heavy chain N-terminal GGGGSGGGGSGGGGS (SEQ ID NO: 118) Human IgG1 (L234A, L235A, G237A) 15 PR200539 PR006429 PR002067 Between the first heavy chain domain and IgG — Human IgG1 (L234A, L235A, G237A) 0 PR200536 PR006429 PR002067 heavy chain C-terminal GGSGGS (SEQ ID NO: 117) Human IgG1 (L234A, L235A, G237A) 6 PR200600 PR006429 PR002063 heavy chain C-terminal GGSGGS (SEQ ID NO: 117) Human IgG1 (L234A, L235A, G237A) 6

Example 4

FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on CHO-K1 cell line overexpressing human PD-1

This example is to study the in vitro binding activity of the PD-1 antibody arm of the PD-1×OX40 bispecific antibody to human PD-1. The CHO-K1 cell line (CHO-K1-hu PD-1, Shanghai ChemPartner) overexpressing human PD-1 was used for antibody binding experiments at the cellular level. Briefly, CHO-K1-hu PD-1 cells were digested and resuspended with DMEM complete medium, and the cell density was adjusted to 1× ¹⁰⁶ cells/mL, respectively. Seed 100 µL cells/well in 96-well V-bottom plate (Corning, Cat#: 3894), then add 100 µL/well, 2 times the final concentration of 5-fold serial dilution of the antibody to be tested. Cells were incubated at 4°C for 1 hour in the dark. Afterwards, add 100 µL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Then add 100 µL/well fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, Cat#:109-545-06, 1:500 dilution), 4°C, dark incubation for 30 minute. Wash the cells twice with 100 µL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 µL/well of pre-cooled PBS, and use the BD Accuri C6 Plus flow cytometer to read the fluorescence signal value.

Figure 7 shows the in vitro binding of the PD-1×OX40 bispecific antibody on the CHO-K1 cell line overexpressing human PD-1.

As shown in Figures 7A and 7B, the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 or their monoclonal antibodies PR001985 and PR006429 in this example are all specific to CHO-K1 cells overexpressing human PD-1 sexual union. And the binding ability of PR003787 to human PD-1 is consistent with the binding ability of PD-1 antibody to human PD-1. The PD-1 antibody arm of the PD-1×OX40 bispecific antibody PR003787 in this implementation has the same binding ability as the PD-1 monoclonal antibody.

Example 5

FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on HEK293 cell line overexpressing OX40

In this example, in order to study the activity of the OX40 arm of the PD-1×OX40 bispecific antibody binding to human OX40, the HEK293 cell line (OX40 / NF-κB Reporter – HEK293, BPS BioScience, Cat# 60482) with high expression of OX40 was used for cell and Human OX40 binding assay. In brief, HEK293 cell suspensions with high expression of OX40 were collected, and the cell density was adjusted to 2×10 ⁶ cells/mL. 50 µL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 µL/well, 2 times the final concentration of 5-fold dilutions of the antibody to be tested was added. Cells were incubated at 4°C for 2 hours in the dark. Afterwards, add 100 µL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant. Then add 100 µL/well fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, Cat#:109-605-098, diluted 1:1000), incubate at 4°C in the dark 1 hour. Wash the cells twice with 100 µL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, pre-cool FACS to resuspend the cells at 200 µL/well, and use the BD Accuri C6 Plus Flowcytometer to read the fluorescence signal value.

Figures 8A and 8B show the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or monoclonal antibodies constituting their structures in human HEK293/OX40/NF-kb reporter cells determined by flow cytometry PR002067 specifically binds to HEK293 cells overexpressing human OX40, and binds to OX40 on the cell surface as the concentration increases. Moreover, the OX40 antibody arms of the double-antibody PR003787 and PR200538 have the same or better binding ability as the OX40 monoclonal antibody.

Example 6

FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on HEK293 cell line overexpressing OX40 and PD-1

This example is to study the activity of PD-1×OX40 bispecific antibody binding to OX40 and PD-1 co-expression cells. HEK293 cell line with high expression of OX40 (OX40 / NF-κB Reporter – HEK293, BPS BioScience, Cat# 60482) was used to construct a cell line stably expressing human PD1 (Origene, Cat# PS100092V) with lentivirus, which was screened for resistance to puromycin Afterwards, HEK293 cell lines stably expressing OX40 and PD-1 were obtained, and the obtained cell lines were used for cell binding experiments. In brief, HEK293 cell suspensions highly expressing OX40 and PD-1 were collected, and the cell density was adjusted to 2×10 ⁶ cells/mL, respectively. 50 µL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 µL/well, 2 times the final concentration of 5-fold dilutions of the antibody to be tested was added. Cells were incubated at 4°C for 2 hours in the dark. Afterwards, add 100 µL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant. Then add 100 µL/well fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, Cat#:109-605-098, diluted 1:1000), incubate at 4°C in the dark 1 hour. Wash the cells twice with 100 µL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, pre-cool FACS to resuspend the cells at 200 µL/well, and use the BD Accuri C6 Plus Flowcytometer to read the fluorescence signal value.

Figure 9 shows that in human HEK293/OX40/PD1 NF-kb reporter cells, the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies constituting their structures were all detected by flow cytometry. It specifically binds to HEK293 cells overexpressing human OX40 and PD-1, and binds to OX40 and PD-1 on the cell surface as the concentration increases. Compared with the OX40 monoclonal antibody, the double-antibody PR003787, PR200538 and PR200539 showed stronger binding ability; compared with the PD1 monoclonal antibody, the double-antibody PR003787, PR200538 and PR200539 showed similar or slightly weaker binding ability.

Example 7

FACS detection of in vitro binding of PD-1×OX40 bispecific antibody on primed T cells

Since the overexpression cells cannot truly reflect the expression of the target under physiological conditions, this example studies the activity of T cells initiated by the binding of the PD-1×OX40 bispecific antibody. Human leukocyte concentrate (Research blood components LLC) was purchased, and human peripheral blood mononuclear cells (PBMC) were isolated, and T cells were stimulated with anti-CD3 antibody coated on a cell culture plate for 48 hours. The stimulated PBMCs were collected, and the cell density was adjusted to 4×10 ⁶ cells/mL. 50 µL cells/well were inoculated in a 96-well V-bottom plate (Corning, Cat#: 3894), and then 50 µL/well, 2 times the final concentration of 5-fold dilutions of the antibody to be tested was added. Cells were incubated at 4°C for 2 hours in the dark. Afterwards, add 100 µL/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500g, 4°C for 5 minutes, and discard the supernatant. Add 100 µL/well fluorescent secondary antibody (Goat anti-Human IgG Fc Secondary Antibody, PE, eBioscience™, Cat#: 12-4998-82, 1:250 dilution) and APC-labeled anti-human CD3 antibody (BioLegend, Cat #317318), incubate at 4°C for 1 hour in the dark. Wash the cells twice with 100 µL/well of pre-cooled PBS, centrifuge at 500 g, 4°C for 5 minutes, and discard the supernatant. Finally, pre-cool FACS to resuspend the cells at 200 µL/well, and use the BD BD LSRFortessa Flowcytometer to read the fluorescent signal value.

Figure 10 shows that in the activated human T cells determined by flow cytometry, the PD-1×OX40 bispecific antibodies PR003787, PR200538, PR200539 of the present invention or the monoclonal antibodies that constitute their structures all bind to the activated T cells , and the binding to T cells increases with the concentration. Compared with the OX40 monoclonal antibody and the PD1 monoclonal antibody, the double antibodies PR003787, PR200538 and PR200539 showed stronger binding abilities. It shows that the double antibody of the present invention can specifically bind PD1 and OX40 positive T cells.

Example 8 Using a reporter gene cell line to detect the stimulating effect of PD-1×OX40 bispecific antibody on OX40 signaling pathway

Spread 100 μL of culture medium per well, or 1.5×10 ⁴ CHO-K1 cells expressing CD32b (CHO-K1/CD32b) (BPS BioScience, #79511) or CHO cells expressing human PD1 (CHO-K1/PD-1) To a 96-well plate (Perkin Elmer, #6005225), add 40 μL of 2-fold dilution of the antigen-binding protein to be tested to each well, the initial concentration is 200 nM, and 4-fold serial dilution, hlgG1 is the negative control group. Add 40 μL 4.5×10 ⁴ HEK293 reporter cells (HEK293/OX40/NF-kb reporter cells, BPS Biosciences, #60482) that can continuously express the luciferase reporter genes of OX40 and NF-kb response elements to each well. Incubate for 6 hours at 37°C in a 5% CO ₂ incubator. Then add ONE-Glo ^TM luciferase reagent (Promega, #E6110), incubate at room temperature for 15 minutes, and detect the luminescence value with a microplate reader.

The results are shown in Figures 11 to 13. The results in Figure 11 to Figure 13 show that the PD-1×OX40 bispecific antibody PR003787 of the present invention is PD-1 cross-linking dependent. The results in Figure 11 show that the PD-1×OX40 bispecific antibody PR003787 has no direct activation effect on HEK293/OX40/NF-kb reporter cells. The results in Figure 12 show that the OX40 parental monoclonal antibody showed a concentration-dependent effect of enhancing the NF-Kb signaling pathway under CHO-K1/CD32b cross-linking conditions, while the PD-1×OX40 bispecific antibody PR003787 did not show NF-Kb signaling pathway The -Kb signal activates the action. The results in Figure 13 show that the PD-1×OX40 bispecific antibody PR003787, with the help of CHO-K1/PD-1 cell cross-linking, specifically caused a concentration-dependent enhancement of the NF-Kb signaling pathway.

The PD-1×OX40 bispecific antibody PR003787 of the present invention can specifically induce PD-1 cross-linking-dependent OX40-mediated NF-Kb signaling pathway promotion, and the signal intensity it causes increases in a positive correlation with its concentration .

Example 9 Using a reporter gene cell line to detect the inhibitory effect of PD-1×OX40 bispecific antibody (PR003787) on PD-1 signaling pathway

Spread HEK293 cells overexpressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) on a 96-well plate with a cell volume of 1.25×10 ⁴ /well, 100 μL/well. Add 50 μL/well of the antigen-binding protein dilution to be tested, the initial concentration is 100 nM, and the concentration is diluted 4 times, and hlgG1 is used as the control group. Add 5×10 ⁴ /well Jurkat reporter cells (built by Shanghai ChemPartner) that can sustainably express PD-1 and NFAT-luciferase reporter genes, 50 μL/well. Incubate for 6 hours at 37°C in a 5% CO ₂ environment. Add ONE-Glo ^TM luciferase reagent (Promega, #E6110), incubate at room temperature for 15 minutes, and detect the luminescence value with a microplate reader.

The result is shown in Figure 14. It shows that the PD-1×OX40 bispecific antibody (PR003787) of the present invention and the parental antibody of PD-1 both have an inhibitory effect on the PD-1 signaling pathway. Example 10 Detection of the activation function of PD-1×OX40 bispecific antibody on T cells

Purchase human concentrated leukocytes (Research blood components LLC), separate human peripheral blood mononuclear cells (PBMC), then co-culture human PBMC cells and mitomycin-treated CHOK1-huPD-L1 cells, add 2.5ug/mL PHA-L (eBioscience, #00-4977-03) stimulation for 3 days. The supernatant was collected, and the secretion of tumor necrosis factors TNFα and IFNγ was detected by MSD (Meso Scale Discovery, U-Plex K15067M-2).

As shown in Figure 15 and Figure 16, the PD-1×OX40 bispecific antibody (PR003787) and the parent antibody of PD-1 (PR001985) of the present invention can both enhance TNFα compared with the human IgG1 isotype (PR000324) and IFNγ secretion. Example 11 PD-1×OX40 bispecific antibody inhibits the production of IL-10 in Tregs cells

Purchase concentrated human leukocytes (Research blood components LLC), isolate Treg cells (StemCell, Cat#18063), add CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF ), according to the state of cell growth, add fresh culture medium containing IL-2 every 2-4 days during the culture period, after 12 days of culture, analyze the ratio of Treg by flow cytometry, and cryopreserve the Treg cells expanded in vitro. Thaw the preserved Treg cells, and culture them with the culture medium containing recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF) for 2 days, then the Treg cells and mitomycin-treated CHOK1- huPD-L1 cells were co-cultured with CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) for 5 days. The cells were collected, and the ratio of FoxP3 and interleukin IL-10 secreting cells was detected with a BD Accuri C6 Plus flow cytometer.

As shown in Figure 17, compared with the human IgG1 isotype (PR000324), the PD-1×OX40 bispecific antibody (PR003787) and the parental antibody of OX40 (PR002067) of the present invention have higher effects on the secretion of IL-10 by Treg cells. inhibition.

Example 12 Functional difference between PD-1×OX40 bispecific antibody (PR003787) and parent monoclonal antibody (IFNγ, Granzyme B, IL-2 secretion)

Purchase human leukocytes concentrated (Research blood components LLC), separate T cells (StemCell, #17951), and cryopreserve. Simultaneously isolate Treg cells (StemCell, Cat#18063), add CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF), during the culture period according to the cell growth status every 2 On day 4, fresh culture medium containing IL-2 was added, and after 12 days of culture, the ratio of Treg was analyzed by flow cytometry.

Purchase the second donor human enriched leukocytes (Research blood components LLC), separate monocytes cells (StemCell, #19359), add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) After 6 days of induction, immature human CD14+ dendritic cells (iDC cells) were obtained.

Inoculate 5×10 ⁴ T cells, 5×10 ⁴ Treg cells from the first donor and 2×10 ⁴ iDC cells from the second donor into a 96-well plate at a ratio of 2.5:2.5:1, and add 2nM Or 10 nM of the PD-1×OX40 bispecific antibody (PR003787) of the present invention, the parental monoclonal antibody or two parental monoclonal antibodies of PD-1 and OX40, and co-culture for 4-5 days. Collect 100 μL of culture supernatant, and detect the secretion of IL-2, Granzyme B and IFNγ by MSD (Meso Scale Discovery, U-Plex K15067M-2).

As shown in Figure 18, Figure 19 and Figure 20, the activity of the PD-1×OX40 bispecific antibody (PR003787) of the present invention is superior to that of PD-1 monoclonal antibody (PR001985) and OX40 monoclonal antibody (PR002067), and is superior to The combination of PD-1 mAb (PR001985) and OX40 mAb (PR002067) showed synergistic activity.

Example 13 Detection of T cell priming effect of PD-1×OX40 bispecific antibody by PHA

Purchase concentrated human leukocytes (Research blood components LLC), separate human peripheral blood mononuclear cells (PBMC), and then add 2.5ug/mL PHA-L (eBioscience, #00-4977-03) to human PBMC cells to stimulate 3 sky. The supernatant was collected, and the secretion of tumor necrosis factor IL-2 was detected by MSD (Meso Scale Discovery, U-Plex K15067M-2).

As shown in Figure 21A and Figure 21B, compared with the PD1 monoclonal antibody, the PD-1×OX40 bispecific antibodies PR003787, PR500531, PR200538, PR200539, PR200536, PR200528 and PR200600 of the present invention have stronger activation of T cells Ability to secrete IL-2. Moreover, compared with PR003787, PR200538, PR200539, and PR200528 exhibit dose-dependent T cell priming ability, and have stronger T cell activation ability under high concentration conditions.

none

Figure 1 shows the PD-1xOX40 bispecific antibody IgG_HC-VH structure (structure 1).

Figure 2 shows the PD-1xOX40 bispecific antibody VH-IgG_HC structure (Structure 2).

Figure 3 shows the PD-1xOX40 bispecific antibody Fab(CL)-VH-Fc structure (Structure 3).

Figure 4 shows the PD-1xOX40 bispecific antibody IgG_HC-VH-VH structure (Structure 4).

Figure 5 shows the PD-1xOX40 bispecific antibody IgG_HC-VH-VH-VH structure (Structure 5).

Figure 6 shows that PD-1 monoclonal antibody blocks the binding of PD-1 and PD-L1.

Fig. 7 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to PD-1 on the cell surface as the concentration increases in CHO cells expressing human PD-1 determined by flow cytometry.

Figure 8 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to OX40 on the cell surface as the concentration increases in HEK 293 cells of human OX40 and NFkB promoter-luc determined by flow cytometry .

Figure 9 shows that in HEK 293 cells of human OX40, PD1 and NFkB promoter-luc determined by flow cytometry, the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure increases with the concentration on the cell surface combined.

Figure 10 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to the cell surface as the concentration increases in activated human T cells determined by flow cytometry.

Figure 11 shows the detection of luciferase reporter gene expression in HEK293 cells expressing human OX40 and NFkB promoter-luc, after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure.

Figure 12 shows the co-culture of CHO cells expressing human CD32b and HEK 293 cells expressing human OX40 and NFκB promoter-luc, after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure, the effect on luciferin Detection of enzyme reporter gene expression.

Figure 13 shows that CHO cells expressing human PD-1 and HEK 293 cells expressing human OX40 and NFκB promoter-luc were co-cultured, and after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure, the effect on fluorescent Detection of luciferase reporter gene expression.

Figure 14 shows the co-culture of HEK 293 cells expressing human PD-L1 and OS8 and Jurkat cells expressing human PD-1 and NFAT promoter-luc, adding different concentrations of the bispecific antibody of the present invention or a single strain of its structure Detection of luciferase reporter gene expression after antibody.

Figure 15 shows that in the co-culture system of human peripheral blood mononuclear cells (PBMC) and CHO cells overexpressing human PD-L1, PHA-L and different concentrations of the bispecific antibody of the present invention or a single strain of its structure are added Secretion of the cytokine TNFα following antibody.

Figure 16 shows that in the co-culture system of human peripheral blood mononuclear cells (PBMC) and CHO cells overexpressing human PD-L1, PHA-L and different concentrations of the bispecific antibody of the present invention or a single strain of its structure are added Secretion of the cytokine IFNγ following antibody.

Fig. 17 shows that the Treg secretion of IL10 was detected by flow cytometry after adding different concentrations of the bispecific antibody of the present invention to the expanded Treg cells in vitro.

Figure 18 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of IFNγ.

Figure 19 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of Granzyme B.

Figure 20 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and isolated DC cells induced in vitro, cytokines after adding different concentrations of the bispecific antibody of the present invention or the monoclonal antibody that constitutes its structure Secretion of IL2.

Figure 21 shows the secretion of cytokine IL2 in human peripheral blood mononuclear cells (PBMC) after adding PHA-L and different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure.

Claims (23)

An antibody or an antigen-binding fragment thereof targeting PD-1 or a fragment thereof, wherein the antibody or an antigen-binding fragment thereof comprises a light chain variable region VL and a heavy chain variable region VH , the VL and the VH includes three CDRs respectively, wherein: said VL includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively shown in SEQ ID NO: 39, 46 and 54; said VH includes amino acid sequences respectively shown by SEQ ID NO HCDR1, HCDR2 and HCDR3 shown in : 8, 18 and 28; or the VL comprises amino acid sequences LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 39, 46 and 54; the VH comprises amino acids The sequences are respectively HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 81, 86 and 91; preferably, the CDR contains 1 to 3 amino acid substitutions. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the VL variable region includes the amino acid sequence shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, or 90% of it %, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; said VH includes the amino acid sequence shown in SEQ ID NO:62 or has at least 80% thereof , 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical amino acid sequences; or The VL variable region comprises the amino acid sequence shown in SEQ ID NO: 67 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% thereof or 100% identical amino acid sequence; the VH includes the amino acid sequence shown in SEQ ID NO: 102 or has at least 80%, 85%, 88%, 90%, 92%, 95%, Amino acid sequences that are 97%, 98%, 99% or 100% identical. The antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the antibody further comprises a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region of the antibody is selected from hIgG1 , hIgG2, hIgG3 or hIgG4, the light chain constant region is selected from κ chain or λ chain; more preferably, amino acid substitution occurs at position 234 and/or 235 of Fc of the antibody, preferably The above amino acids are replaced by L234A and/or L235A. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is selected from full-length antibody, Fab, Fab', F(ab')2, Fv or scFv. The antibody or antigen-binding fragment thereof as claimed in claims 1 to 4, wherein said antibody or antigen-binding fragment thereof further comprises a heavy chain and a light chain, wherein, The light chain comprises the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof % identical amino acid sequence; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 72 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97% thereof %, 98%, 99% or 100% amino acid sequence identity; or The light chain comprises the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof % identical amino acid sequence; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 106 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97% thereof %, 98%, 99% or 100% identical amino acid sequences. A bispecific antibody or antigen-binding fragment thereof comprising: A first domain, which binds to PD-1 or a fragment thereof, said first domain is an antibody targeting PD-1 according to any one of claims 1 to 5; and The second domain, which binds to OX40 or a fragment thereof, preferably, the second domain is a VH structure. The bispecific antibody or antigen-binding fragment thereof according to claim 6, wherein the bispecific antibody or antigen-binding fragment thereof comprises two polypeptide chains, wherein: polypeptide chain 2 has N'-VL ₁ -CL ₁ - The structure shown by C', the polypeptide chain 1 has the structure shown by N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L-VH ₂ -C'; or the polypeptide chain 2 has the structure shown by N'-VL ₁ - the structure shown by CL ₁ -C', the polypeptide chain 1 has the structure shown by N'-VH ₂ -L-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -C'; or the polypeptide chain 2 has the structure shown by N The structure represented by '-VH'-CH ₁ '-C', the polypeptide chain 1 has the structure represented by N'-VL'-CL'-L-VH ₂ -h-CH ₂ -CH ₃ -C'; or Polypeptide chain 2 has the structure shown by N'-VL ₁ -CL ₁ -C', and polypeptide chain 1 has the structure shown by N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L ₁ -VH ₂ -L ₂ - The structure shown by VH ₂ -C'; or the polypeptide chain 2 has the structure shown by N'-VL ₁ -CL ₁ -C', and the polypeptide chain 1 has the structure shown by N'-VH ₁ -CH ₁ -h-CH ₂ -CH _{3 -} the structure represented by _{L 1} -VH ₂ -L ₂ -VH ₂ -L ₃ -VH ₂ -C', wherein the VL ₁ , VH ₁ , VL' and VH' are respectively the VL of the first domain and VH, the VH ₂ is the VH of the second domain, the h is the hinge region, the L, L ₁ , L ₂ and L ₃ are connecting peptides, and the CL ₁ and CL' are the first structure Domain CL, said CH ₁ and CH ₁ ' are CH ₁ of the first structural domain; preferably, said connecting peptide is a peptide with a length of 0-30 amino acids, preferably, its amino acid The sequence is shown in any one of SEQ ID NO: 116-140; preferably, the polypeptide chain 1 and the polypeptide chain 2 form a tetravalent symmetrical structure, a hexavalent symmetrical structure or an octavalent symmetrical structure. The bispecific antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein the second domain comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence composed of HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29; or the heavy chain variable region comprises an amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO HCDR1, HCDR2, HCDR3 shown in :85, SEQ ID NO:90; Preferably, the second domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chain variable regions. The bispecific antibody or antigen-binding fragment thereof according to claim 8, wherein the Fc is a human IgG1 structure, preferably, the Fc comprises L234A, L235A and P329G, or the Fc comprises L234A, L235A and G237A. The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the bispecific antibody or antigen-binding fragment thereof comprises: Polypeptide chain 1 includes or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 79 Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% amino acid sequence identity; or Polypeptide chain 1 includes or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 109 Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% amino acid sequence identity; or Polypeptide chain 1 comprises the amino acid sequence shown in SEQ ID NO: 110 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% thereof Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% amino acid sequence identity; or Polypeptide chain 1 includes or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 111 Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% amino acid sequence identity; or Polypeptide chain 1 includes or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 112 Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% amino acid sequence identity; or Polypeptide chain 1 includes or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 114 Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 113 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% amino acid sequence identity; or Polypeptide chain 1 includes or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 115 Consistent amino acid sequence; polypeptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 77 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, Amino acid sequences with 98%, 99% or 100% identity. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5 or a bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 6 to 10 , Preferably, said nucleic acid molecule is an mRNA molecule. An expression vector, which comprises the isolated nucleic acid molecule as described in claim 11, said expression vector is a eukaryotic cell expression vector or a prokaryotic cell expression vector, preferably a retroviral vector, a lentiviral vector, a phage vector, an adenocarcinogen Viral, adeno-associated, or herpes simplex vectors. The expression vector according to claim 12, wherein the expression vector exists in nanoparticles, liposomes or gene guns. A kind of host cell, it comprises the nucleic acid molecule of separation as described in claim item 11, or the expression vector as described in claim item 12 or 13, described host cell is prokaryotic cell and/or eukaryotic cell, and described prokaryotic cell is relatively Preferably it is E. coli cells, and the eukaryotic cells are preferably HEK293 cells or CHO cells. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment thereof according to any one of claims 6 to 9, comprising: In the case of expressing the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or the expression of the bispecific antibody or antigen-binding fragment thereof as described in any one of claims 6 to 10, culturing The host cell as described in Claim 14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment thereof as described in any one of claims 6-10 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition further comprises a second therapeutic agent, and the second therapeutic agent includes a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressant, and a cytotoxic drug. The pharmaceutical composition as claimed in item 16, wherein the pharmaceutical composition is in the form of solid, semi-solid or liquid; Preferably, the pharmaceutical composition is in the form of aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion. An antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5, a bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 6 to 10, as described in claim 11 The isolated nucleic acid molecule, the expression vector as described in claim item 12 or 13, the host cell as described in claim item 14, or the pharmaceutical composition as described in claim item 16 or 17 in the preparation for prevention, treatment and/or Or use in medicines for the diagnosis of neoplastic diseases, acute and chronic inflammatory diseases and/or immune diseases. The application according to claim 18, wherein the tumor is selected from breast cancer, renal cell carcinoma, melanoma, colon cancer, B cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma , urothelial cancer, liver cancer, esophageal cancer, gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute granulocytic One or more of leukemia, Hodgkin's lymphoma, squamous cell carcinoma of the skin, locally advanced or metastatic malignancy; The inflammatory disease is atopic dermatitis or ulcerative colitis, The immune disease is graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus or asthma. A method for detecting OX40 and/or PD-1 in a sample, the method comprising utilizing the antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5, or as claimed in claims 6 to 10 The step of detecting OX40 and/or PD-1 in any one of the bispecific antibodies or antigen-binding fragments thereof, the samples being whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, Tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and/or nasal secretions; preferably, the detection method is for non-diagnostic purposes. A set of kits comprising one or more kits, the one or more kits including the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, as in claims 6 to 10 The bispecific antibody or antigen-binding fragment thereof according to any one of the claims, or the pharmaceutical composition according to claim 16 or 17. The kit as described in claim 21, wherein the kit includes a first kit, and the first kit includes the bispecific antibody or its antigen-binding fragment; Optionally, the set of kits further includes a second kit, and the second kit includes at least one therapeutic agent selected from chemotherapeutic agents, radiotherapeutic agents, immunosuppressants and cytotoxic drugs. A method for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antibody as described in any one of claims 1 to 5 or Its antigen-binding fragment, such as the bispecific antibody or antigen-binding fragment thereof as described in any one of claims 6 to 10, the isolated nucleic acid molecule as described in claim 11, the expression as described in claim 12 or 13 The carrier, the host cell as described in Claim 14, or the pharmaceutical composition as described in Claim 16 or 17.

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