TWI835166B - Specific binding protein targeting pd-1 and ox40 and application thereof - Google Patents

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof targeting PD-1, as well as bispecific antibodies or fragments thereof targeting PD-1 and OX40. The invention also relates to nucleic acid molecules encoding said antibodies or antigen-binding fragments thereof. The present invention also relates to the antibody or antigen-binding fragment thereof targeting PD-1, the bispecific antibody targeting PD-1 and OX40 or its fragment, and the nucleic acid molecules encoding the same in treatment, prevention and/or diagnosis. Use in diseases such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

Description

Specific binding proteins targeting PD-1 and/or OX40 and their applications

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

The mammalian immune system is a finely balanced system, but it is sometimes disrupted by diseases such as cancer. Immune checkpoint receptors play an important role in the immune system's response to disease by acting as either costimulatory or costinhibitory, 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 excessive immune response. On the other hand, costimulators promote the development of protective immune responses by promoting T cell lineage expansion (clonal expansion), effector differentiation and survival.

An effective approach to cancer immunotherapy could trigger the immune system to recognize and kill tumor cells by targeting these checkpoint receptors with antibodies that block co-inhibitory receptor function or induce co-stimulatory receptor activity (Pardoll, 2012) . Antibodies that block the activity of co-inhibitory receptors have shown good clinical activity and are currently approved for the treatment of cancer (Larkin et al., 2015). Antibodies that induce costimulatory receptor activity have shown great promise in preclinical model systems (Moran et al., 2013; Schaer et al., 2014), and several drugs are currently in clinical trials (Mayes et al., 2018; Melero et al., 2013 ). These antibodies are designed to mimic the ligands of these costimulatory 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 cells. Helper cells (Th1 and Th2) and regulatory T (Treg) cells, and expressed on activated natural killer cells (NK). The interaction of OX40 and its ligand OX40 ligand (OX40L) expressed on antigen-presenting cells (APCs) increases T cell lineage expansion, differentiation, and survival and enhances memory Generation of 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 eliminate the suppressive activity of Treg cells (Takeda et al., 2004). OX40 has been found to be expressed on tumor-infiltrating T cells in 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, with most showing good safety profiles and limited clinical activity. The effect of targeting OX40 as a single therapy is not ideal and has never 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, namely programmed cell death ligand 1 (Programmed cell death 1 ligand 1; PD-L1) and programmed cell 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 results in a decrease in tumor-infiltrating lymphocytes, reduced T-cell receptor-mediated proliferation, and immune evasion of cancer cells.

Although the anti-cancer principle of using agonists to stimulate costimulatory receptors or checkpoint receptor inhibitors is clear: checkpoint receptor inhibitors such as CTLA4 and PD1/PDL1 enhance the anticancer effect of T cells, while costimulatory receptors The body agonists CD28, 4-1BB, OX40, GITR, CD27 and ICOS promote the anti-tumor immunity of T cells. However, the clinical use of costimulatory receptor agonists and checkpoint receptor inhibitors together has not achieved good results. Theoretically, stimulation of these costimulatory receptors with agonists should enhance antitumor immunity in the highly immunosuppressive tumor microenvironment. Extensive mouse data also confirm the therapeutic potential of this class of drugs. For example, Genentech combined an OX40 agonist antibody with a PD-L1 antibody to achieve complete remission in 90% of colorectal cancer tumors in a mouse model. However, while the data from animal studies are promising, the data from clinical trials are less than ideal. About OX40 Agonists at Genentech In a phase Ib trial of MOXR0916 combined with the PD-L1 antibody atezolizumab, only 4% (2 of 51) of patients achieved partial response, so Genentech shut down its OX40 project. In most cases, the combination of chemotherapy drugs may have a synergistic effect, thereby making the tumor treatment more effective. However, the combination of immunotherapy is not simply 1+1>2, and it may be far from the expected results. , or the complete opposite. Data from two groups of mouse models published in 2017 showed that when OX40 agonists and PD-1 inhibitors were given simultaneously, the effects of the two antibodies canceled each other out due to T cell exhaustion or programmed cell death. However, sequential administration enhanced the antitumor activity of these drugs. One hypothesis is that costimulation must first promote tumor-specific T cells to the point where checkpoint suppression of the immune response occurs 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 explore methods that can exert the functions of both at the same time, such as preparing bispecific antibodies.

The present invention provides specific binding proteins that can bind to one or more antigens with high affinity and high specificity, and the antigens are PD-1 or fragments thereof, and/or OX40 or fragments thereof. The present invention also provides nucleic acid molecules encoding the specific binding proteins, expression vectors for producing the specific binding proteins, host cells and methods for preparing the specific binding proteins. The present invention also relates to the use of the 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 includes CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54 respectively, the VH includes CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54 respectively. :8, 18 and 28, or as shown in SEQ ID NOs: 81, 86 and 91 respectively. 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 includes CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54 respectively, and the VH includes 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 includes CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 39, 46 and 54 respectively, and the VH includes CDR1, CDR2 and CDR3, its amino acid sequences are shown in SEQ ID NO: 81, 86 and 91 respectively. In some embodiments, these CDRs may contain amino acid mutations that maintain the antibody's ability to specifically bind PD-1. Preferably, the amino acid mutation is an amino acid substitution, and the number of the amino acid substitutions may 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%, An amino acid sequence that is 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 includes the amino acid sequence shown in SEQ ID NO: 62, or has at least 80%, 85%, 88%, 90%, 92%, An amino acid sequence that is 95%, 97%, 98%, 99% or 100% identical. In a specific embodiment, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof includes 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 includes the amino acid sequence shown in SEQ ID NO: 102, or has at least 80%, 85%, 88%, 90%, 92%, An amino acid sequence that is 95%, 97%, 98%, 99% or 100% identical. In some embodiments, the VH of the above-mentioned specific antibody or antigen-binding fragment thereof includes 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 specific antibody or antigen-binding fragment thereof is IgG. In some embodiments, the above-described specific antibodies or antigen-binding fragments thereof include heavy chain constant regions and/or light chain constant regions. In some embodiments, the heavy chain constant region described above can be derived from human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the light chain constant region can be selected from a kappa chain or a lambda chain. In some embodiments, the above-mentioned specific antibodies or antigen-binding fragments thereof can be prepared polyclonal antibodies or monoclonal antibodies.

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

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

In some embodiments, the above-described specific antibodies or antigen-binding fragments thereof include heavy and light chains. In some embodiments, the light chain includes or is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% identical to the amino acid sequence set forth 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 %, 95%, 97%, 98%, 99% or 100% identity of the amino acid sequence. In some embodiments, the light chain includes or is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% identical to the amino acid sequence set forth 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% identity of the amino acid sequence.

In a second aspect, the present invention provides a specific binding protein comprising at least two structural domains capable of binding to 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 binding PD-1 or a fragment thereof, and The second domain binds OX40 or a fragment thereof. In some embodiments, the second junction The 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 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, and VH is one or more.

In some embodiments, the first domain is in the form of IgG. Preferably, the heavy chain constant region of the IgG 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 of L234A, L235A and P329G, more preferably two mutations of L234A and L235A or three mutations of L234A, L235A and P329G; preferably Preferably, the Fc of IgG in the first domain is the Fc of human IgG1, and more preferably, the Fc includes L234A, L235A and P329G mutations; preferably, the Fc of IgG in the first domain is human IgG4 Fc, more preferably, the Fc contains L234A and L235A and P329G mutations.

In some embodiments, the human IgG preferably comprises one, two or three mutations of L234A, L235A and G237A, more preferably two mutations of L234A and L235A or three mutations of L234A, L235A and G237A; preferably Preferably, the Fc of IgG in the first domain is the Fc of human IgG1, and more preferably, the Fc includes L234A, L235A and G237A mutations; preferably, the Fc of IgG in the first domain is human IgG4 Fc, more preferably, the Fc contains L234A and L235A and G237A mutations.

Preferably, the first structural 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 linking peptide L to form a bispecific binding protein.

In some embodiments, the specific binding protein is a bispecific antibody of IgG_HC-VH tetravalent symmetry structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which contains N'-VL ₁ -CL ₁ -C'; polypeptide chain 1, from the amino terminal to the carboxyl terminal, it contains 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 respectively, 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, and the CH ₁ is CH1 of the first 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 symmetry structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which contains N'-VL ₁ -CL ₁ -C'; Polypeptide chain 1, from the amino terminal to the carboxyl terminal, contains 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 respectively, 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 CH ₁ of a domain, the L is a connecting peptide, and VH ₂ of polypeptide chain 1 is connected to VH ₁ via the connecting peptide L.

In some embodiments, the specific binding protein is a bispecific antibody with a Fab(CL)-VH-Fc tetravalent symmetric structure, which contains two polypeptide chains, polypeptide chain 2, from the amino terminus to the carboxyl terminus, which contains N'-VH'-CH1'-C'; polypeptide chain 1, from the amine terminus to the carboxyl terminus, which contains N'-VL'-CL'-L-VH ₂ -h-CH ₂ -CH ₃ -C' . Wherein, the VL' and VH' are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region, the L is the connecting peptide, and the CL ' is the CL of the first domain, the CH1' is the CH1 of the first domain, and the CL' of the polypeptide chain 1 is directly fused and connected to VH ₂ , that is, the length of L is 0.

In some embodiments, the specific binding protein is a bispecific antibody of IgG_HC-VH-VH hexavalent symmetry structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which contains N'- VL ₁ -CL ₁ -C'; polypeptide chain 1, from the amino terminus to the carboxyl terminus, which contains N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L ₁ -VH ₂ -L2-VH ₂ -C'. Wherein, the VL ₁ and VH ₁ are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region, the L1 and L2 are the connecting peptides, and the The CL ₁ is the CL of the first structural domain, the CH ₁ is the CH1 of the first structural domain, CH3 of the polypeptide chain 1 is connected to VH ₂ via 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 eight-valent symmetry structure, which contains two polypeptide chains: polypeptide chain 2, from the amino terminus to the carboxyl terminus, which contains N '-VL1-CL1-C'; polypeptide chain 1, from the amino terminus to the carboxyl terminus, it contains N'-VH1-CH1-h-CH2-CH3-L1-VH2-L2-VH2-L3-VH2-C' . Wherein, the VL1 and VH1 are the VL and VH of the first domain respectively, the VH2 is the VH of the second domain, the h is the hinge region, the L1, L2 and L3 are connecting peptides, and the CL1 is the CL of the first domain, the CH1 is the CH1 of the first domain, CH3 in the polypeptide chain 1 is connected to the first VH2 from the N-terminus to the C-terminus of the polypeptide chain 1 via the connecting peptide L1, the first VH2 and the second VH2 are connected through the connecting peptide L2, and the second VH2 and the third VH2 are connected through the connecting peptide L3.

Among them, 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, the L is a peptide with a length of 0-30 amino acids, and its amino acid sequence is as 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.

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

In some embodiments, the first domain includes VL and VH. In some embodiments, the amino acid sequence of the VL is as shown in SEQ ID NO: 67 or is 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 amino acid sequence of the VH is such as SEQ ID NO: 62 or has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% therewith Or 100% consistency. In a specific embodiment, the amino acid sequence of the VH is SEQ ID NO: 62. In some embodiments, the amino acid sequence of the VH is as shown in SEQ ID NO: 102, or is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% identical thereto. , 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% similarity to the amino acid set forth in SEQ ID NO: 77 , a light chain that is 99% or 100% identical, and has at least 80%, 85%, 88%, or 90% identity with 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% identity of the heavy chain.

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

In some embodiments, the first domain comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% similarity to the amino acid set forth in SEQ ID NO: 77 , a light chain that is 99% or 100% identical, and is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% identical to the amino acid shown in SEQ ID NO: 72 , 99% or 100% identical heavy chain. In a specific embodiment, the 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% similarity to the amino acid set forth in SEQ ID NO: 77 , a light chain that is 99% or 100% identical, and is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98% identical to the amino acid set forth in SEQ ID NO: 106 , 99% or 100% identical heavy chain. In a specific embodiment, the 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) comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are set forth in SEQ ID NO: 9, 19 and 29, respectively. . In some embodiments, the second domain comprises a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3, the amino acid sequences of which are set forth in SEQ ID NO: 9, 85 and 90, respectively. .

In some embodiments, the second domain comprises a VH whose amino acid sequence is as shown in SEQ ID NO: 63, or is at least 80%, 85%, 88%, 90%, 92% identical thereto. , 95%, 97%, 98%, 99% or 100% consistency. In specific embodiments, the VH of the second domain The amino acid sequence is shown in SEQ ID NO: 63. In some embodiments, the second domain comprises a VH whose amino acid sequence is as shown in SEQ ID NO: 101, or is at least 80%, 85%, 88%, 90%, 92% identical thereto. , 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% similarity to the amino acid sequence set forth in SEQ ID NO: 73 %, 99% or 100% identity of the heavy chain. In specific embodiments, the 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% similarity to the amino acid sequence set forth in SEQ ID NO: 105 %, 99% or 100% identity of the heavy chain. In specific embodiments, the second domain comprises a heavy chain as set forth in SEQ ID NO:105.

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

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

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

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

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

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

In some embodiments, the bispecific binding protein comprises polypeptide chain 1, which is a long chain, and polypeptide chain 2, which 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 with 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 and SEQ ID NO: 113, or has at least 80%, 85%, 90%, 95%, 97%, 98%, Amino acid sequence with 99% or 100% identity.

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

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein polypeptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 109; polypeptide chain 2 includes 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 includes the amino acid sequence shown in SEQ ID NO: 110; polypeptide chain 2 includes 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 an amino acid sequence as shown in SEQ ID NO: 111; polypeptide chain 2 comprises an amino acid sequence as shown in SEQ ID NO: 77.

In a specific embodiment, the bispecific antibody comprises two polypeptide chains: wherein polypeptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 112; polypeptide chain 2 includes 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 includes the amino acid sequence shown in SEQ ID NO: 114; polypeptide chain 2 includes 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 includes the amino acid sequence shown in SEQ ID NO: 115; polypeptide chain 2 includes 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 of the first aspect of the present invention or its antigen-binding fragment, the specific binding protein of the second aspect or its fragment.

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 according to 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 retroviral vector, a lentiviral vector, a phage vector, an adenoviral 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 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 into the antibody of the first aspect or its antigen-binding fragment or the bispecific antibody of the second aspect. sexual antibodies. Preferably, the host cell is a prokaryotic cell and/or a eukaryotic cell, the prokaryotic cell is preferably E.coli cells such as TG1, BL21 (expressing single chain antibodies or Fab antibodies), and the eukaryotic cell is preferably HEK293 cells or CHO cells (expressing full-length IgG antibodies). By transforming the expression vector of the fourth aspect into a host cell, the host cell of the present invention can be obtained. The transformation method is a conventional transformation method in this field, 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 of the first aspect or its antigen-binding fragment or the bispecific antibody of the second aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization technology, etc.

In some embodiments, the preparation method 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 a human immunoglobulin immune repertoire and is able to produce a new "heavy chain" only antibody that is only half the size of traditional IgG antibodies. That The antibody produced has only the human antibody "heavy chain" variable domain and the mouse Fc constant domain.

Preferably, the method of using HCAb transgenic mice to prepare the second domain of the bispecific antibody of the second aspect includes the following steps: (a) immunizing the HCAb transgenic mice with human OX40, more preferably, the human The OX40 antigen is recombinant human OX40-ECD-Fc. Specifically, the antigen is a recombinant fusion protein composed of the extracellular region of OX40 connected to Fc; (b) RNA reverse transcription of splenic B cells of transgenic mice using HCAb after immunization cDNA, 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) purification step (c) ) fully human OX40 monoclonal antibody obtained.

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 of the first aspect or its antigen-binding fragment 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 antibodies it produces have complete human antibody variable domains and rat constant domains.

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 includes the following steps: (a) using human PD-1 immunizes H2L2 transgenic mice. More preferably, the human PD-1 antigen is soluble recombinant human PD-1-hFc. Specifically, the antigen is a recombinant fusion protein composed of PD-1 connected to Fc; (b) After immunization, spleen cells of H2L2 transgenic mice were fused with myeloma cell lines to obtain hybridoma cells. The isolated hybridomas expressed heavy and light chains with complete human variable domains and rat constant domains. Antibody molecule; (c) gene-synthesize and select the antibody light chain variable domain sequence (VL) obtained in step (b) into a mammalian cell expression vector encoding the human antibody kappa light chain constant domain sequence, to Encoding the full-length light chain to produce an antibody; (d) using the antibody heavy chain variable domain sequence (VH) obtained in step (b) through gene synthesis and selecting and cloning mammalian cells encoding the human IgG antibody heavy chain constant domain sequence In the expression vector, the full-length heavy chain encoding the IgG antibody is produced; (e) the expression vectors of steps (c) and (d) are simultaneously transfected into mammalian host cells, and conventional recombinant protein expression and purification techniques can be used to obtain light and heavy chains. Recombinant antibodies assembled with correctly paired chains.

Preferably, 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 IgG4 antibody heavy chain constant domain sequence to encode and produce Full-length heavy chain of IgG4 antibody.

Alternatively, 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 and produce Full-length heavy chain of 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 bind to two targets at the same time, where the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, and the second domain can bind to OX40 molecules on T cells. The specific binding protein After the protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing the 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 bind to two targets at the same time, where the first domain can recognize PD-1 specifically expressed on the surface of tumor cells, and the second domain can bind to OX40 molecules on T cells. The specificity After the binding protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing the tumor cells.

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

Preferably, the dual-specific binding protein has the structure and sequence described in the second aspect.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to the first aspect or the bispecific antibody according to 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 physiological or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipients are conventional pharmaceutical excipients in this field, and preferably include pharmaceutically acceptable excipients, fillers or diluents, etc. More preferably, the pharmaceutical composition includes 0.01 to 99.99% of the specific binding protein and/or other small molecule drugs or antibodies or polypeptides, and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is Quality percentage of the pharmaceutical composition.

The administration route of the pharmaceutical composition may be parenteral, injection or oral administration. The pharmaceutical composition can be prepared in a form suitable for administration, such as a solid, semi-solid or liquid form, an aqueous solution, a non-aqueous solution or a suspension, a powder, a tablet, a capsule, a granule, an injection or an infusion. form. Administration may be via intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation or intrathoracic instillation. The pharmaceutical composition may also be administered in the form of an aerosol or spray, for example nasally; alternatively, intrathecally, intramedullary or intraventricularly, and may also be administered transdermally, percutaneously, topically, enterally, intravaginally. , sublingual or rectal administration. The pharmaceutical composition can be made into various dosage forms as needed, and can be administered by a physician based on the patient's type, age, weight, general disease status, administration method and other factors to determine a dose that is beneficial to the patient.

In some embodiments, the specific binding protein in the pharmaceutical composition is combined with other The active ingredients 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 according to the first aspect, the bispecific antibody according to the second aspect, the isolated nucleic acid molecule according to the third aspect, and the pharmaceutical composition according to the seventh aspect. Application in the preparation of drugs for the prevention, treatment and/or diagnosis of immune diseases, acute and chronic inflammatory diseases, and tumor diseases.

The tumors may be breast cancer, renal cell carcinoma, melanoma, colon cancer, as well as 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 lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignant tumors, etc.

The inflammatory disease may be atopic dermatitis, ulcerative colitis, etc.

The immune disease may be graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma, etc.

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

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

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

In a tenth aspect, the present invention also provides a pharmaceutical kit, which includes one or more pharmaceutical kits, including the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or the dual antibody as described in the second aspect. Specific antibodies or pharmaceutical compositions according to the seventh aspect of the present invention.

In some embodiments, the kit of parts includes a first kit that includes the antibody of the first aspect or an antigen-binding fragment thereof, or the first domain and the second structure of the second aspect. Bispecific antibodies composed of domains.

In some embodiments, the kit may further include a second kit that includes other therapeutic agents including, but not limited to, chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, agents and cytotoxic drugs.

In some embodiments, the first medicine box and the second medicine box can be used at the same time, or the first medicine box can be used first and then the second medicine box, or the second medicine box can be used first and then the third medicine box. A pill box can be determined according to the actual needs of the specific application.

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, which comprise 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 described in the second aspect or the pharmaceutical composition described in 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 monkey PD-1; the second domain can bind to human OX40 protein and cynomolgus monkey OX40 Protein; the second domain shows a concentration-dependent enhancement of the 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; with the assistance of CHO-K1/PD-1 cell cross-linking, it specifically causes concentration-dependent enhancement of the NF-κb signaling pathway. It can specifically induce the promotion of the PD-1 cross-linking-dependent OX40-mediated NF-κb signaling pathway, and the signal intensity caused by it increases in a positive correlation with its concentration;

4. The PD-1xOX40 bispecific antibody of the present invention has an inhibitory effect on the PD-1 signaling pathway; it can Enhances the secretion of TNFα and IFNγ; inhibits the secretion of IL-10 by Treg cells; can promote the secretion of the cytokine Granzyme B in T cells.

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 Tregs; compared with individual PD-1 monoclonal antibodies and OX40 monoclonal antibodies, and PD- The combination of 1 mAb and OX40 mAb showed obvious synergistic activity.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly used in the art to which this invention belongs. For the purposes 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 a measured value given the nature or precision of the measurement. Typically the margin of error is within 20% of the given value or range of values, typically within 10%, and even more typically within 5%.

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

The term "antibody" as used herein encompasses a variety of 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 are sufficient as long as they exhibit the required antigen-binding activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for the possible presence of trace amounts of variant antibodies (e.g. containing naturally occurring mutations or generated during the production of monoclonal antibody preparations). , usually present in small amounts), the individual antibodies included in the antibody population are identical and/or bind to 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 multiple epitopes. Antibodies to the opposite sex. These multispecific antibodies include, but are not limited to: antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; having two or more Antibodies with VL and VH regions, each VH-VL unit binds to different targets or different epitopes of the same target; antibodies with two or more single variable regions, each single variable region binds to Binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), triabodies (triabodies), antibody fragments covalently or non-covalently linked together, etc. .

The term "bispecific binding protein" or "bispecific antibody" of the present invention refers to one that can specifically bind at least two different antigenic determinants, for example, each consisting of an antibody heavy chain variable domain (VH) and an antibody light chain. The two binding sites formed by the chain variable domain (VL) bind to 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 binding site for 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 contain two antigen-binding sites, each specific for a different antigenic determinant.

The term "valency" in the present invention means that the antigen-binding molecule is present with a specified number of binding domains. Therefore, the terms "bivalent", "tetravalent" and "hexavalent" refer to 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 cases, the antibodies have two or more binding sites and are bispecific. That is, an antibody can be bispecific even where more than two binding sites are present (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 antibodies that are structurally substantially similar to native antibodies. "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or a heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) ( Also called heavy chain constant region). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called variable light chain domain or light chain variable domain) and light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), and can be further divided into subtypes. Types, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an antibody can be one of two types, based on the amino acid sequence of its constant domain, namely kappa light chain and lambda light chain.

Within the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. . Each heavy chain consists 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 consists 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 region of an antibody may mediate 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 fragment" includes a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') _2, and Fv; diabodies, tribodies, 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" of the present invention refers to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to that portion of an antibody that contains a region that specifically binds and is complementary to part or all of the antigen. Where the antigen molecule is large, the antigen-binding molecule can bind only to a specific part of the antigen, called an epitope. The antigen binding domain may be provided, for example, by one or more variable domains (also called variable regions). Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

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

1μM、

100nM、

10nM、

1nM、

0.1nM、

0.01nM或

0.001nM(例如10^-7M或更低，例如10^-7M至10^-13M，例如10-⁹M至10^-13M)。 "Specifically binds" means that binding is selective for an antigen and can be distinguished from undesired or non-specific binding. The ability of an antigen-binding molecule to bind to 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) technology as well as traditional binding assays. In one embodiment in which the antigen-binding molecule binds to the unrelated protein to a degree that is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, such as as measured 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

0.001 nM (eg 10 ^-7 M or less, eg 10 ^-7 M to 10 ^-13 M, eg 10 - ⁹ M to 10 ^-13 M).

"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 often be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (k _off and _kon , respectively). Therefore, equivalent affinities can include different rate constants as long as the ratio of the 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" of the present invention means that the Kd of the antibody to the target antigen is 10 ^-9 M or lower, or even 10 ^-10 M or lower. The term "low affinity" of the present invention refers to an antibody with a Kd of 10 ^-8 M or higher.

An "affinity matured" antibody is one that has a hypervariable region (HVR) in one or more hypervariable regions (HVR). An antibody with one or more modifications that result in improved affinity of the antibody for the antigen compared to the parent antibody without such modifications.

The terms "single domain antibody" and "nanobody" of the present invention have the same meaning and refer to having only the variable region of the heavy chain of an antibody. The construction of a single domain antibody consisting of only one heavy chain variable region has a complete The smallest functional antigen-binding fragment.

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

The terms "bispecific antibody comprising a first antigen-binding domain that specifically binds PD1 and a second antigen-binding domain that specifically binds OX40" "bispecific antibody that specifically binds PD1 and OX40" "specific for PD1 and "Bispecific antigen-binding molecule for OX40" 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 The antibodies can be used as bispecific antibodies as diagnostic and/or therapeutic agents targeting PD1 and OX40.

The term "T effector cells" of 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. But it does not include regulatory T cells (Treg cells). The anti-OX40 antibodies of the invention can activate T effector cells, such as CD4+ and CD8+ T effector cells.

The term "regulatory T cells" or "Treg cells" in the present invention refers to a special type of CD4+ T cells that can block the responses of other T cells. Treg cells are characterized by 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 autologous tolerance to 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, which is an immunoglobulin superfamily and was first published in 1992 (Ishida et al., EMBO J., 11(1992), 3887-3895). PD-1 is an expanded CD28/CTLA-4 family T cell regulator A member of the factor and has at least two ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein structure consists of the extracellular IgV domain, followed by the transmembrane region and intracellular tail. The intracellular tail contains two phosphorylation sites located in the inhibitory motif of the immunoreceptor tyrosine base and the switch motif of the immunoreceptor tyrosine base, indicating that PD-1 negatively regulates T cell receptor TCR signaling. This is consistent with the binding of SHP-1 phosphatase and SHP-2 phosphatase to the cytoplasmic tail of PD-1 upon ligand binding. Although PD-1 is not expressed on naive T cells, it is upregulated following 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 treatments mainly block the interaction of PD-1 with its ligands to enhance T cell responses. The terms "programmed cell death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PD1", "PDCD1", "hPD-1" and "hPD-I" are used interchangeably and include human PD Variants, isoforms, species homologs of -1, and analogs that share at least one epitope in common with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) accession number Q15116.

1μM、

100nM、

10nM、

1nM、

0.1nM、

0.01nM或

0.001nM(例如10^-8M或更低，例如10^-13M至10^-8M，例如10^-13M至10^-9M)。在一些實施方案中，在表面電漿共振測定中，使用人PD1(PD1-ECD)測定結合親和力的相應KD值，以獲得PD-1結合親和力。PD-1/PD-L1抗體的治療策略是幾種轉移性腫瘤中的標準治療策略，並已顯示出它們在早期疾病階段和輔助治療中的作用，尤其是黑 色素瘤和非小細胞肺癌。 The term "PD1 antibody" of the present invention is capable of binding to PD1, especially PD1 polypeptide expressed on the cell surface, with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting PD1. Typically, a PD1 antibody has less ability to bind to unrelated non-PD1 proteins than the antibody does, as determined by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance using a biosensor system. Approximately 10% of the binding capacity of PD-1. In certain embodiments, the KD value of the antigen-binding protein that binds human PD1

1μM,

100nM,

10nM,

1nM,

0.1nM,

0.01nM or

0.001 nM (eg 10 ^-8 M or less, eg 10 ^-13 M to 10 ^-8 M, eg 10 ^-13 M to 10 ^-9 M). In some embodiments, the corresponding KD value for binding affinity is determined using human PD1 (PD1-ECD) in a surface plasmon resonance assay to obtain PD-1 binding affinity. Therapeutic strategies with PD-1/PD-L1 antibodies are standard treatment strategies in several metastatic tumors and have shown their role in early disease stages and in adjuvant therapy, particularly in melanoma and non-small cell lung cancer.

The term "OX40" of the present invention, also known as CD134, is expressed in activated CD4+T cells 1-3 days after activation, as well as CD8+T cells, dendritic cells, neutrophils, Treg cells, etc. The ligand of OX40, OX40L, is expressed on the surface of antigen-presenting cells (APCs) such as dendritic cells and B cells. The OX40/OX40L interaction can recruit TRAF molecules within the intracellular region of OX40, and can also activate the classic NF-κB1 pathway or the non-canonical NF-κB2 pathway, PI3k/PKB and NFAT pathways, thereby regulating T cell division and survival. Genes, as well as promoting the transcription of cytokine genes and the expression of cytokine receptors, can also promote the differentiation of B cells into plasma cells, promote antibody production, etc. In other words, it can enhance the activity of effector T cells, NK, and NK-T cells. , releasing the immunosuppressive effect of Treg can enhance both the specific immune response and the innate immune response, thereby enhancing anti-tumor immunity.

1μM，

100nM，

10nM，

1nM，

0.1nM，

0.01nM，或

0.001nM(例如10^-8M或更低，例如10^-13M到10^-8M，例如10^-13M到10^-9M)的解離常數(KD)。 The term "OX40 antibody" of the present invention is capable of binding OX40 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent targeting OX40. Typically, OX40 antibodies have less ability to bind to unrelated OX40 protein than the antibody does to OX40, as determined by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance using a biosensor system. about 10% of the binding capacity. In certain embodiments, an antibody that binds OX40 has

1μM,

100nM,

10nM,

1nM,

0.1nM,

0.01nM, or

A dissociation constant (KD) of 0.001 nM (eg 10 ^-8 M or less, eg 10 ^-13 M to 10 ^-8 M, eg 10 ^-13 M to 10 ^-9 M).

The term "H2L2 transgenic mouse" or "Harbour H2L2 mouse" in the present invention is a transgenic mouse carrying a human immunoglobulin immune repertoire that produces a fully human variable region consisting of two heavy chains and A traditional tetrameric antibody composed of two light chains (H2L2), with a complete human antibody variable domain and a rat constant domain. The antibodies produced by the transgenic mice are affinity matured, have 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 new "heavy chain" only antibody that is only half the size of traditional IgG antibodies. . The antibodies it produces have only the variable structure of the human antibody "heavy chain" domain and the constant domain of mouse Fc. Due to the fact that it does not contain a light chain, this "heavy chain"-only antibody almost solves the problems of light chain mismatching and heterodimerization, allowing 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 domain of an antibody heavy chain or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "variable" as used herein 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, variability is not evenly distributed throughout the variability domain. Instead, it is concentrated in three segments called hypervariable regions (HVR) within the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of native heavy and light chains each contain four FR regions, mostly in a β-sheet configuration, connected by three HVRs, which form loop connections and in some cases form part of the β-sheet structure. The HVRs in each chain are held tightly together by the FR region and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen and has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.

The term "hypervariable region" or "HVR" of the present invention refers to a region within an antibody variable domain region that is hypervariable in sequence and/or forms a structurally defined loop ("hypervariable loop"). Typically, natural four-chain antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)" that 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) occur at amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89-97 (L3), amino acid residues at 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 ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). For comparison, the corresponding amino acid residues comprising the CDRs defined in the references cited above are listed in Table 2. In the present invention, The amino acid sequences of the CDRs listed above are all shown in accordance with the Chothia definition rules (the claims of the present invention are also sequences shown in accordance with the Chothia definition rules). However, it is well known to those in the art that in this field CDRs of antibodies are defined by a variety of methods, such as Kabat's definition rules based on sequence variability and Chothia's definition rules based on the position of structural loop regions (see J Mol Biol 273:927-48, 1997). In the present invention, it is also possible to use The Combined definition rule, which includes the Kabat definition and the Chothia definition, determines the amino acid residues in the variable domain sequence. The Combined definition rule is to combine the ranges of the Kabat definition and the Chothia definition, and based on this, a larger range is taken . It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass the above-mentioned as described by the present invention. The complementarity determining region defined by any of the known schemes. Although the scope of protection claimed in the present invention is based on the sequence shown by the Chothia definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within this scope. within the protection scope of the invention.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain usually consists of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear 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, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

"Humanized antibodies" comprise amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all of the FRs Corresponds to the FR 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, such as a non-human antibody, is an antibody that has been humanized.

A "human antibody" has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by humans or human cells or derived from non-human sources utilizing human antibody libraries or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies containing 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 containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region contains the IgG CH2 domain and the IgG CH3 domain. The CH2 domains herein may be native sequence CH2 domains or variant CH2 domains. The CH3 domains herein may be native sequence CH3 domains or variant CH3 domains. The CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fcγ receptors (eg, FcγRI, FcγRIIa, FcγRIIb, FcγRIII) 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 complement binding would be expected to reduce or eliminate CDC mediated by antibody molecules. Mutations that reduce or eliminate CH2 domain binding 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 mutations" which involve the replacement of leucine residues at positions 1.3 and 1.2 of the IMGT of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, by changing the CH2 structure The asparagine (N) at position 84.4 of the IMGT domain in the domain is mutated to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q), thus converting the conserved N-chain sugar Mutation of the sylation site to generate α-glycosyl antibodies to reduce IgG1 effector function is also known (Wang et al., 2018). As an alternative, it is known that complement initiation (C1q binding) and ADCC can be reduced by mutating proline at position 114 of the IMGT position of the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to generate antibody molecules with further reduced or no 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 retains the ability to specifically bind to a given antigen (eg, PD-1 or OX4). The antigen-binding function of an antibody can be performed by fragments of the intact antibody. Examples of the term antigen-binding portion or antigen-binding fragment of an antibody include, but are not limited to, Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; F(ab) ₂ fragment, a fragment containing two Fab fragments A bivalent fragment, the two Fab fragments are connected by a disulfide bridge located in the hinge region; an Fd fragment, which is composed of VH and CH1 domains; an Fv fragment, which is composed of the VL and VH domains of the single arm of the antibody; A single domain antibody (dAb) fragment consisting of a VH domain or a VL domain; and an isolated complementarity determining region (CDR).

"A region equivalent to the Fc region of an immunoglobulin" includes variants of naturally occurring alleles of the Fc region of an immunoglobulin, as well as variants with substitutions, additions, or deletions that do not substantially reduce immunoglobulin-mediated effector function ( Modified variants of the ability such as antibody-dependent cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants may be selected so as to have minimal impact on activity according to general rules known in the art (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 changes with the isotype of the antibody. 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), cytokines Secreted, immune complex-mediated antigen-presenting cells Antigen uptake by cells, downregulation of cell surface receptors (such as B cell receptors), and B cell priming.

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

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

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 of the encoding molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be made to obtain a final construct having the desired properties, such as antigen-binding activity. Sites used for substitution typically include HVR and framework (FR). See Table 3 for possible substitutions of amino acids.

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

The term "isolated" nucleic acid molecule or polynucleotide as used herein refers to a nucleic acid molecule, DNA or RNA, that 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. Isolated polynucleotides include polynucleotide molecules that are normally present in the cell containing the polynucleotide molecule, but which are present extrachromosomally or in a chromosomal location that is different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, in positive and negative stranded forms, as well as double-stranded forms. The isolated polynucleotides or nucleic acids of the invention further include synthetically produced such molecules. Additionally, a polynucleotide or nucleic acid may be or may include regulatory elements, such as a promoter, a ribosome binding site, or a transcription terminator.

The term "expression cassette" as used herein refers to a recombinantly or synthetically produced polynucleotide having a series of nucleic acid elements that permit the transcription of a specific 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 the expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, expression cassettes of the invention comprise polynucleotide sequences encoding bispecific antigen-binding molecules of the invention, or fragments thereof.

The terms "vector" or "expression vector" and "expression construct" of the present invention are used interchangeably, and are DNA molecules to which a specific gene operably linked is introduced into a target cell and directs expression. Such vectors include vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. The expression vector of the present invention contains an expression cassette. Expression vectors enable the transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the gene is produced by the cellular transcription and/or translation machinery. Encoded ribonucleic acid molecules or proteins. In one embodiment, the expression vector of the invention includes an expression cassette containing 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 cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants/transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom. The nucleic acid of the offspring may not be completely identical to that of the parent cell and may contain mutations. A host cell is any type of cell that can be used to produce the bispecific antigen-binding molecules 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 include cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.

An "effective amount" of a drug is that amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as, for example, the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dosage or dosage regimen that avoids significant side effects or toxic effects.

The bispecific antibodies according to the invention have synergistic effects. "Synergistic effect" means that the combined effect of two drugs is greater than the sum of their individual effects and is statistically different from the control and the single drugs. Additive effect in the present invention means that the combined effect of 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) is that amount, in dosage and administration interval and time, that is effective in achieving the desired therapeutic or preventive 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 animals (such as cattle, sheep, cats, dogs and horses), primates (such as humans and non-human primates such as monkeys), rabbits and rodents (such as mice and rats). In particular, the individual or subject is a human being.

"Pharmaceutical composition" means a mixture containing one or more antibodies or antigen-binding fragments thereof of the disclosure together with other chemical components, such as physiologically/pharmaceutically acceptable carriers or excipients. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.

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

The term "cancer" is used to describe a disease in mammals characterized by unregulated cell growth. Examples of cancers include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of cancer include, but are not limited to, squamous cell carcinoma (eg, 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 cancer, hepatocellular carcinoma, Gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethra 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, cholangiocarcinoma, central nervous system (CNS) tumors , spinal axial tumors, brainstem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma and Ewing sarcoma, 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-transplant lymphoproliferative disorder (PTLD), as well as phakomatoses, edema (such as those associated with brain tumors) and Abnormal blood vessel proliferation, brain tumors and cancers associated with Meigs syndrome, and head or neck cancers and related metastases.

"Treatment" means administering to a patient an internal or external therapeutic agent, such as a composition comprising or encoding a specific binding protein, bispecific antibody, antibody or antigen-binding fragment thereof of the present disclosure. Antibodies, nucleic acid molecules of antibodies or antigen-binding fragments thereof, the patient has one or more diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Generally, a therapeutic agent is administered to a subject or population in an amount effective to alleviate 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" refers to delaying, inhibiting or preventing the onset of cancer in a mammal in which the initiation of carcinogenesis or tumorigenesis has not been proven, but has been identified, for example by genetic screening or other methods. Have cancer susceptibility. The term also includes treatment of a mammal with a precancerous condition to terminate the progression of the precancerous condition to malignancy or to cause regression thereof.

The term "liposome" is meant to include a variety of unilamellar and multilamellar lipid carriers formed by producing closed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess 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 thereof, for example, by oral administration, injection, or topical administration to the subject. who apply.

The sequence numbers (SEQ ID NO:) of the light chain, heavy chain and variable region of the antibody of the present invention are shown in Table 4.

The sequence number (SEQ ID NO:) of the framework region of the antibody of the present invention is shown in Table 5.

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

Figure 2 shows the structure of PD-1xOX40 bispecific antibody VH-IgG_HC (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 antibodies block the binding of PD-1 to PD-L1.

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

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

Figure 9 shows the effect of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure on the cell surface as the concentration increases in HEK 293 cells of human OX40, PD1 and NFkB promoter-luc, measured by flow cytometry. combine.

Figure 10 shows the binding of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure to the cell surface with increasing concentration in primed human T cells as measured 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 that CHO cells expressing human CD32b and HEK 293 cells expressing human OX40 and NFκB promoter-luc are co-cultured, and different concentrations of the bispecific antibody of the present invention or the monoclonal antibody constituting its structure are added to the luciferin Detection of enzyme reporter gene expression.

Figure 13 shows the co-culture of CHO cells expressing human PD-1 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 fluorescence 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 constituting its structure. Detection of luciferase reporter gene expression following antibody.

Figure 15 shows that in a 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 constituting its structure are added Secretion of the cytokine TNFα after antibodies.

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

Figure 17 shows the detection of IL10 secretion by Treg using flow cytometry after adding different concentrations of the bispecific antibody of the present invention to expanded Treg cells cultured in vitro.

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

Figure 19 shows that in the co-culture system of isolated human T cells, Treg cells expanded in vitro and DC cells induced and isolated in vitro, different concentrations of the bispecific antibody of the present invention or the structure thereof are added. Secretion of the cytokine Granzyme B following monoclonal antibodies.

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

The examples shown below are intended to illustrate specific embodiments of the invention and are not intended to limit the scope of the specification or claims in any way. The examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, methods of inserting protein-encoding genes into such vectors and plasmids, or methods of introducing 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 Obtaining anti-OX40 fully human HCAb antibodies

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

1.1 Immunization of HCAb mice

The Harbor HCAb human antibody transgenic mice aged 6 to 8 weeks were immunized for multiple rounds using two groups of immunization regimens. Specifically: immunization protocol 1, using recombinant human OX40-ECD-Fc (ChemPartner, #21127-022) antigen protein for immunization. The total injection dose received by each mouse per immunization was 100 μL by subcutaneous inguinal injection or by intraperitoneal injection. In the first round of immunization, each mouse was immunized with an immunogenic reagent prepared by mixing 50 μg of antigen protein and complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1. In each subsequent round of boosted immunization, each mouse received immunization with an immunogenic reagent mixed with 25 μg of antigenic protein and Ribi adjuvant (Sigma Adjuvant System, Sigma, #S6322). Immunization scheme 2, using HEK293/OX40 (ChemPartner, Shanghai) stable cell line overexpressing human OX40 for immunization. Each mouse was injected intraperitoneally with 2 × 10 ⁶ cell suspension during each immunization. The interval between each round of boosters is at least two weeks and usually no more than five rounds of boosters. The immunization time is day 0, day 14, day 28, day 42, day 56, and day 70; and on day 49 and day 77, the mouse serum antibody titer is detected. Five days before HCAb mouse spleen B cell isolation, a final booster immunization was performed at a dose of 25 μg OX40-ECD-Fc (ChemPartner, #21127-022) antigen 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 place it on an ELISA plate coated with human OX40-ECD-Fc An ELISA test was performed to determine the titer of anti-human OX40 in mouse blood, and flow cytometry was used to detect the response of two concentrations of mouse blood (1:100, 1:1000) to CHO-K1/hOX40 cells with high OX40 expression. (Chempartner, Shanghai) and specific reactivity of CHO-K1 mother cells. The blank control group (PB) was the serum of pre-immune mice.

1.2 Obtain the HCAb antibody sequence against OX40

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

The constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain HCAb antibodies. Detect the binding of the supernatant expressing HCAb to the stable cell line CHO-K1/OX40 overexpressing human OX40 (CHO-K1/hu OX40, Genscript, #M00561), and use a positive antibody (pogazumab) as a positive control , perform mirrorball® fluorescent cytometer ( SPT Labtech Ltd. ) screening. The specific steps are: wash CHO-K1/OX40 cells with serum-free F12K medium (Thermo, #21127022), and resuspend them in 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 in a dark place for 30 minutes. 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 this mixture from each well to the 384-well plate (Greiner Bio One, #781091). Then add 10 μL of positive control or HCAB-expressing supernatant to the 384-well plate and culture for 2 hours. Read fluorescence values on a mirrorball fluorescence flow cytometer. 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. Conventional sequencing methods are used to obtain the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence of the pure antibody. After removing repetitive sequences, the remaining sequenced pure antibody plasmids were transfected into HEK293 cells for expression. The obtained supernatant was again subjected to NF-kb functional test, thus obtaining 64 proteins that simultaneously bind CHO-K1/hu OX40 and cynomolgus monkey OX40. A functional, fully human OX40 monoclonal antibody with a unique sequence. Based on the human-monkey binding ability and NF-Kb functional test results, the top-ranked antibodies were selected for recombinant expression.

1.3 Preparation of fully human recombinant antibodies against OX40

The plasmid encoding the HCAb antibody obtained above is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and conventional recombinant protein expression and purification techniques are used to obtain purified anti-OX40 recombinant heavy chain antibodies. Specifically, HEK293 cells were expanded in FreeStyle ^™ F17 Expression Medium (Thermo, #A1383504). Before starting transient transfection, adjust the cell concentration to 6×10 ⁵ cells/mL and culture it in a 37°C 8% CO ₂ shaker for 24 hours. The cell concentration is 1.2×10 ⁶ cells/mL. Prepare 30 mL of cultured cells, dissolve 30 μg of the above 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)120μL and let stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop in the plasmid-PEI mixed solution while shaking the culture bottle, and culture in a 37°C 8% _CO2 shaker for 5 days. The cell viability was observed after 5 days. Collect the culture, centrifuge at 3300G for 10 minutes and take the supernatant; then centrifuge the supernatant at high speed to remove impurities. Equilibrate a gravity column (Bio-Rad, #7311550) containing MabSelect ^TM (GE Healthcare Life Science, #71-5020-91 AE) with PBS (pH 7.4) and rinse with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. Then use 0.1M glycine at pH 3.5 to elute the target protein, then use Tris-HCl at pH 8.0 to adjust to neutrality, and finally use an ultrafiltration tube (Millipore, #UFC901024) to concentrate and change the medium into PBS buffer to obtain purification. Anti-human OX40 HCAb monoclonal antibody solution. The antibody concentration was determined using NanoDrop detection absorbance at 280 nm, and the purity of the antibody was determined using SEC-HPLC and SDS-PAGE.

OX40 antibodies PR002067 and PR002063 were obtained, and their 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 produces and prepares a positive control antibody Pogalizumab analog (PR003475) against OX40. Its amino acid sequence is derived from IMGT data. The amino acid sequence of the antibody heavy chain can be found in SEQ ID NO: 74. The amino acid sequence of the antibody light chain is shown in SEQ ID NO: 78.

1.4 Analysis of protein purity and polymers using HPLC-SEC

Analytical size exclusion chromatography (SEC) was used to analyze the purity and aggregate form of the antibody protein samples obtained above. The analytical column TSKgel G3000SWx1 (Tosoh Bioscience, 08541, 5 μm, 7.8 mm × 30 cm) was connected to a high-pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II), and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg, and the sample concentration is adjusted to 1 mg/mL) is filtered with a 0.22 μm filter and injected into the system, and the HPLC program is set: use pH 7.4 PBS buffer to flow the sample through the chromatographic column at a flow rate of 1.0 mL/min. , the maximum time is 20 minutes; the detection wavelength is 280nm. After collection, use ChemStation software to integrate the chromatogram and calculate relevant data, generate an analysis report, and report the retention times of different molecular size components in the sample.

1.5 Analysis of protein purity and hydrophobicity using HPLC-HIC

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

1.6 Use DSF to determine the thermal stability of protein molecules

Differential Scanning Fluorimetry (DSF) is a commonly used high-throughput method for determining protein thermal stability. It uses a real-time fluorescence quantitative PCR instrument to reflect the denaturation process of the protein by monitoring changes in the fluorescence intensity of the dye bound to unfolded protein molecules, thus reflecting the thermal stability of the protein molecules. This embodiment uses the DSF method 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), and then buffer was added to make a final volume of 40 μL per well. Seal the PCR plate and place it in the real-time fluorescence quantitative PCR machine (Bio-Rad, model CFX96 PCR System), first incubate at 25°C for 5 minutes, then gradually increase the temperature from 25°C to 95°C with a gradient of 0.2°C/0.2 minutes, and reduce the temperature to 25°C at the end of the test. Use FRET scanning mode and use Bio-Rad CFX Maestro software to analyze the data and calculate the Tm of the sample.

The results of testing the physical and chemical properties of OX40 antibodies PR002067 and PR002063 are shown in Table 6.

1.7 ELISA detects the binding ability of OX40 HCAb monoclonal antibody protein level

This example is to study the activity of the prepared anti-OX40 HCAb monoclonal antibody in vitro binding to human and cynomolgus monkey OX40 protein. 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 1ug/mL human OX40 protein and cynomolgus monkey OX40 protein dissolved in PBS, and incubated at 4°C overnight. The next day, the 384-well plate was washed three times with PBS containing 0.05% Tween (MEDICAGO, #09-9410-100) and blocked with PBS containing 2% milk (Bio-Rad, #170-6404) at 37°C for 1 hour. . The initial concentration of the OX40 antibody and positive antibody (Pogalizumab) to be tested is 10nM, and a 4-fold gradient dilution is performed. The blocked 384-well plate was washed three times with PBST, 10 μL PBS or 10 μL 4-fold gradient dilution of antibody and positive control (Pogalizumab) were added to the plate, and incubated at room temperature for 1 hour. Wash three times, add 20 μL of goat anti-human Fc horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc., #109-035-098) to each well, and incubate at 37°C for 40 minutes. Wash 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 values were analyzed and graphed using 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 is similar to Tab (Pogalizumab), indicating that the antibody can be more effective at lower concentrations. Sensitively binds human OX40.

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

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

Example 2 Preparation of anti-PD-1 H2L2 antibodies

In order to prepare antibody molecules that specifically bind to PD-1, PD-1 antigen can usually be used to immunize experimental animals, which can be mice, rats, rabbits, sheep, camels, etc. Typically, the resulting antibody molecules are of non-human origin. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability. However, the process of humanizing antibodies has its technical complexities, and humanized molecules often have reduced affinity for the antigen. On the other hand, advances in transgenic technology have made it possible to create genetically engineered mice carrying a human immunoglobulin immune repertoire and deleting the endogenous murine immune repertoire. The antibodies produced by this transgenic mouse have fully human sequences, so no further humanization is required, which greatly improves the efficiency of therapeutic antibody development.

Harbor H2L2 mice (Harbour Antibodies BV) are a type of mouse that carries human immune cells Transgenic mice of the protein immune library produce antibodies with complete human antibody variable domains and rat constant domains.

2.1 PD-1 immunized 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, the total injection dose received by each mouse was 100 μL. In the first round of immunization, each mouse received immunization with an immunogenic reagent prepared by mixing 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 boosted immunization, each mouse received immunization with an immunogenic reagent mixed with 25 μg of antigenic protein and Ribi adjuvant (Sigma Adjuvant System, #S6322). The interval between each round of boosters is at least two weeks and usually no more than five rounds of boosters. The immunization time is day 0, day 14, day 28, day 42, day 56, and day 70; and on day 49 and day 77, the mouse serum antibody titer is detected. Three days before cell fusion, a final booster immunization was performed with a dose of 25 μg of antigen 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 coat it with human PD-1-His (Shanghai ChemPartner ) ELISA plate was used to perform ELISA testing to determine the titer of anti-human PD-1 in mouse blood, and flow cytometry was used to detect the high levels of anti-PD-1 in mouse blood at two concentrations (1:100, 1:1000). Specific reactivity of expressed CHO-K1/hPD-1 cells (Shanghai ChemPartner) and CHO-K1 parent cells. The blank control group (PB) was the serum of pre-immune mice.

2.2 Obtain hybridoma monoclonal and antibody sequences

When the PD-1-specific antibody titer in the serum of H2L2 mice reaches a certain level, the spleen cells of the mice are removed and fused with myeloma cell lines to obtain hybridoma cells; the hybridoma cells undergo multiple rounds of screening and selection. After colonization, at least 8 hybridomas expressing anti-PD-1 monoclonal antibody molecules are isolated. The isolated hybridoma cells and the monoclonal antibodies they express are represented by the corresponding selection numbers, for example: 4004_10H9A12, 4004_12H9C1 and so on. The isolated hybridomas express antibody molecules with intact human variable domains and rat constant domains of heavy and light chains. The above monoclonal antibodies were further identified, and several hybridoma clones were selected based on parameters such as their binding ability to human PD-1, cynomolgus monkey PD-1 binding ability, and ability to inhibit the binding of PD-1 and PD-L1. Perform sequencing. Conventional hybridoma sequencing methods are used to obtain the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence. In this example, the sequence of the variable domain of the anti-PD-1 monoclonal antibody molecule obtained from the immunized Harbor H2L2 mice is a human antibody sequence. In the present invention, the CDR sequence is divided by Chothia definition rules.

The heavy chain variable domain sequence of the antibody is derived from events such as gene rearrangements and somatic high-frequency mutations of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement of germline gene V and J gene segments of the light chain gene group and somatic high-frequency mutations. Gene rearrangements and high-frequency somatic mutations are major factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but overall the similarity is high. Use some 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 rearrangements occur can be inferred from the variable domain sequence of the antibody.

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

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

Table 10. Sequence numbers of anti-PD-1 H2L2 antibodies (SEQ ID NO:)

2.3 Preparation of recombinant fully human antibodies

After obtaining the light chain and heavy chain variable domain sequences encoding antibody molecules in the above 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 chains. 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 gene synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody kappa light chain constant domain sequence to encode The full-length light chain of the antibody is produced. The antibody heavy chain variable domain sequence (VH) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG4 antibody heavy chain constant domain sequence (SEQ ID NO: 143) to encode the production of IgG4 antibodies. Full length heavy chain.

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

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

2.4 Analysis of protein purity and multimeric forms using HPLC-SEC

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

2.5 Change the Fc sequence of H2L2 antibody

The Fc of PR000674 was modified, the antibody type was converted from IgG4 to IgG1 and LALA (L234A and L235A mutations, numbered according to EU index) mutations were introduced to obtain PR001985. Table 10 lists the light and heavy chain variables of the PR001985 antibody. Domain amino acid sequence, light chain full-length amino acid sequence, heavy chain (human IgG1) full-length amino acid sequence, and amino acid sequence of CDRs defined according to Chothia definition rules.

2.6 Antibody sequence optimization

In this example, amino acid mutations were performed on the potential PTM site of the PR001985 antibody to obtain a new antibody molecule PR006429 (called the 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 and recombinant antibodies were obtained 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 in binding human/cynomolgus 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) that overexpress human or cynomolgus PD-1. Briefly, CHO-K1-hPD-1 cells were digested and resuspended in F-12K complete medium, and the cell density was adjusted to 1 × ¹⁰ cells/mL. Inoculate 100 μL cells/well in a 96-well V-bottom plate (Corning, Cat#3894), then add 100 μL/well of the antigen-binding protein to be tested, 2 times the final concentration and 5 times the final concentration gradient dilution, and mix evenly, in which the antigen-binding protein The highest final concentration was 100nM or 300nM, with a total of 8 to 11 concentrations, and hIgG was used as a control. Place the cells at 4°C and incubate in the dark for 1 hour. Then, add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well of 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 in the dark for 30 minutes. Wash the cells twice with 200 μL/well pre-cooled PBS, centrifuge at 500 g and 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 protein to CHO-K1 cells overexpressing human PD-1

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

2.8 Antigen-binding protein blocks 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 to human PD-L1 in vitro, the CHO-K1 cell line overexpressing human PD-1 (CHO-K1-hPD-1) was used to conduct cell-level experiments. Human PD-1/human PD-L1 binding blocking experiment. Briefly, CHO-K1-hPD-1 cells were digested and resuspended in F-12K complete medium, and the cell density was adjusted to 1 × ¹⁰ cells/mL. Inoculate 100 μL cells/well in a 96-well V-bottom plate (Corning, Cat#3894), and then add 100 μL/well of the antigen-binding protein to be tested, 2 times the final concentration, 3 times or 5 times the gradient dilution of the antigen-binding protein to be tested, and mix evenly. The highest final concentration of antigen-binding protein is 100nM or 300nM, with a total of 8 concentrations, and hIgG is used as a control. Place the cells at 4°C and incubate in the dark for 1 hour. Afterwards, centrifuge for 5 minutes at 4°C, discard the supernatant, then add 50 μL/well, 1 μg/mL concentration of biotin-labeled human PD-L1 protein (AcroBiosystems, PD1-H82F2), and incubate at 4°C in the dark for 30 minutes. Add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g and 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 in the dark for 30 minutes. Wash the cells twice with 200 μL/well pre-cooled PBS, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well pre-chilled PBS, use BD FACS CANTOII to read the fluorescence 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 the same as that of the positive control. Keytruda (pembrolizumab) is equivalent.

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

The anti-OX40 antibody prepared in Example 1 and the anti-PD-1 antibody prepared in Example 2 are combined to prepare a bispecific antibody, which can bind to two targets at the same time, one end (the first domain) of which can recognize the surface of tumor cells. Specifically expressed PD-1, and the other end (the second domain) can bind to the OX40 molecule on T cells. When the PD-1×OX40 dual-antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing the 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 number), R200538, PR200539, PR200531, PR200536, PR200528 and PR200600 have Fc L234A, L235A and G237A), see Figure 1-5 for its molecular structure.

3.1 Construction of bispecific antibodies with IgG_HC-VH tetravalent symmetric structure (Structure 1)

The anti-PD-1 H2L2 antibody and the anti-OX40 HCAb antibody were used to construct bispecific antibodies PR003787, PR200531, and PR200528 with an IgG_HC-VH tetravalent symmetric structure. The binding protein of the IgG_HC-VH tetravalent symmetry structure (see Figure 1) contains two polypeptide chains: polypeptide chain 2, also called a short chain, from the amino end to the carboxyl end, which contains N'-VL ₁ -CL ₁ -C'; Polypeptide chain 1, also called long chain, from the amino end to the carboxyl end, it contains N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L-VH ₂ -C'. Wherein, the VL ₁ and VH ₁ are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region of the IgG antibody, and the CL ₁ is the first structure CL of the domain, the CH ₁ is the CH 1 of the first domain, the L is a connecting peptide, and CH3 of the polypeptide chain 1 is connected to VH ₂ via L. The linking 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 linking peptide L in the bispecific antibodies PR200531 and PR200528 is (G2S) ₂ , whose amino acid sequence is shown in SEQ ID NO: 116 in Table 15. The sequence is shown in Table 15 as SEQ ID NO: 117.

3.2 Construction of bispecific antibody with VH-IgG_HC quadrivalent symmetric structure (Structure 2)

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

3.3 Construction of bispecific antibodies with Fab(CL)-VH-Fc tetravalent symmetric 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 Fab(CL)-VH-Fc tetravalent symmetric structure. The binding protein of Fab(CL)-VH-Fc tetravalent symmetry structure (see Figure 3) contains two polypeptide chains: polypeptide chain 2, also known as short chain, from the amino end to the carboxyl end, which contains N'- VH'-CH1'-C'; polypeptide chain 1, also called long chain, from the amino end to the carboxyl end, it contains N'-VL'-CL'-L-VH ₂ -h-CH2-CH3-C' . Wherein, the VL' and VH' are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region, the L is the connecting peptide, and the CL ' is the CL of the first domain, and the CH1' is the CH1 of the first domain. The CL' of polypeptide chain 1 in PR200539 is directly fused and connected to VH2, that is, the length of L is 0.

3.4 Construction of bispecific antibody with hexavalent symmetrical structure of IgG_HC-VH-VH (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 an IgG_HC-VH-VH hexavalent symmetric structure. The binding protein of the IgG_HC-VH-VH hexavalent symmetric structure (see Figure 4) contains two polypeptide chains: polypeptide chain 2, also known as the short chain, from the amino end to the carboxyl end, which contains N'-VL ₁ - CL ₁ -C'; Polypeptide chain 1, also called long chain, from the amino end to the carboxyl end, it contains N'-VH1-CH ₁ -h-CH2-CH3-L1-VH ₂ -L2-VH ₂ -C '. Wherein, the VL ₁ and VH ₁ are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region, the L1 and L2 are the connecting peptides, and the The CL1 is the CL of the first structural domain, and the _CH1 is the CH1 of the first structural domain. CH3 of polypeptide chain 1 is connected to VH ₂ via L1, and the two VH _2s are connected via 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 antibodies with IgG_HC-VH-VH-VH eight-valent 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 eight-valent symmetric structure of IgG_HC-VH-VH-VH. The binding protein of the IgG_HC-VH-VH-VH eight-valent symmetry structure (see Figure 5) contains two polypeptide chains: polypeptide chain 2, also known as the short chain, from the amino end to the carboxyl end, which contains N'-VL ₁ -CL ₁ -C'; Polypeptide chain 1, also called long chain, from the amine end to the carboxyl end, it contains N'-VH1-CH ₁ -h-CH ₂ -CH3-L1-VH ₂ -L ₂ - _VH2 -L3- _VH2 -C'. Wherein, the VL ₁ and VH ₁ are the VL and VH of the first domain respectively, the VH ₂ is the VH of the second domain, the h is the hinge region, and the L1, L ₂ and L ₃ are Connecting peptide, the CL ₁ is the CL of the first domain, and the CH ₁ is the CH ₁ of the first domain. CH3 in polypeptide chain 1 is connected to the first VH ₂ via the connecting peptide L1 (ordered from the N-terminal to the C-terminal of the polypeptide chain-1), the first VH ₂ and the second VH ₂ are connected through the connecting peptide L2, The two VH ₂ and the third VH ₂ are connected through the connecting peptide L3, where L1 is (G2S) 2, L2 is (G4S) 3, and L3 is (G4S) _2. The sequences of L1, L2 and L3 are as shown in Table 15 respectively. SEQ117, 118, and 119 are shown.

The amino acid sequence of the polypeptide chain of the bisantibody molecule obtained by the present invention is shown in Table 16 in the sequence listing.

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

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

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 aims 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 overexpressing human PD-1 (CHO-K1-hu PD-1, Shanghai ChemPartner) was used to conduct antibody binding experiments at the cellular level. Briefly, CHO-K1-hu PD-1 cells were digested and resuspended in DMEM complete medium, and the cell density was adjusted to 1 × ¹⁰ cells/mL respectively. Inoculate 100 μL cells/well in a 96-well V-bottom plate (Corning, Cat#: 3894), and then add 100 μL/well of the antibody to be tested in a 5-fold concentration gradient dilution of 2 times the final concentration. Place the cells at 4°C and incubate in the dark for 1 hour. Afterwards, add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well of fluorescent secondary antibody (Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson, Cat#: 109-545-06, 1:500 dilution), and incubate at 4°C in the dark for 30 minutes. . Wash the cells twice with 100 μL/well pre-cooled PBS, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Finally, the cells were resuspended in 200 μL/well pre-cooled PBS, and the fluorescence signal value was read using a BD Accuri C6 Plus flow cytometer.

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 the monoclonal antibodies PR001985 and PR006429 composed of them in this example are 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 antibodies and 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 in binding to human OX40, the HEK293 cell line that highly expresses OX40 (OX40/NF-κB Reporter-HEK293, BPS BioScience, Cat# 60482) was used for cell and Human OX40 binding assay. Briefly, HEK293 cell suspensions that highly express OX40 were collected, and the cell density was adjusted to 2 × 10 ⁶ cells/mL respectively. Inoculate 50 μL cells/well in a 96-well V-bottom plate (Corning, Cat#: 3894), and then add 50 μL/well of the antibody to be tested in a gradient dilution of 2 times the final concentration and 5 times the final concentration. Place the cells at 4°C and incubate in the dark for 2 hours. Afterwards, add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well of fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, Cat#: 109-605-098, 1:1000 dilution), and incubate at 4°C in the dark for 1 hours. Wash the cells twice with 100 μL/well pre-cooled PBS, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well pre-cooled FACS, and use 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 or monoclonal antibodies composing their structures in human HEK293/OX40/NF-kb reporter cells determined by flow cytometry. PR002067 specifically binds to HEK293 cells overexpressing human OX40. combined, and the binding to cell surface OX40 increases as the concentration increases. Moreover, the OX40 antibody arms of double antibodies PR003787 and PR200538 have the same or better binding ability than 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 cells co-expressing OX40 and PD-1. The HEK293 cell line that highly expresses OX40 (OX40/NF-κB Reporter-HEK293, BPS BioScience, Cat# 60482) was used, and lentivirus was further used to construct a stably expressing human PD1 cell line (Origene, Cat# PS100092V), which was screened for puromycin resistance. Finally, a HEK293 cell line stably expressing OX40 and PD-1 was obtained, and the obtained cell line was used for cell binding experiments. Briefly, HEK293 cell suspensions that highly express OX40 and PD-1 were collected, and the cell density was adjusted to 2 × 10 ⁶ cells/mL respectively. Inoculate 50 μL cells/well in a 96-well V-bottom plate (Corning, Cat#: 3894), and then add 50 μL/well of the antibody to be tested in a gradient dilution of 2 times the final concentration and 5 times the final concentration. Place the cells at 4°C and incubate them in the dark for 2 hours. Afterwards, add 100 μL/well pre-cooled PBS to rinse the cells twice, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Then add 100 μL/well of fluorescent secondary antibody (Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific, Jackson ImmunoResearch, Cat#: 109-605-098, 1:1000 dilution), and incubate at 4°C in the dark for 1 hours. Wash the cells twice with 100 μL/well pre-cooled PBS, centrifuge at 500 g and 4°C for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 200 μL/well pre-cooled FACS, and use BD Accuri C6 Plus Flowcytometer to read the fluorescence signal value.

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

Example 7

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

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

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

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

Each well is plated with 100 μL of culture medium, 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 a 2-fold dilution of the antigen-binding protein to be tested to each well, starting with a total concentration of 200 nM, 4-fold gradient dilution, and hlgG1 is the negative control group. 40 μL of 4.5 × 10 ⁴ HEK293 reporter cells that sustainably express luciferase reporter genes for OX40 and NF-kb response elements (HEK293/OX40/NF-kb reporter cells, BPS Biosciences, #60482) were added to each well. Incubate for 6 hours at 37°C in a 5% _CO2 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 of Figures 11 to 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 priming effect on HEK293/OX40/NF-kb reporter cells. The results in Figure 12 show that the OX40 parental monoclonal antibody showed a concentration-dependent enhancement of 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. -Kb signal activates the function. The results in Figure 13 show that the PD-1×OX40 bispecific antibody PR003787 specifically causes a concentration-dependent enhancement of the NF-Kb signaling pathway with the aid of cross-linking of CHO-K1/PD-1 cells.

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

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

HEK293 cells overexpressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were spread on a 96-well plate, the cell volume was 1.25×10 ⁴ /well, 100 μL/well. Add 50 μL/well of the dilution solution of the antigen-binding protein to be tested, with a starting concentration of 100 nM and a 4-fold dilution. hlgG1 is the control group. Add 5×10 ⁴ /well of Jurkat reporter cells (constructed by Shanghai ChemPartner) that can sustainably express PD-1 and NFAT-luciferase reporter genes, 50 μL/well. Incubate at 37°C for 6 hours in a 5% _CO2 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 results are shown in Figure 14. Display the PD-1×OX40 bispecific antibody of the present invention (PR003787) and the parent antibody of PD-1 both have inhibitory effects on the PD-1 signaling pathway.

Example 10 Detection of activation function of T cells by PD-1×OX40 bispecific antibody

Purchase human concentrated leukocytes (Research blood components LLC), isolate human peripheral blood mononuclear cells (PBMC), and then co-culture human PBMC cells and mitomycin-treated CHOK1-huPD-L1 cells, add 2.5ug/mL PHA-L (eBioscience, #00-4977-03) stimulated 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 Figures 15 and 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), and add CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF ), during the culture period, fresh culture medium containing IL-2 is added every 2-4 days according to the cell growth status. After 12 days of culture, the proportion of Treg is analyzed by flow cytometry, and the Treg cells expanded in vitro are cryopreserved. The preserved Treg cells were thawed and cultured with culture medium containing recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF) for 2 days, and then the Treg cells and mitomycin-treated CHOK1- huPD-L1 cells were co-cultured, CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) were added and cultured for 5 days. Cells were collected and the ratio of FoxP3 and interleukin IL-10 secreting cells was detected using BD Accuri C6 Plus flow cytometer.

As shown in Figure 17, the PD-1×OX40 bispecific antibody (PR003787) and the parent antibody of OX40 (PR002067) of the present invention are effective in inhibiting the secretion of IL-10 by Treg cells compared with the human IgG1 isotype (PR000324). inhibitory effect.

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

Purchase concentrated human leukocytes (Research blood components LLC), isolate T cells (StemCell, #17951), and freeze them. At the same time, Treg cells (StemCell, Cat#18063) were isolated, and CD3/CD28 magnetic beads and recombinant human interleukin 2 (IL-2) (R&D, #202-IL-010/CF) were added. During the culture period, the cell growth status was determined every 2 On day -4, fresh culture medium containing IL-2 was added. After 12 days of culture, the proportion of Treg was analyzed by flow cytometry.

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

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 parent monoclonal antibody or the two parental PD-1 and OX40 combined monoclonal antibodies, and co-culture for 4-5 days. 100 μL of culture supernatant was collected and the secretion of IL-2, Granzyme B and IFNγ was detected 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 better than that of the PD-1 monoclonal antibody (PR001985) and the OX40 monoclonal antibody (PR002067), and is superior to The combination of PD-1 monoclonal antibody (PR001985) and OX40 monoclonal antibody (PR002067) showed synergistic activity.

Example 13 PHA detects the T cell priming effect of PD-1×OX40 bispecific antibody

Purchase concentrated human leukocytes (Research blood components LLC), isolate human peripheral blood mononuclear cells (PBMC), and then add 2.5ug/mL PHA-L (eBioscience, #00-4977-03) to the 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 ability to activate T cells. The ability to secrete IL-2. And with Compared with PR003787, PR200538, PR200539, and PR200528 show dose-dependent T cell priming ability and have stronger T cell activation ability under high concentration conditions.

<110> HARBOUR BIOMED(SHANGHAI)CO.,LTD

<120> Specific binding proteins targeting PD-1 and/or OX40 and their applications

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Claims (20)

An antibody or an antigen-binding fragment targeting PD-1 or a fragment thereof, characterized in that the antibody or an antigen-binding fragment thereof includes a light chain variable region VL and a heavy chain variable region VH, the VL and the VH respectively includes three CDRs, wherein: the VL includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively represented by SEQ ID NO: 39, 46 and 54; and the VH includes the amino acid sequence represented by SEQ ID NO: 39, 46 and 54 respectively. HCDR1, HCDR2 and HCDR3 as shown in NO:8, 18 and 28; or the VL comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are respectively represented by SEQ ID NO: 39, 46 and 54; and the VH comprises an amine The amino acid sequences are shown in SEQ ID NO: 81, 86 and 91 respectively for HCDR1, HCDR2 and HCDR3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VL variable region includes the amino acid sequence shown in SEQ ID NO: 67; and the VH includes the amino acid sequence shown in SEQ ID NO: 62 The amino acid sequence; or the VL variable region includes the amino acid sequence shown in SEQ ID NO: 67; and the VH includes the amino acid sequence shown in SEQ ID NO: 102. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody further includes a heavy chain constant region and/or a light chain constant region. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is selected from the group consisting of full-length antibodies, Fab, Fab’, F(ab’)2, Fv or scFv. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein the light chain includes an amine as shown in SEQ ID NO: 77 The amino acid sequence; and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 71; the light chain includes the amino acid sequence shown in SEQ ID NO: 77; and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 77; and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 77. The amino acid sequence shown in SEQ ID NO: 72; or The light chain includes an amino acid sequence as shown in SEQ ID NO:77; and the heavy chain includes an amino acid sequence as shown in SEQ ID NO:106. A bispecific antibody or an antigen-binding fragment thereof, comprising: a first structural domain that binds PD-1 or a fragment thereof, the first structural domain being a target as described in any one of claims 1 to 5 An antibody to PD-1; and a second domain that binds OX40 or a fragment thereof, the second domain being a VH structure, the second domain comprising a heavy chain variable region, wherein the heavy chain variable The region includes the amino acid sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 19, and SEQ ID NO: 29 respectively; or the heavy chain variable region includes the amino acid sequences respectively HCDR1, HCDR2, and HCDR3 represented by SEQ ID NO:9, SEQ ID NO:85, and SEQ ID NO:90. The bispecific antibody or antigen-binding fragment thereof according to claim 6, wherein the bispecific antibody or antigen-binding fragment thereof includes two polypeptide chains, wherein: polypeptide chain 2 has N'-VL ₁ -CL ₁ - The structure shown by C', polypeptide chain 1 has the structure shown by N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L-VH ₂ -C'; or polypeptide chain 2 has N'-VL ₁ -CL ₁ -C', polypeptide chain 1 has the structure N'-VH ₂ -L-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -C'; or polypeptide chain 2 has N _{or ​ ​ ​} Polypeptide chain 2 has the structure shown by N'-VL ₁ -CL ₁ -C', and polypeptide chain 1 has N'-VH ₁ -CH ₁ -h-CH ₂ -CH ₃ -L ₁ -VH ₂ -L ₂ - The structure represented by VH ₂ -C'; or the polypeptide chain 2 has the structure represented by N'-VL ₁ -CL ₁ -C', and the polypeptide chain 1 has the structure represented by N'-VH ₁ -CH ₁ -h-CH ₂ -CH The structure shown in ₃ -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 first structural domain VL 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 CL, CH ₁ and CH ₁ ' of the first domain are CH ₁ of the first domain. The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the polypeptide chain 1 and the polypeptide chain 2 form a four-valent symmetrical structure, a six-valent symmetrical structure or an eight-valent symmetrical structure. The bispecific antibody or antigen-binding fragment thereof as described in claim 6 or 7, wherein the second domain comprises a heavy amino acid sequence as shown in SEQ ID NO: 63 or SEQ ID NO: 101. chain variable region. The bispecific antibody or antigen-binding fragment thereof according to claim 9, wherein Fc is a human IgG1 structure. The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the bispecific antibody or antigen-binding fragment thereof includes: polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO: 79 ; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO: 77; or polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO: 109; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in NO: 77; or polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO: 110; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO: 77 ; or polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO: 111; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO: 77; or polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in NO: 112; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO: 77; or polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO: 114 ; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO: 113; or polypeptide chain 1, including the amino acid sequence shown in SEQ ID NO: 115; and polypeptide chain 2, including the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in NO:77. An isolated nucleic acid molecule encoded as described in any one of claims 1 to 5 An antibody or an antigen-binding fragment thereof or a bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 6 to 11. An expression vector comprising the isolated nucleic acid molecule as described in claim 12, wherein the expression vector is a eukaryotic cell expression vector or a prokaryotic cell expression vector. A host cell comprising the isolated nucleic acid molecule as described in claim 12, or the expression vector as described in claim 13, wherein the host cell is a prokaryotic cell and/or a eukaryotic cell. A method for preparing 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 to 11, including In the case of expressing 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 11, culture 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 to 11 ; and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition further comprises a second therapeutic agent, and the second therapeutic agent includes a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressive agent, or a cytotoxic drug. An antibody or 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 11, as described in claim 12 The isolated nucleic acid molecule, the expression vector as described in claim 13, the host cell as described in claim 14, or the pharmaceutical composition as described in claim 16 or 17 is used in the preparation of prevention, treatment and/or diagnosis. Use in medicines for neoplastic diseases, acute and chronic inflammatory diseases and/or immune diseases. The use according to claim 18, wherein the tumor is selected from the group consisting of breast cancer, renal cell carcinoma, melanoma, colon cancer, B-cell lymphoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urinary tract cancer, Skin 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 lymphoma, cutaneous squamous cell carcinoma, locally advanced or metastatic malignancy or more; the inflammatory disease is atopic dermatitis or ulcerative colitis, and the immune disease is graft versus host disease, rheumatoid arthritis, systemic lupus erythematosus or asthma. A set of pharmaceutical kits, which includes one or more pharmaceutical kits, the one or more pharmaceutical kits including the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, as in claims 6 to 11 The bispecific antibody or antigen-binding fragment thereof according to any one of them, or the pharmaceutical composition according to claim 16 or 17.

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