A bifunctional antibody against pd-1 and ox-40 and preparation method and application thereof

Abstract

The present application relates to an anti-PD-1 and OX-40 bifunctional antibody and a preparation method and application thereof. The bifunctional antibody comprises an antibody domain targeting PD-1 and an antibody domain targeting OX-40. The present application designs a novel anti-PD-1 / OX-40 bifunctional antibody, constructs a specific structure bifunctional antibody by using a PD-1 activating antibody and an OX-40 blocking antibody, and the whole synergizes, has high affinity to PD-1, can activate PD-1 activity and its signal pathway, has high affinity to OX40, can block OX40 / OX40L interaction and its signal transduction, inhibits T cell proliferation, inhibits CD25 or PD-1 positive CD4 + T cell production can be applied to the development of related drugs for treating inflammatory or autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of inflammatory or autoimmune disease technology, and relates to a bifunctional antibody against PD-1 and OX-40, its preparation method and application. Background Technology

[0002] Programmed death receptor 1 (PD-1), also known as CD279, is a 55 kDa transmembrane protein encoded by the PDCD1 gene located in band 3 of region 37 (2q37.3) on the long arm of chromosome 2. PD-1 is a member of the immunoglobulin B7-CD28 family and consists of an extracellular region, a hydrophobic transmembrane region, and an intracellular region. Its intracellular region contains the immunoreceptor tyrosine-based inhibitory motif (ITIM) and the immunoreceptor tyrosine-based switch motif (ITSM). Activation of the ITSM is closely related to effector T cell activity. PD-1 protein is expressed on B lymphocytes, T lymphocytes, natural killer cells (NK cells), monocytes, and dendritic cells (DCs).

[0003] Activation of the PD-1 / PD-L1 signaling pathway can lead to immunosuppressive activity. PD-L1-induced PD-1 aggregation has been found to promote the recruitment of SHP2 phosphatase, which then inhibits T cell activity via CD28 dephosphorylation. Conversely, PD-1 deficiency or lack of activity can lead to autoimmunity. For example, PD-1 knockout in C57BL / 6 mice results in lupus-like syndrome. In humans, mutations in the PD-1 gene are highly associated with the development and progression of systemic lupus erythematosus, type 1 diabetes, rheumatoid arthritis, and multiple sclerosis. Against this backdrop, developing a PD-1 agonist (such as a PD-1 agonist antibody) to enhance PD-1-mediated immunosuppressive signaling, thereby inhibiting or treating inflammatory and autoimmune diseases, has significant clinical implications. This strategy not only opens new avenues for the development of innovative antibody drugs targeting immune checkpoints but also provides new research directions for the treatment of immune-related diseases.

[0004] OX40 (also known as CD134, ACT35, or TNFRSF4) is a member of the TNFR superfamily and is a 50kD membrane glycoprotein composed of a cytoplasmic tail, a transmembrane region, and an extracellular region. OX40 is virtually unexpressed in inactive naive T cells but highly expressed in activated T cells. OX40 ligand (OX40L) is a 34kD type II membrane protein expressed as a trimer on antigen-presenting cells. In OX40 knockout mice, antigen stimulation resulted in reduced proliferation of CD4 and CD8 T cells and the development of a defective immune memory response, indicating that endogenous OX40 plays a crucial role in T cell immune regulation.

[0005] OX40 is essential for T cell proliferation and survival. T cells lacking OX40 cannot survive effectively during and after division, which is associated with decreased expression levels of anti-apoptotic Bcl-2 family members. Using natural ligands (OX40L) or activating antibodies to bind to the OX40 receptor on the T cell surface can activate multiple signaling pathways, including the nuclear factor kappa-light-chain enhancer of activated B (NF-κB) and the activated T cell transcription factor (NFAT) pathway. This enhances the expression of multiple protein molecules, such as survival proteins, cyclin A, cyclin-dependent kinases, and Bcl-2 anti-apoptotic molecules, and increases the survival rate and proliferation of various effector T cell subsets after antigen stimulation. OX40L-OX40 signaling can regulate cytokine secretion from T cells, antigen-presenting cells, and NKT cells, and modulate signal transduction in cytokine receptor transduction pathways. OX40 is mainly found in activated CD4+ cells. + T cells and certain activated CD8 cells + It is expressed in T cells and is highly expressed in memory T cells of some chronic inflammatory and autoimmune diseases. Its ligand (OX40L) is also expressed in presenting cells as the cells are activated.

[0006] Following the clinical trials of various activating OX40 antibodies for cancer immunotherapy, inhibitory antibodies against OX40 have also demonstrated protective effects in animal models of various autoimmune diseases, such as asthma, irritable bowel disease, transplant rejection, autoimmune diabetes, graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis, and atopic dermatitis (AD). Atopic dermatitis, a chronic, relapsing inflammatory skin disease characterized by intense itching (e.g., pruritus) and scaly and dry eczematous lesions, significantly impacts patients' quality of life. Currently, treatment options for AD remain limited, while blocking the OX40 / OX40L signaling pathway can specifically inhibit the function of disease-inducing effector T cells without causing widespread immunosuppression.

[0007] In recent years, the construction of bifunctional antibodies has created a new avenue for antibody drug development. Many bifunctional antibody drugs have entered clinical trials, demonstrating stronger therapeutic effects than their corresponding monoclonal antibodies. Therefore, constructing bifunctional antibodies using humanized PD-1 activating antibodies and OX-40 blocking antibodies could further enhance immunosuppression against autoimmune diseases and improve the therapeutic efficacy of antibodies for these diseases. However, the number of publicly available anti-PD-L1 and OX40 bispecific antibodies is currently limited. Therefore, developing novel anti-PD-L1 and OX40 bispecific antibodies is of great significance for the treatment of autoimmune diseases. Summary of the Invention

[0008] To address the shortcomings of existing technologies and practical needs, this invention provides a bifunctional antibody against PD-1 and OX-40, its preparation method, and its applications. By designing and constructing a bifunctional antibody comprising a PD-1 activating antibody and an OX-40 blocking antibody, this invention provides a novel bifunctional antibody, offering new ideas for the development of drugs for the treatment of chronic inflammation or autoimmune diseases.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a bifunctional antibody against PD-1 and OX-40, the bifunctional antibody comprising an antibody domain targeting PD-1 and an antibody domain targeting OX-40; the amino acid sequences of the heavy chain complementarity-determining regions CDR1-CDR3 of the antibody domain targeting PD-1 sequentially include the sequences shown in SEQ ID NO.1 to SEQ ID NO.3, and the amino acid sequences of the light chain complementarity-determining regions CDR1-CDR3 sequentially include the sequences shown in SEQ ID NO.4 to SEQ ID NO.6; the amino acid sequences of the heavy chain complementarity-determining regions CDR1-CDR3 of the antibody domain targeting OX-40 sequentially include the sequences shown in SEQ ID NO.9 to SEQ ID NO.11, and the amino acid sequences of the light chain complementarity-determining regions CDR1-CDR3 sequentially include the sequences shown in SEQ ID NO.12 to SEQ ID NO.14.

[0011] This invention designs a novel bifunctional antibody against PD-1 and OX-40, which possesses high PD-1 and OX-40 binding activity. It can activate PD-1 and its downstream signal transduction, thereby significantly inhibiting immune activity, blocking the interaction and signal transduction activity of OX40 / OX40L, inhibiting and regulating the effect and level of OX40, and significantly inhibiting the body's immune inflammatory response. It has broad application prospects in the field of developing drugs for the treatment of chronic inflammation or autoimmune diseases.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the antibody domain targeting PD-1 includes the sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8.

[0013] Preferably, the amino acid sequence of the heavy chain variable region of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO. 16.

[0014] Preferably, the antibody domain targeting OX-40 is an scFv fragment.

[0015] Preferably, the amino acid sequence of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO.17 and / or SEQ ID NO.18.

[0016] Preferably, the C-terminus of the heavy chain of the antibody domain targeting PD-1 is connected to the N-terminus of the antibody domain targeting OX-40.

[0017] Preferably, the amino acid sequence of the heavy chain variable region of the antibody domain targeting PD-1 in the anti-PD-1 and OX-40 bifunctional antibody includes the sequence shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8, and the amino acid sequence of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO.17.

[0018] Preferably, the amino acid sequence of the heavy chain variable region of the antibody domain targeting PD-1 in the anti-PD-1 and OX-40 bifunctional antibody includes the sequence shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8, and the amino acid sequence of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO.18.

[0019] Preferably, the antibody domain targeting PD-1 and the antibody domain targeting OX-40 are linked by a linker peptide.

[0020] Preferably, the amino acid sequence of the linker peptide includes the sequence shown in SEQ ID NO.19.

[0021] Preferably, the heavy chain amino acid sequence of the anti-PD-1 and OX-40 bifunctional antibody includes the sequence shown in SEQ ID NO.20 and / or SEQ ID NO.21.

[0022] Table 1

[0023]

[0024]

[0025]

[0026] In a second aspect, the present invention provides a nucleic acid molecule that encodes the anti-PD-1 and OX-40 bifunctional antibody described in the first aspect.

[0027] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the second aspect.

[0028] Fourthly, the present invention provides a host cell containing the nucleic acid molecules described in the second aspect, or transformed by the recombinant vector described in the third aspect.

[0029] Fifthly, the present invention provides a method for preparing the anti-PD-1 and anti-OX-40 bifunctional antibody as described in the first aspect, the method comprising culturing the host cells as described in the fourth aspect, and recovering the bifunctional antibody from the cell culture.

[0030] In a sixth aspect, the present invention provides an antibody conjugate comprising the anti-PD-1 and anti-OX-40 bifunctional antibody described in the first aspect and conjugates thereto; the conjugate comprises a therapeutic agent and / or a detection agent.

[0031] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the anti-PD-1 and OX-40 bifunctional antibody as described in the first aspect and / or the antibody conjugate as described in the sixth aspect.

[0032] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients, such as carriers, excipients, or stabilizers.

[0033] Eighthly, the present invention provides the use of the anti-PD-1 and OX-40 bifunctional antibody of the first aspect and / or the antibody conjugate of the sixth aspect or the pharmaceutical composition of the seventh aspect in the preparation of a medicament for treating inflammatory and / or autoimmune diseases.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] This invention designs a novel anti-PD-1 / OX-40 bifunctional antibody. It utilizes a PD-1 activating antibody and an OX-40 blocking antibody to construct a specific structural bifunctional antibody. The antibody works synergistically, exhibiting high affinity for PD-1, activating PD-1 activity and its signaling pathway, and high affinity for OX40, blocking OX40 / OX40L interaction and its signal transduction. This also inhibits CEF-induced CD8+.+ Memory T cell proliferation, inhibition of T cell proliferation, and inhibition of CD25 or PD-1 positive CD4+. + The generation of T cells has broad application prospects in the development of drugs for treating inflammation or autoimmune diseases. Attached Figure Description

[0036] Figure 1 A schematic diagram of the structure of a humanized PD-1 / OX-40 bifunctional antibody;

[0037] Figure 2 The figure shows the results of ELISA determination of the binding characteristics of humanized PD-1 / OX-40 to PD-1 protein;

[0038] Figure 3 The figure shows the results of ELISA determination of the binding characteristics of humanized PD-1 / OX-40 to OX-40 protein;

[0039] Figure 4 The figure shows the results of FACS determination of the binding characteristics of humanized PD-1 / OX-40 to PD-1 protein;

[0040] Figure 5 The figure shows the results of FACSA determination of the binding characteristics of humanized PD-1 / OX-40 to OX-40 protein;

[0041] Figure 6 The figure shows the results of PD-1 / OX-40 significantly activating the PD-1 signaling pathway in the PD-1 reporter gene assay.

[0042] Figure 7 The figure shows the results of PD-1 / OX-40 significantly blocking the OX-40L / OX-40 signaling pathway in the OX40 reporter gene assay.

[0043] Figure 8 For PD-1 / OX-40 bifunctional antibody against CEF-induced CD8 + The proliferation of memory T cells was significantly inhibited (see diagram).

[0044] Figure 9 The PD-1 / OX-40 bifunctional antibody has significant inhibitory effects on CD4. + The results of the role of T cell proliferation;

[0045] Figure 10A The PD-1 / OX-40 bifunctional antibody inhibits CD25-positive CD4. + T cell results diagram;

[0046] Figure 10BA PD-1 / OX-40 bifunctional antibody to inhibit PD-1-positive CD4 + T cell results diagram. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0048] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0049] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0050] In this invention, the technical term "antibody" refers to a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments containing a complementarity-determining region (CDR), particularly full-length antibodies or antibody functional fragments. The term "full-length antibody" includes both polyclonal and monoclonal antibodies. The term "antibody functional fragment" refers to a substance containing part or all of the antibody's CDR, lacking at least some amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope.

[0051] In some specific implementations, "antibody" specifically refers to a bifunctional antibody. The term "bifunctional" herein refers to the specific binding to two different target sites; as is customary to those skilled in the art, it may also be called a bispecific antibody (BiAb) or a bifunctional antibody.

[0052] In this invention, the term "humanized antibody," also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse or other animal-derived CDR sequences into the variable region framework of a human antibody, i.e., in different types of human lineage antibody framework sequences; the term "humanized antibody" also includes surface-remodeled antibodies and fully humanized antibodies, etc.

[0053] The term "complementarity-determining region" or "CDR" refers to a highly variable region of the heavy and light chains of an immunoglobulin, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy-chain CDRs and three light-chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of antibodies to the antigens or epitopes they recognize. In another specific embodiment, the CDR region or CDR refers to a highly variable region of the heavy and light chains of an immunoglobulin as defined by the IMGT.

[0054] As used herein, the phrase "therapeutic agent" generally refers to any agent that, when administered to an organism, elicits the desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent when it demonstrates a statistically significant effect in an appropriate population. In some embodiments, an appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as an age group, sex, genetic background, pre-existing clinical condition, etc. In some embodiments, a therapeutic agent is any substance that can be used to relieve, improve, reduce, suppress, prevent, delay the onset, reduce the severity, and / or reduce the morbidity of one or more symptoms or features of a disease, condition, and / or symptom. In some embodiments, a "therapeutic agent" is an agent that has been or requires approval by a government agency before it can be commercially available for human administration. In some embodiments, a "therapeutic agent" is an agent required for a medical prescription for human administration.

[0055] As used herein, the term "detector" refers to any detectable component, molecule, functional group, compound, fragment, or portion. In some embodiments, the detection entity is provided or used alone. In some embodiments, the detection entity is provided and / or used in combination (e.g., conjugation) with another reagent. Examples of detection entities include (but are not limited to): various ligands, radionuclides (e.g.,... 3 H, 14 C 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu、 187 Re、 111 In、 90 Y、 99 mTc,177 Lu、 89 Zr, etc.), fluorescent dyes (see below for specific exemplary fluorescent dyes), chemiluminescent agents (such as acridinium esters, stabilized dioxane, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (such as dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins for which antiserum or monoclonal antibodies may be applicable.

[0056] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0057] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0058] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0059] This invention relates to concentration and molecular weight values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may allow fluctuations within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may allow fluctuations within the ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations within ±10% are allowed.

[0060] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0061] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0062] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0063] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0064] This invention relates to a bifunctional antibody against PD-1 and OX-40, comprising an antibody domain targeting PD-1 and an antibody domain targeting OX-40; the C-terminus of the heavy chain IgG constant region of the antibody domain targeting PD-1 is linked to the N-terminus of the antibody domain targeting OX-40 (scFv) via a linker peptide.

[0065] The heavy chain complementarity-determining regions CDR1-CDR3 of the antibody targeting PD-1 are shown in SEQ ID NO.1 to SEQ ID NO.3, and the light chain complementarity-determining regions CDR1-CDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6.

[0066] The heavy chain complementarity-determining regions CDR1-CDR3 of the antibody targeting OX-40 are shown in SEQ ID NO.9 to SEQ ID NO.11, and the light chain complementarity-determining regions CDR1-CDR3 are shown in SEQ ID NO.12 to SEQ ID NO.14.

[0067] In some implementations, the antibody domain also includes a humanized variable region.

[0068] In some embodiments, the antibody domain targeting PD-1 includes the heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.8.

[0069] In some embodiments, the antibody domain targeting OX-40 includes the heavy chain variable region shown in SEQ ID NO.15 and the light chain variable region shown in SEQ ID NO.16.

[0070] In some embodiments, the antibody domain targeting OX-40 is scFv as shown in SEQ ID NO.17 and / or SEQ ID NO.18.

[0071] In some embodiments, the antibody domain targeting PD-1 further includes a light chain constant region and a heavy chain constant region Fc, wherein the Fc includes CH2, CH3 and optionally CH4.

[0072] The heavy chain constant region IgG can be further divided into IgG1, IgG2, IgG3, and IgG4 subtypes. In some preferred embodiments, the heavy chain constant region IgG is the IgG1 constant region, more preferably the human IgG1 constant region.

[0073] In this invention, the term "IgG constant region" includes its natural sequence and variants, particularly the human IgG1 constant region and its variants. Those skilled in the art can also easily avoid heavy chain mismatches or improve its stability by introducing mutations into the Fc region. For example, the Fc mutation can be based on KiH technology, ART-lg technology, SEED technology, or a combination thereof.

[0074] In some specific embodiments, the heavy chain of the bifunctional antibody against PD-1 and OX-40 is as shown in either SEQ ID NO. 20 or SEQ ID NO. 21.

[0075] In some specific implementations, the light chain constant region of the antibody domain can be a kappa (κ) or lambda (λ) light chain, preferably of human origin.

[0076] In some embodiments, the amino acid sequence of the linker peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu.

[0077] In some embodiments, the C-terminus of the antibody heavy chain targeting the PD-1 domain and the N-terminus of the antibody domain targeting OX-40 are linked by a linker peptide.

[0078] There can be two sc-Fvs for the antibody domain targeting OX-40, which are respectively connected to the C-terminus of the two constant regions of the antibody domain targeting PD-1.

[0079] In some embodiments, the linker peptide has 1 to 30 amino acids; it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.

[0080] In some embodiments, the amino acids of the linker peptide are meaningless polypeptides that do not have any additional functions other than linking (e.g., protein localization, enzyme cleavage sites, etc.).

[0081] In some embodiments, the linker peptide is a flexible linker peptide;

[0082] In some embodiments, the amino acid sequence of the linker peptide is selected from one or more of Gly, Ser, Pro, Ala, and Glu;

[0083] In some embodiments, the amino acid sequence of the flexible linker peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n, or (G)n, wherein n is selected from 1, 2, 3, 4, 5, or 6.

[0084] In some embodiments, the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.19.

[0085] In some embodiments, the amino acid sequence of the bifunctional antibody heavy chain obtained by fusing the antibody domain heavy chain targeting PD-1 and the antibody domain targeting OX-40 in the bifunctional antibody against PD-1 and OX-40 is shown in SEQ ID NO. 20 or / and SEQ ID NO. 21.

[0086] Variants of the above-mentioned amino acid sequences are also within the scope of this invention. Each variant, compared to any polypeptide in SEQ ID NO.1 to SEQ ID NO.6, contains a mutation of up to three amino acids. Variants, compared to any polypeptide in SEQ ID NO.9 to SEQ ID NO.14, may contain three or more mutations. For example, variants may have at least 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.15, and SEQ ID NO.16. Mutations can be amino acid substitutions, deletions, additions, or any combination thereof; preferably, the mutations are conserved substitutions. A "conserved substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity.

[0087] Substitutions generally considered conserved are substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; interchange of hydroxyl residues Ser and Thr; exchange of acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange of basic residues Lys and Arg; and substitution between aromatic residues Phe and Tyr. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0088] The present invention also relates to isolated nucleic acids that encode bifunctional antibodies against PD-1 and OX-40 as described above.

[0089] The term "isolated nucleic acid" herein refers to deoxyribonucleic acid or ribonucleic acid polymers existing in single-stranded or double-stranded form. The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA), or RNA sequences transcribed from DNA, and, unless specifically indicated, the polypeptide also includes natural polynucleotides, sugars, or base-modified analogs. According to one aspect of the invention, the polynucleotide is a light-chain polynucleotide.

[0090] The isolated nucleic acids include nucleotide sequences encoding the amino acid sequence of a protein complex, as well as complementary nucleotide sequences. The complementary sequences include fully complementary sequences and substantially complementary sequences, meaning sequences that can hybridize with the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.

[0091] Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex can be altered or mutated. Such alterations include addition, deletion, or non-conservative or conserved substitution. A polynucleotide encoding the amino acid sequence of the protein complex can be interpreted as including a nucleotide sequence that has substantial identity with respect to the isolated nucleic acid. This substantial identity involves aligning the nucleotide sequence with another random sequence in a manner that maximizes their correspondence, such that when the aligned sequences are analyzed using algorithms commonly used in the art, the sequences show greater than 80%, greater than 90%, or greater than 95% homology.

[0092] The present invention also relates to a vector comprising the nucleic acid as described above.

[0093] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0094] In this invention, the vector can be a composition, such as a mixture of multiple plasmids, with different plasmids loading a portion of a bifunctional antibody.

[0095] The present invention also provides a host cell containing the nucleic acid as described above, or transformed by the vector as described above.

[0096] Suitable host cells or cell lines for expressing the bifunctional antibodies of the present invention include mammalian cells such as NSO, Sp2 / O, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells can be used, thus allowing the molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be used. The selection of suitable mammalian host cells, and methods for transformation, culture, amplification, screening, and product generation and purification, are known in the art.

[0097] It can be demonstrated that bacterial cells can be used as host cells, suitable for expressing the humanized antibodies of the present invention or other embodiments. However, since proteins expressed in bacterial cells tend to be in unfolded, incorrectly folded, or non-glycosylated forms, any humanized antibodies produced in bacterial cells must be screened to retain antigen-binding capacity. If the molecule expressed by the bacterial cell is produced in an appropriately folded form, the bacterial cell will be the desired host; alternatively, in an alternative embodiment, the molecule can be expressed in a bacterial host and subsequently refolded. For example, various *Escherichia coli* strains used for expression are well-known host cells in the field of biotechnology. Various strains of *Bacillus subtilis*, *Streptomyces*, and other *Bacillus* species can also be used in this method.

[0098] If necessary, yeast cell strains known to those skilled in the art, as well as insect cells, such as fruit flies and lepidopteran insects, and virus expression systems, may also be used as host cells.

[0099] In some embodiments, the nucleic acid is inserted into the cell genome and can be stably expressed.

[0100] The insertion method can be the vector described above, or the nucleic acid can be directly transferred into the cell without being linked to the vector (e.g., liposome-mediated transfection technology).

[0101] The present invention also relates to a method for preparing bifunctional antibodies, comprising culturing host cells as described above under suitable conditions, and recovering the bifunctional antibodies from the cell culture.

[0102] For example, nucleic acids encoding one or both polypeptide chains of a bifunctional antibody can be introduced into cultured host cells using a variety of known methods, such as transformation, transfection, electroporation, bombardment with nucleic acid-coated microparticles, etc. In some embodiments, the nucleic acid encoding the bifunctional antibody may be inserted into a vector suitable for expression in the host cells before being introduced into the host cells. A typical such vector may contain sequence elements that enable the inserted nucleic acid to be expressed at both the RNA and protein levels.

[0103] The present invention also relates to conjugates, which are bifunctional antibodies as described above that bind to a therapeutic agent or a detection agent.

[0104] Therapeutic agents can be or comprise any class of chemical entities, including, for example (but not limited to), proteins, carbohydrates, lipids, nucleic acids, small organic molecules, non-biological polymers, metals, ions, radioactive isotopes, etc. In some embodiments, the therapeutic agents used according to the invention may have biological activities related to treating one or more symptoms or causes of cancer. In some embodiments, the therapeutic agents used according to the invention may have one or more other activities.

[0105] In some embodiments of the invention, the combined therapeutic agent is a radioactive isotope, a drug conjugate, nanoparticles, an immunotoxin, or any other therapeutic load.

[0106] The detection agent comprises any component that can be detected using analysis, for example, due to its specific functional properties and / or chemical characteristics. Non-limiting examples of such agents include enzymes, radiolabeled substances, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (such as biotin). In some embodiments of the invention, the incorporated detection agent is a diagnostic agent or an imaging agent.

[0107] The present invention also relates to pharmaceutical compositions comprising the bifunctional antibody or antibody-drug conjugate as described above, and their pharmaceutically acceptable carrier, excipient, or stabilizer.

[0108] The bifunctional antibody or antibody-drug conjugate of the present invention can be used to prepare pharmaceutical compositions or sterile compositions, for example, by mixing the bifunctional antibody with a pharmaceutically acceptable carrier, excipient or stabilizer.

[0109] The term "pharmaceutically acceptable" means that when the molecular bulk, molecular fragment, or composition is properly administered to an animal or human, it will not produce adverse, allergic, or other adverse reactions. Specific examples of substances that can serve as pharmaceutically acceptable carriers or components include phosphoric acid, citric acid, and other organic acids; antioxidants (e.g., ascorbic acid and methionine); antibacterial agents (e.g., octadecyl dimethylbenzene ammonium chloride, hexachlorocyclohexane quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, or m-cresol); low molecular weight (less than about 10 kDa) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; and hydrophilic substances. Polymers, such as polyvinylpyrrolidone; amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including, for example, glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions; metal complexes; and / or nonionic surfactants (e.g., including TWEENTM, PLURONICS™, or polyethylene glycol). Furthermore, depending on the formulation method, commonly used fillers, diluents, binders, humectants, disintegrants, and / or surfactants can be appropriately selected by those skilled in the art.

[0110] The bifunctional antibody contained in the pharmaceutical composition can be contained in microcapsules, or in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. The microcapsules can be prepared by techniques such as coacervation or interfacial polymerization, examples of which are hydroxymethyl cellulose or gelatin microcapsules and poly(methyl isobutyl ester) microcapsules.

[0111] The pharmaceutical compositions of the present invention can be administered via any route, as those skilled in the art will understand. In some embodiments, the pharmaceutical compositions of the present invention are administered orally (PO), intravenously (IV), intramuscularly (IM), intra-arterially, intramedullaryly, intrathecally, subcutaneously (SQ), intracardiacly, percutaneously, intradermally, intradermally, rectally (PR), vaginally, intraperitoneally (IP), intragastrically (IG), topically (e.g., using powders, ointments, creams, gels, lotions and / or drops), via mucosa, intranasally, intrabuccally, via the intestine, via the vitreous body, sublingually; via tracheal instillation, bronchial instillation and / or inhalation; as an oral spray, nasal spray and / or aerosol and / or via a portal vein catheter.

[0112] The pharmaceutical composition is preferably an injectable preparation, such as a lyophilized powder for injection.

[0113] The present invention also relates to the use of the bifunctional antibody or conjugate described above in the preparation for the treatment of inflammatory or autoimmune diseases.

[0114] In this invention, "tumor" includes inflammation or autoimmune diseases.

[0115] In some embodiments, the T cells of the disease express PD-1.

[0116] Patients with inflammatory or autoimmune diseases can be selected from animals, preferably mammals, such as groups of humans, dogs, cats, chimpanzees, orangutans, gibbons, macaques, marmosets, pigs, horses, pandas, and elephants.

[0117] The present invention also relates to a method for treating autoimmune diseases, comprising administering to a patient a safe and effective amount of the bifunctional antibody-drug conjugate or pharmaceutical composition as described above.

[0118] The phrase "safe and effective amount" means, as used herein, a dosage of a drug within a reasonable pharmaceutical modulation that is large enough to significantly and effectively relieve the symptoms or condition being treated, but small enough to avoid serious side effects (with a reasonable benefit / risk ratio). The safe and effective amount of the active ingredient in the bifunctional antibody or conjugate or pharmaceutical composition used in the methods of this invention varies depending on the specific symptoms being treated, the patient's age and physical condition, the severity of the disease, the duration of treatment, concurrent treatments, the specific active ingredient used, the specific pharmaceutically acceptable excipients used, and factors including the knowledge and skills of the physicians involved in the treatment.

[0119] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0120] This invention constructs a bifunctional antibody against PD-1 / OX-40. A schematic diagram of the construction of the bifunctional antibody is shown below. Figure 1 As shown in the figures, the portion involving the PD-1 antibody (heavy chain variable region sequence SEQ ID NO.7, light chain variable region sequence SEQ ID NO.8) is represented by ILB2110; the portion involving the OX-40 antibody (scFV, SEQ ID NO.17 or SEQ ID NO.18) is represented by ILB2107. Compared with the corresponding PD-1 and OX-40 monoclonal antibodies, the bifunctional PD-1 / OX-40 antibodies retain their binding ability to their respective antigens, while exhibiting higher biological activity in promoting the production and secretion of cytokines by human immune cells.

[0121] Example 1

[0122] This embodiment describes the construction of a bifunctional anti-PD-1 / OX-40 antibody.

[0123] The scFv sequence of the OX-40 antibody (ILB2107) (e.g., SEQ ID NO.17 and SEQ ID NO.18) was linked to the C-terminus of the heavy chain of the PD-1 antibody (ILB2110) via Linker (G4S)4. Figure 1 As shown, the heavy chain sequences are SEQ ID NO.20 or SEQ ID NO.21, and are co-transfected with the light chain (SEQ ID NO.8) expression vector into 293 cells. After 6 days of shaking culture at 37°C, the supernatant was collected by centrifugation and purified with Protein A. After purification, various tests were performed, including binding and functional activities, to obtain two anti-PD-1 / OX-40 bifunctional antibodies, named ILB2110 / ILB2107scFV-HL (heavy chain SEQ ID NO.20, light chain SEQ ID NO.8) or ILB2110 / ILB2107scFV-LH (heavy chain SEQ ID NO.21, light chain SEQ ID NO.8), respectively.

[0124] Example 2

[0125] In this embodiment, the binding characteristics of the PD-1 / OX-40 bifunctional antibody to human PD-1 or human OX-40 protein were determined using the ELISA method.

[0126] Coat a 96-well ELISA plate (Corning, Cat No: 9018) with PD-1 protein (human PD-1-ECD-mFc) or OX-40 protein (human OX-40-His), incubate overnight at 4°C, wash three times with washing buffer (PBS + 0.05% Tween 20), add blocking buffer (PBS + 1% BSA (Sigma, Cat No: V90093)) and incubate for 1 hour; wash the 96-well plate three times; add serially diluted samples to be tested, including positive controls (ILB2110, ILB2107) and negative controls (hIgG1), then incubate for 1 hour and wash three times; add 100 μL of 1:3000 diluted secondary antibody HRP Anti-Human IgG (BD, Cat No: 555788) to each well, incubate at room temperature for 1 hour, wash three times; add 100 μL of TMB (Beijing Bio-Science, Cat) to each well. After developing the color with No. ES-002 for 3 minutes, add 100 μL / well of stop solution (2N H2SO4) to terminate the reaction. Measure the OD of each sample using a TecanSpark microplate reader. 450 Signal. Experimental results show that the PD-1 / OX-40 bifunctional antibody interacts with human PD-1 protein (…). Figure 2 ) or human OX-40 protein ( Figure 3 They exhibit significant binding affinity. Overall, ILB2110 / ILB2107-HL or ILB2110 / ILB2107-LH showed similar binding activity to the PD-1 activating monoclonal antibody ILB2110 in terms of PD-1 antigen binding activity; and compared to the OX-40 antibody (ILB2107), they showed similar (ILB2110 / ILB2107scFV-LH) or better (ILB2110 / ILB2107scFV-HL) binding activity to the OX40 antigen.

[0127] Example 3

[0128] In this embodiment, the binding of the humanized PD-1 / OX-40 bifunctional antibody to Jurkat-PD-1 and 293T-OX-40 and cells was determined using the FACS method.

[0129] Add 50 μL of serially diluted sample to be tested, including positive and negative controls, to 50 μL of Jurkat-PD-1 or 293T-OX-40 cells (2 × 10⁻⁶). 5 Cells / well were added to U-bottom 96-well plates and incubated for 1 hour. After washing twice with FACS buffer (PBS + 3% FCS), PE goat anti-human secondary antibody (eBioscience, Cat No: 12-4998-82) diluted 1:400 was added and incubated for 30 minutes. After washing with FACS buffer, the PE signal of Jurkat-PD-1 or 293T-OX-40 cells was detected using a flow cytometer from Sinopharm Medical.

[0130] Experimental results are as follows Figure 4 and Figure 5 As shown, the PD-1 / OX-40 bifunctional antibody maintained its binding activity with Jurkat-PD-1 and 293T-OX-40 cells. Overall, ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH showed better activity in binding to the PD-1 antigen on cells compared to the PD-1 activating monoclonal antibody ILB2110. Regarding binding to OX40 on cells, ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH, compared to ILB2107, although showing better activity in binding to EC40 cells... 50 While the binding activity was somewhat reduced, it remained strong overall.

[0131] Example 4

[0132] This embodiment uses reporter gene-based functional assays to demonstrate that the humanized PD-1 / OX-40 bifunctional antibody has a significant activating effect on PD-1 and a significant inhibitory effect on OX-40.

[0133] The activation activity of humanized PD-1 / OX-40 bifunctional antibodies (ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH) on PD-1 was determined using a reporter gene assay. 50 μL (5 × 10⁻⁶) of the antibody was added to each well of a 96-well plate. 4 Jurkat-PD-1-NFAT-luc cells (cells / well) and 50 μL of serially diluted test samples, including positive and negative controls, were added, followed by 25 μL of OKT3 and THP1 cells (5 × 10⁻⁶ cells / well). 4 Cells / well), mixed and incubated at 37°C for 4 hours. Add 25 μL Bright Glo (Promega, Cat No: E2620), incubate at room temperature for 5 minutes, and measure the chemiluminescence signal of each sample using a Tecan Spark microplate reader.

[0134] Experimental results are as follows Figure 6 As shown, when the PD-1 / OX-40 bifunctional antibody (ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH) was added, ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH showed a significant inhibitory effect on the T cell activity signal of the assay. This inhibition or reduction of the signal is achieved through the activation of PD-1. That is, as the concentration of PD-1 / OX-40 bifunctional antibody increases, the activity of PD-1 is promoted, thereby inhibiting the activity of T cells.

[0135] In addition, the inhibitory effect of humanized PD-1 / OX-40 bifunctional antibodies (ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH) on OX-40 activation by OX-40L was determined using a reporter gene assay. 50 μL of PD-1 / OX-40 bifunctional antibody was mixed with 25 μL of Jurkat-OX40 reporter cells (5 × 10⁻⁶ cells). 4Mix the samples (samples / well) and add them to a 96-well half-zone plate. Then add 25 μL of 50 ng / mL OX40L, mix well, and incubate at 37°C for 4 hours. Add 25 μL of preheated Bright-Glo solution (Promega, Cat. No.: E2620) to each well, incubate at room temperature in the dark for 3 minutes, and measure the luminescence signal value of each sample well using a Tecan Spark microplate reader. The Jurkat-OX40 cells used in this experiment are integrated with the NF-κB-luciferase gene.

[0136] Experimental results are as follows Figure 7 As shown, when the PD-1 / OX-40 bifunctional antibody (ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH) was added, ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH showed a significant inhibitory effect on the signal that T cells are activated by OX-40 ligand (OX-40L). This inhibition or reduction of the signal is because the PD-1 / OX-40 bifunctional antibody acts on OX-40, blocking the activation of T cells by OX-40L through OX-40. Therefore, as the concentration of the PD-1 / OX-40 bifunctional antibody increases, the blocking effect on OX-40 ligand is correspondingly enhanced, thereby increasing the inhibitory effect on T cell activity.

[0137] Example 5

[0138] This embodiment measures the effect of the PD-1 / OX-40 bifunctional antibody on CEF-induced CD8+. + Inhibitory effect on the proliferation of memory T cells.

[0139] The testing procedure was as follows: Frozen PBMCs (purchased from Shanghai Aoneng Biotechnology Co., Ltd. and Miaoshun (Shanghai) Biotechnology Co., Ltd.) were thawed and added to pre-warmed RPMI 1640 complete medium. After mixing, the cells were centrifuged at 1500 rpm for 5 minutes. After labeling with CFSE cell proliferation dye, pre-cooled complete medium was added to quench the free dye, and the cells were washed three times. The PBMCs were resuspended in complete medium and counted. 100 μL of CFSE-labeled PBMCs (2 × 10⁶ cells / well) were added to each well of a 96-well plate. 5 / well); Add 50 μL of diluted PD-1 / OX-40 bifunctional antibody (ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH) or monoclonal antibody (ILB2110, ILB2107, GBR830-DE or KHK4083-DE) at different concentrations to the corresponding wells (final concentration: 66.5 nM, 4-fold serial dilution); At the same time, add 50 μL of LCEF peptide (final concentration: 200 ng / mL) to the corresponding wells, mix well, and incubate the 96-well cell culture plate at 37°C, 5% CO2 for 7 days. After centrifugation, collect the cells. Resuspend the cells in flow cytometry buffer (PBS + 3% FCS), add fluorescently labeled anti-human CD8 antibody and cell viability dye, incubate at 4°C for 1 hour, wash the cells twice with flow cytometry buffer, and detect and analyze the dividing CD8 cells using flow cytometry. + T cells (CD8) + CFSElow / dim) accounts for a portion of the total effect CD8 + T cells (CD8) + CFSE + The PD-1 / OX-40 bifunctional antibody of the present invention exhibited significant inhibition of CEF-induced memory CD8 in the ratio of 0.5 to 0.5%. + T cell proliferation, experimental results as follows Figure 8 As shown, both ILB2110 / ILB2107scFV-HL and ILB2110 / ILB2107scFV-LH exhibited strong resistance to CD8. + Inhibitory effect on the proliferation of memory T cells.

[0140] Example 6

[0141] This embodiment measures the inhibitory effect of the PD-1 / OX-40 bifunctional antibody on T cell proliferation.

[0142] During the assay, PD-1 / OX-40 bifunctional antibodies (ILB2110 / ILB2107scFV-HL or ILB2110 / ILB2107scFV-LH) or monoclonal antibodies (ILB2110 and ILB2107) were serially diluted to prepare 5-fold antibody dilutions. 50 μL of each dilution was transferred to a 96-well plate, followed by 150 μL of cell culture medium containing 1e5 / well CFSE-labeled humanized PBMC cells and a final concentration of 1 ng / mL of the stimulating factor SEB (Toxin Technology, cat.no:BT202). The plates were then incubated at 37°C for 3–4 days. Activated PBMC cells were collected, and 2.5 μL / well of CD4-PE-cy7 (Biolegend, Cat.No:300512) was added. After incubation at 4°C for 30 min, the cells were washed, and CD4 counts were detected using flow cytometry. + The percentage of T cells proliferating within the T cell group. See the results below. Figure 9 The results showed that the PD-1 / OX-40 bifunctional antibody of the present invention generally exhibited a relatively significant inhibitory effect on T cell proliferation. Compared with PD-1 monoclonal antibody (ILB2110) or OX40 monoclonal antibody (ILB2107), the bifunctional antibodies ILB2110 / ILB2107scFV-HL and ILB2110 / ILB2107scFV-LH had a more significant effect in inhibiting T cell proliferation.

[0143] Example 7

[0144] This embodiment tests the inhibition of CD25 or PD-1-positive CD4 by a PD-1 / OX-40 bifunctional antibody. + The production of T cells.

[0145] PBMC cell activation and antibody treatment were performed as described in Example 6. After collecting activated PBMC cells, 2.5 μL / well of CD4-PE-cy7 (Biolegend, Cat. No: 300512), APC-Anti-PD1 (Biolegend, Cat# 621610), and PEAnti-CD25 (Biolegend Cat# 302606) were added. After incubation at 4°C for 30 min, the cells were washed, and CD4+ was detected using flow cytometry. + T cells expressing CD25 and PD-1. The results are shown in Figure 10. The results indicate that the PD-1 / OX-40 bifunctional antibody of the present invention is effective against CD25-expressing cells. Figure 10A ) or PD-1 ( Figure 10BThe T cells showed a relatively significant inhibitory effect overall. Compared with PD-1 monoclonal antibody (ILB2110) or OX40 monoclonal antibody (ILB2107), the bifunctional antibodies ILB2110 / ILB2107scFV-HL and ILB2110 / ILB2107scFV-LH had a more significant effect in reducing T cells expressing CD25 or PD-1.

[0146] In summary, this invention designs a novel anti-PD-1 / OX-40 bifunctional antibody. It utilizes a PD-1 activating antibody and an OX-40 blocking antibody to construct a specific structural bifunctional antibody, exhibiting synergistic effects. This antibody possesses high affinity for PD-1, activating PD-1 activity and its signaling pathway, and high affinity for OX40, blocking OX40 / OX40L interaction and its signal transduction. Furthermore, it can inhibit CEF-induced CD8+. + Memory T cell proliferation, inhibition of T cell proliferation, and inhibition of CD25 or PD-1 positive CD4+. + The generation of T cells has broad application prospects in the development of drugs for the treatment of chronic inflammation or autoimmune diseases.

[0147] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A bifunctional antibody against PD-1 and OX-40, characterized in that, The bifunctional antibody includes an antibody domain that targets PD-1 and an antibody domain that targets OX-40. The amino acid sequences of the heavy chain complementarity-determining regions CDR1-CDR3 of the antibody targeting PD-1 include the sequences shown in SEQ ID NO.1 to SEQ ID NO.3, and the amino acid sequences of the light chain complementarity-determining regions CDR1-CDR3 include the sequences shown in SEQ ID NO.4 to SEQ ID NO.

6. The amino acid sequences of the heavy chain complementarity-determining regions CDR1-CDR3 of the antibody targeting OX-40 domain include the sequences shown in SEQ ID NO.9 to SEQ ID NO.11, and the amino acid sequences of the light chain complementarity-determining regions CDR1-CDR3 include the sequences shown in SEQ ID NO.12 to SEQ ID NO.

14.

2. The anti-PD-1 and anti-OX-40 bifunctional antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody targeting PD-1 includes the sequence shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.

8.

3. The anti-PD-1 and OX-40 bifunctional antibody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody targeting OX-40 includes the sequence shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.16; Preferably, the antibody domain targeting OX-40 is an scFv fragment; Preferably, the amino acid sequence of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO.17 and / or SEQ ID NO.18; Preferably, the C-terminus of the heavy chain of the antibody domain targeting PD-1 is connected to the N-terminus of the antibody domain targeting OX-40; Preferably, the amino acid sequence of the heavy chain variable region of the antibody domain targeting PD-1 in the anti-PD-1 and OX-40 bifunctional antibody includes the sequence shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8, and the amino acid sequence of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO.

17. Preferably, the amino acid sequence of the heavy chain variable region of the antibody domain targeting PD-1 in the anti-PD-1 and OX-40 bifunctional antibody includes the sequence shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.8, and the amino acid sequence of the antibody domain targeting OX-40 includes the sequence shown in SEQ ID NO.

18. Preferably, the antibody domain targeting PD-1 and the antibody domain targeting OX-40 are linked by a linker peptide; Preferably, the amino acid sequence of the linker peptide includes the sequence shown in SEQ ID NO.19; Preferably, the heavy chain amino acid sequence of the anti-PD-1 and OX-40 bifunctional antibody includes the sequence shown in SEQ ID NO.20 and / or SEQ ID NO.

21.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-PD-1 and OX-40 bifunctional antibody as described in any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 4, or is transformed by the recombinant vector of claim 5.

7. A method for preparing the anti-PD-1 and OX-40 bifunctional antibody according to any one of claims 1-3, characterized in that, The method includes culturing the host cells of claim 6 and recovering the bifunctional antibody from the cell culture.

8. An antibody conjugate, characterized in that, The antibody conjugates include the anti-PD-1 and OX-40 bifunctional antibody as described in any one of claims 1-3 and the conjugates thereto; The conjugates include therapeutic agents and / or detection agents.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-PD-1 and OX-40 bifunctional antibody as described in any one of claims 1-3 and / or the antibody conjugate as described in claim 8.

10. The use of the anti-PD-1 and OX-40 bifunctional antibody according to any one of claims 1-3 and / or the antibody conjugate according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating inflammatory and / or autoimmune diseases.

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