Pd-1 agonist antibodies

Abstract

In some embodiments, provided herein are PD-1 agonist antibodies that bind to PD-1. The PD-1 agonist antibodies of the present disclosure are useful in the treatment of autoimmune and inflammatory diseases by promoting PD-1 signaling. Also provided herein are methods of using the PD-1 agonist antibodies.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511302746.2 (22) Application Date 2023.09.14 (30) Priority Data 63 / 375,676 2022.09.14 US 63 / 515,448 2023.07.25 US (62) Divisional Application Data 202380065677.0 2023.09.14 (71) Applicant Otsuka Pharmaceutical Co., Ltd. Address Tokyo, Japan (72) Inventors Matthew P. Graffin, Cody Allen Moore, Alexander Tomomi Taguchi (74) Patent Agency Junhe Law Firm, Beijing 11517 Patent Attorney Wu Yu, Gu Yunfeng (51) Int.Cl. C07K 16 / 28 (2006.01) A61K 39 / 395 (2006.01) A61P 37 / 02 (2006.01) A61P 29 / 00 (2006.01) (54) Invention Title: PD-1 Agonist Antibody (57) Abstract: This document provides a PD-1 agonist antibody that binds to PD-1. The PD-1 agonist antibody disclosed herein can be used to treat autoimmune diseases and inflammatory diseases by promoting PD-1 signaling. This document also provides methods for using the PD-1 agonist antibody. Claims 2 pages, Description 28 pages, Sequence Listing (Electronic Publication), Drawings 32 pages, CN 121159694 A 2025.12.19 CN 1 21 15 96 94 A 1. A PD-1 agonist antibody, wherein the antibody comprises: a. a heavy chain variable domain (VH) complementarity-determining region (CDR) 1, the VH CDR 1 comprising the amino acid sequence of SEQ ID NO:32; VH CDR 2, the VH CDR 2 comprising the amino acid sequence of SEQ ID NO:35; VH CDR 3, the VH CDR 3 comprising the amino acid sequence of SEQ ID NO:34; a light chain variable domain (VL) CDR 1, the VL CDR 1 comprising the amino acid sequence of SEQ ID NO:42; VL CDR 2, the VL CDR 2 comprising the amino acid sequence of SEQ ID NO:43; and VL CDR 3, the VL CDR 3 comprising the amino acid sequence of SEQ ID NO:45; b. VH CDR 1, the VH CDR1 contains the amino acid sequence of SEQ ID NO:10; VH CDR2, which contains the amino acid sequence of SEQ ID NO:11; VH CDR3, which contains the amino acid sequence of SEQ ID NO:12; VLCDR1, the VL CDR1 comprising the amino acid sequence of SEQ ID NO:13; VL CDR2, the VL CDR2 comprising the amino acid sequence of SEQ ID NO:14; and VL CDR3, the VL CDR3 comprising the amino acid sequence of SEQ ID NO:15; c. VH CDR1, the VH CDR1 comprising the amino acid sequence of SEQ ID NO:29; VH CDR2, the VH CDR2 comprising the amino acid sequence of SEQ ID NO:30; VH CDR3, the VH CDR3 comprising the amino acid sequence of SEQ ID NO:25; VL CDR1, the VL CDR1 comprising the amino acid sequence of SEQ ID NO:26; VL CDR2, the VL CDR2 comprising the amino acid sequence of SEQ ID NO:27; and VL CDR3, the VL CDR3 comprising the amino acid sequence of SEQ ID NO:28; d. VH CDR1, the VH CDR1 comprising the amino acid sequence of SEQ ID NO:16; VH CDR2, the VH CDR2 contains the amino acid sequence of SEQ ID NO:17; VH CDR3 contains the amino acid sequence of SEQ ID NO:18; VL CDR1 contains the amino acid sequence of SEQ ID NO:19; VL CDR2 contains the amino acid sequence of SEQ ID NO:20; and VL CDR3 contains the amino acid sequence of SEQ ID NO:21; e. VH CDR1 contains the amino acid sequence of SEQ ID NO:22; VH CDR2 contains the amino acid sequence of SEQ ID NO:23; VH CDR3 contains the amino acid sequence of SEQ ID NO:24; VL CDR1 contains the amino acid sequence of SEQ ID NO:26; VL CDR2 contains the amino acid sequence of SEQ ID NO:27; and VL CDR3 contains the amino acid sequence of SEQ ID NO:27. The amino acid sequence of NO:28; f. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:22; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:23; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:25; VL CDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:26; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:28.The amino acid sequence of SEQ ID NO:27; and VL CDR3, which contains the amino acid sequence of SEQ ID NO:28; g. VH CDR1, which contains the amino acid sequence of SEQ ID NO:22; VH CDR2, which contains the amino acid sequence of SEQ ID NO:23; VH CDR3, which contains the amino acid sequence of SEQ ID NO:25; VL CDR1, which contains the amino acid sequence of SEQ ID NO:26; VL CDR2, which contains the amino acid sequence of SEQ ID NO:31; and VL CDR3, which contains the amino acid sequence of SEQ ID NO:28; h. VH CDR1, which contains the amino acid sequence of SEQ ID NO:37; VH CDR2, which contains the amino acid sequence of SEQ ID NO:38; VH CDR3, which contains the amino acid sequence of SEQ ID NO:39; VL CDR1, the VL CDR1 comprising the amino acid sequence of SEQ ID NO:40; VL CDR2, the VL CDR2 comprising the amino acid sequence of SEQ ID NO:27; and VL CDR3, the VL CDR3 comprising the amino acid sequence of SEQ ID NO:41; i. VH CDR1, the VH CDR1 comprising the amino acid sequence of SEQ ID NO:32; VH CDR2, the VH CDR2 comprising the amino acid sequence of SEQ ID NO:33; VH CDR3, the VH CDR3 comprising the amino acid sequence of SEQ ID NO:34; VL CDR1, the VL CDR1 comprising the amino acid sequence of SEQ ID NO:42; VL CDR2, the VL CDR2 comprising the amino acid sequence of SEQ ID NO:43; and VL CDR3, the VL CDR3 comprising the amino acid sequence of SEQ ID NO:44; j. VH CDR1, the VH CDR1 comprising the amino acid sequence of SEQ ID NO:32; VH CDR2, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:33 as stated on page 1 / 2 of CN 121159694 A; VH CDR3, which comprises the amino acid sequence of SEQ ID NO:34; VL CDR1, which comprises the amino acid sequence of SEQ ID NO:42; VL CDR2, which comprises the amino acid sequence of SEQ ID NO:43; and VL CDR3, which comprises the amino acid sequence of SEQ ID NO:43.CDR3 contains the amino acid sequence of SEQ ID NO:45; k. VH CDR1, which contains the amino acid sequence of SEQ ID NO:32; VH CDR2, which contains the amino acid sequence of SEQ ID NO:33; VH CDR3, which contains the amino acid sequence of SEQ ID NO:34; VL CDR1, which contains the amino acid sequence of SEQ ID NO:46; VL CDR2, which contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, which contains the amino acid sequence of SEQ ID NO:45; l. VH CDR1, which contains the amino acid sequence of SEQ ID NO:32; VH CDR2, which contains the amino acid sequence of SEQ ID NO:33; VH CDR3, which contains the amino acid sequence of SEQ ID NO:34; VL CDR1, which contains the amino acid sequence of SEQ ID NO:35; The amino acid sequence of SEQ ID NO:46; VL CDR2, which contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, which contains the amino acid sequence of SEQ ID NO:47; or m.VH CDR1, which contains the amino acid sequence of SEQ ID NO:36; VH CDR2, which contains the amino acid sequence of SEQ ID NO:33; VH CDR3, which contains the amino acid sequence of SEQ ID NO:34; VL CDR1, which contains the amino acid sequence of SEQ ID NO:42; VL CDR2, which contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, which contains the amino acid sequence of SEQ ID NO:45. 2. The antibody of claim 1, wherein the antibody comprises: a. VH, wherein the VH comprises the amino acid sequence of any one of SEQ ID NO: 1-5, 48-54, and b. VL, wherein the VL comprises the amino acid sequence of any one of SEQ ID NO: 6-9, 55-61. 3. The antibody of any one of claims 1 to 2, wherein the antibody is: a. a monoclonal antibody; b. a full-length antibody; or c. an antibody fragment, optionally wherein the antibody is fused with the Fc domain of any one of: human IgG1, human IgG2, human IgG3, and human IgG4. 4. A tandem scFv-Fc PD-1 agonist antibody, wherein the antibody comprises tandem Fc domains on each antibody arm.5. The antibody of claim 4, wherein the antibody has a total of four scFv binding sites in a single scFv-Fc form. 6. The antibody of claim 4 or 5, wherein the scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and wherein the scFv2 of each antibody arm comprises a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2). 7. The antibody of any one of claims 4 to 6, wherein the VH1 region and the VH2 region each comprise an amino acid sequence of any one of SEQ ID NO: 1-5 and 48-54, more preferably SEQ ID NO: 1 or 53; and wherein the VL1 region and the VL2 region each comprise an amino acid sequence of any one of SEQ ID NO: 6-9 and 55-61, or preferably SEQ ID NO: 6 or 60. 8. The antibody according to any one of claims 4 to 7, wherein the linker comprises the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64). Claims 2 / 2 Page 3 CN 121159694 A PD-1 agonist antibody

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 375,676, filed September 14, 2022, and U.S. Provisional Application Serial No. 63 / 515,448, filed July 25, 2023, the entire contents of which are incorporated herein by reference.

[0003] Statement of federal government-funded research

[0004] Not applicable.

[0005] The contents of the electronic sequence listing (IBIO_1036P2_SL_ST26.xml; size: 69,377 bytes; and creation date: September 12, 2022) are incorporated herein by reference in their entirety. Background Art

[0007] Programmed cell death protein 1 (PD-1) is a cell surface receptor that plays a key role as an immune checkpoint inhibitor. PD-1 belongs to the immunoglobulin superfamily and is known to be expressed on T cells, B cells, monocytes, natural killer T cells, and dendritic cells. The transmembrane protein programmed death ligand 1 (PD-L1) acts as the natural ligand for PD-1. The PD-1:PD-L1 interaction is used to inhibit immune cells by phosphorylation of a cytoplasmic immune receptor tyrosine-based inhibitory motif (ITIM), which activates downstream signaling that can inhibit T cell activation, cytokinesIt can produce and also promote apoptosis. Therefore, PD-1 is essential for preventing autoimmune and overstimulated immune responses (which, if left unchecked, can be harmful to the body).

[0008] It is well known that the PD-1:PD-L1 checkpoint is exploited by upregulated PD-L1 cancer cells to evade detection, and therefore many PD-1 antagonist agents and antibodies have been developed as tumor therapeutics to counteract this exploitation. Despite the focus on PD-1 antagonist agents, PD-1 agonist agents and antibodies are still needed in the treatment of various autoimmune and inflammatory diseases, ideally without interfering with the innate PD-1:PD-L1 interaction. Such antibodies are provided herein. Summary of the Invention

[0009] This invention provides PD-1 agonist antibodies that bind to PD-1. As embodied and broadly described herein, one aspect of this disclosure relates to PD-1 agonist antibodies, wherein the antibody comprises: a heavy chain variable domain (VH) complementarity-determining region (CDR) 1, the VH CDR 1 comprising the amino acid sequence of any one of SEQ ID NO: 10, 16, 22, 29, 32, 36, 37; and a VH CDR 2, the VH CDR 2 comprising the amino acid sequence of any one of SEQ ID NO: 11, 17, 23, 30, 33, 35, 38; and a VH CDR 3, the VH CDR 3 comprising the amino acid sequence of any one of SEQ ID NO: 12, 18, 24, 25, 34, 39; and a light chain variable domain (VL) CDR 1, the VL CDR 1 comprising the amino acid sequence of any one of SEQ ID NO: 13, 19, 26, 40, 42, 46; and a VL CDR 2, the VL CDR 2 comprising the amino acid sequence of any one of SEQ ID NO: 10, 16, 22, 29, 32, 36, 37; The amino acid sequence of any one of SEQ ID NO: 14, 20, 27, 31, 43; and VL CDR3, the VL CDR3 comprising the amino acid sequence of any one of SEQ ID NO: 15, 21, 28, 41, 44, 45, 47. On one hand, the antibody comprises: VH, the VH comprising the amino acid sequence of any one of SEQ ID NO: 1-5, 48-54; and VL, the VL comprising the amino acid sequence of any one of SEQ ID NO: 6-9, 55-61. On the other hand, the antibody is a monoclonal antibody. On the other hand, the antibody is a full-length antibody. On the other hand, the antibody is an antibody fragment. On the other hand, the antibody is fused with the Fc domain of any one of the following described on page 1 / 28 of CN 121159694 A: human IgG1, human IgG2, human IgG3, and human IgG4.

[0010] As embodied and broadly described herein, one aspect of this disclosure relates to a treatment for a subject in need of a disease.A method for treating an autoimmune disease, the method comprising administering a therapeutically effective amount of the antibody described above to the subject. On one hand, the disease is an autoimmune disease. On the other hand, the disease is an inflammatory disease. On the other hand, the subject is a human.

[0011] As embodied and broadly described herein, one aspect of this disclosure relates to a tandem scFv-Fc PD-1 agonist antibody, wherein the antibody comprises tandem scFv1 binding sites and scFv2 binding sites on each antibody arm, and wherein the scFv1 and the scFv2 are linked by a linker, optionally a flexible linker. On one hand, the antibody has a total of four scFv binding sites in a single scFv-Fc form of the antibody. On the other hand, the scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and wherein the scFv2 of each antibody arm comprises a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2). On the other hand, the VH1 region and the VH2 region each comprise an amino acid sequence of any one of SEQ ID NO: 1-5 and 48-54, more preferably SEQ ID NO: 1 or 53; and wherein the VL1 region and the VL2 region each comprise an amino acid sequence of any one of SEQ ID NO: 6-9 and 55-61, or preferably SEQ ID NO: 6 or 60. On the other hand, the linker comprises the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).

[0012] As embodied and broadly described herein, one aspect of this disclosure relates to a method of treating a disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 15. On one hand, the disease is an autoimmune disease. On the other hand, the disease is an inflammatory disease. On the other hand, the subject is a human.

[0013] As embodied and broadly described herein, one aspect of this disclosure relates to a nucleic acid encoding a PD-1 agonist antibody described above. On the one hand, the nucleic acid sequence is selected from nucleic acid sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:66 and 67, 68 and 69, 70 and 71, 72 and 73, 74 and 75, 76 and 77, 78 and 79, 80 and 81, 82 and 83, 84 and 85, 86 and 87, 88 and 89, or 90 and 91. On the other hand, the nucleic acid sequence is selected from variable heavy chains selected from 92, 94, 96, 98, 100, 102, or 104, and sequences selected from SEQ ID NO:93, 95, 97, 99, 101, ...The light chains 103, 104, or 105 have sequences with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0014] As embodied and broadly described herein, one aspect of this disclosure relates to a nucleic acid vector comprising the nucleic acid sequences described above. As embodied and broadly described herein, one aspect of this disclosure relates to a host cell comprising the nucleic acid vector described above. Brief Description of the Drawings

[0015] This patent or application contains at least one color drawing. A copy of this patent or application disclosure with color drawings will be provided upon request and payment of the necessary fees. This application can be understood by referring to the following description in conjunction with the drawings.

[0016] Figure 1A illustrates a PD-1 agonist epitope-directed MEM-nanoparticle B cell activation discovery strategy, as further described in Example 1.

[0017] Figure 1B shows the MEM-nanoparticle titer and PD1AB6 binding via Coomassie blue and surface plasmon resonance (SPR) binding, respectively.

[0018] Figure 1C illustrates a mouse immunization regimen using alternating doses of MEM-nanoparticles and full-length PD-1, and the final combination enhanced.

[0019] Figure 1D shows the binding of the resulting mouse serum to PD-1 as measured by enzyme-linked immunosorbent assay (ELISA).

[0020] Figure 2A shows the PD-1 binding of antibodies generated from monoclonal hybridomas as measured by ELISA.

[0021] Figure 2B shows the binding of exemplary antibody 27A5 to PD-1 (left) and competitive PD-1 binding between 27A5 (or lack thereof) and PD1AB6 and pembrolizumab (right).

[0022] Figure 2C shows the resulting PD-1 binding affinity curve for 27A5 as measured by SPR.

[0023] Figure 2D shows the PD-1 agonist (left) and antagonist (right) responses of 27A5 as measured by checkpoint signal transduction assay.

[0024] Figure 3A illustrates the phage panning strategy for full-length PD-1 and MEM-nanoparticles in in vitro scFv libraries, as described in Example 3.

[0025] Figure 3B shows the resulting SPR binding screening of isolated antibodies generated by phage panning.

[0026] Figure 3C shows that isolated antibodies generated by phage panning do not compete with pembrolizumab for PD-1 binding.

[0027] Figure 3D shows the SPR binding affinity and affinity curves of exemplary antibodies generated by the phage panning strategy.

[0028] Figure 4A shows the PD-1 agonist curve of exemplary antibodies measured by checkpoint signal transduction assay.

[0029] Figure 4B shows the PD-1 antagonist curve of exemplary antibodies measured by checkpoint signal transduction assay.

[0030] Figure 4C shows the competitive PD-1 binding between an exemplary antibody and PD1AB6 (left) or pembrolizumab (right) via SPR.

[0031] Figure 4D shows the affinity, affinity, and agonist EC50 values ​​of the pooled exemplary antibodies generated by the phage panning strategy.

[0032] Figure 5 shows the PD-1 SPR binding curves of the two resulting exemplary antibodies.

[0033] Figure 6A shows the PD-1 agonist curve of the exemplary antibody as measured by checkpoint signal transduction assay.

[0034] Figure 6B shows the PD-1 antagonist curve of the exemplary antibody as measured by checkpoint signal transduction assay.

[0035] Figure 7A illustrates the AI ​​model / mammalian antibody discovery strategy as described in Example 7.

[0036] Figure 7B shows the resulting PD-1 SPR binding curve of the exemplary antibody.

[0037] Figure 7C shows the thermostability measurement of the exemplary antibody.

[0038] Figure 8A shows the complete blockade of PD1AB6 binding to PD-1 by the exemplary antibody.

[0039] Figure 8B shows the PD-1 agonist curve of an exemplary antibody as measured by checkpoint signal transduction assay.

[0040] Figure 8C shows the PD-1 antagonist deficiency of an exemplary antibody as measured by checkpoint signal transduction assay.

[0041] Figure 9 shows the PD-1 agonist activity of an exemplary antibody as measured by expression of human primary CD4 T cell cytokine release and activation markers.

[0042] Figure 10 shows epitope grouping of three exemplary clones exhibiting effective PD-1 agonist activity.

[0043] Figure 11 illustrates the structure of an exemplary tandem scFv-Fc antibody of this disclosure.

[0044] Figure 12 shows the melting temperature of the tandem scFv-Fc antibody.

[0045] Figure 13 shows the PD-1 agonist activity of an exemplary tandem scFv-Fc antibody as measured by checkpoint signal transduction assay.

[0046] Figure 14 shows the PD-1 antagonist deficiency of an exemplary tandem scFv-Fc antibody as measured by checkpoint signal transduction assay.

[0047] Figure 15 shows the PD-1 agonist activity of an exemplary tandem scFv-Fc form antibody as measured by expression of human primary CD4 T cell cytokine release and activation markers. Specification 3 / 28 pages 6 CN 121159694 A Detailed Description

[0048] While the preparation and use of various embodiments of the invention are discussed in detail below, it should be understood that the invention provides many applicable inventive concepts that may be embodied in various specific contexts. The specific embodiments discussed herein only illustrate specific ways of preparing and using the invention and do not limit the scope of the invention.

[0049] To facilitate understanding of the invention, many terms are defined below. The terms defined herein have the same meaning as those of the invention.The meaning is as commonly understood by one of ordinary skill in the art. Terms such as “a / an” and “the” are not intended to refer only to a singular entity, but to include general categories from which specific examples may be used for illustration. The terms used herein are used to describe specific embodiments of the invention, but their use is not limiting of the invention except as set forth in the claims.

[0050] This document provides agonist antibodies that bind to PD-1 at sites not recognized by PD-L1. Methods for preparing and using such antibodies are also provided. These antibodies can be used to downregulate an individual’s immune response. For example, in some embodiments, the antibodies can be used to treat diseases involving autoimmunity and / or excessive inflammation.

[0051] Where elements are presented in list form (e.g., as Markush groups), it should be understood that each possible subgroup of the elements is also disclosed, and any one or more elements may be removed from the list or group.

[0052] It should be understood that, unless explicitly stated otherwise, in any method described or disclosed herein that includes more than one action, the order of actions is not necessarily limited to the order in which the actions of the method are narrated, but this disclosure covers exemplary embodiments in which the order of actions is thus limited.

[0053] Unless otherwise limited in specific instances, the terms used throughout this specification are defined as follows. Unless the context otherwise clearly requires, the singular forms “a / an” and “the” as used in this specification and claims include a plurality of indicators. Unless otherwise defined or stated, all technical and scientific terms used in this specification and claims have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise stated or defined, all numerical ranges include the values ​​within the defined range and all integer values ​​therebetween.

[0054] As used throughout, the term “antibody” is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, non-human antibodies, chimeric antibodies, monovalent antibodies, antibody fragments, and tandem scFv-Fc antibodies.

[0055] The antibody fragments of this disclosure retain PD-1 antigen-binding specificity. Antibody fragments include antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL sequences, single-chain variable fragments (scFv) containing VH and VL sequences linked together in one chain, single-chain antibody fragments (scAb), or other antibody variable region fragments, such as those retaining antigen-binding specificity.

[0056] The tandem scFv-Fc antibodies of this disclosure contain two or more tandem scFv binding sites on each antibody arm, said binding sites optionally linked by a linker (optionally a flexible linker), thereby enabling antibody binding in a single scFv-Fc form.A total of four or more scFv binding sites are generated. Figure 11 illustrates an exemplary tandem scFv-Fc antibody structure of this disclosure.

[0057] As used throughout, the term “mesoscale molecule (MEM)” includes engineered peptides and polypeptides ranging from about 1 kDa to about 10 kDa. As used throughout, the term “MEM-nanoparticle” includes MEMs that have been conjugated to nanoparticles (e.g., ferritin nanoparticles).

[0058] As used throughout, “subject” can be a mammalian subject. Mammal subjects include humans, non-human primates, rodents (e.g., rats, mice), rabbits (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., a cat, a dog).

[0059] All disclosures, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as specifically and individually indicated that each individual disclosure, patent or patent application is incorporated by reference.

[0060] I. PD-1 Agonist Antibodies

[0061] This document provides antibodies that bind to PD-1 and elicit an agonistic response. These antibodies are referred to herein as PD-1 agonist antibodies. Many discovery strategies have been employed to obtain exemplary antibodies of this disclosure, which are discussed further below.

[0062] The amino acid sequence of full-length human PD-1 is provided as SEQ ID NO:63 (refer to UniProtKB ID Q15116).

[0063] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN

[0064] ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQ

[0065] LPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRA

[0066] EVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTI

[0067] GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSA DGPRSAQPLRPEDGHCSWPL(SEQ ID NO:63)

[0068] In some embodiments, the PD-1 agonist antibody specifically binds to a PD-1 epitope, which is obtained through...Identified using the amino acid sequence provided by SEQ ID NO:62, RFRVTQLPNGRDFHMSVV.

[0069] Referring to FIG1A, the MEM of this disclosure is prepared to mimic the agonist epitope identified by SEQ ID NO:62 and subsequently used for antibody screening. An advantage of this method is its ability to guide antibodies to discover locations away from the PD-L1 binding site and toward the desired epitope.

[0070] In some embodiments, the MEM-nanoparticles, together with full-length PD-1, are used to immunize a subject to generate antibodies specific to the MEM epitope. Monoclonal hybridomas are then generated to produce epitope-specific PD-1 antibodies. In other embodiments, mouse serum is collected and used to generate an in vitro scFv library. The full-length PD-1 and MEM-nanoparticles are then subjected to phage panning to isolate epitope-specific clones. In other embodiments, antibodies are generated using phage panning of a primary antibody library targeting full-length PD-1 and MEM-nanoparticles. In other embodiments, humanized PD-1 CDRs are generated from AI model predictions based on a reference antibody. Then, a mammalian display library was created, and single cells were sorted to select epitope-specific PD-1 antibodies.

[0071] Those skilled in the art will recognize that antibodies exhibiting little or no binding to the target antigen can be described as having low affinity and a high equilibrium dissociation constant (KD) against the target antigen. Those skilled in the art will also recognize that antibodies exhibiting little or no binding to a collective assembly of the target antigen epitope can be described as having low affinity and a high equilibrium dissociation constant (KD) against a collective assembly of the target antigen epitope.

[0072] In some embodiments, this document provides PD-1 agonist antibodies having binding affinity (KD) to PD-1 of about 5 μM to about 5 pM, about 1 μM to about 5 pM, about 0.5 μM to about 5 pM, about 0.1 μM to about 5 pM, about 50 nM to about 5 pM, about 10 nM to about 5 pM, about 5 nM to about 5 pM, about 1 nM to about 5 pM, about 0.5 nM to about 5 pM, about 0.1 nM to about 5 pM, about 50 pM to about 5 pM, and about 10 pM to about 5 pM.

[0073] In some embodiments, the binding affinity (KD) of the PD-1 agonist antibody to PD-1 is about 500 nM to about 0.1 pM, about 100 nM to about 0.1 pM, about 50 nM to about 0.1 pM, about 10 nM to about 0.1 pM, about 5 nM to about 0.1 pM, about 1 nM to about 0.1 pM, about 0.5 nM to about 0.1 pM, about 0.1 nM to about 0.1 pM, about 50 pM to about 0.1 pM, about 10 pM to about 0.1 pM, about 5 pM to about 0.1 pM, about 1 pM to about 0.1 pM, and about 0.5 pM to about 0.1 pM.

[0074] In some embodiments, the half-maximum effective concentration (EC50) of the PD-1 agonist antibody against PD-1 is about 500 nM to about 0.001 nM, about 100 nM to about 0.001 nM, about 50 nM to about 0.001 nM, about 10 nM to about 0.001 nM, about 5 nM to about 0.001 nM, about 1 nM to about 0.001 nM, about 0.5 nM to about 0.001 nM, about 0.1 nM to about 0.001 nM, about 0.05 nM to about 0.001 nM, about 0.01 nM to about 0.001 nM, about 0.005 nM to about 0.001 nM.

[0075] Those skilled in the art will recognize that binding specificity can be determined through a series of competitive binding paradigms in which the desired antibody demonstrates its ability to prevent known reference antibodies from binding to their target epitopes at different concentrations. In some embodiments, the reference PD-1:PD-L1 antagonist antibody is pembrolizumab. In some embodiments, the reference agonist antibody binding to the epitope identified by SEQ ID NO:62 is PD1AB6 (reference patent number: US10428145B2). Those skilled in the art will also recognize that PD1AB6 and pembrolizumab can be used as control antibodies in agonist and antagonist assays.

[0076] In some embodiments, the PD-1 agonist antibodies of this disclosure do not disrupt the binding of pembrolizumab to PD-1. Exemplary antibodies of this disclosure that do not disrupt the binding of pembrolizumab to PD-1 include antibodies 27A5, 1-CO9-1, 1-CO9-3, 1-F09-1, 1-F12-1, 1-H01-1, and 2-D11-1. Figures 2B and 4C. The PD-1 agonist antibody of this disclosure disrupts the binding of PD1AB6 to the PD-1 epitope identified by SEQ ID NO:62.

[0077] In some embodiments, the PD-1 agonist antibody is a full-length antibody (meaning an antibody having two heavy chains and two light chains attached to an Fc domain, thus obtaining a "Y" shape). In some embodiments, the Fc domain (or simply Fc) is a human Fc domain. In some embodiments, the Fc domain of the PD-1 agonist antibody is derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0078] A. Exemplary PD-1 Agonist Antibody - CDR Sequence

[0079] Sequences of exemplary PD-1 agonist antibodies of this disclosure are provided herein. These include complementarity-determining region (CDR) sequences and variable heavy domain sequences and light domain sequences (VH, VL) constituting the PD-1 antigen-binding domain of this disclosure. The discovery of these antibodies is detailed in the Examples section.

[0080] As described below, the light chain variable (VL) structural domain CDR1 region is referred to as CDR-L1; the VL CDR2 region is referred to as CDR-L2; VLThe CDR3 region is referred to as CDR-L3; the heavy chain variable (VH) domain CDR1 region is referred to as CDR-H1; the VH CDR2 region is referred to as CDR-H2; and the VH CDR3 region is referred to as CDR-H3. Table 1 provides exemplary CDR combinations of antibodies of the present disclosure.

[0081] Table 1: Exemplary PD-1 agonist antibody CDR combination specification 6 / 28 pages 9 CN 121159694 A

[0082] Specification 7 / 28 pages 10 CN 121159694 A

[0083]

[0084] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0085] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

[0086] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0087] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0088] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:28.

[0089] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:25; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0090] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:40, SEQ ID NO:27, and SEQ ID NO:41.

[0091] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.

[0092] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.

[0093] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:46, SEQ ID NO:43, and SEQ ID NO:45.

[0094] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:46, SEQ ID NO:43, and SEQ ID NO:47.

[0095] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:34; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.

[0096] In some embodiments, this document provides a PD-1 agonist antibody, wherein the antibody comprises the amino acid sequences of the following three VH CDRs: SEQ ID NO:36, SEQ ID NO:33, SEQ ID NO:34; and / or comprises the amino acid sequences of the following three VL CDRs: SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.

[0097] B. Exemplary PD-1 agonist antibody - variable region sequence.

[0098] The terms variable domain and variable region are used interchangeably and refer to portions of the light and heavy chains of the antibody including complementarity-determining regions and framework regions (FRs).

[0099] Table 2 provides the amino acid sequences of the variable domains of exemplary PD-1 agonist antibodies of this disclosure. Therefore, in some embodiments, the PD-1 agonist antibody of this disclosure comprises a variable heavy chain containing amino acid sequences selected from SEQ ID NO: 1-5, 48-54; and / or in some embodiments, the PD-1 agonist antibody of this disclosure comprises a variable light chain containing amino acid sequences selected from SEQ ID NO: 6-9, 55-61.

[0100] In some embodiments, the PD-1 agonist antibody of this disclosure comprises a combination of VH / VL variable chain sequences of any of the combinations listed in Table 2.

[0101] Table 2: Exemplary variable heavy and variable light chain amino acid sequences of PD-1 agonist antibodies. Specification 9 / 28 page 12 CN 121159694 A

[0102] Specification 10 / 28 page 13 CN 121159694 A

[0103] Specification 11 / 28 page 14 CN 121159694 A

[0104]

[0105] Table 3 - Exemplary variable heavy chain and variable light chain nucleic acid sequences of PD-1 agonist antibodies.

[0106] Specification 12 / 28 page 15 CN 121159694 A

[0107] Specification 13 / 28 page 16 CN 121159694 A

[0108] Specification 14 / 28 page 17 CN 121159694 A

[0109] Specification 15 / 28 page 18 CN 121159694 A

[0110]

[0111] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:1; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:6.

[0112] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:2; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:7.

[0113] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:3; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:8.

[0114] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:4; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:8.

[0115] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:4; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:9. Specification 16 / 28 pages 19 CN 121159694 A

[0116] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:5; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:8.

[0117] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:48; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:55.

[0118] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:49; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:56.

[0119] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:49.The amino acid sequence of SEQ ID NO:50; and / or the light chain variable domain (VL) of said antibody contains the amino acid sequence of SEQ ID NO:57.

[0120] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of said antibody contains the amino acid sequence of SEQ ID NO:51; and / or the light chain variable domain (VL) of said antibody contains the amino acid sequence of SEQ ID NO:58.

[0121] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of said antibody contains the amino acid sequence of SEQ ID NO:52; and / or the light chain variable domain (VL) of said antibody contains the amino acid sequence of SEQ ID NO:59.

[0122] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of said antibody contains the amino acid sequence of SEQ ID NO:53; and / or the light chain variable domain (VL) of said antibody contains the amino acid sequence of SEQ ID NO:60.

[0123] In some embodiments, this document provides a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:54; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:61.

[0124] C. Exemplary tandem scFv-Fc PD-1 agonist antibody.

[0125] In some embodiments, this disclosure provides a tandem scFv antibody having a plurality of PD-1 binding sites. The tandem scFv-Fc antibody of this disclosure comprises two or more tandem scFv binding sites on each antibody arm, the binding sites optionally being connected by a linker (optionally a flexible linker). In some embodiments, the tandem scFv antibody has a total of four or more scFv binding sites in a single scFv-Fc antibody. Figure 11 illustrates an exemplary tandem scFv-Fc antibody structure of this disclosure.

[0126] Without being bound by theory or mechanism, it is believed that PD-1 activation is driven by the clustering of multiple PD-1 receptors, and therefore tandem scFv-Fc antibodies with multiple PD-1 binding sites can exhibit stronger PD-1 activation compared to similar conventional two-binding-site antibodies, or even scFvs with a single VH and a single VL.

[0127] More specifically, an exemplary tandem scFv-Fc PD-1 agonist antibody comprises two antibody arms, and scFv1 and scFv2 binding sites arranged in tandem on each antibody arm, wherein the scFv1 and the scFv2 are connected by a linker, anyFlexible connectors are selected for connection. Figure 11 illustrates an exemplary tandem scFv-Fc antibody structure of this disclosure. Such antibodies have a total of four scFv binding sites in a single tandem scFv-Fc antibody.

[0128] In some embodiments, scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and scFv2 of each antibody arm comprises a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2).

[0129] In some embodiments, the VH1 region comprises the amino acid sequence of any one of SEQ ID NO: 1-5 and 48-54, more preferably SEQ ID NO: 1 or 53; and the VL1 region comprises the amino acid sequence of any one of SEQ ID NO: 6-9 and 55-61, or preferably SEQ ID NO: 6 or 60, thereby generating scFv1. Similarly, in some embodiments, the VH2 region contains the amino acid sequence of any one of SEQ ID NO: 1-5 and 48-54, more preferably SEQ ID NO: 1 or 53; and the VL2 region contains the amino acid sequence of any one of SEQ ID NO: 6-9 and 55-61, or preferably SEQ ID NO: 6 or 60, thereby generating scFv2.

[0130] The VH1 and VL1 of each scFV1 can be connected by a connector (e.g., a flexible connector).

[0131] The VH2 and VL2 of each scFV2 ​​can be connected by a connector (e.g., a flexible connector).

[0132] The scFv on each antibody arm can be connected by a connector (e.g., a flexible connector). An exemplary connector contains the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).

[0133] Table 4: Exemplary tandem scFv-Fc amino acid sequences.

[0134] Specification 18 / 28 pages 21 CN 121159694 A

[0135]

[0136] Table 3: Exemplary Tandem scFv-Fc Nucleic Acid Sequence Specification 19 / 28 pages 22 CN 121159694 A

[0137] Specification 20 / 28 pages 23 CN 121159694 A

[0138] Specification 21 / 28 pages 24 CN 121159694 A

[0139] Specification 22 / 28 pages 25 CN 121159694 A

[0140]

[0141] In an exemplary embodiment, this document provides a tandem scFv-Fc PD-1 agonist antibody, said antibody inEach antibody arm has scFv1 and scFv2, wherein the first heavy chain variable domain (VH1) of the antibody contains the amino acid sequence of SEQ ID NO:1; wherein the second heavy chain variable domain (VH2) of the antibody contains the amino acid sequence of SEQ ID NO:1; wherein the first light chain variable domain (VL1) of the antibody contains the amino acid sequence of SEQ ID NO:6; and wherein the second light chain variable domain (VL2) of the antibody contains the amino acid sequence of SEQ ID NO:6; wherein scFv1 and scFv2 are connected to a linker containing the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:64).

[0142] In an exemplary embodiment, this document provides a tandem scFv-Fc PD-1 agonist antibody having scFv1 and scFv2 on each antibody arm, wherein a first heavy chain variable domain (VH1) of the antibody comprises the amino acid sequence of SEQ ID NO:53; wherein a second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO:53; wherein a first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO:60; and wherein a second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO:60; wherein scFv1 and scFv2 are connected to a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:64).

[0143] In an exemplary embodiment, this document provides a tandem scFv-Fc PD-1 agonist antibody having scFv1 and scFv2 on each antibody arm, wherein a first heavy chain variable domain (VH1) of the antibody comprises the amino acid sequence of SEQ ID NO:1; wherein a second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO:53; wherein a first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO:6; and wherein a second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO:60; wherein scFv1 and scFv2 are connected to a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:64).

[0144] In an exemplary embodiment, this document provides a tandem scFv-Fc PD-1 agonist antibody, the antibody having scFv1 and scFv2 on each antibody arm, wherein the first heavy chain variable domain (VH1) of the antibody comprises the amino acid sequence of SEQ ID NO:53; wherein the second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO:53.The amino acid sequence of NO:1; wherein the first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO:60; and wherein the second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO:6; wherein scFv1 and scFv2 are connected to a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:64).

[0145] As depicted in FIG11, the Fc domain of the tandem scFv-Fc PD-1 agonist antibody of this disclosure may be human IgG1, human IgG2, human IgG3 or human IgG4. Specification 23 / 28 pages 26 CN 121159694 A

[0146] II. Use of PD-1 agonist antibody.

[0147] A. Therapeutic PD-1 agonist antibody.

[0148] In some embodiments, the PD-1 agonist antibody provided herein may be used to treat diseases or conditions involving immune responses.

[0149] In some embodiments, the PD-1 agonist antibodies provided herein may be used to treat autoimmune diseases. Autoimmune diseases consist of a potentially harmful immune response to self-antigens. Examples of autoimmune diseases include: alopecia, ankylosing spondylitis, atopic dermatitis, celiac disease, Crohn's disease, cutaneous lupus erythematosus (CLE), lupus nephritis, multiple sclerosis, neuromyelitis optica, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, systemic lupus erythematosus (SLE), temporal arteritis, type I diabetes, ulcerative colitis, uveitis, and vitiligo.

[0150] In some embodiments, the PD-1 agonist antibodies provided herein may be used to treat hyperinflammatory diseases. Hyperinflammatory diseases consist of a potentially harmful, overstimulated immune response. Examples of hyperinflammatory diseases include: chronic allergies, hypersensitivity vasculitis, and T-cell hypersensitivity diseases.

[0151] B. Administration of therapeutic PD-1 agonist antibodies.

[0152] The in vivo administration of the therapeutic PD-1 agonist antibody described herein can be performed intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraventricularly, intranasally, transmucosally, by implantation, or by inhalation. Intravenous administration can be performed by injection or infusion. In some embodiments, the PD-1 agonist antibody of this disclosure is administered intravenously. In some embodiments, the PD-1 agonist antibody of this disclosure is administered subcutaneously. The administration of the therapeutic PD-1 agonist antibody can be performed with any suitable excipient, carrier, or other agent to provide suitable or improved tolerability, transfer, delivery, etc.

[0153] Examples

[0154] Example 1: Discovery of a PD-1 agonist based on engineered MEM-nanoparticle immunization.

[0155] MEM was designed based on the epitope identified by SEQ ID NO:62 and then conjugated with nanoparticles to guide B cell antibody production toward the epitope and away from the PD-L1 binding site, Fig. 1A. The MEM conjugated with ferritin nanoparticles was identified by Coomassie-based Western blotting and found to contain approximately 20–30 MEMs per nanoparticle, Fig. 1B. Using surface plasmon resonance (SPR), the MEM-nanoparticles showed nanomolar binding affinity to PD1AB6. BALB / c mice were then immunized with alternating doses of engineered MEM-nanoparticles suspended in adjuvant and / or full-length PD-1 over a 5-week period, ultimately enhancing the combination containing both, Fig. 1C. Mouse serum was collected, yielding strong PD-1 binding as measured by ELISA, Fig. 1D.

[0156] Example 2: Antibodies generated by the first monoclonal hybridoma showed strong PD-1 binding and activation.

[0157] Hybridomas were generated from immunized mouse B cells using a standard electrocyte fusion method. The resulting 27A5 antibody and several other antibodies were generated from the monoclonal hybridomas and showed strong PD-1 binding by ELISA, Figure 2A. The PD-1 binding and competitive binding of 27A5 were then further evaluated in vitro using SPR, Figure 2B. 27A5 showed a 55 nM KD and blocked the PD1AB6 binding site but did not block the pembrolizumab binding site. The PD-1 affinity of 27A5 was also measured by SPR, showing strong binding at a 5 pM KD, Figure 2C. The in vitro function of 27A5 at PD-1 was then evaluated. Checkpoint signal transduction assays were performed to measure PD-1 agonism and antagonism, producing the resulting concentration / response curves, Figure 2D. 27A5 showed comparable agonism to the control antibody and no antagonism.

[0158] Example 3: Antibodies generated from an in vitro scFv library showed strong PD-1 binding.

[0159] Additionally, mouse serum collected from previous immunizations was used to construct an in vitro scFv library using standard molecular cloning techniques and phage display (see page 24 / 28 of the specification, CN 121159694 A). The library was subjected to three rounds of phage panning against full-length PD-1 and MEM-nanoparticles, in the order listed in Figure 3A. The isolated clones were then screened using SPR for PD-1 binding affinity and pembrolizumab competitive binding (Figures 3B-3C). Exemplary antibodies binding to PD-1 with high affinity and co-linked affinity were identified from the scFv in vitro library using SPR (Figure 3D).

[0160] Example 4: The selected antibodies generated from the in vitro scFv library showed activating activity at the expected epitopes.

[0161] The PD-1 activating and antagonistic effects of the selected antibodies were then evaluated using checkpoint signal transduction assays.Figures 4A-4B. The resulting agonist concentration-response curves were used to obtain the EC50 value for each antibody, thereby producing agonist activity comparable to or higher than that of the control antibody. The resulting antagonist concentration-response curves were used to obtain the IC50 value, which was undetectable for all six antibodies, indicating a lack of PD-1 antagonism. The competitive binding of the PD1AB6 binding site and the pembrolizumab binding site of the six antibodies was then further evaluated, Figure 4C. All six antibodies showed complete blocking of PD1AB6 binding and no blocking of pembrolizumab binding. Figure 4D lists the pooled agonist, affinity, and affinity values ​​for all six antibodies.

[0162] Example 5: The selected antibodies generated by the PD-1 / MEM-nanoparticle panning strategy of the original library showed strong PD-1 binding.

[0163] The MEM-nanoparticles and full-length PD-1 were then used in the phage panning strategy of the original antibody library. Selected antibodies 7 and 43 were generated, and their PD-1 binding affinity was further evaluated in vitro. Figure 5 shows the SPR response curves of the two antibodies at three concentrations. KD values ​​were calculated from the SPR response curves, and both antibodies were found to be in the nanomolar range.

[0164] Example 6: PD-1 agonist activity of the first antibody generated by the primary library panning strategy.

[0165] PD-1 agonism of antibodies 7 and 43 was then evaluated in vitro. Figure 6A shows the results of the agonist assay in the form of log-scale concentration-response curves. EC50 values ​​were calculated, showing higher relative potency compared to the control antibody. PD-1 antagonism of the two antibodies was also evaluated in vitro. Figure 6B shows the results of the antagonist assay in the form of log-scale concentration-response curves. For either antibody showing a lack of equivalent antagonism when compared to the negative control antibody, the IC50 value was undetectable. Pembrolizumab was used as a positive control, showing an IC50 of 0.06 nM.

[0166] Example 7: PD-1 binding affinity of selected antibodies generated via an AI / mammalian display strategy.

[0167] Fully humanized PD-1 agonist antibody CDRs were generated from AI-model predictions (starting with a reference CDR antibody template of PD1AB6), Figure 7A. A mammalian display library was then created, and single cells were sorted using FACS to select antibodies with favorable binding properties and exploitability. The PD-1 binding affinity of the selected antibodies was evaluated in vitro. Figure 7B shows the SPR response binding curves of the selected antibodies at five concentrations. KD values ​​were calculated from the SPR response curves and it was found that the KD values ​​were in the picomolar and nanomolar range, showing higher affinity compared to PD1AB6. Figure 7C shows the calculated thermostability of the selected antibodies, which showed comparable Tm (°C) values ​​compared to PD1AB6.

[0168] Example 8: AI / mammalian display of antibodies showing PD-1 activating at the expected epitope.

[0169] The ability of the selected antibodies to compete with PD1AB6 for PD-1 binding was then tested in vitro. Figure 8A shows the SPR response curves, indicating the lack of PD1AB6 binding, showing that all four antibodies showed specific binding to the expected PD-1 epitope. The PD-1 activating effect of the selected antibodies was then evaluated in vitro. Figure 8B shows the results of the agonist assay in the form of log-scale concentration-response curves. EC50 values ​​were calculated to show equivalent or higher relative potency compared to PD1AB6. The PD-1 antagonism of the selected antibodies was then evaluated in vitro. Figure 8C shows the results of the antagonist assay in the form of log-scale concentration-response curves. For all four antibodies showing the lack of equivalent antagonism compared to PD1AB6, the IC50 value was undetectable. Pembrolizumab was used as a positive control, showing an IC50 value of 0.06 nM.

[0170] Example 9: First in vitro reporter gene assay cloned human primary cell assay. Instructions for Use, pages 25 / 28, CN 121159694 A

[0171] Antibody clones 7, 43, 1-H01-1, and D4-A7-7_201 were selected for the assay of cytokine release and activation biomarkers in human primary CD4 T cells. These four antibodies were selected based on their EC50 values ​​as PD-1 agonist reporter genes in vitro. Human PBMCs were isolated from the 7-donor and activated with 5 μg / mL PHA for 48 hours to upregulate PD-1 expression. CD4 T cells were then purified from the pre-stimulated PBMCs and plated at a uniform density on a series of 96-well plates sequentially coated with 3 μg / mL OKT3 and each test item. The supernatant was collected, and IL-2 was measured at 24 hours and IFN-γ at 72 hours. CD4 T cells were collected at 72 hours, and PD-1 and CD69 activation biomarkers were analyzed by flow cytometry. Figure 9 shows the results of the assay of cytokine release and activation biomarkers in 7-donor CD4 T cells. Clones 7, 43, 1-H01-1, and D4-A7-7_201 significantly attenuated IL-2, IFN-γ cytokines, and CD69 T cell activation markers more significantly than PD1AB6. Compared to the isotype control, clones 7, 43, and 1-H01-1 showed significant downregulation of PD-1 expression, comparable to PD1AB6, indicating that these clones effectively activated the PD-1 pathway, thereby triggering PD-1 downregulation.

[0172] Example 10: Epitope Grouping.

[0173] Based on in vitro reporter gene and human primary cell assays of antibody clones 7, 1-H01-1, and D4-A7-7_201, the antibody clones were selected for epitope grouping. Clones 7, 1-H01-1, and D4-A7-7_201 were tested against each other and those with pre-existing epitopes.Epitope grouping was performed by competing for SPR binding ability among the baseline antibodies PD1AB6, nivolumab, pembrolizumab, and UCB949, which are known to bind to PD-1 epitopes. Figure 10 shows the epitope grouping results, indicating that clones 7, 1-H01-1, and D4-A7-7_201 bind to different regions on PD-1.

[0174] Example 11: Tandem scFv-Fc antibody form.

[0175] Antibody clones 7 and 1-H01-1 were selected for testing in the form referred to herein as tandem scFv-Fc, which has two tandem scFv binding sites on each antibody arm connected by a adapter (in this example, a flexible adapter), and a total of four scFv binding sites in a single scFv-Fc antibody form, as shown in the exemplary structure in Figure 11.

[0176] Without being bound by theory or mechanism, it is believed that PD-1 activation is driven by clusters of multiple PD-1 receptors, and therefore tandem scFv-Fc antibodies with more than two PD-1 binding sites can exhibit stronger PD-1 activation compared to similar two-binding-site antibodies.

[0177] Three tandem scFv-Fc conformations were tested using clones 7 and 1-H01-1: tandem clone 7 monospecific, tandem clone 1-H01-1 monospecific, and tandem clone 7+1-H01-1 bispecific. Thermostatic stability (Tm) of the tandem scFv-Fc antibodies was tested using differential scanning fluorometry. Figure 12 shows the thermostatic stability of the following tandem scFv-Fc clones: 7 monospecific, 1-H01-1 monospecific, and 7+1-H01-1 bispecific. The ability of the three tandem scFv-Fc antibody conformations to activate and antagonize the PD-1 pathway was tested using the same in vitro reporter gene assay used to identify the non-tandem bivalent 7 and 1-H01-1 clones. Figure 13 shows the results of PD-1 agonist reporter gene assays, and Figure 14 shows the results of PD-1 antagonist reporter gene assays for the three tandem scFv-Fc antibodies tested. In each case, the tandem scFv-Fc configuration with four scFv binding sites showed a 2-fold or greater improvement in PD-1 agonist EC50 compared to the bivalent non-tandem configuration, and a 3-fold or greater improvement in PD-1 agonist EC50 compared to the PD1AB6 baseline. No PD-1 antagonism was observed in the tandem scFv-Fc antibodies tested.

[0178] Example 12: Human primary cell assays of tandem scFv-Fc clones.

[0179] Three tandem scFv-Fc configurations were tested using clone 7 and 1-H01-1: tandem clone 7 monospecific, tandem clone 1-H01-1 monospecific, and tandem clone 7+1-H01-1 bispecific were selected for human primary CD4 T cell cytokine release and activation marker assays. Human PBMCs were isolated from a 6-donor and administered at 5 μg / mL.PHA activation was performed for 48 hours, upregulating PD-1 expression. CD4 T cells were then purified from pre-stimulated PBMCs and plated at a uniform density on 96-well plates sequentially coated with 3 μg / mL OKT3 and each test item. The supernatant was collected, and IL-2 was measured at 24 hours and IFN-γ at 72 hours. CD4 T cells were collected at 72 hours, and PD-1 and CD69 activation biomarkers were analyzed by flow cytometry.

[0180] Figure 15 shows the results of 6-donor CD4 T cell cytokine release and activation biomarker assays. Tandem clone 7 single-specific, tandem clone 1-H01-1 single-specific, and tandem clone 7+1-H01-1 bispecific significantly attenuated IL-2 and IFN-γ cytokines compared to PD1AB6. Tandem clone 7+1-H01-1 bispecific showed significant downregulation of PD-1 expression relative to the isotype control and PD1AB6. Tandem clone 7 monospecific showed significant downregulation of CD69 expression relative to the isotype control and PD1AB6.

[0181] It is contemplated that any of the embodiments discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to implement the methods of the invention.

[0182] It should be understood that the specific embodiments described herein are shown by way of illustration and not as a limitation of the invention. The main features of the invention may be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to use many equivalents of the specific procedures described herein without departing from conventional experiments. Such equivalents are considered to be within the scope of the invention and are covered by the claims.

[0183] All publications and patent applications referenced in this specification are intended to be at the level of a person skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference as if specifically and individually indicated that each individual publication or patent application is to be incorporated by reference.

[0184] When used in conjunction with the term “comprising” in the claims and / or description, the word “a / an” may mean “one,” but it is also consistent with the meanings of “one or more,” “at least one,” and “one or more than one.” The term “or” in the claims is used to mean “and / or,” unless explicitly stated to refer only to alternatives or mutually exclusive alternatives, but this disclosure supports referring only to alternatives and…Definition of “and / or”. Throughout this application, the term “about” is used to indicate that the value includes inherent error variations with respect to the apparatus or method used to determine the value, or variations that exist between the subjects under study.

[0185] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional unlisted elements or method steps. In any embodiments of compositions and methods provided herein, “comprising” may be replaced by “consistently composed of” or “composed of”. As used herein, the phrase “consistent with…” requires the specified integer or steps and the integer or steps that do not substantially affect the characteristics or function of the claimed invention. As used herein, the term “consistent with…” is used only to indicate the presence of an enumerated set of integers (e.g., features, elements, characteristics, properties, method / process steps, or limitations) or an integer group (e.g., (multiple) features, (multiple) elements, (multiple) properties, (multiple) properties, (multiple) method / process steps, or limitations).

[0186] As used herein, the term “or a combination thereof” refers to all permutations and combinations of the items listed preceding the term. For example, “A, B, C or a combination thereof” is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is important in the particular context. Continuing with this example, combinations containing one or more repeated items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. Those skilled in the art will understand that, unless otherwise apparent from the context, there is generally no limitation on the number of items or terms in any combination. Specification 27 / 28 pages 30 CN 121159694 A

[0187] As used herein, approximations such as, but not limited to, “about,” “basically,” or “essentially” refer to conditions that, when modified in this way, are not necessarily absolute or perfect, but would be considered sufficiently close to what a person of ordinary skill in the art would know.The certificate specifies the conditions as conditions that exist. The extent to which this specification may vary will depend on the extent to which changes can be implemented, and will still possess the desired features and ability that a person skilled in the art would recognize as still having the unmodified features. Generally, but depending on the foregoing discussion, numerical values ​​modified herein by approximate terms such as “about” may vary from said values ​​by at least ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, 12%, or 15%.

[0188] Additionally, the section headings herein are intended to be consistent with the recommendations under 37 CFR 1.77, or to provide organizational clues. These headings should not limit or characterize one or more inventions that may be set forth in any of the claims of this disclosure. Specifically and by way of example, although the headings refer to “technical field,” such claims should not be limited by the language used to describe the so-called technical field under this heading. Furthermore, the description of the technology in the “Background Art” section should not be construed as an admission that the technology is prior art to any one or more inventions of this disclosure. “Summary of the Invention” should also not be regarded as a feature of one or more inventions set forth in the published claims. Furthermore, any reference to the singular form of “invention” in this disclosure should not be used to argue that only a single novelty exists in this disclosure. Multiple inventions may be formulated according to the limitations of multiple claims published from this disclosure, and such claims accordingly define the invention protected thereunder and its equivalents. In all cases, the scope of these claims should be considered in accordance with the advantages of this disclosure itself, but should not be limited by the headings set forth herein.

[0189] For each claim in the claims, each dependent claim may depend both on the independent claim and on each of the preceding dependent claims of each claim, provided that the preceding claims provide a suitable premise basis for the claim item or element.

[0190] To assist the Patent Office and any reader of any patent published on the basis of this application in interpreting the appended claims, the applicant wishes to note that unless the terms “means for…” or “steps for…” are expressly used in a particular claim, they do not wish any of the appended claims to invoke paragraph 6 of 35 U.S.C. §112, paragraph (f) of USC §112, or their equivalents, as they already existed on the filing date of this application.

[0191] According to this disclosure, all compositions and / or methods disclosed and claimed herein can be prepared and performed without excessive experimentation. While the compositions and methods of the present invention have been described in conjunction with preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the steps or the order of steps in the compositions and / or methods and the methods described herein without departing from the concept, spirit, and scope of the invention. It will be apparent to those skilled in the art that...It is evident that all such substitutions and modifications are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims. Instruction manual page 28 / 28, page 31, CN 121159694 A, Figure 1A; Instruction manual figure 1 / 32, page 32, CN 121159694 A, Figure 1B; Instruction manual figure 2 / 32, page 33, CN 121159694 A, Figure 1C; Instruction manual figure 3 / 32, page 34, CN 121159694 A, Figure 1D; Instruction manual figure 4 / 32, page 35, CN 121159694 A, Figure 2A; Instruction manual figure 5 / 32, page 36, CN 121159694 A, Figure 2B; Instruction manual figure 6 / 32, page 37, CN 121159694 A, Figure 2C; Instruction manual figure 7 / 32, page 38, CN 121159694 A, Figure 2D; Instruction manual figure 8 / 32, page 39, CN 121159694 A, Figure 3A; Instruction manual figure 9 / 32, page 40, CN 121159694 Figure 3B (Instruction Manual Illustration, Page 10 / 32, 41 CN 121159694) Figure 3C (Instruction Manual Illustration, Page 11 / 32, 42 CN 121159694) Figure 3D (Instruction Manual Illustration, Page 12 / 32, 43 CN 121159694) Figure 4A (Instruction Manual Illustration, Page 13 / 32, 44 CN 121159694) Figure 4B (Instruction Manual Illustration, Page 14 / 32, 45 CN 121159694) Figure 4C (Instruction Manual Illustration, Page 15 / 32, 46 CN 121159694) Figure 4D (Instruction Manual Illustration, Page 16 / 32, 47 CN 121159694) Figure 5 (Instruction Manual Illustration, Page 17 / 32, 48 CN 121159694) Figure 6A (Instruction Manual Illustration, Page 18 / 32, 49 CN 121159694) Figure 6B (Instruction Manual Illustration, Page 19 / 32) Page 50 CN 121159694 A Figure 7A Description Drawing 20 / 32 Page 51 CN 121159694 A Figure 7B Description Drawing 21 / 32 Page 52 CN 121159694 A Figure 7C Description Drawing 22 / 32 Page 53 CN 121159694 A Figure 8A Description Drawing 23 / 32 Page 54 CN 121159694 A Figure 8B DescriptionFigure 24 / 32, page 55, CN 121159694 A, Figure 8C; Figure 25 / 32, page 56, CN 121159694 A, Figure 9; Figure 26 / 32, page 57, CN 121159694 A, Figure 10; Figure 27 / 32, page 58, CN 121159694 A, Figure 11; Figure 28 / 32, page 59, CN 121159694 A, Figure 12; Figure 29 / 32, page 60, CN 121159694 A, Figure 13; Figure 30 / 32, page 61, CN 121159694 A, Figure 14; Figure 31 / 32, page 62, CN 121159694 A, Figure 15; Figure 32 / 32, page 63, CN 121159694 A, 1 Our Ref.: 023236-8156HK02 Invention Title: PD-1 Agonist Antibodies Abstract This document provides PD-1 agonist antibodies that bind to PD-1. The PD-1 agonist antibodies disclosed herein can be used to treat autoimmune and inflammatory diseases by promoting PD-1 signaling. Methods of using the PD-1 agonist antibodies are also provided herein.

Claims

1. A PD-1 agonist antibody, wherein the antibody comprises: a. Heavy chain variable domain (VH) complementarity-determining region (CDR) 1, wherein the VH CDR1 contains the amino acid sequence of SEQ ID NO:32; VH CDR2, wherein the VH CDR2 contains the amino acid sequence of SEQ ID NO:35; VH CDR3, wherein the VH CDR3 contains the amino acid sequence of SEQ ID NO:34; light chain variable domain (VL) CDR1, wherein the VL CDR1 contains the amino acid sequence of SEQ ID NO:42; VL CDR2, wherein the VL CDR2 contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, wherein the VLCDR3 contains the amino acid sequence of SEQ ID NO:45; b. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:10; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:11; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:12; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:13; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:14; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:15; c. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:29; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:30; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:25; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:26; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:27; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:28; d. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:16; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:17; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:18; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:19; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:20; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:21; e. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:22; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:23; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:24; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:26; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:27; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:28; f. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:22; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:23; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:25; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:26; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:27; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:28; g. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:22; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:23; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:25; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:26; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:31; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:28; h. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:37; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:38; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:39; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:40; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:27; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:41; i. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:32; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:33; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:34; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:42; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:44; j. VH CDR1, wherein VH CDR1 contains the amino acid sequence of SEQ ID NO:32; VH CDR2, wherein VH CDR2 contains the amino acid sequence of SEQ ID NO:33; VH CDR3, wherein VH CDR3 contains the amino acid sequence of SEQ ID NO:34; VLCDR1, wherein VL CDR1 contains the amino acid sequence of SEQ ID NO:42; VL CDR2, wherein VL CDR2 contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, wherein VL CDR3 contains the amino acid sequence of SEQ ID NO:45; k. VH CDR1, wherein the VH CDR1 contains the amino acid sequence of SEQ ID NO:32; VH CDR2, wherein the VH CDR2 contains the amino acid sequence of SEQ ID NO:33; VH CDR3, wherein the VH CDR3 contains the amino acid sequence of SEQ ID NO:34; VLCDR1, wherein the VL CDR1 contains the amino acid sequence of SEQ ID NO:46; VL CDR2, wherein the VL CDR2 contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, wherein the VL CDR3 contains the amino acid sequence of SEQ ID NO:45; l. VH CDR1, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO:32; VH CDR2, wherein VH CDR2 comprises the amino acid sequence of SEQ ID NO:33; VH CDR3, wherein VH CDR3 comprises the amino acid sequence of SEQ ID NO:34; VLCDR1, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO:46; VL CDR2, wherein VL CDR2 comprises the amino acid sequence of SEQ ID NO:43; and VL CDR3, wherein VL CDR3 comprises the amino acid sequence of SEQ ID NO:47; or m.VH CDR1, wherein the VH CDR1 contains the amino acid sequence of SEQ ID NO:36; VH CDR2, wherein the VH CDR2 contains the amino acid sequence of SEQ ID NO:33; VH CDR3, wherein the VH CDR3 contains the amino acid sequence of SEQ ID NO:34; VLCDR1, wherein the VL CDR1 contains the amino acid sequence of SEQ ID NO:42; VL CDR2, wherein the VL CDR2 contains the amino acid sequence of SEQ ID NO:43; and VL CDR3, wherein the VL CDR3 contains the amino acid sequence of SEQ ID NO:

45.

2. The antibody according to claim 1, wherein the antibody comprises: a.VH, wherein VH comprises the amino acid sequence of any one of the following SEQ ID NO:1-5, 48-54, and b.VL, wherein the VL comprises the amino acid sequence of any one of the following SEQ ID NO:6-9, 55-61.

3. The antibody according to any one of claims 1 to 2, wherein the antibody is: a. Monoclonal antibodies; b. Full-length antibody; or c. An antibody fragment, optionally wherein the antibody is fused with the Fc domain of any of the following: human IgG1, human IgG2, human IgG3, and human IgG4.

4. A tandem scFv-Fc PD-1 agonist antibody, wherein the antibody comprises tandem scFv1 binding sites and scFv2 binding sites on each antibody arm, and wherein the scFv1 and the scFv2 are connected by a linker, optionally a flexible linker.

5. The antibody of claim 4, wherein the antibody has a total of four scFv binding sites in a single scFv-Fc form.

6. The antibody according to claim 4 or 5, wherein the scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and wherein the scFv2 of each antibody arm comprises a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2).

7. The antibody according to any one of claims 4 to 6, wherein the VH1 region and the VH2 region each comprise the amino acid sequence of any one of SEQ ID NO: 1-5 and 48-54, more preferably SEQ ID NO: 1 or 53; and wherein the VL1 region and the VL2 region each comprise the amino acid sequence of any one of SEQ ID NO: 6-9 and 55-61, or preferably SEQ ID NO: 6 or 60.

8. The antibody according to any one of claims 4 to 7, wherein the linker comprises the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).