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

[0001] Cross-reference to related applications

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

[0003] Statement on Federally Sponsored Research

[0004] not applicable.

[0005] Incorporation by Reference of Materials Submitted on CD-ROM

[0006] 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) serves as a natural ligand for PD-1. The PD-1:PD-L1 interaction is used to inhibit immune cells by phosphorylation of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which activates downstream signaling, which can inhibit T cell activation, cytokine production, and can also promote apoptosis. Therefore, PD-1 is essential for preventing autoimmunity and overstimulated immune responses (which may be harmful to the body if not constrained).

[0008] It is well known that the PD-1:PD-L1 checkpoint is exploited by cancer cells that upregulate PD-L1 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, there is still a need for PD-1 agonist agents and antibodies in the treatment of various autoimmune and inflammatory diseases, which ideally do not interfere with the innate PD-1:PD-L1 interaction. Such antibodies are provided herein. Summary of the invention

[0009] Provided herein are PD-1 agonist antibodies that bind to PD-1. As embodied and broadly described herein, one aspect of the present disclosure relates to a PD-1 agonist antibody, wherein the antibody comprises: a heavy chain variable domain (VH) complementarity determining region (CDR) 1, wherein the VH CDR1 comprises the amino acid sequence of any one of the following SEQ ID NOs: 10, 16, 22, 29, 32, 36, 37; and a VH CDR2, wherein the VH CDR2 comprises the amino acid sequence of any one of the following SEQ ID NOs: 11, 17, 23, 30, 33, 35, 38; and a VH CDR3, wherein the VH CDR3 comprises the amino acid sequence of any one of the following SEQ ID NOs: 12, 18, 24, 25, 34, 39; and a light chain variable domain (VL) CDR1, wherein the VL CDR1 comprises the amino acid sequence of any one of the following SEQ ID NOs: 13, 19, 26, 40, 42, 46; and a VL CDR2, wherein the VL CDR2 comprises the amino acid sequence of any one of the following SEQ ID NOs: NO:14, 20, 27, 31, 43; and VL CDR3, the VL CDR3 comprises the amino acid sequence of any one of the following SEQ ID NO:15, 21, 28, 41, 44, 45, 47. In one aspect, the antibody comprises: VH, the VH comprises the amino acid sequence of any one of the following SEQ ID NO:1-5, 48-54; and VL, the VL comprises the amino acid sequence of any one of the following SEQ ID NO:6-9, 55-61. In another aspect, the antibody is a monoclonal antibody. In another aspect, the antibody is a full-length antibody. In another aspect, the antibody is an antibody fragment. In another aspect, the antibody is fused to the Fc domain of any one of the following: human IgG1, human IgG2, human IgG3 and human IgG4.

[0010] As embodied and broadly described herein, one aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody as described above. In one aspect, the disease is an autoimmune disease. In another aspect, the disease is an inflammatory disease. In another aspect, the subject is a human.

[0011] As embodied and broadly described herein, one aspect of the present disclosure relates to a tandem scFv-Fc PD-1 agonist antibody, wherein the antibody comprises a tandem scFv1 binding site and a scFv2 binding site on each antibody arm, and wherein the scFv1 and the scFv2 are connected by a linker, optionally a flexible linker. On the one hand, the antibody has a total of four scFv binding sites in a single scFv-Fc format. 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 NOs: 1-5 and 48-54, more preferably SEQ ID NOs: 1 or 53; and wherein the VL1 region and the VL2 region each comprise an amino acid sequence of any one of SEQ ID NOs: 6-9 and 55-61, or preferably SEQ ID NOs: 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 the present disclosure relates to a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 15. In one aspect, the disease is an autoimmune disease. In another aspect, the disease is an inflammatory disease. In another aspect, the subject is a human.

[0013] As embodied and broadly described herein, one aspect of the present disclosure relates to a nucleic acid encoding a PD-1 agonist antibody as described above. In one aspect, the nucleic acid sequence is selected from a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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 a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a variable heavy chain selected from 92, 94, 96, 98, 100, 102 or 104 and a light chain selected from SEQ ID NO:93, 95, 97, 99, 101, 103, 104 or 105.

[0014] As embodied and broadly described herein, one aspect of the present disclosure relates to a nucleic acid vector comprising the nucleic acid sequence described above. As embodied and broadly described herein, one aspect of the present disclosure relates to a host cell comprising the nucleic acid vector described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] This patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. The present application may be understood by reference to the following description in conjunction with the drawings.

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

[0017] Figure 1B MEM-nanoparticle potency and PD1AB6 binding were shown by Coomassie blue and surface plasmon resonance (SPR) binding, respectively.

[0018] Figure 1C A mouse immunization regimen using alternating doses of MEM-nanoparticles and full-length PD-1 and a final combination boost was demonstrated.

[0019] Figure 1D The binding of the resulting mouse sera to PD-1 measured by enzyme-linked immunosorbent assay (ELISA) is shown.

[0020] Figure 2A Shown is PD-1 binding of antibodies produced from monoclonal hybridomas measured by ELISA.

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

[0022] Figure 2C The resulting PD-1 binding affinity curve of 27A5 measured by SPR is shown.

[0023] Figure 2D Shown are PD-1 agonist (left) and antagonist (right) responses of 27A5 measured by checkpoint signaling assays.

[0024] Figure 3A The in vitro scFv library full-length PD-1 and MEM-nanoparticle phage panning strategy was demonstrated, as described in Example 3.

[0025] Figure 3BThe resulting SPR binding screen of isolated antibodies generated by phage panning is shown.

[0026] Figure 3C It was shown that the isolated antibodies generated by phage panning did not compete with pembrolizumab for PD-1 binding.

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

[0028] Figure 4A Shown are PD-1 agonist curves of exemplary antibodies measured by a checkpoint signaling assay.

[0029] Figure 4B Shown are PD-1 antagonist curves of exemplary antibodies measured by a checkpoint signaling assay.

[0030] Figure 4C Shown is the competitive PD-1 binding between exemplary antibodies and PD1AB6 (left) or pembrolizumab (right) by SPR.

[0031] Figure 4D Shown are pooled avidity, affinity and agonist EC50 values ​​for exemplary antibodies generated by the phage panning strategy.

[0032] Figure 5 The resulting PD-1 SPR binding curves for two exemplary antibodies are shown.

[0033] Fig. 6A Shown are PD-1 agonist curves of exemplary antibodies measured by a checkpoint signaling assay.

[0034] Figure 6B Shown are PD-1 antagonist curves of exemplary antibodies measured by a checkpoint signaling assay.

[0035] Fig. 7A The AI ​​model / mammalian display antibody discovery strategy as described in Example 7 is demonstrated.

[0036] Figure 7B The resulting PD-1 SPR binding curves for exemplary antibodies are shown.

[0037] Figure 7C Thermal stability measurements of exemplary antibodies are shown.

[0038] Fig. 8A Complete blocking of PD1AB6 binding to PD-1 by the exemplary antibodies is shown.

[0039] Figure 8BShown are PD-1 agonist curves of exemplary antibodies measured by a checkpoint signaling assay.

[0040] Figure 8C Exemplary antibodies are shown to lack PD-1 antagonism as measured by a checkpoint signaling assay.

[0041] Fig. 9 Shown are PD-1 agonist activities of exemplary antibodies as measured by human primary CD4 T cell cytokine release and activation marker expression.

[0042] Fig.10 Epitope grouping of three exemplary clones exhibiting potent PD-1 agonist activity is shown.

[0043] Fig.11 Antibody structures of exemplary tandem scFv-Fc formats of the present disclosure are shown.

[0044] Fig.12 Melting temperatures of antibodies in tandem scFv-Fc format are shown.

[0045] Fig.13 Shown is the PD-1 agonist activity of an exemplary tandem scFv-Fc format antibody measured by a checkpoint signaling assay.

[0046] Fig.14 Shown is the lack of PD-1 antagonism of an exemplary tandem scFv-Fc format antibody as measured by a checkpoint signaling assay.

[0047] Fig.15 Shown are the PD-1 agonist activities of exemplary tandem scFv-Fc format antibodies as measured by human primary CD4 T cell cytokine release and activation marker expression. DETAILED DESCRIPTION

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

[0049] To facilitate understanding of the present invention, a number of terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention relates. Terms such as "a / an" and "the" are not intended to refer only to singular entities, but to include general categories whose specific examples may be used for illustration. The terms herein are used to describe specific embodiments of the present invention, but their use does not limit the present invention except as outlined in the claims.

[0050] Provided herein are agonist antibodies that bind to PD-1 at sites that are not recognized by PD-L1. Also provided are methods for preparing and using such antibodies. 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 (eg, in Markush groups), it is understood that every 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, in any method described or disclosed herein that includes more than one act, the order of the acts is not necessarily limited to the order in which the acts of the method are recited, but the present disclosure covers exemplary embodiments in which the order of acts is so limited.

[0053] Unless otherwise limited in specific examples, the terms used throughout this specification are defined as follows. Unless the context clearly dictates otherwise, when used in this specification and claims, the singular forms "a / an" and "the" include plural referents. Unless otherwise defined or indicated, all technical and scientific terms used in this specification and claims have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Unless otherwise indicated or defined, all numerical ranges include the values ​​defining the ranges and all integer values ​​therebetween.

[0054] As used throughout this document, 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 disclosed herein 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 retaining antigen binding specificity.

[0056] The tandem scFv-Fc antibodies of the present disclosure comprise two or more scFv binding sites in tandem on each antibody arm, optionally connected by a linker (optionally a flexible linker), thereby generating a total of four or more scFv binding sites in a single scFv-Fc format of the antibody. Fig.11 Antibody structures of exemplary tandem scFv-Fc formats of the present disclosure are shown.

[0057] As used throughout this article, the term "mesoscale molecules (MEM)" includes engineered peptides and polypeptides between about 1 kDa and about 10 kDa. As used throughout this article, the term "MEM-nanoparticles" includes MEMs that have been conjugated to nanoparticles (e.g., ferritin nanoparticles).

[0058] As used herein, a "subject" can be a mammalian subject. Mammalian subjects include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.), 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 publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0060] I. PD-1 agonist antibodies

[0061] Provided herein are 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 the present disclosure, which are discussed further below.

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

[0063] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDN

[0064] ATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQ

[0065] LPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRA

[0066] EVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTI

[0067] GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO:63)

[0068] In some embodiments, the PD-1 agonist antibody specifically binds to a PD-1 epitope identified by the amino acid sequence provided as RFRVTQLPNGRDFHMSVV SEQ ID NO:62.

[0069] refer to Figure 1A MEMs of the present disclosure were prepared to mimic the agonist epitope identified by SEQ ID NO: 62 and subsequently used to screen antibodies. The advantage of this approach is the ability to direct antibody discovery away from the PD-L1 binding site and toward the desired epitope.

[0070] In some embodiments, MEM-nanoparticles together with full-length PD-1 are used to immunize subjects in order to produce antibodies specific to MEM epitopes. Monoclonal hybridomas are then produced to produce epitope-specific PD-1 antibodies. In other embodiments, mouse serum is collected and used to produce an in vitro scFv library. Full-length PD-1 and MEM-nanoparticles are then subjected to phage panning to isolate epitope-specific clones. In other embodiments, antibodies are produced using phage panning of the original antibody library for full-length PD-1 and MEM-nanoparticles. In other embodiments, humanized PD-1 CDRs are generated based on reference antibodies predicted from an AI model. A mammalian display library is then created, and single cells are sorted to select epitope-specific PD-1 antibodies.

[0071] The skilled person will recognize that antibodies that exhibit little or no binding to the target antigen can be described as having low affinity and a high equilibrium dissociation constant (KD) for the target antigen. The skilled person will also recognize that antibodies that exhibit little or no binding to the collective assembly of target antigen epitopes can be described as having low affinity and a high equilibrium dissociation constant (KD) for the collective assembly of target antigen epitopes.

[0072] In some embodiments, provided herein are PD-1 agonist antibodies having a 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, about 10 pM to about 5 pM.

[0073] In some embodiments, the PD-1 agonist antibody binds to PD-1 with a binding affinity (KD) of 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, about 0.5 pM to about 0.1 pM.

[0074] In some embodiments, the half-maximal effective concentration (EC50) of the PD-1 agonist antibody for 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] The skilled person will recognize that binding specificity can be determined by a series of competitive binding paradigms, in which the desired antibody shows its ability to prevent a known reference antibody from binding to its target epitope at different concentrations. In some embodiments, the reference PD-1:PD-L1 antagonist antibody is pembrolizumab. In some embodiments, the reference agonist antibody that binds to the epitope identified by SEQ ID NO: 62 is PD1AB6 (reference patent number: US10428145B2). The skilled person 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 the present disclosure do not disrupt the binding of pembrolizumab to PD-1. Exemplary antibodies of the present disclosure that do not disrupt the binding of pembrolizumab to PD-1 include antibodies 27A5, 1-C09-1, 1-C09-3, 1-F09-1, 1-F12-1, 1-H01-1, and 2-D11-1. Figure 2B and 4C The PD-1 agonist antibodies of the present disclosure disrupt 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 (referring to an antibody having two heavy chains and two light chains attached to an Fc domain, thereby 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 from human IgG1, human IgG2, human IgG3, or human IgG4.

[0078] A. Exemplary PD-1 agonist antibody-CDR sequences

[0079] Provided herein are sequences of exemplary PD-1 agonist antibodies of the present disclosure. Including complementary determining region (CDR) sequences and variable heavy domain sequences and light domain sequences (VH, VL) constituting the PD-1 antigen binding domain of the present disclosure. The discovery of these antibodies is described in detail in the Examples section.

[0080] As described below, the light chain variable (VL) domain CDR1 region is referred to as CDR-L1; the VL CDR2 region is referred to as CDR-L2; the VL 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 for antibodies of the present disclosure.

[0081] Table 1: Exemplary PD-1 agonist antibody CDR combinations

[0082]

[0083]

[0084] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0085] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0086] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0087] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0088] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:28.

[0089] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0090] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 40, SEQ ID NO: 27, and SEQ ID NO: 41.

[0091] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44.

[0092] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45.

[0093] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 46, SEQ ID NO: 43, and SEQ ID NO: 45.

[0094] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 46, SEQ ID NO: 43, and SEQ ID NO: 47.

[0095] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45.

[0096] In some embodiments, provided herein is 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 VLCDRs: SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 45.

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

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

[0099] Table 2 provides the amino acid sequences of the variable domains of exemplary PD-1 agonist antibodies of the present disclosure. Thus, in some embodiments, the PD-1 agonist antibodies of the present disclosure comprise a variable heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 1-5, 48-54; and / or in some embodiments, the PD-1 agonist antibodies of the present disclosure comprise a variable light chain comprising an amino acid sequence selected from SEQ ID NOs: 6-9, 55-61.

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

[0101] Table 2: Exemplary variable heavy chain and variable light chain amino acid sequences of PD-1 agonist antibodies.

[0102]

[0103]

[0104]

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

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] In some embodiments, provided herein is 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, provided herein is 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, provided herein is 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, provided herein is 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, provided herein is 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.

[0116] In some embodiments, provided herein is 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, provided herein is 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, provided herein is 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, provided herein is a PD-1 agonist antibody, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 50; and / or wherein the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 57.

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

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

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

[0123] In some embodiments, provided herein is 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, the present disclosure provides a series of scFv antibodies with multiple PD-1 binding sites. The series of scFv-Fc antibodies disclosed herein include two or more scFv binding sites in series on each antibody arm, and the binding sites are optionally connected by a joint (optionally a flexible joint). In some embodiments, the series of scFV antibodies has a total of four or more scFv binding sites in a single scFv-Fc form of antibody. Fig.11 Antibody structures of exemplary tandem scFv-Fc formats of the present disclosure are shown.

[0126] Without being bound by theory or mechanism, it is believed that PD-1 agonism is driven by clustering of multiple PD-1 receptors, and thus tandem scFv-Fc antibodies with multiple PD-1 binding sites can exhibit more potent PD-1 agonism when compared to similar traditional 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, and wherein the scFv1 and the scFv2 are connected by a linker, optionally a flexible linker. Fig.11 The antibody structures of such exemplary tandem scFv-Fc formats of the present disclosure are shown. Such antibodies have a total of four scFv binding sites in a single tandem scFv-Fc format of the antibody.

[0128] In some embodiments, the scFv1 of each antibody arm comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); and the 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 an amino acid sequence of any one of SEQ ID NOs: 1-5 and 48-54, more preferably SEQ ID NOs: 1 or 53; and the VL1 region comprises an amino acid sequence of any one of SEQ ID NOs: 6-9 and 55-61, or preferably SEQ ID NOs: 6 or 60, thereby generating scFv1. Likewise, in some embodiments, the VH2 region comprises an amino acid sequence of any one of SEQ ID NOs: 1-5 and 48-54, more preferably SEQ ID NOs: 1 or 53; and the VL2 region comprises an amino acid sequence of any one of SEQ ID NOs: 6-9 and 55-61, or preferably SEQ ID NOs: 6 or 60, thereby generating scFv2.

[0130] The VH1 and VL1 of each scFV1 may be connected by a linker (eg, a flexible linker).

[0131] The VH2 and VL2 of each scFV2 ​​may be connected by a linker (eg, a flexible linker).

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

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

[0134]

[0135]

[0136] Table 3: Exemplary tandem scFv-Fc nucleic acid sequences

[0137]

[0138]

[0139]

[0140]

[0141] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist 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: 1; wherein the second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO: 1; wherein the first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO: 6; and wherein the second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO: 6; wherein the scFv1 and scFv2 are connected to a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).

[0142] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist 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; 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: 60; wherein the scFv1 and scFv2 are connected to a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).

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

[0144] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist 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: 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 the scFv1 and scFv2 are connected to a linker comprising the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).

[0145] like Fig.11As depicted, the Fc domain of the tandem scFv-Fc PD-1 agonist antibodies of the present disclosure may be human IgG1, human IgG2, human IgG3, or human IgG4.

[0146] II. Uses of PD-1 agonist antibodies.

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

[0148] In some embodiments, the PD-1 agonist antibodies provided herein can be used to treat diseases or conditions involving an immune response.

[0149] In some embodiments, the PD-1 agonist antibodies provided herein can be used to treat autoimmune diseases. Autoimmune diseases consist of potentially harmful immune responses 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, 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 can be used to treat hyperinflammatory diseases. Hyperinflammatory diseases consist of potentially harmful overstimulated immune responses. Examples of hyperinflammatory diseases include: chronic allergies, hypersensitivity vasculitis, and T-cell hypersensitivity diseases.

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

[0152] In vivo administration of the therapeutic PD-1 agonist antibodies 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 antibodies of the present disclosure are administered intravenously. In some embodiments, the PD-1 agonist antibodies of the present disclosure are administered subcutaneously. Administration of the therapeutic PD-1 agonist antibodies can be performed with any suitable excipient, carrier, or other agent to provide suitable or improved tolerance, transfer, delivery, etc.

[0153] Examples

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

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

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

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

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

[0159] Additionally, mouse sera collected from previous immunizations were used to construct an in vitro scFv library using standard molecular cloning techniques and phage display. Figure 3A The library was subjected to three rounds of phage panning against full-length PD-1 and MEM-nanoparticles in the order listed in . The resulting isolated clones were then screened using SPR for PD-1 binding affinity and pembrolizumab competitive binding. Figure 3B-3C. Using SPR to identify exemplary antibodies that bind to PD-1 with high affinity and avidity from a scFv in vitro library, Figure 3D .

[0160] Example 4: Selected antibodies generated from an in vitro scFv library show agonism at the expected epitope.

[0161] Then use Checkpoint signaling assays evaluate PD-1 agonism and antagonism of selected antibodies, Figure 4A-4B The resulting agonist concentration-response curves were used to obtain EC50 values ​​for each antibody, resulting in agonist activity comparable to or greater than that of the control antibody. The resulting antagonist concentration-response curves were used to obtain IC50 values, which were undetectable for all six antibodies, indicating a lack of PD-1 antagonism. The six antibodies were then further evaluated for competitive binding of both the PD1AB6 binding site and the pembrolizumab binding site. Figure 4C All six antibodies showed complete blockade of PD1AB6 binding and did not block pembrolizumab binding. Figure 4D Pooled agonism, avidity, and affinity values ​​for all six antibodies are listed.

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

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

[0164] Example 6: PD-1 agonist activity of the top antibodies generated by the naive library panning strategy.

[0165] Antibodies 7 and 43 were then evaluated for PD-1 agonism in vitro. Fig. 6A The results of the agonist assays are shown in the form of logarithmic scale concentration-response curves. EC50 values ​​were calculated, indicating higher relative potency compared to the control antibody. The two antibodies were additionally evaluated in vitro for PD-1 antagonism. Figure 6B The results of the antagonist assays are presented in the form of logarithmic scale concentration-response curves. For any antibody that showed a lack of equivalent antagonism when compared to the negative control antibody, the IC 50 The value was undetectable. Pembrolizumab was used as a positive control, which showed an IC of 0.06 nM 50 .

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

[0167] Fully humanized PD-1 agonist antibody CDRs were generated from AI-model predictions (starting from the reference CDR antibody template of PD1AB6). Fig. 7A Mammalian display libraries were then created and single cells were sorted using FACS to select antibodies with favorable binding properties and developability. The selected antibodies were evaluated in vitro for PD-1 binding affinity. Figure 7B SPR response binding curves of selected antibodies at five concentrations are shown. KD values ​​were calculated from the SPR response curves and found to be in the picomolar and nanomolar range, showing higher affinity when compared to PD1AB6. Figure 7C Calculated thermal stabilities of selected antibodies are shown, which showed comparable Tm (°C) values ​​when compared to PD1AB6.

[0168] Example 8: Antibodies generated by AI / mammalian display show PD-1 agonism at the expected epitope.

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

[0170] Example 9: Human primary cell assay of the first in vitro reporter gene assay clone.

[0171] Antibody clones 7, 43, 1-H01-1 and D4-A7-7_201 were selected for human primary CD4 T cell cytokine release and activation marker assays. These four antibodies were selected based on their PD-1 agonist in vitro reporter gene assay EC50 values. Human PBMCs were isolated from 7-donors and activated with 5ug / mL PHA for 48 hours to upregulate PD-1 expression. CD4 T cells were then purified from pre-stimulated PBMCs and plated at uniform density on 96-well plates continuously coated with 3ug / mL OKT3 and a titration series of each test article. Supernatants were collected, IL-2 was measured at 24 hours, and IFN-γ was measured at 72 hours. CD4 T cells were collected at 72 hours and PD-1 and CD69 activation biomarkers were analyzed by flow cytometry. Fig. 9 The results of 7-donor CD4 T cell cytokine release and activation marker assays are shown. 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 than PD1AB6. Relative to isotype controls, clones 7, 43 and 1-H01-1 showed significant downregulation of PD-1 expression and were comparable to PD1AB6, indicating that these clones effectively agitate the PD-1 pathway, thereby triggering downregulation of PD-1.

[0172] Example 10: Epitope grouping.

[0173] Based on the in vitro reporter gene and human primary cell assay results of antibody clones 7, 1-H01-1 and D4-A7-7_201, the antibody clones were selected for epitope grouping. Epitope grouping was performed by testing the ability of clones 7, 1-H01-1 and D4-A7-7_201 to compete for SPR binding with each other and with benchmark antibodies PD1AB6, nivolumab, pembrolizumab and UCB949 with known PD-1 binding epitopes. Fig.10 Epitope grouping results are shown, indicating that clone 7, 1-H01-1, and D4-A7-7_201 bind to different regions on PD-1.

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

[0175] Antibody clones 7 and 1-H01-1 were selected for testing in what is herein referred to as a tandem scFv-Fc format, which have two scFv binding sites in tandem on each antibody arm connected by a linker (in this example, a flexible linker) and a total of four scFv binding sites in the antibody in a single scFv-Fc format, see Fig.11 An exemplary structure in .

[0176] Without being bound by theory or mechanism, it is believed that PD-1 agonism is driven by clustering of multiple PD-1 receptors, and thus tandem scFv-Fc antibodies with more than two PD-1 binding sites may exhibit more potent PD-1 agonism when compared to similar two binding site antibodies.

[0177] 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. Tandem scFv-Fc antibody thermal stability Tm (°C) was tested using differential scanning fluorimetry. Fig.12 Thermostability is shown for the following tandem scFv-Fc clones: 7 monospecific, 1-H01-1 monospecific, 7+1-H01-1 bispecific. The three tandem scFv-Fc antibody configurations were tested for their ability to agonize and antagonize the PD-1 pathway using the same in vitro reporter gene assay used to identify the non-tandem bivalent 7 and 1-H01-1 clones. Fig.13 The results of the PD-1 agonist reporter gene assay are shown. Fig.14 The results of the PD-1 antagonist reporter assay for the three tandem scFv-Fc antibodies tested are shown. In each case, the tandem scFv-Fc configuration with four scFv binding sites showed a 2-fold or more improvement in PD-1 agonism EC50 relative to the bivalent non-tandem configuration, and a 3-fold or more improvement in PD-1 agonism EC50 relative to the PD1AB6 benchmark. No PD-1 antagonism was observed in the tandem scFv-Fc antibodies tested.

[0178] Example 12: Human primary cell assay 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 6-donors and activated with 5ug / mL PHA for 48 hours to upregulate PD-1 expression. CD4 T cells were then purified from pre-stimulated PBMCs and plated at uniform density on 96-well plates continuously coated with 3ug / mL OKT3 and a titration series of each test article. Supernatants were collected, IL-2 was measured at 24 hours, and IFN-γ was measured at 72 hours. CD4 T cells were collected at 72 hours and analyzed for PD-1 and CD69 activation biomarkers by flow cytometry.

[0180] Fig.15Results of 6-donor CD4 T cell cytokine release and activation marker assays are shown. Tandem clone 7 monospecific, tandem clone 1-H01-1 monospecific, and tandem clone 7+1-H01-1 bispecific significantly attenuated IL-2, IFN-γ cytokines more than PD1AB6. Tandem clone 7+1-H01-1 bispecific showed significant downregulation of PD-1 expression relative to isotype control and PD1AB6. Tandem clone 7 monospecific showed significant downregulation of CD69 expression relative to isotype control and PD1AB6.

[0181] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent or composition of the invention, and vice versa. In addition, 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 rather than as limitations to the present invention. Without departing from the scope of the present invention, the main features of the present invention may be adopted in various embodiments. Those skilled in the art will recognize or be able to determine many equivalents of the specific procedures described herein using no more than routine experiments. Such equivalents are considered to be within the scope of the present invention and are covered by the claims.

[0183] All publications and patent applications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0184] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the words "a / an" can mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". The term "or" used in the claims is used to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, but the present disclosure supports a definition referring only to alternatives and "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method used to determine the value, or the variation that exists between the subjects of 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 unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of". As used herein, the phrase "consisting essentially of" requires the specified integer or step and the integer or step that does not materially affect the characteristic or function of the claimed invention. As used herein, the term "consisting of" is used only to indicate the presence of a listed integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature(s), element(s), property(s), property(s), method / process step(s), or limitation).

[0186] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if the order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. The skilled person will understand that the number of items or terms in any combination is generally not limited unless otherwise apparent from the context.

[0187] As used herein, approximating words such as, but not limited to, "about," "substantially," or "substantially" refer to conditions that, when so modified, are not necessarily absolute or perfect, but would be considered close enough to warrant designation of the condition as existing by one of ordinary skill in the art. The degree to which this specification may vary will depend on the degree to which changes can be implemented and still have the desired characteristics and ability for one of ordinary skill in the art to recognize the modified features as still having the unmodified features. In general, but depending on the foregoing discussion, numerical values ​​modified herein by approximating words such as "about" may vary from the stated value by at least ±1%, 2%, 3%, 4%, 5%, 6%, 7%, 10%, 12%, or 15%.

[0188] In addition, the section titles of this article are intended to be consistent with the recommendations under 37CFR 1.77, or to provide organizational clues. These titles should not limit or characterize the one or more inventions that can be set forth in any claim issued from this disclosure. Specifically and by way of example, although the title mentions the "technical field", such claims should not be limited by the language describing the so-called technical field under this title. Further, the description of the technology in the "background technology" section should not be understood as an admission that the technology is the prior art of any one or more inventions in this disclosure. "Invention content" should also not be considered as a feature of one or more inventions set forth in the issued claims. In addition, any reference to the singular "invention" in this disclosure should not be used to argue that there is only a single novel point in this disclosure. Multiple inventions can be set forth according to the limitations of multiple claims issued from this disclosure, and such claims accordingly define the inventions protected by them and their equivalents. In all cases, the scope of these claims should be considered according to their own merits in accordance with this disclosure, but should not be limited by the titles set forth herein.

[0189] For each claim in the claims, each dependent claim may depend on both the independent claim and each of the previous dependent claims of each claim, as long as the previous claims provide proper antecedent basis for the claim item or element.

[0190] To assist the Patent Office and any reader of any patent issuing based on this application in interpreting the appended claims, applicants wish to note that unless the words "means for" or "step for" are expressly used in a particular claim, they do not intend that any of the appended claims invoke 35 U.S.C. §112, paragraph 6, USC §112, paragraph (f), or their equivalents as they exist on the filing date of this application.

[0191] All compositions and / or methods disclosed and claimed herein can be prepared and performed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in conjunction with preferred embodiments, it is apparent to those skilled in the art that the steps or sequence of steps in the compositions and / or methods and methods described herein may be altered without departing from the concept, spirit and scope of the present invention. It is apparent to those skilled in the art that all such similar substitutions and modifications are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A PD-1 agonist antibody, wherein the antibody comprises: a. a heavy chain variable domain (VH) complementarity determining region (CDR) 1, wherein the VH CDR1 comprises the amino acid sequence of any one of the following SEQ ID NOs: 10, 16, 22, 29, 32, 36, 37; and b. VH CDR2, wherein the VH CDR2 comprises the amino acid sequence of any one of the following SEQ ID NOs: 11, 17, 23, 30, 33, 35, 38; and c. VH CDR3, wherein the VH CDR3 comprises the amino acid sequence of any one of the following SEQ ID NOs: 12, 18, 24, 25, 34, 39; and d. a light chain variable domain (VL) CDR1, wherein the VL CDR1 comprises the amino acid sequence of any one of the following SEQ ID NOs: 13, 19, 26, 40, 42, 46; and e. VL CDR2, wherein the VL CDR2 comprises the amino acid sequence of any one of the following SEQ ID NOs: 14, 20, 27, 31, 43; and f. VL CDR3, comprising the amino acid sequence of any one of the following SEQ ID NOs: 15, 21, 28, 41, 44, 45, 47.

2. The antibody according to claim 1, wherein the antibody comprises: a. VH, the VH comprising the amino acid sequence of any one of the following SEQ ID NOs: 1-5, 48-54, and b. VL, comprising the amino acid sequence of any one of the following SEQ ID NOs: 6-9, 55-61.

3. The antibody according to any one of claims 1 to 2, wherein the antibody is a monoclonal antibody.

4. The antibody according to any one of claims 1 to 3, wherein the antibody is a full-length antibody.

5. The antibody according to any one of claims 1 to 3, wherein the antibody is an antibody fragment. 6 . The antibody fragment of claim 5 , wherein the antibody is fused to the Fc domain of any one of the following: human IgG1, human IgG2, human IgG3, and human IgG4.

7. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody according to any one of claims 1 to 6.

8. The method of claim 7, wherein the disease is an autoimmune disease.

9. The method of claim 7, wherein the disease is an inflammatory disease.

10. The method according to any one of claims 7 to 9, wherein the subject is a human.

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

12. The antibody of claim 11, wherein the antibody has a total of four scFv binding sites in a single scFv-Fc format of the antibody.

13. The antibody of claim 12, 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).

14. The antibody according to claim 13, wherein the VH1 region and the VH2 region each comprise an amino acid sequence of any one of SEQ ID NOs: 1-5 and 48-54, more preferably SEQ ID NOs: 1 or 53; and wherein the VL1 region and the VL2 region each comprise an amino acid sequence of any one of SEQ ID NOs: 6-9 and 55-61, or preferably SEQ ID NOs: 6 or 60.

15. The antibody of claim 14, wherein the linker comprises the following amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 64).

16. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of claims 1 to 15.

17. The method of claim 16, wherein the disease is an autoimmune disease.

18. The method of claim 16, wherein the disease is an inflammatory disease.

19. The method according to any one of claims 16 to 18, wherein the subject is a human.

20. A nucleic acid encoding the PD-1 agonist antibody according to any one of claims 1 to 6 or claims 11 to 15.

21. The nucleic acid of claim 20, wherein the nucleic acid sequence is selected from a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to 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.

22. A nucleic acid according to claim 20, wherein the nucleic acid sequence is selected from a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a variable heavy chain selected from 92, 94, 96, 98, 100, 102 or 104 and a light chain selected from SEQ ID NO: 93, 95, 97, 99, 101, 103, 104 or 105. A nucleic acid vector comprising the nucleic acid sequence according to claim 20 . A host cell comprising the nucleic acid vector according to claim 21 .

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