PD-1 agonist antibody

Abstract

Provided herein are PD-1 agonist antibodies that bind to PD-1. The PD-1 agonist antibodies of the present disclosure are useful for treating autoimmune and inflammatory diseases through promotion of PD-1 signaling. Methods of using the PD-1 agonist antibodies are also provided herein. Programmed cell death protein 1 (PD-1) is a cell surface receptor that plays a critical 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.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 375,676, filed September 14, 2022, and U.S. Provisional Patent Application No. 63 / 515,448, filed July 25, 2023, the entire contents of which are incorporated herein by reference.

[0002] Statement of Federally Sponsored Research Not applicable.

[0003] Incorporation by reference of material filed on a compact disc 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 technology]

[0004] Programmed cell death protein 1 (PD-1) is a cell surface receptor that plays a crucial 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. PD-1:PD-L1 interaction acts to suppress immune cells through phosphorylation of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which activate downstream signaling pathways that can inhibit T cell activation, cytokine production, and promote apoptosis. Therefore, PD-1 is crucial for preventing autoimmunity and overstimulated immune responses, which, if left unchecked, can be harmful to the body.

[0005] The PD-1:PD-L1 checkpoint is well known to be exploited by cancer cells to upregulate PD-L1 to evade detection, and therefore many PD-1 antagonists and antibodies have been developed as tumor therapeutics to combat this exploitation. Despite the focus on PD-1 antagonists, there is a need for PD-1 agonists and antibodies in the treatment of various autoimmune and inflammatory diseases, which ideally do not interfere with the innate PD-1 and PD-L1 interaction. Such antibodies are provided herein. Summary of the Invention [Means for solving the problem]

[0006] Provided herein are PD-1 agonist antibodies that bind to PD-1. As embodied and broadly described herein, embodiments of the disclosure relate to PD-1 agonist antibodies, the antibodies comprising a heavy chain variable domain (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of any one of SEQ ID NOs: 10, 16, 22, 29, 32, 36, or 37 below, a VH CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 11, 17, 23, 30, 33, 35, or 38 below, and a VH CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 12, 18, 24, 25, 34, or 39 below, and a light chain variable domain (VL) CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 13, 19, 26, 40, 42, or 46 below, a VL CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 14, 20, 27, 31, or 43 below, and a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15, 21, 28, 41, 44, 45, or 47 below. The antibody comprises a CDR3. In one embodiment, the antibody comprises a VH comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 5, 48 to 54 below, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 6 to 9, 55 to 61 below. In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a full-length antibody. In another embodiment, the antibody is an antibody fragment. In another embodiment, the antibody is fused to the Fc domain of any one of human IgG1, human IgG2, human IgG3, and human IgG4 below.

[0007] As embodied and broadly described herein, aspects of the present disclosure relate to a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody described herein 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 human.

[0008] As embodied and broadly described herein, embodiments of the present disclosure relate to tandem scFv-Fc PD-1 agonist antibodies, which comprise scFv1 and scFv2 binding sites in tandem on each antibody arm, with the scFv1 and scFv2 linked by a linker, optionally a flexible linker. In one embodiment, the antibody has a total of four scFv binding sites in a single scFv-Fc format antibody. In another embodiment, 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). In another embodiment, the VH1 region and the VH2 region each comprise the amino acid sequence of any one of SEQ ID NOs: 1 to 5 and 48 to 54, more preferably SEQ ID NO: 1 or 53, and the VL1 region and the VL2 region each comprise the amino acid sequence of any one of SEQ ID NOs: 6 to 9 and 55 to 61, or preferably SEQ ID NO: 6 or 60. In another embodiment, the linker comprises the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 64).

[0009] As embodied and broadly described herein, an embodiment of the present disclosure relates to a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody according to any one of claims 1 to 15. In one embodiment, the disease is an autoimmune disease. In another embodiment, the disease is an inflammatory disease. In another embodiment, the subject is human.

[0010] As embodied and broadly described herein, embodiments of the present disclosure relate to nucleic acids encoding the PD-1 agonist antibodies described hereinabove. In one embodiment, the nucleic acid sequence is selected from sequences having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NOs: 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. In another embodiment, the nucleic acid sequence is selected from a sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to a variable heavy chain selected from 92, 94, 96, 98, 100, 102, or 104, and a light chain selected from SEQ ID NOs: 93, 95, 97, 99, 101, 103, 104, or 105.

[0011] As embodied and broadly described herein, aspects of the present disclosure relate to nucleic acid vectors comprising the nucleic acid sequences described hereinabove.As embodied and broadly described herein, aspects of the present disclosure relate to host cells comprising the nucleic acid vectors described hereinabove. [Brief explanation of the drawings]

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. This application can be understood by reference to the following description in conjunction with the accompanying drawings.

[0013] [Figure 1A] FIG. 1A illustrates the PD-1 agonist epitope-directed MEM nanoparticle B cell activation probing strategy, further described in Example 1.

[0014] [Figure 1B] Figure 1B shows MEM nanoparticle titer and PD1AB6 binding via Coomassie blue and surface plasmon resonance (SPR) binding, respectively.

[0015] [Figure 1C] FIG. 1C illustrates the mouse immunization protocol with alternating doses of MEM nanoparticles and full-length PD-1, with a final combination boost.

[0016] [Figure 1D] Figure 1D shows the binding of the resulting mouse sera to PD-1 as measured by enzyme-linked immunosorbent assay (ELISA).

[0017] [Figure 2A] Figure 2A shows PD-1 binding of antibodies generated from monoclonal hybridomas by ELISA.

[0018] [Figure 2B] Figure 2B shows the binding of exemplary antibody 27A5 to PD-1 (left) and the competitive PD-1 binding (or lack thereof) of 27A5 to PD1AB6 and pembrolizumab (right).

[0019] [Figure 2C] Figure 2C shows the PD-1 binding activity curve obtained for 27A5 as measured by SPR.

[0020] [Figure 2D] FIG. 2D shows PD-1 agonist (left) and antagonist (right) responses by 27A5 as measured by checkpoint signaling assays.

[0021] [Figure 3A] FIG. 3A illustrates the full-length PD-1 and MEM nanoparticle phage panning strategy of an in vitro scFv library, as described in Example 3.

[0022] [Figure 3B] Figure 3B illustrates the resulting SPR binding screen of isolated antibodies generated by phage panning.

[0023] [Figure 3C] Figure 3C shows that isolated antibodies generated by phage panning do not compete with pembrolizumab for PD-1 binding.

[0024] [Figure 3D] FIG. 3D shows the avidity and affinity curves of SPR binding of exemplary antibodies generated by a phage panning strategy.

[0025] [Figure 4A] FIG. 4A shows the PD-1 agonism curves of exemplary antibodies measured by checkpoint signaling assays.

[0026] [Figure 4B] FIG. 4B shows the PD-1 antagonist curves of exemplary antibodies as measured by checkpoint signaling assays.

[0027] [Figure 4C] Figure 4C shows competitive PD-1 binding of exemplary antibodies to PD1AB6 (left) or pembrolizumab (right) via SPR.

[0028] [Figure 4D] FIG. 4D shows the collected avidity, affinity, and agonist EC50 values ​​of exemplary antibodies generated by the phage panning strategy.

[0029] [Figure 5] Figure 5 shows the resulting PD-1 SPR binding curves for two exemplary antibodies.

[0030] [Figure 6A] FIG. 6A shows the PD-1 agonism curves of exemplary antibodies measured by checkpoint signaling assays.

[0031] [Figure 6B] FIG. 6B shows the PD-1 antagonist curves of exemplary antibodies as measured by checkpoint signaling assays.

[0032] [Figure 7A] FIG. 7A illustrates the AI ​​model / mammalian display antibody discovery strategy described in Example 7.

[0033] [Figure 7B] Figure 7B shows the resulting PD-1 SPR binding curves for exemplary antibodies.

[0034] [Figure 7C] FIG. 7C shows thermostability measurements of an exemplary antibody.

[0035] [Figure 8A] FIG. 8A shows complete blockade of PD1AB6 binding to PD-1 by an exemplary antibody.

[0036] [Figure 8B] FIG. 8B shows the PD-1 agonism curves of exemplary antibodies measured by checkpoint signaling assays.

[0037] [Figure 8C] FIG. 8C shows the lack of PD-1 antagonism of exemplary antibodies as measured by checkpoint signaling assays.

[0038] [Figure 9] FIG. 9 shows PD-1 agonist activity by exemplary antibodies as measured by human primary CD4 T cell cytokine release and activation marker expression.

[0039] [Figure 10] Figure 10 shows epitope binning of three exemplary clones that exhibit potent PD-1 agonist activity.

[0040] [Figure 11] FIG. 11 illustrates an exemplary tandem scFv-Fc format antibody structure of the present disclosure.

[0041] [Figure 12] FIG. 12 shows the melting temperatures of tandem scFv-Fc format antibodies.

[0042] [Figure 13] FIG. 13 shows PD-1 agonist activity by exemplary tandem scFv-Fc format antibodies as measured by a checkpoint signaling assay.

[0043] [Figure 14] FIG. 14 shows PD-1 antagonism by exemplary tandem scFv-Fc format antibodies as measured by a checkpoint signaling assay.

[0044] [Figure 15] Figure 15 shows PD-1 agonist activity by exemplary tandem scFv-Fc format antibodies as measured by human primary CD4 T cell cytokine release and activation marker expression. DETAILED DESCRIPTION OF THE INVENTION

[0045] While the making and use of various embodiments of the invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0046] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art relevant to the present invention. Terms such as "a," "an," and "an" are not intended to refer to a singular entity only, but include general classifications that may be used to describe specific examples. While the terminology herein is used to describe particular embodiments of the present invention, their use does not define the present invention except as outlined in the claims.

[0047] Provided herein are agonistic antibodies that bind to PD-1 at a site that is not recognized by PD-L1. Methods for producing and using such antibodies are also provided. These antibodies may be useful for downregulating immune responses in individuals. For example, in some embodiments, the antibodies may be used to treat diseases involving autoimmunity and / or hyperinflammation.

[0048] When elements are presented in list form (e.g., Markush groups), it is to be understood that each possible subgroup of the elements is also disclosed, and that any one or more elements can be removed from the list or group.

[0049] Unless expressly indicated, in methods described or disclosed herein that include more than one act, the order of the acts is not necessarily limited to the order in which the acts of the method are described, but it will be understood that the present disclosure encompasses example embodiments in which the order of the acts is so limited.

[0050] Terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. All technical and scientific terms, acronyms, and abbreviations 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 belongs, unless otherwise defined or stated. All numerical ranges include the values ​​defining the range and all integer values ​​therebetween, unless otherwise indicated or defined.

[0051] As used throughout this specification, 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.

[0052] Antibody fragments of the present disclosure retain PD-1 antigen-binding specificity and 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 a single chain, single-chain antibody fragments (scAb), or other antibody variable region fragments, e.g., other antibody variable region fragments that retain antigen-binding specificity.

[0053] Tandem scFv-Fc antibodies of the present disclosure are composed of two or more scFv binding sites in tandem on each antibody arm, optionally linked by a linker, optionally a flexible linker, resulting in a total of four or more scFv binding sites in a single scFv-Fc format antibody. Figure 11 illustrates an exemplary tandem scFv-Fc format antibody structure of the present disclosure.

[0054] As used throughout this specification, the term "mesoscale molecules (MEMs)" includes engineered peptides and polypeptides of about 1 kDa to about 10 kDa. As used throughout this specification, the term "MEMs nanoparticles" includes MEMs conjugated to nanoparticles (e.g., ferritin nanoparticles).

[0055] As used herein, a "subject" may be a mammalian subject. Mammalian subjects include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, e.g., a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., a cat, a dog).

[0056] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0057] I.PD-1 agonist antibodies

[0058] Provided herein are antibodies that bind to PD-1 and elicit an agonist response. These antibodies are referred to herein as PD-1 agonist antibodies. A number of discovery strategies utilized to obtain exemplary antibodies of the present disclosure are further discussed below.

[0059] The amino acid sequence of full-length human PD-1 is provided as SEQ ID NO: 63 (see UniProtKB ID Q15116). MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 63)

[0060] In some embodiments, the PD-1 agonist antibody specifically binds to the PD-1 epitope identified by the amino acid sequence provided as RFRVTQLPNGRDFHMSVV SEQ ID NO:62.

[0061] Referring to Figure 1A, MEMs of the present disclosure are engineered to mimic the agonist epitope identified by SEQ ID NO: 62 and then used to screen antibodies. An advantage of this approach is the ability to direct antibody discovery away from the PD-L1 binding site and towards the desired epitope.

[0062] In some embodiments, subjects are immunized with MEM nanoparticles together with full-length PD-1 to generate antibodies specific to MEM epitopes. Monoclonal hybridomas are then generated to produce epitope-specific PD-1 antibodies. In other embodiments, mouse sera are collected and used to generate an in vitro scFv library. Phage panning is then performed against full-length PD-1 and MEM nanoparticles to isolate epitope-specific clones. In other embodiments, antibodies are generated using phage panning of a naive antibody library against 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. A mammalian display library is then created, and single-cell sorting is performed to select epitope-specific PD-1 antibodies.

[0063] Those skilled in the art will recognize that an antibody that exhibits little or no binding to a target antigen can be described as having a low affinity for the target antigen and a high equilibrium dissociation constant (KD). Those skilled in the art will recognize that an antibody that exhibits little or no binding to a collection of target antigen epitopes can be described as having low avidity for a collection of target antigen epitopes and a high equilibrium dissociation constant (KD).

[0064] In some embodiments, provided herein are PD-1 agonist antibodies that have a binding affinity (KD) for 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, or about 10 pM to about 5 pM.

[0065] In some embodiments, the PD-1 agonist antibody has a binding activity (KD) for binding to PD-1 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, or about 0.5 pM to about 0.1 pM.

[0066] In some embodiments, the PD-1 agonist antibody has a half-maximal effective concentration (EC50) against PD-1 of 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, or about 0.005 nM to about 0.001 nM.

[0067] Those skilled in the art will recognize that binding specificity can be determined through a series of competitive binding paradigms, with the desired antibody demonstrating its ability to prevent binding of a known reference antibody to its target epitope at various 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 recognized by SEQ ID NO: 62 is PD1AB6 (see Patent No. US10428145B2). Those skilled in the art will also recognize that PD1AB6 and pembrolizumab can be utilized as control antibodies in agonist and antagonist assays.

[0068] In some embodiments, the PD-1 agonist antibodies of the present disclosure do not interfere with the binding of pembrolizumab to PD-1. Exemplary antibodies of the present disclosure that do not interfere with pembrolizumab binding 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 (see Figures 2B and 4C). The PD-1 agonist antibodies of the present disclosure interfere with PD1AB6 binding to the PD-1 epitope identified by SEQ ID NO:62.

[0069] In some embodiments, the PD-1 agonist antibody is a full-length antibody (referring to an antibody in which two heavy chains and two light chains are linked to an Fc domain, forming a "Y" shape). In some embodiments, the Fc domain (or simply referred to as 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.

[0070] A. Exemplary PD-1 Agonist Antibody-CDR Sequences

[0071] The sequences of exemplary PD-1 agonist antibodies of the present disclosure are provided herein, including the complementarity-determining region (CDR) sequences and variable heavy and light chain domain sequences (VH, VL) that make up the PD-1 antigen-binding domain of the present disclosure. The discovery of these antibodies is described in detail in the Examples section.

[0072] 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 disclosure.

[0073] [Table 1-1] [Table 1-2]

[0074] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0075] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

[0076] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0077] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the amino acid sequences of the following three VH CDRs: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, and / or the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0078] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the amino acid sequences of the following three VH CDRs: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, and / or the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:31, and SEQ ID NO:28.

[0079] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the amino acid sequences of the following three VH CDRs: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:25, and / or the amino acid sequences of the following three VL CDRs: SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0080] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:40, SEQ ID NO:27, and SEQ ID NO:41.

[0081] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.

[0082] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.

[0083] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:46, SEQ ID NO:43, and SEQ ID NO:45.

[0084] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:46, SEQ ID NO:43, and SEQ ID NO:47.

[0085] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:34, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.

[0086] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the antibodies comprise the following three VH CDRs: the amino acid sequences of SEQ ID NO:36, SEQ ID NO:33, SEQ ID NO:34, and / or the following three VL CDRs: the amino acid sequences of SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:45.

[0087] B. Exemplary PD-1 Agonist Antibodies—Variable Region Sequences

[0088] The terms variable domain and variable region are used interchangeably and refer to the portions of the light and heavy chains of an antibody that contain the complementarity determining regions and framework regions (FR).

[0089] Table 2 provides the amino acid sequences of the variable domains of exemplary PD-1 agonist antibodies of the disclosure. Thus, in some embodiments, a PD-1 agonist antibody of the disclosure comprises a variable heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 1-5, 48-54, and / or in some embodiments, a PD-1 agonist antibody of the disclosure comprises a variable light chain comprising an amino acid sequence selected from SEQ ID NOs: 6-9, 55-61.

[0090] In some embodiments, the PD-1 agonist antibodies of the disclosure comprise any one of the VH / VL variable chain sequence combinations listed in Table 2.

[0091] [Table 2-1] [Table 2-2]

[0092] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0093] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 1, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 6.

[0094] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:2, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:7.

[0095] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:3, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:8.

[0096] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:4, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:8.

[0097] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:4, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:9.

[0098] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:5, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:8.

[0099] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 48, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 55.

[0100] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO:49, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO:56.

[0101] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 50, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 57.

[0102] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 51, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 58.

[0103] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 52, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 59.

[0104] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 53, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 60.

[0105] In some embodiments, provided herein are PD-1 agonist antibodies, wherein the heavy chain variable domain (VH) of the antibody comprises the amino acid sequence of SEQ ID NO: 54, and / or the light chain variable domain (VL) of the antibody comprises the amino acid sequence of SEQ ID NO: 61.

[0106] C. Exemplary tandem scFv-Fc PD-1 agonist antibodies.

[0107] In some embodiments, the present disclosure provides tandem scFv antibodies with multiple PD-1 binding sites. The tandem scFv-Fc antibodies of the present disclosure are composed of two or more scFv binding sites in tandem on each antibody arm, optionally linked 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 format antibody. Figure 11 illustrates an exemplary tandem scFv-Fc format antibody structure of the present disclosure.

[0108] Without being bound by theory or mechanism, it is believed that PD-1 agonism is driven by the clustering of multiple PD-1 receptors, and therefore, tandem scFv-Fc antibodies with multiple PD-1 binding sites may exhibit stronger PD-1 agonism when compared to similar conventional two-binding site antibodies or scFvs with a single VH and a single VL.

[0109] 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, with the scFv1 and scFv2 linked by a linker, optionally a flexible linker. Figure 11 illustrates such an exemplary tandem scFv-Fc format antibody structure of the present disclosure. Such an antibody has a total of four scFv binding sites in a single tandem scFv-Fc format antibody.

[0110] 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).

[0111] In some embodiments, the VH1 region comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 5 and 48 to 54, more preferably SEQ ID NO: 1 or 53, and the VL1 region comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 9 and 55 to 61, or preferably SEQ ID NO: 6 or 60, resulting in scFv1. Similarly, in some embodiments, the VH2 region comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 5 and 48 to 54, more preferably SEQ ID NO: 1 or 53, and the VL2 region comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 9 and 55 to 61, or preferably SEQ ID NO: 6 or 60, resulting in scFv2.

[0112] The VH1 and VL1 of each scFV1 may be connected by a linker, for example a flexible linker.

[0113] The VH2 and VL2 of each scFV2 ​​may be connected by a linker, for example a flexible linker.

[0114] The scFvs on each antibody arm may be linked by a linker, e.g., a flexible linker. An exemplary linker comprises the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 64).

[0115] [Table 4]

[0116] [Table 5-1] [Table 5-2] [Table 5-3]

[0117] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist antibody having an scFv1 and an 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, the second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO: 1, the first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO: 6, and the second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO: 6, and wherein scFv1 and scFv2 are linked by a linker comprising the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 64).

[0118] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist antibody having an scFv1 and an 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, the second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO: 53, the first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO: 60, and the second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO: 60, and wherein scFv1 and scFv2 are linked by a linker comprising the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 64).

[0119] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist antibody having an scFv1 and an 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, the second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO: 53, the first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO: 60, and the second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO: 6, and wherein scFv1 and scFv2 are linked by a linker comprising the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 64).

[0120] In an exemplary embodiment, provided herein is a tandem scFv-Fc PD-1 agonist antibody having an scFv1 and an 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, the second heavy chain variable domain (VH2) of the antibody comprises the amino acid sequence of SEQ ID NO: 1, the first light chain variable domain (VL1) of the antibody comprises the amino acid sequence of SEQ ID NO: 60, and the second light chain variable domain (VL2) of the antibody comprises the amino acid sequence of SEQ ID NO: 6, and wherein scFv1 and scFv2 are linked by a linker comprising the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 64).

[0121] As shown in FIG. 11, the Fc domain of the tandem scFv-Fc PD-1 agonist antibodies of the disclosure may be human IgG1, human IgG2, human IgG3, or human IgG4.

[0122] II. Use of PD-1 agonist antibodies.

[0123] A. Therapeutic PD-1 agonist antibodies.

[0124] In some embodiments, the PD-1 agonist antibodies provided herein are useful for treating diseases or conditions that involve an immune response.

[0125] In some embodiments, the PD-1 agonist antibodies provided herein are useful for treating 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.

[0126] In some embodiments, the PD-1 agonist antibodies provided herein are useful for treating 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.

[0127] B. Administration of therapeutic PD-1 agonist antibodies.

[0128] In vivo administration of the therapeutic PD-1 agonist antibodies described herein can be intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, intrathecal, intracerebroventricular, intranasal, transmucosal, via implantation, or via inhalation. Intravenous administration can be via 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. The therapeutic PD-1 agonist antibodies can be administered with any suitable excipient, carrier, or other agent to provide suitable or improved tolerance, migration, delivery, etc. [Example]

[0129] Example 1: Engineered MEM nanoparticle immunization-based PD-1 agonist discovery.

[0130] MEMs were engineered based on the epitope identified by SEQ ID NO:62 and then conjugated to nanoparticles to direct B cell antibody production to the epitope and away from the PD-L1 binding site (Figure 1A). MEMs conjugated to ferritin nanoparticles were identified by Coomassie-based Western blot and found to contain approximately 20-30 MEMs per nanoparticle (Figure 1B). MEM nanoparticles demonstrated nanomolar binding affinity to PD1AB6 using surface plasmon resonance (SPR). BALB / c mice were then immunized over a 5-week period with alternating doses of engineered MEM nanoparticles and / or full-length PD-1 suspended in adjuvant, with a final boost containing a combination of the two (Figure 1C). Mouse sera were collected and strong PD-1 binding measured via ELISA (Figure 1D).

[0131] Example 2: The top monoclonal hybridoma-produced antibody shows potent PD-1 binding and agonism.

[0132] Hybridomas were generated from immunized mouse B cells using standard electrofusion methods. The resulting 27A5 antibody and several other antibodies were generated from the monoclonal hybridomas and demonstrated strong PD-1 binding via ELISA (FIG. 2A). 27A5 was then further evaluated in vitro for PD-1 binding and competitive binding for PD-1 using SPR (FIG. 2B). 27A5 exhibited a KD of 55 nM and blocked the PD1AB6 binding site but not the pembrolizumab binding site. PD-1 binding activity was also measured for 27A5 via SPR, which demonstrated strong binding with a KD of 5 pM (FIG. 2C). 27A5 was then evaluated for in vitro functionality at PD-1. PathHunter® checkpoint signaling assays were performed to measure PD-1 agonism and antagonism, and the resulting concentration / response curves were generated (FIG. 2D). 27A5 exhibited agonism comparable to that of the control antibody and no antagonism.

[0133] Example 3: Antibodies generated from an in vitro scFv library exhibit potent PD-1 binding.

[0134] Using additional mouse serum collected from previous immunizations, an in vitro scFv library was constructed using standard molecular cloning techniques and phage display. 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 resulting isolated clones were then screened for PD-1 binding affinity and competitive binding to pembrolizumab using SPR (Figures 3B-3C). Exemplary antibodies were identified from the in vitro scFv library that bound to PD-1 with high affinity and avidity using SPR (Figure 3D).

[0135] Example 4: Selected antibodies generated from an in vitro scFv library exhibit agonism at the intended epitope.

[0136] Selected antibodies were then evaluated for PD-1 agonism and antagonism using the PathHunter® checkpoint signaling assay (Figures 4A-4B). The resulting agonist concentration-response curves were used to obtain EC50 values ​​for each antibody, demonstrating 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 and pembrolizumab binding sites (Figure 4C). All six antibodies demonstrated complete blockade of PD1AB6 binding and no blockade of pembrolizumab binding. Figure 4D lists the collected agonism, avidity, and affinity values ​​for all six antibodies.

[0137] Example 5: Selected antibodies generated by a PD-1 / MEM nanoparticle panning strategy of a naive library exhibit potent PD-1 binding.

[0138] The MEM nanoparticles and full-length PD-1 were then used in a phage panning strategy of a naive antibody library. Selected antibodies 7 and 43 were purified and further evaluated in vitro for PD-1 binding affinity. Figure 5 shows the SPR response curves for both antibodies at three concentrations. KD values ​​were calculated from the SPR response curves and found to be in the nanomolar range for both antibodies.

[0139] Example 6: PD-1 agonist activity of the top antibodies generated by a naive library panning strategy.

[0140] Antibodies 7 and 43 were then evaluated for PD-1 agonism in vitro. Figure 6A shows the results of the agonist assay as a logarithmic concentration-response curve. EC50 values ​​were calculated, indicating higher relative potency compared to the control antibody. Both antibodies were additionally evaluated for PD-1 antagonism in vitro. Figure 6B shows the results of the antagonist assay as a logarithmic concentration-response curve. IC 50 An IC value of 0.06 nM was not detectable for any of the antibodies, which showed a comparable lack of antagonism when compared to the negative control antibody. Pembrolizumab was used as a positive control and had an IC value of 0.06 nM. 50 showed.

[0141] Example 7: PD-1 binding affinity of selected antibodies generated by AI / mammalian display strategy.

[0142] Fully humanized PD-1 agonist antibody CDRs were generated from AI model predictions starting from the PD1AB6 reference CDR antibody template (Figure 7A). A mammalian display library was then created and single-cell 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 shows SPR response binding curves for the selected antibodies at five concentrations. KD values ​​were calculated from the SPR response curves and found to be in the picomolar and nanomolar range, indicating higher affinity compared to PD1AB6. Figure 7C shows the calculated thermal stability for the selected antibodies compared to PD1AB6, which showed comparable Tm (°C) values.

[0143] Example 8: AI / mammalian display-generated antibodies exhibit PD-1 agonism at intended epitopes.

[0144] Selected antibodies were then tested in vitro for their ability to compete with PD1AB6 binding to PD-1. Figure 8A shows SPR response curves, which demonstrate a lack of PD1AB6 binding, indicating that all four antibodies exhibit specific binding to the intended PD-1 epitope. Selected antibodies were then evaluated for PD-1 agonism in vitro. Figure 8B shows the results of the agonist assay as logarithmic concentration-response curves. EC50 values ​​were calculated, indicating equivalent or greater relative potency compared to PD1AB6. Selected antibodies were then evaluated for PD-1 antagonism in vitro. Figure 8C shows the results of the antagonist assay as logarithmic concentration-response curves. IC50 values ​​were undetectable for all four antibodies, demonstrating an equivalent lack of antagonism compared to PD1AB6. Pembrolizumab was used as a positive control and exhibited an IC50 value of 0.06 nM.

[0145] Example 9: Human primary cell assay for the best in vitro reporter assay clones.

[0146] 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 EC50 values ​​in PD-1 agonist in vitro reporter assays. Human PBMCs were isolated from seven donors 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 seeded at uniform densities onto 96-well plates coated with 3 μg / mL OKT3 and a titration series of each test item. Supernatants were collected to measure IL-2 at 24 hours and IFN-gamma at 72 hours. CD4 T cells were collected at 72 hours and analyzed by flow cytometry for PD-1 and CD69 activation biomarkers. Figure 9 shows the results of the CD4 T cell cytokine release and activation marker assays for the seven donors. Clones 7, 43, 1-H01-1, and D4-A7-7_201 attenuated IL-2, IFN-gamma cytokines, and the CD69 T cell activation marker significantly more than PD1AB6. Clones 7, 43, and 1-H01-1 showed significant downregulation of PD-1 expression compared with the isotype control and comparable to PD1AB6, suggesting that these clones potently stimulate the PD-1 pathway, thereby inducing downregulation of PD-1.

[0147] Example 10: Epitope binning.

[0148] Antibody clones 7, 1-H01-1, and D4-A7-7_201 were selected for epitope binning based on their in vitro reporter and human primary cell assay results. Epitope binning was performed by testing the ability of clones 7, 1-H01-1, and D4-A7-7_201 to compete with each other for SPR binding, as well as benchmark antibodies PD1AB6, nivolumab, pembrolizumab, and UCB949, which have known PD-1 binding epitopes. Figure 10 shows the epitope binning results, demonstrating that clones 7, 1-H01-1, and D4-A7-7_201 bind to distinct regions on PD-1.

[0149] Example 11: Tandem scFv-Fc antibody format.

[0150] Antibody clones 7 and 1-H01-1 were selected for testing, which are herein designated as tandem scFv-Fc format, with two scFv binding sites in tandem on each antibody arm connected by a linker (in this example, a flexible linker), for a total of four scFv binding sites in a single scFv-Fc format antibody; see exemplary structure in Figure 11.

[0151] Without being bound by theory or mechanism, it is believed that PD-1 agonism is driven by clustering of multiple PD-1 receptors, and therefore, tandem scFv-Fc antibodies with more than two PD-1 binding sites may exhibit stronger PD-1 agonism when compared to similar two-binding site antibodies.

[0152] Three tandem scFv-Fc configurations 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. Differential scanning fluorimetry was used to test the tandem scFv-Fc antibody thermal stability Tm (°C). Figure 12 shows the thermal stability of tandem scFv-Fc clones: 7 monospecific, 1-H01-1 monospecific, and 7 + 1-H01-1 bispecific. The three tandem scFv-Fc antibody configurations were tested for their ability to stimulate and antagonize the PD-1 pathway using the same in vitro reporter assay used to identify the non-tandem bivalent 7 and 1-H01-1 clones. Figure 13 shows the results of a PD-1 agonist reporter assay, and Figure 14 shows the results of a PD-1 antagonist reporter assay for the three tandem scFv-Fc antibodies tested. In each case, the tandem scFv-Fc configuration with four scFv binding sites shows a greater than two-fold improvement in PD-1 agonism EC50 compared to the bivalent non-tandem configuration, and a greater than three-fold improvement in PD-1 agonism EC50 compared to the PD1AB6 benchmark. No PD-1 antagonism was observed with any of the tandem scFv-Fc antibodies tested.

[0153] Example 12: Human primary cell assay for tandem scFv-Fc clones.

[0154] Using clones 7 and 1-H01-1, three tandem scFv-Fc configurations were tested, and the tandem clone 7 monospecific, tandem clone 1-H01-1 monospecific, and tandem clone 7 + 1-H01-1 bispecific configurations were selected for human primary CD4 T cell cytokine release and activation marker assays. Human PBMCs were isolated from six donors 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 seeded at uniform density onto 96-well plates coated with 3 μg / mL OKT3 and a titration series of each test item. Supernatants were collected to measure IL-2 at 24 hours and IFN-gamma at 72 hours. CD4 T cells were harvested at 72 hours and analyzed by flow cytometry for PD-1 and CD69 activation biomarkers.

[0155] Figure 15 shows the results of CD4 T cell cytokine release and activation marker assays for six donors. Tandem Clone 7 monospecific, Tandem Clone 1-H01-1 monospecific, and Tandem Clone 7+1-H01-1 bispecific attenuate IL-2 and IFN-gamma cytokines significantly more than PD1AB6. Tandem Clone 7+1-H01-1 bispecific shows significant downregulation of PD-1 expression compared to the isotype control and PD1AB6. Tandem Clone 7 monospecific shows significant downregulation of CD69 expression compared to the isotype control and PD1AB6.

[0156] It is contemplated that any embodiment discussed herein 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 achieve the methods of the invention.

[0157] It will be understood that the specific embodiments described herein are shown for purposes of illustration of the invention and not as limitations of the invention. The principal features of this invention can be utilized in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific techniques described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0158] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0159] When used in conjunction with the term "comprising" in the claims and / or this specification, the use of the terms "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only, or the alternatives are not mutually exclusive, although the present disclosure supports a definition that refers 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, the method utilized to determine the value, or the variation that exists among testing subjects.

[0160] As used in the specification and claims, "comprising" (and any form of "comprising" such as "include" and "comprise"), "having" (and any form of "having" such as "having" and "having"), "comprising" (and any form of "comprising" such as "comprising" and "comprise"), or "containing" (and any form of "containing" such as "containing" and "containing") are inclusive or open-ended and do not exclude additional, unrecited components or method steps. In any embodiment 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 specific integers or steps as well as integers or steps that do not substantially affect the properties or function of the claimed invention. As used herein, the term "consisting of" is used to indicate only the presence of a recited integer (e.g., a feature, component, property, property, method / process step, or limitation) or group of integers (e.g., a feature, component, property, property, method / process step, or limitation).

[0161] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding 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, or, where order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repeats of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination unless otherwise clear from the context.

[0162] As used herein, without limitation, approximation terms such as "about," "substantial," or "substantially" refer to a state that, when so modified, is understood not to be necessarily absolute or complete, but that would be considered close enough to a person of ordinary skill in the art to justify specifying that the state exists. The extent to which the description may vary depends on how large a change can be initiated, yet one of ordinary skill in the art would recognize that the modified feature has the required properties and capabilities of the unmodified feature. Generally, subject to the foregoing considerations, numerical values ​​herein modified by approximation terms such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0163] Furthermore, the section headings herein are provided for consistency with suggestions under 37 CFR 1.77 or to otherwise provide organizational guidance. These headings do not limit or characterize the inventions described in any claims that may issue from this disclosure. Specifically, and by way of example, headings refer to the "Field of the Invention," but such claims should not be limited by language under this heading to describe the so-called technical field. Furthermore, the description of technology in the "Background of the Invention" section should not be construed as an admission that the technology is prior art to any invention in this disclosure. No "Abstract" is intended to characterize the inventions described in the issued claims. Furthermore, any reference in this disclosure to "the invention" in the singular should not be used to assert that there is only a single novelty in this disclosure. Multiple inventions may be set forth subject to the limitations of the multiple claims that may issue from this disclosure, and such claims, accordingly, define the inventions and their equivalents that are protected thereby. In all cases, such claims should be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.

[0164] For each claim, each dependent claim may depend on both the independent claim and each preceding dependent claim, so long as the preceding claim provides adequate precedent for a term or element of the claim.

[0165] To assist the Patent Office, and any reader of any patent issued based on this application, in interpreting the appended claims, applicants are advised that, unless the phrase "means for" or "steps for" is expressly used in a particular claim, none of the appended claims intends to invoke 35 U.S.C. § 112, paragraph 6, U.S.C. § 112(f), or any equivalent thereto, as in existence at the filing date of this application.

[0166] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods and the steps or order of steps of the methods described herein without departing from the concept, spirit and scope of the present invention.All such similar substitutes and modifications apparent to those skilled in the art are deemed 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, the antibody comprising: a. a heavy chain variable domain (VH) complementarity determining region (CDR) 1 comprising any one of the amino acid sequences set forth in SEQ ID NOs: 10, 16, 22, 29, 32, 36, and 37; and b. a VH CDR2 comprising any one of the amino acid sequences set forth in SEQ ID NOs: 11, 17, 23, 30, 33, 35, and 38; and c. A VH CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 12, 18, 24, 25, 34, and 39; and d. a light chain variable domain (VL) CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 13, 19, 26, 40, 42, and 46; and e. A VL CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 14, 20, 27, 31, and 43, and f. A PD-1 agonist antibody comprising a VL CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 15, 21, 28, 41, 44, 45, and 47.

2. The antibody a. A VH comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 5, 48 to 54 below; and b. The antibody of claim 1, comprising a VL comprising any one of the amino acid sequences of SEQ ID NOs: 6 to 9 and 55 to 61 below.

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 an Fc domain of any one of human IgG1, human IgG2, human IgG3, and human IgG4.

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

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

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

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

11. A tandem scFv-Fc PD-1 agonist antibody comprising a scFv1 and a scFv2 binding site in tandem on each antibody arm, wherein said scFv1 and scFv2 are linked 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 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 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 of claim 13, wherein the VH1 region and the VH2 region each comprise the amino acid sequence of any one of SEQ ID NOs: 1 to 5 and 48 to 54, more preferably SEQ ID NO: 1 or 53, and the VL1 region and the VL2 region each comprise the amino acid sequence of any one of SEQ ID NOs: 6 to 9 and 55 to 61, or preferably SEQ ID NO: 6 or 60.

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

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

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

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

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

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

21. 21. The nucleic acid of claim 20, wherein the nucleic acid sequence is selected from sequences having at least 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NOs: 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. 21. The nucleic acid of claim 20, wherein the nucleic acid sequence is selected from a sequence having at least 95, 96, 97, 98, 99, or 100% sequence identity to a variable heavy chain selected from 92, 94, 96, 98, 100, 102, or 104, and a light chain selected from SEQ ID NOs: 93, 95, 97, 99, 101, 103, 104, or 105.

23. A nucleic acid vector comprising the nucleic acid sequence of claim 20.

24. A host cell comprising the nucleic acid vector of claim 21.

Images