WO2020021061A1 - Humanized anti-pd-1 antibodies and uses thereof - Google Patents

Abstract

The disclosure provides, among other things, humanized antibodies that bind to programmed cell death protein 1 (PD-1) and compositions comprising such antibodies, whereby the antibodies and/or compositions can be useful for generating a durable anti-tumor or anti-infection response. Such antibodies can be used in many pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as a variety of tumors, or as anti-infection agents. The present disclosure also concerns methods of making the antibodies described herein as well as compositions comprising such antibodies. The present disclosure further relates to nucleic acid molecules encoding such antibodies and to methods for generation of such antibodies and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of such antibodies as well as compositions comprising one or more of such antibodies.

Description

Humanized anti-PD-1 antibodies and uses thereof

I. BACKGROUND

[0001] Programmed cell death protein 1 , or PD-1 (also known as cluster of differentiation 279 or CD279) is a member of the cluster of differentiation 28 (CD28) gene family and is expressed on activated T, B, and myeloid lineage cells (Sharpe et al., Nat Immunol, 2007, Greenwald et al., Annu Rev Immunol, 2005). PD-1 interacts with two ligands, programmed cell death 1 ligand 1 (PD-L1 ) and programmed cell death 1 ligand 2 (PD-L2). Interaction of these ligands with PD-1 plays an important role in downregulating the immune system by limiting overly-active T cells locally, which in turn prevents autoimmunity and maintains peripheral tolerance during infection or inflammation in normal tissues.

[0002] PD-1 negatively modulates T cell activation, and the inhibitory function of PD-

1 on T cell activation is linked to an immunoreceptor tyrosine-based inhibitory motif (ITIM) of its cytoplasmic domain (Parry et al., Mol Cell Biol, 2005, Greenwald et al., Annu Rev Immunol, 2005). Disruption of the inhibitory function of PD-1 can lead to autoimmunity. On the other hand, sustained negative signals by PD-1 have been implicated in T cell dysfunctions in many pathologic situations, such as chronic viral infections and tumor immune evasion.

[0003] In many cancers, PD-1 is expressed by tumor-infiltrating lymphocytes (TILs), associated with host anti-tumor immunity (Galon et al., Science, 2006). Multiple lines of evidence have indicated that TILs are subject to PD-1 inhibitory regulation and the anti-tumor immunity is modulated by PD-1/PD-L1 signaling. First, PD-L1 expression has been confirmed in a number of human and mouse tumor lines and that expression can be further upregulated by IFN-g in vitro (Dong et al., Nat Med, 2002). Second, expression of PD-L1 by tumor cells has been directly associated with their resistance to lysis by anti-tumor T cells in vitro (Blank et al., Cancer Res, 2004, Dong et al., Nat Med, 2002). Third, PD-1 knockout mice are resistant to tumor challenge (Iwai et al., Int Immunol, 2005) and T cells from PD-1 knockout mice are highly effective in tumor rejection when adoptively transferred to tumor- bearing wild-type mice (Blank et al., Cancer Res, 2004). Fourth, blocking PD-1 inhibitory signals by a monoclonal antibody can potentiate host anti-tumor immunity in mice (Hirano et al., Cancer Res, 2005, Iwai et al., Int Immunol, 2005). Fifth, high degrees of PD-L1 expression in tumors (detected by immunohistochemical staining) are associated with poor prognosis for many human cancer types (Hamanishi et al., Proc Natl Acad Sci U S A, 2007). Early-phase studies, accordingly, paved the way for the development of PD-1 inhibitors approved by the Food and Drug Administration (FDA) with for the treatment of several malignancies. The first PD-1 inhibitors, nivolumab and pembrolizumab, were approved in 2014. Since then, the clinical development of PD-1/PD-L1 inhibitors has rapidly broadened. PD-L1 inhibitors including atezolizumab and avelumab have also been granted FDA approvals.

[0004] There still exists a need for new compounds, including antibodies, that can modulate PD-1 activity for the treatment of various diseases and disorders, including cancer, inflammation, and autoimmune diseases. Many anti-PD-1 antibodies are known in the art, for example, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106, marketed as Opdivo®), pembrolizumab (also referred to as lambrolizumab or MK03475, trade name Keytruda®), PDR001 , MEDI0680 (formerly AMP-514), pidilizumab (CT-011 ), ENUM-388D4 (including the 388D4-1 , 388D4-2 and 388D4-3 variants), and ENUM-244C8 (including all the variants). Various patent applications disclose anti-PD-1 antibodies, production thereof, and/or methods of enhancing immune responses with such anti-PD-1 antibodies, including U.S. Patent Application Publication NOs. US 2003/0039653, US 2004/0213795, US 2006/01 10383, US 2007/0065427, US 2007/0122378, US 2009/0217401 , US 2011/0008369, and US2015/0203579 and PCT International Application Publication NOs. WO 2003/099196, WO 2006/121 168, WO 2007/005874, WO 2008/156712, WO 20091 14335, WO 2010/027423, W02 011/1 10604, WO 2012/145493, WO 2013/014668, WO 2014/194302, WO 2015/035606, and WO 2016/106159. There is a need for improved anti-PD-1 antibodies that endow drug-like properties as clinical candidates, including high efficacy and specificity and favorable pharmacokinetic properties, half-life, thermostability, and formulation properties, which are provided in the present invention.

[0005] The identification of anti-PD-1 antibodies as clinical candidates often requires optimization practices including humanization of a murine antibody of early antibody candidates (“hits”). CDR grafting, or antibody reshaping, where murine frameworks are replaced with those of the selected human antibodies, is one of the most clinically validated routes to a humanized antibody (Almagro and Fransson, Front Biosci, 2008). However, merely grafting does not always reconstitute the binding affinity and specificity of the original murine antibody but often results in partial or complete loss of antigen-binding affinity and/or specificity due to incompatibilities between murine CDRs and human donor frameworks. For example, WO 2016/106159 discloses murine anti-PD-1 antibody 388D4 and humanized antibodies thereof, and certain humanized 388D4 shows impaired affinity and specificity and undesired off-target binding.

[0006] Encompassed within the present disclosure is the recognition that, with a given set of mouse CDRs (specific for PD-1 ), careful selection of appropriate human donor frameworks and consideration of the substitution of some key residues from human donor frameworks into the murine original frameworks (backmutation) to restore antigen-binding affinity are required in order to retain a comparable antigen-binding affinity and specificity after CDR-g rafting. In this regard, it is an object of the present invention to provide humanized anti-PD-1 antibodies with intact antigen-binding affinity and specificity. Particularly, in some embodiments, provided methods and compositions encompass the recognition that an increase in antigen specificity often helps eliminate undesired off-target antibody interactions. Off-target binding of an antibody can significantly affect the pharmacokinetics, tissue distribution, efficacy and toxicity of a therapeutic antibody. Thus, in some embodiments, provided anti-PD-1 antibodies exhibit diminished non-specific binding as compared to previously known PD-1 antibodies, e.g., humanized 388D4 as described in WO 2016/106159.

[0007] Favorable pharmacokinetic properties, including adequate half-life and bio- distribution for the intended use and proper bioavailability of selected route of administration, are another selection criteria for drug candidates. With a poor pharmacokinetic profile, it can be difficult to achieve the dose profile required for therapeutic efficacy and result in patient noncompliance (Alavijeh and Palmer, IDrugs, 2004). Antibodies, particularly IgG antibodies, generally have a longer half-life compared to other therapeutics. Nevertheless, further improvement of the pharmacokinetics of antibodies can still be beneficial and/or required, e.g., for some chronic diseases. Improving the pharmacokinetics allows the dosage to be lowered, enabling a subcutaneous formulation to be developed and COGs to be reduced, and prolongs the dosing interval, which is more convenient for the patients (Ryman and Meibohm, CPT Pharmacometrics Syst Pharmacol, 2017). In this regard, in some embodiments, provided anti-PD-1 antibodies exhibit favorable pharmacokinetic properties as compared to previously known PD-1 antibodies, e.g., humanized 388D4 as described in WO 2016/106159.

[0008] Additionally, poor stability may cause various problems such as aggregation, resulting in reduced biological activity and increased immunogenicity and low expression yield, resulting in increased cost of goods (Igawa et al., MAbs, 2011 ). Accordingly, in some embodiments, provided anti-PD-1 antibodies exhibit increased stability and, as a result, improved expression profile as compared to previously known PD-1 antibodies, e.g., humanized 388D4 as described in WO 2016/106159.

II. DEFINITIONS

[0009] The following list defines terms, phrases, and abbreviations used throughout the instant specification. All terms listed and defined herein are intended to encompass all grammatical forms.

[0010] As used herein, unless otherwise specified, “PD-1” means human PD-1

(huPD-1 ) and includes variants, isoforms, and species homologs of human PD-1. PD-1 is also known as“programmed cell death protein 1”,“cluster of differentiation 279” or“CD279”, which are used interchangeably. Human PD-1 means a full-length protein defined by UniProt Q151 16, a fragment thereof, or a variant thereof. Human PD-1 is encoded by the PDCD1 gene.

[0011] As used herein,“antibody” includes whole antibodies and any antigen binding fragment (i.e.,“antigen-binding portion”) or single chain thereof. An“antibody” refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter- connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable domain (V_H or HCVR) and a heavy chain constant region (C_H). The heavy chain constant region is comprised of three domains, C_Hi, C_H2 and C_H3- Each light chain is comprised of a light chain variable domain (V_L or LCVR) and a light chain constant region (C_L). The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V_H and V_L is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy- terminus: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen (PD-1 in the present case). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. [0012] As used herein, an“antibody fragment” can be an antigen-binding fragment or a fragment crystallizable region.

[0013] As used herein, “antigen-binding fragment,” “antigen-binding portion” or

“antigen-binding domain” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-1 ). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment consisting of the V_H, V_L, C_L and C_Hi domains; (ii) a F(ab')₂ fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment comsisting of the V_H, V_L, C_L and C_Hi domains and the region between C_Hi and C_H2 domains; (iv) a Fd fragment consisting of the V_H and C_Hi domains; (v) a single-chain Fv fragment consisting of the V_H and V_L domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., Nature, 1989) consisting of a V_H domain; and (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker; (viii) a“diabody” comprising the V_H and V_L connected in the same polypeptide chain using a short linker (see, e.g., patent documents EP 404,097; WO 93/1 1 161 ; and Holliger et al., Proc Natl Acad Sci U S A, 1993); (ix) a“domain antibody fragment” containing only the V_H or V_L, where in some instances two or more V_H regions are covalently joined.

[0014] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, multispecific). Antibodies may also be fully human.

[0015] As used herein,“framework” or“FR” refers to the variable domain residues other than the hypervariable region (CDR) residues.

[0016] “Fragment crystallizable region” or“Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine (residue 447 according to EU index of Kabat) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the invention include human lgG1 , lgG2 (lgG2A, lgG2B), lgG3, and lgG4.

[0017] Fc receptor” or“FcR” refers to a receptor that binds to the Fc region of an antibody.

[0018] As used herein,“isolated antibody” refers to an antibody that is substantially free of its natural environment. For instance, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An “isolated antibody” further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In the present case, an isolated antibody that binds specifically PD-1 is substantially free of antibodies that specifically bind antigens other than PD-1. However, an isolated antibody that specifically binds PD-1 may have cross-reactivity to other antigens, such as PD-1 molecules from other species.

[0019] As used herein, “monoclonal antibody” refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0020] As used herein,“humanized antibody” refers to an antibody that consists of the CDR of antibodies derived from mammals other than human, and the FR region and the constant region of a human antibody or derived from a human antibody. In some embodiments a humanized antibody comprises a variable domain that has a variable region amino acid sequence which, analyzed as a whole, is closer to human than to other species as assessed using the Immunogenetics Information System (IMGT) DomainGapAlign tool, as described by Ehrenmann et al. (2010). A humanized antibody is useful as an effective component in a therapeutic agent due to the reduce antigenicity. The term “therapeutic agent” or “therapeutically active agent”, as used herein, refers to an agent which is therapeutically useful. A therapeutic agent may be any agent for the prevention, amelioration, or treatment of a diseases, a physiological condition, a symptom, or for the evaluation or diagnosis thereof.

[0021] As used herein,“human antibody” includes antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0022] As used herein,“binding affinity” describes the ability of a biomolecule (e.g., a polypeptide or a protein) of the disclosure (e.g., an antibody) to bind a selected target and form a complex. Binding affinity is measured by a number of methods known to those skilled in the art including, but are not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays, including direct and competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well-established in the art and some examples of such methods are further described herein. Binding affinity is thereby reported as a value of dissociation constant ( K_d ), half maximal effective concentration (EC₅₀), or half maximal inhibitory concentration (IC₅₀) measured using such these methods. A lower K_d, EC₅₀, or IC₅₀ value reflects better (higher) binding ability (affinity). Accordingly, the binding affinities of two biomolecules toward a selected target can be measured and compared. When comparing the binding affinities of two biomolecules toward the selected target, the tearm“about the same,” “substantially the same” or“substantially similar” means one biomolecule has a binding affinity reported as a K_d, an EC₅₀, or an IC₅₀ value that is identical or similar to another molecule within the experimental variability of the binding affinity measurement. The experimental variability of the binding affinity measurement is dependent upon the specific method used and is known to those skilled in the art.

[0023] As used herein, the term“detect,”“detection,”“detectable,” or“detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative, and qualitative measurements performed on a biomolecule of the disclosure.

[0024] As used herein, “detectable affinity” generally means the binding ability between a biomolecule and its target, reported by a K_d, EC₅o, or IC₅o value, is at most about 10⁵ M or lower. A binding affinity, reported by a K_d, EC₅₀, or IC₅₀ value, higher than 10⁵ M is generally no longer measurable with common methods such as ELISA and SPR and is therefore of secondary importance.

[0025] It is noted that the complex formation between the biomolecule of the disclosure and its target is influenced by many different factors such as the concentrations of the respective target, the presence of competitors, pH and the ionic strength of the buffer system used, the experimental method used for determination of the binding affinity (e.g., fluorescence titration, competitive ELISA (also called competition ELISA), and surface plasmon resonance), and even the mathematical algorithm used for evaluation of the experimental data. Therefore, it is clear to the skilled person that binding affinity reported by a K_d, EC₅₀, or IC₅₀ value may vary within a certain experimental range, depending on the method and experimental setup. This means that there may be a slight deviation in the measured_d, EC₅o, or IC₅o values or a tolerance range depending, for example, on whether such values were determined by ELISA (including direct or competition ELISA), by SPR, or by another method.

[0026] As used herein, “specific for,” “specific binding,” “specifically bind,”

“specificity,” or“binding specificity” relates to the ability of a biomolecule to discriminate between the desired target (for example, PD-1 ) and one or more reference targets (for example, cytochromes). It is understood that such specificity is not an absolute but a relative property and can be determined, for example, in accordance with SPR, western blots, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), ImmunoHistoChemistry (IHC), and peptide scans. When used herein in the context of the anti-PD-1 antibody of the present disclosure, the term“specific for,”“specific binding,”“specifically bind,”“specificity,” or“binding specificity” means that the anti-PD-1 antibody binds to, reacts with, or is directed against PD-1 , as described herein, but does not essentially bind another target. The term “another target” includes any proteins or molecules that are not PD-1 or proteins closely related to or being homologous to PD-1. However, PD-1 from species other than human and fragments and/or variants of PD-1 are not excluded by the term“another target.” The term “does not essentially bind” means that the anti-PD-1 antibody of the present disclosure binds another protein with lower binding affinity than PD-1 , i.e., shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6, or 5%. Whether the anti-PD-1 antibody specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of a anti-PD-1 antibody of the present disclosure with PD-1 and the reaction of said fusion protein with (an)other target(s). [0027] Accordingly, the term“off-target” or“off-target binding” as used herein means that a biomolecule does not specifically bind the desired target (e.g., PD-1 ), i.e., the biomolecule cannot discriminate between the desired target and one or more other targets.

[0028] The term “fragment” as used herein with respect to PD-1 relates to N- terminally and/or C-terminally shortened protein or protein domains, which retain the capability of the full-length antigen to be recognized and/or bound by an antibody according to the disclosure.

[0029] As used herein, “mutagenesis” refers to experimental conditions that are chosen such that modifications are introduced into a nucleotide or an amino acid sequences. The term "mutagenesis" also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. It is however preferred that mutagenesis does not significally affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence.

[0030] The term“random mutagenesis” means that no predetermined single amino acid (mutation) is present at a certain sequence position and mutations can be introduced randomly along all or part of an anti-PD-1 antibody coding sequence. The resulting modified antibodies can be screened for binding specificities and affinities.

[0031] “Identity” is a property of sequences that measures their similarity or relationship. The term“sequence identity” or“identity” as used in the present disclosure means the percentage of pair-wise identical residues— following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question— with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[0032] The term “homology” is used herein in its usual meaning and includes identical amino acids as well as amino acids which are regarded to be conservative substitutions (for example, exchange of a glutamate residue by an aspartate residue) at equivalent positions in the linear amino acid sequence of a polypeptide of the disclosure (e.g., any lipocalin mutein of the disclosure).

[0033] As used herein, the term“substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. Accordingly, the term“substantially” may be used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0034] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002) (cf. Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cut-off value set to 10³). It is calculated as the percentage of numbers of “positives” (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0035] “Gaps” are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of sequence identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990), and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981 ).

[0036] The word“detect”,“detection”,“detectable”, or“detecting” as used herein is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative and qualitative measurements of a molecule of interest.

[0037] A“subject” is a vertebrate, preferably a mammal, more preferably a human.

The term“mammal” is used herein to refer to any animal classified as a mammal, including, without limitation, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples. Preferably, the“mammal” herein is human.

[0038] An“effective amount” is an amount sufficient to effect beneficial or desired results. For example, an effective amount of a composition may be one that would be sufficient to enhance or diminish the immune response to a desired level of a therapy. The results of a therapy (e.g., activation of a suppressed or deficient immune response, increased cytolytic activity of T cells, increased T cell effector function, alteration of PD-1 activity associated with the negative regulation of T-cell mediated immune response, or reduction in tumor growth) can be determined by suitable methods known in the art. An effective amount can be administered in one or more administrations. An effective amount, for example, of a composition, can be administered alone, with one additional therapeutic agent, or in combination with two or more additional therapeutic agents.

[0039] A“sample” is defined as a biological sample taken from any subject. By way of non-limiting example, biological samples include blood, serum, urine, feces, semen, or tissue.

III. DESCRIPTIONS OF FIGURES

[0040] Figure 1 : depicts the results of an enzyme-linked immunosorbent assay

(ELISA) in which the binding to human PD-1 (Figure 1A) and cynomolgus PD-1 (Figure 1 B) of humanized PD-1 specific antibodies as described in Example 4. Recombinant PD-1 -His (PD-1 with a C-terminal polyhistidine tag) was coated on a microtiter plate, and the tested antibodies were titrated starting with the highest concentration of 100 nM and a 1 :3 dilution series. Bound samples under study were detected via an anti-human IgG Fc antibody. The data was fit with a 1 :1 binding model with EC₅₀ value and the maximum signal as free parameters, and a slope that was fixed to one. The resulting EC₅₀ values are provided in Table 3.

[0041] Figure 2: depicts the target binding of the humanized PD-1 specific antibodies assessed by flow cytometry using human PD-1 -expressing Flp-ln-CHO cells (Figure 2A) or cynomolgus PD-1 -expressing Flp-ln-CHO cells (Figure 2B), as described in Example 5. The geometric means of the fluorescence intensity were used to calculate EC₅₀ values using nonlinear regression (shared bottom, SLOPE =1 ). The resulting EC₅₀ values are provided in Table 4.

[0042] Figure 3: shows that the humanized PD-1 specific antibodies compete with

PD-L1 and PD-L2 for binding to PD-1 , depicted in competitive ELISA studies conducted as described in Example 6. A constant concentration of PD-L1-Fc or PD-L2-Fc fusion was coated on a microtiter plate, and a mixture of biotinylated human PD-1-Fc fusion at a constant centration and testing antibodies at different concentrations were titrated. Bound biotinylated PD-1 was detected via Extravidin-HRP. The data was fit with a 1 :1 binding model with IC₅₀ value and the maximum signal as free parameters, and a slope that was fixed to one. The resulting IC₅₀ values are provided in Table 5.

[0043] Figure 4: depicts the results of fluorescence-activated cell sorting (FACS) studies assessing the off-target or non-specific binding of exemplary PD-1 specific antibodies to human endothelial cells (HUVEC) that are PD-1 negative (do not express PD-1 ). The previously known humanized PD-1 antibody with the sequences set forth in SEQ ID NOs: 4 and 9 shows strong, non-specific adhesion to HUVEC cells, while such non-specific adhesion is significantly reduced in humanized PD-1 antibodies provided herein with the sequences set forth in SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 115, and SEQ ID NOs: 109 and 116.

[0044] Figure 5: provides the results of a pharmacokinetic analysis of the exemplary humanized PD-1 specific antibodies in mice as described in Example 9. Male CD-1 mice (2 mice per timepoint) were injected intravenously with humanized PD-1 specific antibodiesat a dose of 2 mg/kg. Drug levels were detected using an ELISA at the indicated time points. The data were plotted in a time vs. drug concentration graph. Selected humanized antibodies (SEQ ID NOs: 52 and 82, SEQ ID NOs: 52 and 83, SEQ ID NOs: 109 and 1 15, SEQ ID NOs: 109 and 1 16, SEQ ID NOs: 61 and 9, SEQ ID NOs:4 and 75, SEQ ID NOs: 4 and 78, SEQ ID NOs: 4 and 82, SEQ ID NOs: 4 and 83, SEQ ID NOs: 4 and 98, SEQ ID NOs: 58 and 75, SEQ ID NOs: 58 and 96, SEQ ID NOs: 72 and 108, SEQ ID NOs: 73 and 108, SEQ ID NOs: 4 and 406, and SEQ ID NOs: 405 and 108) described herein display favorable pharmacokinetic profiles or antibody-like pharmacokinetics.

[0045] Figure 6: The ability of representative humanized PD-1 specific antibodies to release the inhibitory signal through PD-1 via PD-L1 using a PD-1/PD-L1 blockade bioassay as described in Example 10. PD-1-NFAT-luc Jurkat T cells (a Jurkat cell line expressing PD- 1 and a NFAT-mediated luciferase gene) were co-cultured with PD-L1 expressing Raji cells in presence of various concentrations of humanized PD-1 specific antibodies. After 4 hours, luciferase assay reagent was added and luminescent signals measured. The humanized PD- 1 specific antibodies inhibitese PD-1/PD-L1 blockade and led to T-cell activation.

[0046] Figure 7: shows the results of representative experiments in which the ability of exemplary humanized PD-1 specific antibodies to induce T cell activation was investigated as described in Example 11. In the experiment, human peripheral blood mononuclear cells (PBMCs) stimulated with 0.1 ng/mL staphylococcal enterotoxin B (SEB) were incubated with tested antibody. Levels of secreted interleukin 2 (IL-2), reflective of T cell activation, were determined by an electrochemoluminescence-based assay as readout for T cell activation. The humanized PD-1 specific antibodies increased IL-2 secretion in a dose-dependent manner. [0047] Figure 8: shows the tumor volume after treatment with selected humanized

PD-1 speicfic antibodies in humanized mouse tumor model as described in Example 12. NOG mice were engrafted with s.c. HCC827 tumors which were allowed to grow for 10 days. Mice were randomized into treatment groups and received 5^c 10⁶ fresh human PBMCs intravenously as well as intraperitoneal injections of the humanized PD-1 speicfic antibodies (or control molecules) at the doses indicated on day 1 , day 4, day 8, and day 12. Tumor growth was recorded every 3-4 days. The PD-1 specific antibody of SEQ ID NOs: 109 and 115 successfully inhibited the tumor growth.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

[0048] The present disclosure provideshumanized antibodies that binds PD-1 with high affinity and specificity and that exhibit favorable stability and pharmackenetic profiles as well as useful application and methods of characterizing such antibodies. Additionally, in some embodiments, the disclosed humanized PD-1 specific antibodies are cross-reactive with human and cynomolgus PD-1. No such humanized antibodies having these features attendant to the uses provided by present disclosure have been previously described.

A. Exemplary humanized PD-1 specific antibodies.

[0049] Some embodiments of the current disclosure relate to humanized PD-1 specific antibodies obtained by CDR-grafting of a murine antibody specific for human PD-1 , e.g., 388D4 antibody. The technology of CDR-grafting is well-known in the art, where complementarity-determining regions (CDRs) of the antibody light and heavy chain variable regions are grafted into frameworks (FRs) from another species. For example, murine CDRs can be grafted into human FRs. In some embodiments, the CDRs of the light and heavy chain variable regions of a murine anti-PD-1 antibody (e.g., the light and the heavy chain variable regions of SEQ ID NO: 3 and SEQ ID NO: 2, respectively, or of 388D4 antibody) are grafted into human FRs or consensus human FRs. CDR-grafting is described in, e.g., U.S. Patent Nos. 7,022,500; 6,982,321 ; 6,180,370; 6,054,297; 5,693,762; 5,859,205; 5,693,761 ; 5,565,332; 5,585,089; and 5,530,101 and PCT International Application Publication Nos.: WO 2016/102716.

[0050] The selection of human light and heavy variable regions in generating humanized antibodies is important to retain the affinity and the specificity of the original murine antibody and reduce antigenicity. As is further discussed herein, such selection can be particularly influential since the antigen specificity often helps eliminate undesired off- target antibody interactions, which in turn affects the pharmacokinetics, tissue distribution, efficacy and toxicity of an antibody. Online databases of mature human IgG sequences can be searched for comparison to the murine light and heavy chain variable regions. In general, the human sequence that is closest to that of the murine is then accepted as the FR for the humanized antibody (Sims et al., J Immunol, 1993, Chothia and Lesk, J Mol Biol, 1987).

[0051] Another method for making humanized antibodies includes uses of a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Presta et al., J Immunol, 1993, Carter et al., Proc Natl Acad Sci U S A, 1992).

[0052] Since it is important for humanized antibodies to retain affinity for the antigen and other desirable biological properties, humanized antibodies can be further analyzed and engineered against parental sequences and using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are known to those skilled in the art. Computer programs that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Inspection of these displays permits analyses of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0053] Accordingly, in some embodiments of the current disclosure, an isolated antibody or antigen-binding fragment thereof has at least one of a heavy chain variable domain comprising complementarity determining regions (CDRs) from the heavy chain variable region sequence set forth in SEQ ID NO: 2, and a light chain variable domain comprising CDRs from the light chain variable region sequence set forth in SEQ ID NO: 3. In some preferred embodiments, such an isolated antibody or antigen-binding fragment thereof contains the heavy chain and light chain CDRs of the monoclonal antibody 388D4 but a different framework from the 388D4 antibody.

[0054] In some preferred embodiments, the heavy chain variable region of a provided humanized PD-1 specific antibody or the antigen-binding fragment thereof comprise CDRs with the following sequences: GYTFTDYE (HCDR1 , SEQ ID NO: 28), IDPGTGGT (HCDR2, SEQ ID NO: 29), TSEKFGSNYYFDY (HCDR3, SEQ ID NO: 30). The light chain variable region of a provided humanized PD-1 specific antibody or the antigen-binding domain thereof comprise CDRs with the following sequences: QTIVHSDGNTY (LCDR1 , SEQ ID NO: 31 ), KVS (LCDR2), FQGSHVPLT (LCDR3, SEQ ID NO: 33).

[0055] Unless otherwise indicated, all CDR sequences disclosed herein are defined according to the IMGT method as described in Lefranc (1999). CDR1 consists of positions 27 to 38, CDR2 consists of positions 56 to 65, CDR3 for germline V-genes consists of positions 105 to 1 16, and CDR3 for rearranged V-J-genes or V-D-J-genes consists of positions 105 to 1 17 (position preceding J-PHE or J-TRP 118) with gaps at the top of the loop for rearranged CDR3-IMGT with less than 13 amino acids, or with additional positions 1 12.1 , 11 1.1 , 112.2, 1 11.2, etc. for rearranged CDR3-IMGT with more than 13 amino acids. The positions given in this paragraph are according to the IMGT numbering described in Lefranc (1999).

[0056] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 119-159 and 195-199.

[0057] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 144, 147, 158, 159, and 195.

[0058] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 144 and 195.

[0059] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a light chain variable domain selected from the group consisting of SEQ ID NOs: 160-194, 200-204, and 407.

[0060] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a light chain variable domain selected from the group consisting of SEQ ID NOs: 161 , 165, 168, 169, 182, 184, 194, 201 , 202, and 407.

[0061] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a light chain variable domain selected from the group consisting of SEQ ID NOs: 168, 169, 201 , and 202.

[0062] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 119-159 and 195-199 and a light chain variable region selected from the group consisting of SEQ ID NOs: 160-194, 200-204, and 407.

[0063] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 144, 147, 158, 159, and 195 and a light chain variable region selected from the group consisting of SEQ ID NOs 161 , 165, 168, 169, 182, 184, 194, 201 , 202, and 407.

[0064] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain selected from the group consisting of SEQ ID NOs: 144 and 195 and a light chain variable region selected from the group consisting of SEQ ID NOs: 168, 169, 201 , and 202.

[0065] Examples of pairings of the heavy chain variable domain and light chain variable domain are provided throughout the present disclosure. However, additional functional pairings are within the scope of the invention. In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain and light chain variable domain pair having the amino acid sequences selected from the group consisting of SEQ ID NOs:144 and 168, SEQ ID NOs: 144 and 169, SEQ ID NOs: 195 and 201 , SEQ ID NOs: 195 and 202, SEQ ID NOs: 147 and 21 , SEQ ID NOs:16 and 161 , SEQ ID NOs:16 and 165, SEQ ID NOs: 16 and 168, SEQ ID NOs: 16 and 169, SEQ ID NOs: 16 and 184, SEQ ID NOs: 144 and 161 , SEQ ID NOs: 144 and 182, SEQ ID NOs: 158 and 194, SEQ ID NOs: 159 and 194, SEQ ID NOs: 156 and 195, and SEQ ID NOs: 16 and 407.

[0066] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof have a heavy chain variable region with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 119-159 and 195-199, and a light chain variable region with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 98% to an amino acid sequence selected from the group consisting of SEQ ID NOs: 160- 194, 200-204, and 407.

[0067] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof have a heavy chain variable region with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 147, 158, 159, and 195, and a light chain variable region with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 98% to an amino acid sequence selected from the group consisting of SEQ ID NOs: 161 , 165, 168, 169, 182, 184, 194, 201 , 202, and 407.

[0068] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof have a heavy chain variable region with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 144 and 195, and a light chain variable region with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 98% to an amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 169, 201 , and 202.

[0069] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding fragment thereof comprises a heavy chain variable domain and light chain variable domain pair having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to the amino acid sequences selected from the group consisting of SEQ ID NOs:144 and 168, SEQ ID NOs: 144 and 169, SEQ ID NOs: 195 and 201 , SEQ ID NOs: 195 and 202, SEQ ID NOs: 147 and 21 , SEQ ID NOs:16 and 161 , SEQ ID NOs:16 and 165, SEQ ID NOs: 16 and 168, SEQ ID NOs: 16 and 169, SEQ ID NOs: 16 and 184, SEQ ID NOs: 144 and 161 , SEQ ID NOs: 144 and 182, SEQ ID NOs: 158 and 194, SEQ ID NOs: 159 and 194, SEQ ID NOs: 156 and 195, and SEQ ID NOs: 16 and 407.

[0070] In some embodiments, a provided isolated antibody that binds to PD-1 is or comprises a full-length immunoglobulin. The immunoglobulin, for example, may be lgG1 , lgG2 or lgG4.

[0071] Since the Fc region of an antibody interacts with a number of Fc receptors resulting in different functional capabilities (which are referred to as "effector functions"), a provided antibody is, in some embodiments, a full-length antibody (also called a full-length immunoglobulin) or an antibody that contains a portion of the Fc region. The choice of the Fc region depends on the desired effector functions like complement fixation or antibody- dependent cell-mediated cytotoxicity are desirable features.

[0072] In some embodiments, a provided isolated antibody that binds to PD-1 may comprise an Fc region that allows for extended in vivo half-life. Such Fc region is preferably from human origin, more preferably a human Fc region of an lgG1 or lgG4 antibody, even more preferably an engineered human Fc region of an lgG1 or lgG4 with activating or silencing effector functions, wherein silencing effector functions are preferred over activating effector functions.

[0073] In some particular embodiments, a provided isolated antibody that binds to

PD-1 may comprise an Fc region which allows for reduced or fully suppressed Fc receptor binding. This may be achieved, for example, by using an lgG4 backbone or switching from the lgG1 backbone to lgG4, as lgG4 is known to display reduced Fc-gamma receptor interactions, including as compared to lgG1. In some particular embodiments, mutations may be introduced into the lgG4 backbone, such as F234A and L235A, to further reduce the residual binding to Fc-gamma receptors. In some other embodiments, an S228P mutation may be introduced into the lgG4 backbone to minimize the exchange of lgG4 half-antibody. In some further embodiments, M428L and N434S or M252Y, S254T, and T256E may be introduced into the lgG4 backbone for enhanced binding to the neonatal Fc receptor and extended serum half-life. In some still further embodiments, an additional N297A mutation may be present in the immunoglobulin heavy chain of the antibody in order to remove the natural glycosylation motif resulting in decreased binding to Fc-gamma receptor and reduced or eliminated antibody cytotoxicity. Numbering for all sets of these potential mutations is according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000).

[0074] Accordingly, in some embodiments, a provided isolated antibody that binds to

PD-1 or the antigen-binding domain thereof comprises a heavy chain having the sequence selected from SEQ ID NOs: 33-73, 109-113, and 405. In some further embodiments, the isolated antibody that binds to PD-1 or the antigen-binding domain thereof comprises a light chain having the sequence selected from SEQ ID NOs: 74-108, 114-118, and 406.

[0075] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding domain thereof comprises a heavy chain having the sequence selected from SEQ ID NOs: 58, 61 , 72, 73, 109, and 405. In some further embodiments, the isolated antibody that binds to PD-1 or the antigen-binding domain thereof comprises a light chain having the sequence selected from SEQ ID NOs: 75, 78, 82, 83, 96, 98, 108, 115, 116, and 406. [0076] In some embodiments, a provided isolated antibody that binds to PD-1 or the antigen-binding domain thereof comprises a heavy chain having the sequence selected from SEQ ID NOs: 58 and 109. In some further embodiments, the isolated antibody that binds to PD-1 or the antigen-binding domain thereof comprises a light chain having the sequence selected from SEQ ID NOs: 82, 83, 115, and 116.

[0077] In some preferred embodiments, a provided isolated antibody that binds to

PD-1 or the antigen-binding fragment thereof comprises a heavy chain and light chain pair having the amino acid sequences selected from the group consisting of SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 115, SEQ ID NOs: 109 and 116, SEQ ID NOs: 61 and 9, SEQ ID NOs:4 and 75, SEQ ID NOs: 4 and 78, SEQ ID NOs: 4 and 82, SEQ ID NOs: 4 and 83, SEQ ID NOs:4 and 98, SEQ ID NOs: 58 and 75, SEQ ID NOs: 58 and 96, SEQ ID NOs: 72 and 108, SEQ ID NOs: 73 and 108, SEQ ID NOs: 4 and 406, SEQ ID NOs: 405 and 108.

[0078] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof have a heavy chain with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-73, 109-113, and 405 and a light chain with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5, at lest 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 74-108, 114-118, and 406.

[0079] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof have a heavy chain with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61 , 72, 73, 109, 405, and a light chain with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5, at lest 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 78, 82, 83, 96, 98, 108, 1 15, 116, and 406.

[0080] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof have a heavy chain with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 and 109 and a light chain with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5, at lest 98%, at least 98.5%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 83, 115, and 116.

[0081] In some preferred embodiments, a provided isolated antibody that binds to

PD-1 or the antigen-binding fragment thereof comprises a heavy chain and light chain pair having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 98%, at least 98.5%, or at least 99% sequence identity to the amino acid sequences selected from the group consisting of SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 115, SEQ ID NOs: 109 and 116, SEQ ID NOs: 61 and 9, SEQ ID NOs:4 and 75, SEQ ID NOs: 4 and 78, SEQ ID NOs: 4 and 82, SEQ ID NOs: 4 and 83, SEQ ID NOs:4 and 98, SEQ ID NOs: 58 and 75, SEQ ID NOs: 58 and 96, SEQ ID NOs: 72 and 108, SEQ ID NOs: 73 and 108, SEQ ID NOs: 4 and 406, SEQ ID NOs: 405 and 108.

[0082] In some embodiments, a provided PD-1 antibody or antigen-binding fragment thereof binds to a sequence in PD-1 selected from the group consisting of SEQ ID NOs: 355- 385.

[0083] A provided isolated PD-1 antibody or an antigen-binding fragment thereof that competitively inhibits the binding of a reference antibody to a target or binds to the same epitope of the target as a reference antibody or an antigen-binding domain thereof is specifically contemplated as within the scope of the present disclosure. In some embodiments, a provided antibody competitively inhibits the binding or binds to the same epitope of a target as a reference antibody that comprises an antigen-binding region, such as any one of the three heavy chain CDRs and the three light chain CDRs, from any one of the anti-PD-1 antibodies disclosed in above mentioned applications. In some further embodiments, the antibody competitively inhibits the binding or binds to the same epitope of the antibody that comprises a heavy chain variable domain and light chain variable domain pair selected from the group consisting of SEQ ID NOs:144 and 168, SEQ ID NOs: 144 and 169, SEQ ID NOs: 195 and 201 , and SEQ ID NOs: 195 and 202. In some further embodiments, a provided antibody competitively inhibits the binding or binds to the same epitope of the target of a reference antibody that comprises a heavy chain and light chain pair selected from the group consisting of SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 115, and SEQ ID NOs: 109 and 1 16.

[0084] Methods for determining whether two or more antibodies compete for binding to the same target or bind to the same epitopes are known in the art. For example, a competitive binding, or competition, assay can be used to determine whether one antibody blocks the binding of another antibody to the target.

[0085] Further, some embodiments of the current disclosure relate to an isolated antibody binding to PD-1 or an antigen-binding fragment thereof that binds PD-1 , preferably human PD-1 , with an affinity measured by a K_d of about 20 nM, 15 nM, 5 nM, 3 nM, or lower. Such affinity can be determined, for example, by surface plasmon resonance (SPR) analysis essentially described in Example 3.

[0086] In other embodiments, a provided PD-1 specific antibody or the antigen- binding fragment thereof may be cross-reactive with cynomolgus PD-1 , and in some further embodiments, binds cynomolgus PD-1 with an affinity measured by a K_d of about 50 nM, 40 nM, 10 nM, or lower. Such affinity can be determined, for example, by SPR analysis essentially described in Example 3.

[0087] In some preferred embodiments, a provided humanized PD-1 specific antibody or antigen-binding fragment thereof binds PD-1 with substantially the same or lower K_d as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9 or as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0088] In some embodiments, a provided isolated antibody or an antigen-binding fragment thereof binds PD-1 with an EC₅₀ value of at most about 10 nM or even lower, such as about 5 nM, about 1 nM, about 0.5 nM, about 0.2 nM, about 0.15 nM, about 0.1 nM, or even lower. In some embodiments, a provided isolated antibody or an antigen-binding domain thereof binds cynomolgus PD-1 with an EC₅₀ value of at most about 10 nM or even lower, such as about 5 nM, about 1 nM, about 0.5 nM, about 0.2 nM, about 0.15 nM, about 0.1 nM, or even lower. In some embodiments, a provided isolated antibody or an antigen- binding domain thereof binds PD-1 with substantially the same or lower EC₅₀ value as an antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 4 and 9 or SEQ ID NOs: 16 and 21. The ability of such antibody to bind PD-1 may be determined by, for example, and an enzyme-linked immunosorbent assay (ELISA) essentially described in Example 4.

[0089] In some embodiments, a provided isolated antibody or an antigen-binding fragment thereof binds PD-1 expressed on a cell with an EC₅₀ value of at most about 10 nM or even lower, such as about 8 nM, about 5 nM, about 2 nM, about 1 nM, or even lower. In some embodiments, a provided isolated antibody or an antigen-binding domain thereof binds PD-1 expressed on a cell with substantially the same or lower EC₅₀ value as an antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 4 and 9 or SEQ ID NOs: 16 and 21 . The ability to bind PD-1 expressed on a cell may be determined by, for example, a fluorescence activated cell sorting (FACS) assay, such as the FACS assay as essentially described in Example 5. The cell expressing PD-1 may, for example, be a CHO cell transfected with human PD-1 or cynomolgus PD-1.

[0090] In some still further embodiments, a provided isolated antibody or an antigen- binding fragment thereof competes with PD-L1 or PD-L2 for binding to PD-1 . In some embodiments, a provided isolated antibody or an antigen-binding domain thereof of the disclosure may be able to compete with PD-L1 or PD-L2 for binding to PD-1 with an IC₅₀ value of about 10 nM, 6 nM, 5 nM, 4 nM, 3 nM, 1 nM, or even lower. In some embodiments, a provided isolated antibody or an antigen-binding domain thereof may be able to compete with PD-L1 or PD-L2 for binding to PD-1 with an IC₅₀ value that is substantially equal to or lower than the IC₅₀ value of the antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 4 and 9 or SEQ ID NOs: 16 and 21 . The ability to compete with PD-L1 or PD-L2 may be determined by, for example, an enzyme-linked immunosorbent assay (ELISA) essentially described in Example 6.

[0091] In some embodiments, a provided isolated antibody or an antigen-binding fragment thereof does not essentially bind targets other than PD-1 . Accordingly, a provided isolated antibody or an antigen-binding domain thereof has reduced to targets other than the designated target PD-1 (also called off-target binding) compared to an antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 16 and 21 or SEQ ID NOs: 4 and 9. Such evaluation of off target binding can be performed by ELISA as described in Example 7.

[0092] In some further embodiments, a provided isolated antibody or an antigen- binding domain thereof does not essentially bind cells that do not express PD-1 . Accordingly, a provided isolated antibody or an antigen-binding domain thereof has reduced nonspecific binding to cells that do not express PD-1 compared to an antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 16 and 21 or SEQ ID NOs: 4 and 9. Such evaluation of non-specific binding can be performed by fluorescence- activated cell sorting (FACS) as described in Example 8. [0093] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof have favorable stability and pharmacokinetics profiles. In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof have comparable pharmacokinetics profile compared to an antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 16 and 21 or SEQ ID NOs: 4 and 9. In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof have antibody-like pharmacokinetics. As described herein, a favorable pharmacokinetic profile or an antibody-like pharmacokinetics may be considered to be achieved for provided antibodies if the percentage of c_max was above 15 % after 168 h. Pharmacokinetics profiles of provided antivodies may be analyzed as described in Example 9.

[0094] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof blocks the inhibitory signal of PD-1. In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof releases a brake for T cell activation or leads to successful T cell activation. The blockade of PD-1 inhibitory signal or T cell activation may be measured, for example, in a PD-1/PD-L1 blockade bioassay as described in Example 10.

[0095] In some embodiments, a provided PD-1 antibody or antigen-binding domain thereof can enhance IL-2 secretion. In some preferred embodiments, a provided PD-1 antibody or antigen-binding domain thereof may demonstrate a tendency to induce more enhanced IL-2 secretion at higher concentrations. In some still preferred embodiments, a provided PD-1 antibody or antigen-binding domain thereof induces dose-dependent increase of IL-2 secretion, comparable to an antibody having a heavy chain and light chain pair comprising the amino acid sequences of SEQ ID NOs: 4 and 9. In some related embodiments, a provided PD-1 antibody or antigen-binding domain thereof can lead to successful T cell activation. The IL-2 secretion or T cell activation may be measured, for example, in in a functional T-cell activation assay as described in Example 11.

[0096] In some embodiment, a provided PD-1 antibody or antigen-binding domain thereof can inhibit tumor growth. The tumor growth inhibition may be demonstrated in humanized mouse tumor model as described in Example 12.

[0097] The present disclosure additionally encompasses nucleic acid molecules and host cells encoding a probided anti-PD-1 antibodies or antigen-binding domains thereof as described herein. [0098] An anti-PD-1 antibody of the present disclosure, for example, can be prepared by recombinant expression of immunoglobulin heavy and light chain genes. In some embodiments, to generate nucleic acids encoding an anti-PD-1 antibody of the disclosure, nucleotide sequence encoding the heavy and light chain variable regions are first obtained. In some embodiments, the presentdisclosure also provides isolated nucleic acids comprising a nucleotide sequence encoding a heavy or light chain variable region as disclosed herein, or a fragment thereof.

[0099] Once nucleic acid fragments encoding the heavy and light chain variable regions are obtained, resultant fragments can be further manipulated or ligated to other appropriate necleotide sequences by standard recombinant techniques, for example, to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes, or to a scFv gene. In these manipulations, a nucleic acid fragments encoding the heavy and light chain variable region is operatively linked to another fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used herein refers to that the two nucleic acid fragments are joined such that the amino acid sequences encoded by the two remain in-frame.

[00100] A nucleic acid according to the present invention may comprise DNA or RNA, and may be wholly or partially synthetic. Production of defined gene constructs is within routine skill in the art. See, e.g., Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

[00101] Alternatively, nucleotide sequences of a provided anti-PD-1 antibody or the fragment thereof can be cloned out of hybridomas, for example, by conventional hybridization techniques or polymerase chain reaction (PCR) techniques, using synthetic nucleic acid probes or primers whose sequences are based on sequence information provided herein, or known sequence information regarding genes encoding the heavy and light chains of murine antibodies in hybridoma cells.

[00102] In some embodimens, a provided nucleotide sequence(s) encoding an anti- PD-1 antibody or the fragment thereof can be operably linked to a promoter to effect expression of the antibody in a host cell. The sequence may include at its 5' end a leader sequence to facilitate expression in a host cell and/or secretion of the antibody from a host cell. Suitable leader sequences are known in the art and can be selected by the skilled person, taking account of the host cell. [00103] In some embodiments, the nucleic acid is incorporated into a vector. Suitable vectors containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, can be obtained commercially or constructed by persons of skill in the art. For further details see, e.g., Molecular cloning: a laboratory manual, 3rd edition, Sambrook et al., 2001. Examples of vectors include plasmids, phages, phagemids, and cosmids, as well as transcription and expression cassettes.

[00104] In some embodiments, a provided nucleotide sequence encoding an anti-PD- 1 antiobdy or a fragment thereof is incorporated into an expression vector, which is introduced into host cells through conventional transfection or transformation techniques. Accordingly, a host cell can be transformed with an expression vector comprising a nucleotide sequence encoding an anti-PD-1 antiobdy or a fragment thereof. Examples of host cells include E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells.

[00105] Methods of producing an anti-PD-1 antibody or a fragment thereof as disclosed herein are within the scope of the present disclosure. In some embodiments, a provided method comprises: (a) growing a host cell containing an expression vector encoding the anti-PD-1 antiobdy or a fragment thereof under conditions so that the host cell expresses the antibody or a fragment thereof; and (b) isolating the antibody or a fragment thereof.

[00106] Suitable conditions for antibody expression and isolation or purification depend on the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Tip or Tac, and a prokaryotic signal sequence. The expressed secreted protein accumulates in refractile or inclusion bodies, and can be harvested after disruption of the cells by French press or sonication. The refractile bodies then are solubilized, and the proteins refolded and cleaved by methods known in the art.

[00107] If an engineered gene is to be expressed in eukaryotic host cells, e.g., CHO (Chinese hamster ovary) cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, a provided vector or gene construct contains enhancers and introns. [00108] In some embodiments, a provided expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed. A provided gene construct can be introduced into eukaryotic host cells using conventional techniques. Provided host cells may express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, portions thereof, and any combination thereof each of which may be attached to a moiety having another function {e.g., cytotoxicity).

[00109] In some embodiments, a host cell may be transfected with a vector containing the entire or part of a heavy chain or a light chain (e.g., a heavy or light chain variable region). In some other embodiments, a host cell may be transfected with a vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain.

[00110] In some embodiments, a host cell may be co-transfected with more than one expression vector (e.g., one expression vector expressing a polypeptide comprising an entire or part of a heavy chain or heavy chain variable region, and another expression vector expressing a polypeptide comprising an entire or part of a light chain or light chain variable region).

[00111] A polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced, for example, by growing (culturing) a host cell transfected with an expression vector encoding such a variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as Protein A, Protein G, glutathione-S-transferase (GST), or histidine tags.

[00112] Anti-PD-1 antibodies as described hereincan be produced by growing (culturing) a host cell transfected with, for example: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial immunoglobulin light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains), under conditions that permit expression of both chains. The intact antibody (or antigen-binding fragment) can be harvested and purified or isolated using techniques known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) or histidine tags. It is within ordinary skill in the art to express a heavy chain and a light chain from a single expression vector or from two separate expression vectors.

[00113] In some embodiments, an anti-PD-1 antibody or a fragment thereof of the disclosure may belinked to one or moreadditional functional molecules or moieties, e.g., a peptide, protein, toxin, radioisotope, or cytostatic agent, for various purposes such as in vivo diagnostic imaging or a diagnostic assay. Provided antibodies can be linked by one or more chemical cross-linking or by recombinant methods. In some embodiments, provided antibodies also can be linked to any of various nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192; or 4,179,337. Provided antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Examples of polymers and methods to attach them are described in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.

B. Pharmaceutical Formulations

[00114] In some embodiments, provided anti-PD-1 antibodies of the present disclosure are formulated into pharmaceutical compositions suitable for administration to a mammal, e.g., a human patient. Provided compositions typically comprise one or more antibodies described herein and a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable excipient” includes suitable solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Provided compositions also can contain one or more other active compounds providing supplemental, additional, or enhanced therapeutic functions. The pharmaceutical compositions also can be included in a container, pack, or dispenser together with instructions for administration.

[00115] In accordance with various embodiments, provided pharmaceutical compositions may be formulated to be compatible with an intended route of administration. Methods to accomplish the administration are known in the art. The administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, intradermal, topical, inhalation, transmucosal, rectal or transdermal.

[00116] Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, as necessary. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00117] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Preferably, a pharmaceutical composition is stable under the conditions of manufacture and storage and is preserved against contamination by microorganisms such as bacteria and fungi. Avoidance of microorganisms can be achieved by inclusion of antibacterial and/or antifungal agents. Examples include: parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. A carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol such as glycerol, propylene glycol, liquid polyethylene glycol, and the like, and suitable mixtures thereof. In some embodiments, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, e.g., aluminum monostearate or gelatin.

[00118] In some embodiments, oral compositions generally include an inert diluent or an edible carrier. By way of non-limiting examples, provided oral compositions can be enclosed in gelatin capsules or compressed into tablets. For oral administration, provided antibodies can be combined with excipients and used in the form of tablets, troches, or capsules.

[00119] In some embodiments, for transmucosal or transdermal administration, one or more penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished, for example, through the use of lozenges, nasal sprays, inhalers, or suppositories. For example, in case of antibodies that comprise an Fc portion, compositions may be capable of transmission across mucous membranes in intestine, mouth, or lungs (e.g., via the FcRn receptor-mediated pathway as described in U.S. Pat. No. 6,030,613). For transdermal administration, provided antibodies may be formulated into ointments, salves, gels, or creams as generally known in the art. For administration by inhalation, provided antibodies may be delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00120] In some embodiments, provided antibodies and/or antigen-binding domains thereof are formulated with carriers that protect the antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used. Exemplary polymers include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions containing the presently disclosed antibodies can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known in the art. See, e.g., U.S. Patent No. 4,522,811.

[00121] In some embodiments, pharmaceutical compositions contain, in addition to a provided antibody, at least one of a cytotoxic agent, cytostatic agent, anti-angiogenic agent, a tumor targeted agent, an immune stimulating agent or immune modulating agent, or an antibody conjugated to a cytotoxic, cytostatic, or otherwise toxic agent. The pharmaceutical composition optionally can be employed with other therapeutic modalities such as surgery, chemotherapy, and radiation.

[00122] Toxicity and therapeutic efficacy of certein embodiments can be determined by conventional pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/ED₅₀. Compositions that exhibit large therapeutic indices are preferred.

[00123] A therapeutically effective dose of a provided antibody can be estimated initially, e.g., from cell culture assays. Examples of suitable bioassays include but are not limited to, DNA replication assays, cytokine release assays, transcription-based assays, PD- 1/PD-L1 binding assays, creatine kinase assays, assays based on the differentiation of pre- adipocytes, assays based on glucose uptake in adipocytes, immunological assays other assays as, for example, described in the Examples. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosing regimen for use in humans. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the antibody that achieves a half-maximal inhibition of symptoms). Circulating levels in plasma may be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage lies preferably within a range of circulating concentrations with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration.

[00124] Generally, a therapeutically effective amount of an antibody or a composition described herein is in the range of 0.1 mg/kg to 100 mg/kg, preferably 0.1 mg/kg to 50 mg/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation, the serum half-life of the antibody, and the route of administration.

[00125] Administration frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody or fusion protein, and the disease being treated.

B. Exemplary uses and applications of humanized PD-1 specific antibodies

[00126] PD-1 is a cell surface signaling receptor that plays a critical role in the regulation of T cell activation and tolerance (Keir et al., Annu Rev Immunol, 2008). It is a type I transmembrane protein and together with BTLA, CTLA-4, ICOS, and CD28, comprise the CD28 family of T cell co-stimulatory receptors. PD-1 is primarily expressed on activated T cells, B cells, and myeloid cells (Dong et al., Nat Med, 1999). It is also expressed on natural killer (NK) cells (Terme et al., Cancer Res, 2011 ).

[00127] Binding of PD-1 by its ligands, PD-L1 and PD-L2, results in phosphorylation of the tyrosine residue in the proximal intracellular immune receptor tyrosine inhibitory domain, followed by recruitment of the phosphatase SHP-2, eventually resulting in down-regulation of T cell activation. One important role of PD-1 is to limit the activity of T cells in peripheral tissues at the time of an inflammatory response to infection, thus limiting the development of autoimmunity (Pardoll, Nat Rev Cancer, 2012). Evidence of this negative regulatory role comes from the finding that PD-1 -deficient mice develop lupus-like autoimmune diseases including arthritis and nephritis, along with cardiomyopathy (Nishimura et al., Science, 2001 , Nishimura et al., Immunity, 1999). In the tumor setting, the consequence is the development of immune resistance within the tumor microenvironment. PD-1 is highly expressed on tumor-infiltrating lymphocytes, and its ligands are up-regulated on the cell surface of many different tumors (Dong et al., Nat Med, 2002). Multiple murine cancer models have demonstrated that binding of ligand to PD-1 results in immune evasion. In addition, blockade of this interaction results in anti-tumor activity (Hamid et al., N Engl J Med, 2013, Topalian et al., N Engl J Med, 2012).

[00128] By targeting the immune checkpoint PD-1 , humanized antibodies of the disclosure may generate a durable anti-tumor and/or anti-infection response, increase anti- tumor lymphocyte cell activity, and/or enhance anti-tumor immunity. Numerous possible applications for humanized anti-PD1 antibodies of the disclosure, therefore, exist in medicine.

[00129] In some embodiments, the present disclosure relates to the use of the humanized anti-PD1 antibodies disclosed herein for detecting or binding PD-1 in a sample as well as a corresponding method of diagnosis. Such use may include the steps of contacting one or more provided humanized anti-PD1 antibodies, under suitable conditions, with a sample suspected of containing PD-1 , thereby allowing the formation of a complex between the humanized anti-PD1 antibodies and PD-1 , and detecting the complex by a suitable signal. The detectable signal can be caused by a label, as explained above, or by a change of physical properties due to the binding, i.e., the complex formation, itself. One example is surface plasmon resonance, the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold foil.

[00130] In another aspect, the present disclosure provdies a diagnostic or analytical kit comprising a humanized anti-PD1 antibody according to the disclosure.

[00131] In a particular aspect, provided humanized anti-PD1 antibodies disclosed herein are used in diagnosing a PD-1 -mediated adaptive immune resistance in a patient who has cancer. Such use comprises contacting a tumor microenvironment in the patient with an antibody disclosed herein that has been labeled with a detectable moiety and detecting expression of PD-1 on immune cells, e.g., CD⁸⁺ T cells, B cells, and macrophages, within the tumor microenvironment. Such use may further comprise an agent for detecting expression of PD-L1 on immune cells within the tumor microenvironment.

[00132] Adaptive immune resistance includes suppression of a host immune response as a result of activation of a PD-1 signaling pathway in immune cells of the host. For example, cancer tissue suppresses a host immune response by upregulation of PD-L1 and its binding to PD-1 on immune cells on T cells (such as CD⁸⁺ T cells), B cells, and macrophages.

[00133] In some embodiments, diagnostic uses of provided anti-PD-1 antibodies can be performed in vivo, or on a biopsy sample from a patient, wherein the tumor microenvironment is present in a tumor biopsy.

[00134] Modifications of antibodies for diagnostic uses are well known in the art. For example, antibodies may be modified with a ligand group such as biotin, or a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme. Antibodies of the invention can be labeled using conventional techniques. Suitable detectable labels include: fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes typically are detected by their reaction products. For example, horseradish peroxidase can be detected through conversion of tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. For detection, suitable binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art.

[00135] In addition to their use in diagnostics, in yet another aspect, the disclosure contemplates pharmaceutical composition comprising one or more provided humanized anti- PD1 antibodies and a pharmaceutically acceptable excipient. In some embodiments, a provided pharmaceutical composition comprising one or more humanized anti-PD1 antibodies of the disclosure has satisfactory or improved safety profile. In some further embodiments, the satisfactory or improved safety profile results from the the high specificity or reduced or diminished off-target binding of provided anti-PD1 antibodies of the disclosure.

[00136] Accordingly, the present disclosure encompasses the recognition that a provided anti-PD-1 antibody or a fragment thereof may be used in methods for reactivating an exhausted T cell, comprising contacting a population of T cells with such an antibody or a fragment thereof in vitro, ex vivo, or in vivo.

[00137] Furthermore, the present disclosure provides humanized antibodies or fragments thereof that bind PD-1 for use as anti-infection and/or anti-cancer agents and immune modulators. In some embodiments, humanized antibodies of the present disclosure are envisaged to be used in a method of treatment or prevention of human diseases, such as a variety of tumors and autoinflammation in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of one or more humanized anti-PD1 antibodies of the disclosure.

[00138] Examples of cancers that may be treated using provided antibodies or fragments of the disclosure, include liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular 20 cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of 25 childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies 30 including, for example, multiple myeloma, B cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non- Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers. In some embodiments, provided methods, antibodies and compositions are also applicable to the treatment of metastatic cancers.

[00139] In some embodiments, a human patient suffers from non-small cell lung cancer (NSCLC) or virally-related cancer (e.g., a human papillomavirus (HPV)-related tumor) or gastric adenocarcinoma. In some particular embodiment, an HPV-related tumor is HPV+ head and neck cancer (HNC). In another particular embodiment, gastric adenocarcinoma is associated with Epstein-Barr virus (EBV) infection. [00140] As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the subjects, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[00141] In some embodiments, when used to treat human diseases, anti-PD-1 antibodies of the disclosure can be used alone or in combination with one or more other therapeutic agent. When used together with another therapeutic agent, a provided antibody can be linked to, e.g., conjugated to or fused to, an agent (as a complex or fusion protein) or in combination with an agent. Examples of other therapeutic agents include other checkpoint inhibitors, immunogenic agents, attenuated cancerous cells, tumor antigens (e.g., recombinant proteins, peptides, and carbohydrate molecules), antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., JL-2, IFNa2, GM-CSF), and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF); chemotherapy, radiotherapy, surgery and any combination thereof.

[00142] In some embodiments, one or more anti-PD-1 antibodies of the disclosure are administered in combination with one or more additional checkpoint inhibitors. In some embodiments, an additional checkpoint inhibitor can be targeted against PD-1 or against a different checkpoint molecule, e.g., TIM3, CEACAMI, TIGIT, LAG-3, VISTA, and 0X40. In some embodiments, an additionalcheckpoint inhibitor can be a small molecule or a monoclonal antibody.

[00143] In some embodiments, one or more anti-PD-1 antibodies of the disclosure are administered with one or more additional therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. In some embodiments, a provided antibody can be linked to, e.g., conjugated to or fused to, the agent (as a complex or fusion protein) or administered separately. In some embodiments, an additional therapeutic agent is or comprises an immunomodulatory agent or an anti-cancer agent (e.g., a chemotherapeutic agent). In separate administration, an antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies.

[00144] In some other embodiments, an antibody disclosed herein is used as a targeting agent for delivery of a payload, e.g., a toxin, to a cell expressing PD-1. In some embodiments, such use may include administering a provided anti-PD-1 antibody conjugated to a payload moiety. Suitable conjugation methods are known in the art.

[00145] In some further embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof of the disclosure may be fused or conjugated to a moiety that may confer new characteristics to the antibody, such as enzymatic activity or binding affinity for other targets. Examples of suitable moiety include radionuclides, toxins, organic molecules, nucleic acids, therapeutic peptides, antibodies or antibody fragments, and lipocalin muteins (Anticalin®). The resulting fusion proteins or conjugates can be produced by methods well known in the art.

[00146] In some still further embodiments, an anti-PD-1 antibody or antigen-binding fragment thereof of the disclosure may be included in a bi-, tri-, or multi-specific antibody or an antibody-drug conjugate. Accordingly, the present disclosure encompasses bi-, tri-, or multi- specific antibodies and antibody-drug conjugates comprising a provided anti-PD-1 antibody or antigen-binding fragment thereof.

[00147] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present disclosure specifically discloses certain exemplary embodiments and optional features, modification and variation of the disclosures embodied herein may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this disclosure.

V. EXAMPLES

[00148] Example 1 : Exemplary humanized anti-PD-1 antibodies and Fc- engineered humanized anti-PD-1 antibodies

[00149] To humanize the heavy chain variable domain of 388D4 antibody, a murine antibody specific for PD-1 , CDR-grafting technologies coupled with the latest research on antibody structure and up-to-date database of mature human IgG sequences were utilized.

[00150] Online databases of Human IgG sequences were searched for comparison to the murine 388D4 heavy chain variable domain using BLAST search algorithms, and candidate human variable domains were selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of framework homology, maintaining key framework residues and canonical loop structure. Only one of these candidates was chosen as the others were very similar to the previous humanization attempt. The consensus of the top 200 results from the BLAST search served as another framework. Some modifications were involved to maintain key residues and structure during CDR-grafting.

[00151] Frameworks were derived from different human germlines including IGHV1- 3^*01 (SEQ ID NO: 392), IGHV7-4-1^*02 (SEQ ID NO: 393), IGHV4-4^*01 (SEQ ID NO: 394), IGHV5-10-1^*01 (SEQ ID NO: 395), and IGHV3-1 1^*03 (SEQ ID NO: 396), with some key murine residues substituted back in order to maintain the structure.

[00152] Similar approaches were taken to humanize the light chain variable domain of 388D4 antibodies. Online databases of Human IgK sequences were searched for comparison to the murine 388D4 light chain variable domain using BLAST search algorithms, and candidate human variable domains selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of framework homology, maintaining key framework residues and canonical loop structure, and two of these candidates were chosen. The consensus of the top 200 results from the BLAST search served as another framework with some modifications. Frameworks were derived from different human germlines including IGKV2-29^*02 (SEQ ID NO: 397), IGKV2-30^*02 (SEQ ID NO: 398), IGKV4-1^*01 (SEQ ID NO: 399), IGKV3-1 1^*01 (SEQ ID NO: 400), IGKV1 -6^*01 (SEQ ID NO: 401 ), and IGKV3-20^*01 (SEQ ID NO: 402), with some key murine residues substituted back in to maintain the structure.

[00153] As quality controls, all humanized variants were checked to determine whether they had been humanized in accordance with WHO’s definition of humanized antibodies and analyzed for potential T-cell epitopes that may lead to immunogenic response. The sequences of the humanized variants were also analyzed for post-translational modifications including Fv glycosylation and deamidation. DNASTAR NovaFold, a protein structure prediction software, was then used to model the binding sites formed by a pair of humanized heavy chain and light chain variable domains and compare to the murine 388D4 variable domains.

[00154] All antibodies described here had an engineered lgG4 backbone, which contained a S228P mutation to minimize lgG4 half-antibody exchange in-vitro and in-vivo (Silva et al., J Biol Chem, 2015). One or more additional mutations in the lgG4 backbones may exist in all antibodies described here, including F234A and L235A mutations for decreased ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010) and/or M428L and N434S mutations or M252Y, S254T, and T256E mutations for extended serum half-life (Dall'Acqua et al., J Biol Chem, 2006, Zalevsky et al., Nat Biotechnol, 2010).

[00155] Example 2: Expression and analysis of humanized anti-PD-1 antibodies

[00156] In this Example, certain provided humanized 388D4 antibodies were generated by gene synthesis and cloned into a mammalian expression vector. They were then transiently expressed in Expi293F™ cells (Life Technologies). The concentration of antibodies in the cell culture medium was measured by HPLC (Agilent Technologies) employing a POROS® protein A affinity column (Applied Biosystems).

[00157] Antibodies were purified using Protein A chromatography followed by size- exclusion chromatography (SEC) in phosphate-buffered saline (PBS). After SEC purification, the fractions containing monomeric protein are pooled and analyzed again using analytical SEC.

[00158] The expression profiles of exemplary antibodies are described in Table 1. Comparable expression titers were obtained for all humanized 388D4 antibodies. However, the monomer contents of the humanized antibodies in this Example have been improved over the certain previously known humanized antibody SEQ ID NOs: 4 and 9 and their calculated isoelectric points (pis) have been increased.

[00159] Table 1 - Expression titers

[00160] Example 3: Affinity of humanized antibodies binding to human and cynomolgus PD-1 determined by surface plasmon resonance (SPR)

[00161] The binding of exemplary humanized 388D4 antibodies to recombinant human and cynomolgus PD-1 -His (PD-1 with a C-terminal polyhistidine tag, ACROBiosystems) was determined by Surface Plasmon Resonance (SPR) using a Biacore T200 or 8K instrument (GE Healthcare).

[00162] The anti-human IgG Fc antibody (GE Healthcare) was immobilized on a CM5 sensor chip using standard amine chemistry: the carboxyl groups on the chip were activated using 1 -ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDO) and N-hydroxysuccinimide (NHS). Subsequently, anti-human IgG Fc antibody solution (GE Healthcare) at a concentration of 25 pg/mL in 10 mM sodium acetate (pH 5) was applied at a flow rate of 5 pL/min until an immobilization level of 9000-14000 resonance units (RU) was achieved. Residual non-reacted NHS-esters were blocked by passing a solution of 1 M ethanolamine across the surface. The reference channel was treated in an analogous manner. Subsequently, test antibodies at 0.25 pg/mL in HBS-EP+ (pH 7.4) buffer was captured by the anti-human IgG-Fc antibody at the chip surface for 180 s at a flow rate of 10 pL/min.

[00163] For affinity determination, dilutions of human and cynomolgus PD-1 -His between 400 nM and 6.25 nM were prepared in HBS-EP+ buffer and applied to the prepared chip surface with a contact time of 180 s and a dissociation time of 900 s at flow rate of 30 pL/min. All measurements were performed at 25°C. Regeneration of the chip surface was achieved with injections of 3 M MgCI₂ for 60 s and 10 mM glycine-HCI (pH 1 .7) for 180 s at a flow rate of 10 pL/min followed by an extra wash with running buffer (HBS-EP+ buffer) and a stabilization period of 120 s. Prior to the protein measurements, three startup cycles were performed for conditioning purposes. Data were evaluated with Biacore T200 or 8K Evaluation software (v2.0/v1.1 ). Double referencing was used and the 1 :1 binding model was used to fit the raw data.

[00164] The values determined for k_on, k_off and the resulting equilibrium dissociation constant ( K_d ) for a previously known humanized 388D4 (SEQ ID NOs: 4 and 9) and exemplary humanized 388D4 provided herein are summarized in Table 2. All provided humanized 388D4 bind human as well as cynomolgus PD-1 with low nanomolar affinities and such affinities show up to 7-fold improvement as compared to the certain previously known humanized 388D4 SEQ ID NOs: 4 and 9.

[00165] Table 2 - Kinetic constants and affinities of PD-1 specific antibodies determined by surface-plasmon-resonance (SPR).

[00166] Example 4: Binding of humanized antibodies toward human and cynomolgus PD-1 in enzyme-linked immunosorbent assay (ELISA)

[00167] An enzyme-linked immunosorbent assay (ELISA) was employed to determine the binding potency of exemplary humanized 388D4 antibodies from Example 3 to recombinant human and cynomolgus PD-1 -His (PD-1 with a C-terminal polyhistidine tag). PD-1 -His at the concentration of 1 pg/mL in PBS was coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1 %T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 h at room temperature. After washing with 100 pl_ PBS-0.05%T five times, PD-1 specific antibodies at different concentrations were added to the wells and incubated for 1 h at room temperature, followed by another wash step. Bound antibodies under study were detected by incubation with 1 :5000 diluted anti-human IgG Fc-HRP (Jackson Laboratory) in PBS-0.1 %T-2%BSA. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Pierce) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

[00168] The results of the experiment are shown in Figure 1 , together with the fit curves resulting from a 1 :1 binding sigmoidal fit, where the EC₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The resulting EC₅₀ values are provided in Table 3. The observed EC₅₀ values for all tested antibodies were in the sub- nanomolar range and comparable to the certain previously known humanized antibody SEQ ID NOs: 4 and 9.

[00169] Table 3 - ELISA data for PD-1 binding

[00170] Example 5: Binding of humanized antibodies toward human and cynomolgus PD-1 analyzed by flow cytometry

[00171] Target specific binding of humanized 388D4 antibodies to human and cynomolgus PD-1 -expressing cells was assessed by flow cytometry.

[00172] Chinese hamster ovary (CHO) cells were stably transfected with human PD-1 , cynomolgus PD-1 , or a mock control using the Flp-ln system (Invitrogen) according to the manufacturer^'s instructions. Transfected CHO cells were maintained in Ham^'s F12 medium (Invitrogen) supplemented with 10% Fetal Calf Serum (FCS, Biochrom) and 500 pg/ml Hygromycin B (Roth). Cells were cultured in cell culture flasks according to manufacturer’s instruction (37°C, 5% C02 atmosphere).

[00173] For flow cytometric analysis, respective cell lines were incubated with the PD- 1 specific antibodies and detected using a fluorescently labeled anti-human IgG antibody as described in the following: 5^c 10⁴ cells per well were incubated for 1 h in ice-cold PBS containing 5% fetal calf serum (PBS-FCS). Subsequently, a dilution series of humanized 388D4 antibodies were added to the cells and incubated on ice for 1 h. Cells were washed twice with PBS and then incubated with a goat anti-hlgG Alexa647-labeled antibody for 30 min on ice. Cells were subsequently washed and analyzed using iQue Flow cytometer (Intellicyte Screener). Mean geometric fluorescent signals were plotted and fitted using Graphpad software using nonlinear regression (shared bottom, SLOPE =1 ).

[00174] The ability of exemplary humanized 388D4 antibodies to bind human and cynomolgus PD-1 is depicted in Figure 2. Binding affinities (EC₅₀s) to PD-1 expressing cells of provided humanized 388D4 antibodies of the present disclosure are in the single digit nanomolar range, comparable to the certain previously known humanized antibody SEQ ID NOs: 4 and 9 (summarized in Table 4).

[00175] Table 4 - Binding affinities of the humanized 388D4 antibodies to PD-1 expressing cells

[00176] Example 6: ELISA analysis of competitive binding of humanized anti-PD- 1 antibodies with PD-L1 and PD-L2

[00177] An ELISA-based competitive assay was employed to analyze the inhibition of the interaction of PD-1 with PD-L1 and PD-L2 by the humanized 388D4 antibodies. Recombinant human PD-L1-Fc (R&D) or recombinant human PD-L2-Fc (R&D) at a concentration of 1 pg/mL in PBS was coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1 %T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 h at room temperature. After washing with 100 pl_ PBS-0.05%T five times, a mixture of biotinylated human PD-1-Fc (R&D, in-house biotinylation via primary amines) at a constant concentration of 20 nM and PD-1 specific antibodies at different concentrations were added to the wells and incubated for 20 min at room temperature. This mixture of human PD-1 and PD-1 specific antibodies had previously been incubated at room temperature for 1 h. Another wash step followed. Biotinylated PD-1 that still bound to PD-L1/PD-L2 after previous incubation with antibodies, i.e.,“free PD-1”, was detected by incubation with 1 :5000 diluted Extravidin-HRP (Sigma-Aldrich) in PBS-0.1 %T-2%BSA for 1 h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Pierce) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

[00178] In addition, calibration curves for the quantification of free PD-1 were prepared by applying different concentrations of biotinylated human PD-1-Fc alone to the same assays. The concentration of free (non-neutralized) PD-1 after incubation with PD-1 specific antibodies was calculated from these calibration curves.

[00179] The results are shown in Figure 3. The data were analyzed by using a 1 :1 sigmoidal binding fit, where the IC₅₀ value and the maximum signal were free parameters, and the slope was fixed to one. The resulting IC₅₀ values are provided in Table 5. Provided humanized 388D4 (SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 1 15, and SEQ ID NOs: 109 and 116) all clearly inhibited the interaction between PD-1 and PD-L1 and PD-L2, in a similar manner to the certain previously known humanized antibody SEQ ID NOs: 4 and 9.

[00180] Table 5 - PD-1 specific antibodies compete with binding of PD-1 to its ligand PD-L1 and PDL-2.

[00181] Example 7: Off-target binding of humanized antibodies analyzed by ELISA [00182] An ELISA-based assay was employed to assess the off-target binding of humanized antibodies toward 32 different targets including PD-1 , cell surface receptor, and cytokine. Targets at the concentration of 5 pg/mL in PBS were coated overnight on microtiter plates at 4°C. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1%T (PBS supplemented with 0.1 % (v/v) Tween 20) for 1 h at room temperature. After washing with 100 mI_ PBS-0.05%T five times, testing antibodies at 100 nM or 10 nM were added to the wells and incubated for 1 h at room temperature, followed by another wash step. Bound antibodies under study were detected by incubation with 1 :5000 diluted F(ab')2 Fragment goat anti-human IgG F(ab')2 HRP (Jackson Laboratory) in PBS-0.1 %T-2%BSA. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and incubated for 1 h. The fluorescence intensity was detected using a fluorescence microplate reader and normalized to the signal of a previously know humanized 388D4 anti-PD-1 antibody (SEQ ID NOs: 4 and 9). The normalized signals of each testing antibody binding to the 32 targets were summed up to yield the cumulated binding ratio for the antibody at a given concentration, i.e., 100 nM or 10 nM. The cumulated binding ratios at 100 nM and 10 nM for each testing antibody were further summed to yield the sum of cumulated binding ratios. For example, the previously know humanized 388D4 anti-PD-1 antibody (SEQ ID NOs: 4 and 9), to which the fluorescence intensity resulting from binding to each of the 32 targets was normalized, has a cumulated binding ratio of 32 when tested at either 100 nM or 10 nM and a sum of cumulated binding ratios of 64. The results are shown in Table 6. Higher the cumulated binding ratio correlates to stronger off-target binding.

[00183] Certain previously known humanized 388D4 (SEQ ID NOs: 4 and 9) bind strongly to additional targets including cell surface receptor, cytokine, and cytokine mediator. Such undesired off-target binding has been significantly reduced or eliminated in the humanized 388D4 provided herein (SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 1 15, and SEQ ID NOs: 109 and 116).

[00184] Table 6 - Off-target binding of PD-1 specific antibodies

[00185] Example 8: Off-target binding of PD-1 specific antibodies analyzed by fluorescence-activated cell sorting (FACS)

[00186] Fluorescence-activated cell sorting (FACS) studies were employed in order to assess the off-target or non-specific binding of PD-1 specific antibodies to human endothelial cells (HUVEC) that are PD-1 negative.

[00187] To perform the experiment, HUVEC at a confluency of 80% were detached using a trypsin solution (Promocell) following the vendor guidelines and counted. 2.5 x10⁴ HUVEC per well were added to each well of a 384 well plate for FACS (V bottom). Cells were then washed twice with PBS and live-death marker (Invitrogen) was added in a 1/500 dilution to each well. The cells were then incubated with humanized 388D4 antibodies or a human lgG4 control antibody (all at 1000 nM or 100 nM) at 4°C for 30 minutes followed by two washing steps using PBS. An anti-human IgG secondary antibody (Life technologies) was then added to each well, followed by a 20 minutes incubation at at 4°C. After two final washing steps, the plate was inserted into the Intellicyt and measurement was performed. Raw data were then analysed using the Forecyt software allowing to extract Geomean of fluorescence.

[00188] Exemplary data are shown in Figure 4. The certain previously known humanized antibody SEQ ID NOs: 4 and 9 showed signiticant adhersion to HUVEC cells, while such non-specific binding to HUVEC cells were largely diminished in the currently provided humanized antibodies to the same level as the controls.

[00189] Example 9: Pharmacokinetics of humanized antibodies in mice

[00190] An analysis of the pharmacokinetics of exemplary PD-1 specific antibodies was performed in mice. Male CD-1 mice approximately 5 weeks of age (2 mice per timepoint; Charles River Laboratories, Research Models and Services, Germany GmbH) were injected into a tail vein with a PD-1 specific antibody at a dose of 2 mg/kg. Plasma samples from the mice were obtained at the timepoints of 5 min, 24 h, 168 h, and 336 h. Sufficient whole blood - taken under isoflurane anaesthesia - was collected to obtain at least 30-50 mI_ Li-Heparin plasma per animal and time.

[00191] Plasma drug levels were then analyzed with ELISA. Human PD-1-His was dissolved in PBS (1 pg/mL) and coated overnight on microtiter plates at 4°C. The plate was washed after each incubation step with 80 pL PBS supplemented with 0.05% (v/v) Tween 20 five times. The plates were blocked with PBS/BSA/Tween (PBS containing 2% BSA (w/v) and 0.1 % (v/v) Tween 20) for 1 h at room temperature and subsequently washed. Plasma samples were diluted in PBS/BSA/Tween to 20% plasma concentration, added to the wells, and incubated for 1 h at room temperature. Another wash step followed. Bound agents under study were detected after 1 h incubation with SULFO-TAG Anti-Human Antibody (Mesoscale Discovery) at 1 pg/mL diluted in PBS containing 2% BSA (w/v) and 0.1 % (v/v) Tween 20. After an additional wash step, 25 pL reading buffer was added to each well and the electrochemiluminescence (ECL) signal of every well was read using a Mesoscale Discovery reader. Data were transferred to Excel for analysis and quantification. A calibration curve with standard protein dilutions was prepared.

[00192] Figure 5 shows plots of the plasma concentration over time for exemplary previously known humanized 388D4 (SEQ ID NOs: 4 and 9 and SEQ ID NOs: 6 and 7), selected presently provided humanized 388D4. Selected humanized 388D4 (SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 1 15, SEQ ID NOs: 109 and 1 16, SEQ ID NOs: 61 and 9, SEQ ID NOs:4 and 75, SEQ ID NOs: 4 and 78, SEQ ID NOs: 4 and 82, SEQ ID NOs: 4 and 83, SEQ ID NOs: 4 and 98, SEQ ID NOs: 58 and 75, SEQ ID NOs: 58 and 96, SEQ ID NOs: 72 and 108, SEQ ID NOs: 73 and 108, SEQ ID NOs: 4 and 406, and SEQ ID NOs: 405 and 108) described herein display comparable pharmacokinetic profiles as the previously known humanized antibody SEQ ID NOs: 4 and 9 and typical antibody pharmacokinetic profiles. As described herein, a favorable pharmacokinetic profile or an antibody-like pharmacokinetics may be considered to be achieved for provided antibodies if % of c_max was above 15 % after 168 h.

[00193] Example 10: Assessment of T cell activation using a PD-1/PD-L1 blockade bioassay

[00194] The potential of selected humanized 388D4 antibodies to block the inhibitory signal through PD-1 via PD-L1 was assessed using PD-1-NFAT-luc Jurkat T cells (a Jurkat cell line expressing PD-1 with a luc gene (firefly luciferase gene) driven by a NFAT- responsive element) co-cultured with Raji cells over-expressing PD-L1. PD-1/PD-L1 interaction between both cell types inhibits TCR signaling and NFAT-mediated luciferase activity in PD-1-NFAT-luc Jurkat T cells. Addition of a PD-1/PD-L1 blocking agent, such as humanized 388D4, results in releasing PD-1/PD-L1 block on NFAT-mediated luminescence.

[00195] Raji cells over-expressing PD-L1 were grown under standard conditions, and were plated at 1.25 x 10⁴ cells per well and allowed to adhere overnight at 37°C in a humidified 5% C0₂ atmosphere. On the next day, plates were washed with culture media, various concentration of testing antibodies were added to each well, followed by the addition of 3.75 x 10⁴ PD-1 -NFAT-luc Jurkat T cells and Ix CytoStim. Plates were covered with a gas permeable seal and incubated at 37°C in a humidified 5% C0₂ atmosphere. After 4h, 30 mI_ Bio-Glo™ Reagent was added to each well and the bioluminescent signal was quantified using a luminometer (PHERAstar). Four-parameter logistic curve analysis was performed with GraphPad Prism® to calculate EC₅₀ values (shared bottom, fixed slope), which are summarized in Table 7. The assay was performed in triplicates.

[00196] The results of a representative experiment for humanized 388D4 antibodies are depicted in Figure 6. The data demonstrate that all tested antibodies inhibit PD-1/PD-L1 block leading to T-cell activation.

[00197] Table 7 - Assessment of T cell activation using a PD-1/PD-L1 blockade bioassay.

[00198] Example 11 : Assessment of T cell activation using human peripheral blood mononuclear cells (PBMCs)

[00199] A T cell assay was employed to assess the ability of exemplary humanized 388D4 to revert the inhibitory signaling of the negative checkpoint molecule PD-1 by blocking the interaction between PD-1 and its ligands. For this purpose, antibodies at different concentrations were added to staphylococcal enterotoxin B (SEB) stimulated human peripheral blood mononuclear cells (PBMCs) and incubated for 4 days at 37°C. As a readout, secreted IL-2 levels in the supernatants were assessed. [00200] PBMCs from healthy volunteer donors were isolated from buffy coats by centrifugation through a polysucrose density gradient (Biocoll, 1.077 g/mL, Biochrom), following Biochrom^'s protocols. The purified PBMCs were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down using liquid nitrogen and stored in liquid nitrogen until further use. For the assay, PBMCs were thawed and rested for 16 h in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% Penicillin-Streptomycin (Life Technologies).

[00201] The following procedure was performed using triplicates for each experimental condition.

[00202] 2.5x10⁴ PBMCs were incubated in each well of a 384 well flat-bottom tissue culture plates in culture media supplemented or not with SEB at 0.1 ng/mL. Subsequently, a dilution series of the humanized antibodies, benchmark PD-1 antibody, and negative controls typically ranging from 100 to 0.001 nM were added to the cells. Plates were covered with a gas permeable seal and incubated at 37°C in a humidified 5% C0₂ atmosphere for four days. Subsequently, IL-2 levels in the supernatant were assessed.

[00203] Human IL-2 in the cell culture supernatants were quantified using the IL-2 DuoSet kit from R&D Systems.

[00204] The following procedure describes the IL-2 quantification.

[00205] In the first step, a 384 well plate was coated at room temperature for 2 h with 1 pg/mL“Human IL-2 Capture Antibody” (R&D Systems) in PBS. Subsequently, wells were washed 5 times with 80 pi PBS-0.05%T. After 1 h blocking in PBS-0.05%T additionally containing 1% casein (w/w), pooled supernatants and a concentration series of an IL-2 standard diluted in culture medium was incubated in the 384-well plate overnight at 4°C. To allow for detection and quantitation of captured IL-2, a mixture of 100 ng/mL goat anti-hlL-2- Bio detection antibody (R&D Systems) and 1 pg/mL Sulfotag-labelled streptavidin (Mesoscale Discovery) in PBS-T containing 0.5% casein were added and incubated at room temperature for 1 h. After washing, 25 pL reading buffer was added to each well, and the electrochemiluminescence (ECL) signal of every well was read using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[00206] The result of a representative experiment is depicted in Figure 7. It shows the dose-dependent increase of IL-2 secretion level induced by humanized 388D4 antibodies, comparable to the certain previously known humanized antibody SEQ ID NOs: 4 and 9.

[00207] Example 12: Tumor growth inhibition by humanized antibodies in mouse tumor model

[00208] In order to investigate the activity of selected PD-1 specific antibody SEQ ID NOs: 109 and 115 in an in vivo mouse model, we employed immune deficient NOG mice (Taconic, NOD/Shi-scid/IL-2Rynull) engrafted with human HCC827 tumor cells and human PBMC.

[00209] 4-6 week-old NOG mice were subcutaneously (s.c.) injected with 5^c 10⁶

HCC827 cells in a matrigel/PBS (1 :1 ) solution. Tumors were allowed to grow for 10 days and on day 0 of the experiment mice were randomized into treatment groups according to tumor size and animal weight. Mice were given 5^c 10⁶ fresh human PBMC intravenously (i.v.) into a tail vein. Mice received treatment or control into the intraperitoneal cavity after PBMC injection on day 1 , and again on day 4, day 8, and day 12. Tumor growth was recorded every 3-4 days.

[00210] Figure 8 compares the changes in tumor volumes on day 4, day 8, day 11 , and day 14 of the study. Tumor growth inhibition was achieved by the PD-1-specific antibody SEQ ID NOs: 109 and 1 15.

[00211] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms“comprising,”“including,”“containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed, for example, by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this invention. All patents, patent applications, textbooks, and peer- reviewed publications described herein are hereby incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will become apparent from the following claims.

[00212] Equivalents: those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

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Claims

1. A humanized antibody that binds to programmed cell death protein 1 (PD-1 ) or an antigen binding fragment thereof, comprising:

(a) a heavy chain variable domain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 144, 195, 119-143, 145-159, and 196-199; and

(b) a light chain variable domain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 168, 169, 201 , 202, 160-167, 170-194, 200, 203, 204, and 407.

2. A humanized antibody that binds to programmed cell death protein 1 (PD-1 ) or an antigen binding fragment thereof, comprising:

(a) a heavy chain variable domain having complementary determining regions of sequences GYTFTDYE (HCDR1 , SEQ ID NO: 28), IDPGTGGT (HCDR2, SEQ ID NO: 29), and TSEKFGSNYYFDY (HCDR3, SEQ ID NO: 30); and

(b) a light chain variable domain having complementary determining regions of sequences QTIVHSDGNTY (LCDR1 , SEQ ID NO: 31 ), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 33);

wherein the antibody binds PD-1 with an affinity measured by K_d of about 20 nM or lower.

3. A humanized antibody that binds to programmed cell death protein 1 (PD-1 ) or an antigen binding fragment thereof, comprising:

(a) a heavy chain variable domain having complementary determining regions of GYTFTDYE (HCDR1 , SEQ ID NO: 28), IDPGTGGT (HCDR2, SEQ ID NO: 29), and TSEKFGSNYYFDY (HCDR3, SEQ ID NO: 30); and

(b) a light chain variable domain having complementary determining regions of sequences QTIVHSDGNTY (LCDR1 , SEQ ID NO: 31 ), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 33);

wherein the antibody binds PD-1 with reduced off-target binding than an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9.

4. A humanized antibody that binds to programmed cell death protein 1 (PD-1 ) or an antigen binding fragment thereof, comprising: (a) a heavy chain variable domain having complementary determining regions of sequences GYTFTDYE (HCDR1 , SEQ ID NO: 28), IDPGTGGT (HCDR2, SEQ ID NO: 29), and TSEKFGSNYYFDY (HCDR3, SEQ ID NO: 30); and

(b) a light chain variable domain having complementary determining regions of sequences QTIVHSDGNTY (LCDR1 , SEQ ID NO: 31 ), KVS (LCDR2), and FQGSHVPLT (LCDR3, SEQ ID NO: 33);

wherein the antibody comprises

(i) a heavy chain framework region derived from IGHV1-3^*01 (SEQ ID NO: 392) and a light chain framework region derived from IGKV2-29^*02 (SEQ ID NO: 397) or IGKV2-30^*02 (SEQ ID NO: 398);

(ii) a heavy chain framework region derived from IGHV1-3^*01 (SEQ ID NO: 392) or IGHV3-11^*03 (SEQ ID NO: 396) and a light chain framework region derived from IGKV2-29^*02 (SEQ ID NO: 397), IGKV2-30^*02 (SEQ ID NO: 398), or IGKV1-6^*01 (SEQ ID NO: 401 ); or

(ii) a heavy chain framework region derived from IGHV1-3^*01 (SEQ ID NO: 392), IGHV7-4-1^*02 (SEQ ID NO: 393), IGHV4-4^*01 (SEQ ID NO: 394), IGHV5-10-1^*01 (SEQ ID NO: 395), and IGHV3-11^*03 (SEQ ID NO: 396) and a light chain framework region derived from IGKV2-29^*02 (SEQ ID NO: 397), IGKV2-30^*02 (SEQ ID NO: 398), IGKV4-1^*01 (SEQ ID NO: 399), IGKV3-11^*01 (SEQ ID NO: 400), IGKV1-6^*01 (SEQ ID NO: 401 ), and IGKV3-20^*01 (SEQ ID NO: 402);

5. The antibody or fragment of any one of claims 1-4, wherein the antibody has reduced off- target binding as compared to an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9.

6. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment has reduced nonspecific binding to cells that do not express PD-1 compared to an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9.

7. The antibody or fragment of any one of claims 1-4, wherein the antibody has a comparable serum half-life as compared to an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9.

8. 3c. The antibody or fragment of any one of claims 1-4, wherein the antibody has an improved monomer content after expression and/or purification as compared to an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9.

9. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment binds human PD-1 with an affinity measured by K_d of about 20 nM, 15 nM, 5 nM, 3 nM, or lower.

10. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment is cross-reactive with cynomolgus PD-1.

1 1. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment binds cynomolgus PD-1 with an affinity measured by K_d of about 50 nM, 40 nM, 10 nM, or lower.

12. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment binds PD-1 with substantially the same or lower K_d as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 9.

13. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment binds PD-1 with substantially the same or lower K_d as an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 21.

14. The antibody or fragment of any one of claims 1-13, wherein the K_d values are determined by surface plasmon resonance as described in Example 3.

15. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment binds PD-1 with an EC₅₀ value of at most about 1 nM, 0.5 nM, 0.2 nM, 0.15 nM, 0.1 nM, or lower, wherein the EC₅₀ value is determined by by an enzyme-linked immunosorbent assay (ELISA) assay essentially as described in Example 4.

16. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment binds PD-1 expressed on a cell with an EC₅₀ value of at most about 8 nM, 5 nM, 2 nM, 1 nM, or lower, wherein the EC₅₀ value is determined by a fluorescence activated cell sorting (FACS) assay essentially as described in Example 5.

17. The antibody or fragment of any one of claims 1-16, wherein the antibody or fragment competes with PD-L1 for binding to PD-1.

18. The antibody or fragment of any one of claims 1-17, wherein the antibody or fragment is capable of blocking the inhibitory signal of PD-1.

19. The antibody or fragment of any one of claims 1-18, wherein the antibody or fragment is capable of re-activating T cell responses.

20. The antibody or fragment of any one of claims 1-19, wherein the antibody or fragment is capable of inducing enhanced IL-2 secretion.

21. The antibody or fragment of any one of claims 1-20, wherein the antibody or fragment is capable of inhibiting tumor growth in a subject.

22. The antibody or fragment of any one of claims 1-21 , wherein the antibody or fragment has a heavy chain variable domain and light chain variable domain pair comprising the amino acid sequences selected from the group consisting of SEQ ID NOs:144 and 168, SEQ ID NOs: 144 and 169, SEQ ID NOs: 195 and 201 , and SEQ ID NOs: 195 and 202.

23. The antibody or fragment of any one of claims 1-22, wherein the antibody or fragment has a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 33-73, 109-113 and 405, and/or a light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 74-108, 114- 118, and 406.

24. The antibody or fragment of any one of claims 1-23, wherein the antibody or fragment has a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 58 and 109, and/or a light chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 83, 115, and 116.

25. The antibody or fragment of any one of claims 1-24, wherein the antibody or fragment has a heavy chain and light chain pair comprising the amino acid sequences selected from the group consisting of SEQ ID NOs: 58 and 82, SEQ ID NOs: 58 and 83, SEQ ID NOs: 109 and 1 15, and SEQ ID NOs: 109 and 1 16.

26. The antibody or fragment of any one of claims 1-25, wherein the antibody is an IgG molecule.

27. The antibody or fragment of claim 26, wherein the antibody is an lgG1 or lgG4 molecule.

28. The antibody or fragment of claim 27, wherein the lgG4 backbone has the mutation S228P.

29. The antibody or fragment of claim 28, wherein the lgG4 backbone has one or more mutations selected from the group consisting of N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

30. The antibody or fragment of claim 27, wherein the lgG1 backbone has one or more mutations selected from the group consisting of N297A, L234A and L235A.

31. A pharmaceutical composition comprising the antibody or fragment of any one of claims 1-30 and a pharmaceutically acceptable excipient.

32. A method for re-activating T cell response, comprising administering an effective amount of one or more antibodies or fragments of any one of claims 1-30 or one or more compositions comprising such antibodies or fragments.

33. A method for inhibiting tumor growth in a subject, comprising administering an effective amount of one or more antibodies or fragments of any one of claims 1 -30 or one or more compositions comprising such antibodies or fragments.

34. A method for treating a subject having cancer, inflammatory diseases, or autoimmune diseases, comprising administering an effective amount of one or more antibodies or fragments of any one of claims 1-30 or one or more compositions comprising such antibodies or fragments.