Patent Description:
Increasing evidences from preclinical and clinical results have shown that targeting immune checkpoints is becoming the most promising approach to treat patients with cancers. The protein Programmed Death <NUM> (PD-<NUM>), an inhibitory member of the immunoglobulin super-family with homology to CD28, is expressed on activated B cells, T cells, and myeloid cells (Agata et al, supra; <NPL>;<NPL>) and has a critical role in regulating stimulatory and inhibitory signals in the immune system (<NPL>). PD-<NUM> was discovered through screening for differential expression in apoptotic cells (<NPL>).

The PD-<NUM> is a type I transmembrane protein that is part of the Ig gene superfamily (<NPL>) and the structure of PD-<NUM> consists of one immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Although structurally similar to CTLA-<NUM>, PD-<NUM> lacks the MYPPPY motif that is critical for B7-<NUM> and B7-<NUM> binding. PD-<NUM> has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), which are cell surface expressed members of the B7 family (<NPL>; <NPL>; <NPL>). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-<NUM>, but do not bind to other CD28 family members.

PD-<NUM>, as one of the immune-checkpoint proteins, is an inhibitory member of the CD28 family expressed on activated B cells, T cells, and myeloid cells (Agata et al, supra; <NPL>; <NPL>) plays a major role in limiting the activity of T cells that provide a major immune resistance mechanism by which tumor cells escaped immune surveillance. PD-<NUM> induces a state of anergy or unresponsiveness in T cells, resulting in the cells being unable to produce optimal levels of effector cytokines. PD-<NUM> may also induce apoptosis in T cells via its ability to inhibit survival signals. PD-<NUM> deficient animals develop various autoimmune phenotypes, including autoimmune cardiomyopathy and a lupus-like syndrome with arthritis and nephritis (<NPL>; <NPL>). Additionally, PD-<NUM> has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I diabetes, and rheumatoid arthritis (<NPL>: <NPL>; <NPL>). In a murine B cell tumor line, the ITSM of PD-<NUM> was shown to be essential to block BCR-mediated Ca<NUM>+-flux and tyrosine phosphorylation of downstream effector molecules (<NPL>).

The interaction of PD-<NUM> expressed on activated T cells, and PD-L1 expressed on tumor cells negatively regulates immune response and damps anti-tumor immunity. PD-L1 is abundant in a variety of human cancers (<NPL>). Expression of PD-L1 on tumors is correlated with reduced survival in esophageal, pancreatic and other types of cancers, highlighting this pathway as a new promising target for tumor immunotherapy. Several groups have shown that the PD-<NUM>-PD-L interaction exacerbates disease, resulting in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells (<NPL>; <NPL>; <NPL>). Immune suppression can be reversed by inhibiting the local interaction of PD-<NUM> with PD-L1, and the effect is additive when the interaction of PD-<NUM> with PD-L2 is blocked as well.

Anti-PD-<NUM> antibody nivolumab has been characterized in vitro and tested in non-human primates (<NPL>). Multiple agents targeting PD-<NUM> pathway have been developed by several pharmaceutical companies, such as Bristol-Myers Squibb (BMS), Merck, Roche and GlaxoSmithKline (GSK). Data from clinical trials demonstrated early evidence of durable clinical activity and an encouraging safety profile in patients with various tumor types. Nivolumab, an anti-PD-<NUM> drug developed by BMS, is being put at center stage of the next-generation field. Now in <NUM> late-stage studies, the treatment spurred tumor shrinkage in three out of five cancer groups studied, including <NUM>% of lung cancer patients (n=<NUM>), close to one third of melanoma patients (n=<NUM>) and <NUM>% of patients with kidney cancer (n=<NUM>). Developed by Merck, Pembrolizumab is a humanized monoclonal IgG4 antibody that acts against PD-<NUM>, which grabbed the FDA's new breakthrough designation after impressive IB data came through for skin cancer. The results from a phase IB study have shown an objective anti-tumor response in <NUM>% of the cancer patients (n=<NUM>), and a complete response in <NUM>% of the patients. Roche's experimental MPDL3280A (Atezolizumab) demonstrated an ability to shrink tumors in <NUM> of <NUM> (<NUM>%) advanced cancer patients with various tumor sizes.

Further anti-PD-<NUM> antibodies have been disclosed in <CIT>.

There are some spaces for improvement for antibody against PD-<NUM> as a therapeutic agent. Most of monoclonal antibodies against PD-<NUM> currently tested in clinical trials are only against to human PD-<NUM> which limits preclinical in vivo assay and diminished efficacy owing to the immunogenicity of the mouse-derived protein sequences. Humanized antibody with cross-reactivity to mouse PD-<NUM> overcome these shortages and showed more tolerability and higher efficiency in vivo. Thus there is still a need for novel anti-PD-<NUM> antibody.

The present invention provides isolated antibodies, in particular monoclonal antibodies.

The present invention provides an antibody or antigen binding fragment thereof that specifically binds to PD-<NUM>, wherein the antibody or antigen binding fragment thereof comprises:.

The aforesaid antibody or the antigen binding fragment thereof, wherein the PD-<NUM> is murine PD-<NUM> which is mouse or rat PD-<NUM>.

The aforesaid antibody or antigen binding fragment thereof, wherein the antibody.

The aforesaid antibody, wherein the antibody.

wherein the antibody exhibits at least one of the following properties:.

The present invention provides an antibody or an antigen binding fragment thereof, comprising:.

wherein the antibody specifically binds to PD-<NUM>;.

wherein the antibody specifically binds to PD-<NUM>;
or the antibody comprises:.

wherein the antibody specifically binds to PD-<NUM>.

The sequence of the said antibody is shown in Table <NUM> and Sequence Listing. Antibodies other than 1H6, 2E5, 2G4, and 2C2 do not form part of the present invention.

The antibody 1H6 which is an antibody according to the present invention comprises:.

The antibody 2E5 which is another antibody according to the present invention comprises:.

The antibody 2G4 which is another antibody according to the present invention comprises:.

The antibody 2C2 which is yet another antibody according to the present invention comprises:.

The CDR sequence of the said antibody is shown in Table <NUM> and Sequence Listing. Antibodies other than 1H6, 2E5, 2G4, and 2C2 are not part of the present invention.

The antibodies of the invention can be humanized antibody.

The antibodies of the invention can exhibit at least one of the following properties:.

In a further aspect, the invention provides a nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.

The invention provides a cloning or expression vector comprising the nucleic acid molecule encoding the antibody, or antigen binding fragment thereof.

The invention also provides a host cell comprising one or more cloning or expression vectors.

In yet another aspect, the invention provides a process, comprising culturing the host cell of the invention and isolating the antibody, wherein the antibody is prepared through immunization in SD rat with human PD-<NUM> extracellular domain and mouse PD-<NUM> extracellular domain.

The invention provides a transgenic mouse comprising human immunoglobulin heavy and light chain transgenes, wherein the mouse expresses the antibody of this invention.

The invention provides hybridoma prepared from the mouse of this invention, wherein the hybridoma produces said antibody.

In a further aspect, the invention provides pharmaceutical composition comprising the antibody, or the antigen binding fragment of said antibody in the invention, and one or more of a pharmaceutically acceptable excipient, diluent or carrier.

The invention provides an immunoconjugate comprising the said antibody, or antigen-binding fragment thereof in this invention, linked to a therapeutic agent.

Wherein, the invention provides a pharmaceutical composition comprising the said immunoconjugate and a pharmaceutically acceptable excipient, diluent or carrier.

Also disclosed but not forming part of the invention is a method for preparing an anti-PD-<NUM> antibody or an antigen-binding fragment thereof comprising:.

The invention also provides the antibody according to the present invention for use in a method for the treatment or prophylaxis of an immune disorder or cancer.

In one embodiment, the method is a method for the treatment or prophylaxis of cancer by inhibiting growth of tumor cells in a subject, said method comprising administering to the subject a therapeutically effective amount of the said antibody to inhibit growth of the tumor cells.

In one embodiment, the method is a method, wherein the tumor cells are of a cancer selected from a group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.

In one embodiment, the method is a method, wherein the antibody is a humanized antibody.

The inventors have generated a humanized antibody against PD-<NUM> utilizing the proprietary hybridoma technology. The antibodies reported in this invention have high binding affinity, specifically binding to both human and mouse PD-<NUM> protein without cross-family reactions; and potent modulating immune responses, including enhancing T cell proliferation and increasing cytokine IFN-γ and interleukin-<NUM> production.

New anti-PD-<NUM> antibodies binding to mouse PD-<NUM> are derived from immuned rats, which overcomes the disadvantage that is anti-PD-<NUM> antibodies can not be used in pre-clinical mouse model; and the humanized level is close to <NUM>% after sequence humanization, greatly reducing the adverse effects of drugs used in the human body.

Antibodies other than 1H6, 2E5, 2G4, and 2C2 do not form part of the present invention.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The terms "Programmed Death <NUM>", "Programmed Cell Death <NUM>", "Protein PD-<NUM>", "PD-<NUM>", "PD1", "PDCD1", "hPD-<NUM>" and "hPD-F" are used interchangeably, and include variants, isoforms, species homologs of human PD-<NUM>, and analogs having at least one common epitope with PD-<NUM>.

The term "antibody" as referred to herein includes whole antibodies and any antigen- binding fragment (i.e., "antigen-binding portion") or single chains thereof. An "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: 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.

The term "antibody," as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')<NUM>, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind PD-<NUM> specifically. Typically, such fragments would comprise an antigen-binding fragment.

The terms "antigen-binding fragment," "antigen-binding domain," and "binding fragment" refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding fragment may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding fragment is referred to as "epitope" or "antigenic determinant.

An antigen-binding fragment typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.

In line with the above the term "epitope" defines an antigenic determinant, which is specifically bound/identified by a binding fragment as defined above. The binding fragment may specifically bind to/interact with conformational or continuous epitopes, which are unique for the target structure, e.g. the human and murine PD-<NUM>. A conformational or discontinuous epitope is characterized for polypeptide antigens by the presence of two or more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen. The two or more discrete amino acid residues contributing to the epitope are present on separate sections of one or more polypeptide chain(s). These residues come together on the surface of the molecule when the polypeptide chain(s) fold(s) into a three-dimensional structure to constitute the epitope. In contrast, a continuous or linear epitope consists of two or more discrete amino acid residues, which are present in a single linear segment of a polypeptide chain.

The term "binds to an epitope of PD-<NUM>" refers to the antibodies have specific binding for a particular epitope of PD-<NUM>, which may be defined by a linear amino acid sequence, or by a tertiary, i.e., three-dimensional, conformation on part of the PD-<NUM> polypeptide. Binding means that the antibodies affinity for the portion of PD-<NUM> is substantially greater than their affinity for other related polypeptides. The term "substantially greater affinity" means that there is a measurable increase in the affinity for the portion of PD-<NUM> as compared with the affinity for other related polypeptides. Preferably, the affinity is at least <NUM>-fold, <NUM>-fold, <NUM>-fold <NUM>-fold, <NUM>-fold, <NUM><NUM>-fold, <NUM><NUM>-fold, <NUM><NUM>-fold, <NUM><NUM>-fold or greater for the particular portion of PD-<NUM> than for other proteins. Preferably, the binding affinity is determined by enzyme-linked immunoabsorbent assay (ELISA), or by fluorescence-activated cell sorting (FACS) analysis or surface Plasmon resonance (SPR). More preferably, the binding specificity is obtained by fluorescence-activated cell sorting (FACS) analysis.

The term "cross-reactivity" refers to binding of an antigen fragment described herein to the same target molecule in human and murine (mouse or rat). Thus, "cross-reactivity" is to be understood as an interspecies reactivity to the same molecule X expressed in different species, but not to a molecule other than X. Cross-species specificity of a monoclonal antibody recognizing e.g. human PD-<NUM>, to a murine (mouse or rat) PD-<NUM>, can be determined, for instance, by FACS analysis.

As used herein, the term "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms "patient" or "subject" are used interchangeably.

The terms "treatment" and "therapeutic method" refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.

DNAs encoding the ECD or full length of PD-<NUM> and PD-L1 were synthesized and inserted into the expression vector pcDNA3. Max-prep the plasmid DNAs and the inserted DNA sequences were verified by sequencing. Fusion proteins PD-<NUM> ECD and PD-L1 ECD containing various tags, including human Fc, mouse Fc and His tags, were obtained by transfection of human PD-<NUM> ECD gene into CHO-S or HEK293 cells. After <NUM> days, supernatants were harvested from the culture of transient transfected cells. The fusion proteins were purified and quantified for usage of immunization and screening.

In order to obtain tools for antibody screening and validation, we generated PD-<NUM> and PD-L1 transfecting cell lines. Briefly, CHO-K1 or 293F cells were transfected with pcDNA3. <NUM> expression vector containing full-length PD-<NUM> or PD-L1 using Lipofectamine <NUM> Transfection kit according to manufacturer's protocol. <NUM>-<NUM> hours post transfection; the transfected cells were cultured in medium containing Blasticidin or G418 to select the cells that had PD-<NUM> or PD-L1 genes stably incorporated into their genomic DNAs. Meanwhile the cells were checked for interested genes PD-<NUM> and PD-L1 expression. Once the expression verified, single clones of interested were picked by limited dilution and scaled up to large volumes. The established monoclonal cell lines were then maintained in medium containing lower dose of antibiotics Blasticidin or G418.

Female SD rats, at <NUM>-<NUM> weeks of age, were immunized with <NUM>µg/animal of human PD-<NUM> ECD protein and <NUM>µg/animal of mouse PD-<NUM> ECD protein in TiterMax by footpad injection for prime, and were boosted twice a week with human PD-<NUM> ECD protein or mouse PD-<NUM> ECD protein in Aluminium alternately. The serum antibody titers were measured by ELISA or FACS every two weeks.

When the serum antibody titer was sufficiently high, rats were given a final boost with both human and mouse PD-<NUM> ECD protein in the equal volume of D-PBS (Dulbecco's Phosphate Buffered Saline) without adjuvant. The cell fusion was performed as follows: preparing myeloma cells SP2/<NUM>, myeloma cells were thawed the week before the fusion, and were split at <NUM>:<NUM> each day until the day before the fusion to keep in logarithmic growth. B lymphocytes isolated from lymph node of immunized SD rats were combined with myeloma cells (at <NUM>:<NUM> ratio). The cells were treated with Trypsin and the reaction was stopped by FBS. Cell mixture was then washed and re-suspended in ECF solution at <NUM>×<NUM><NUM> cells/ml for ECF. After electronic cell fusion (BTX2000), cell suspension from the fusion chamber was immediately transferred into a sterile tube containing more medium, and incubated for at least <NUM> hours in a <NUM> incubator. The cell suspension was then mixed and transferred into <NUM>-well plates (<NUM>×<NUM><NUM> cells/well). The <NUM>-well plates were cultured at <NUM>, <NUM>% CO<NUM>, and were monitored periodically. When the clones were big enough (after <NUM>-<NUM> days), <NUM>µL of supernatant were transferred from the tissue culture plates to <NUM>-well assay plates for antibody screening.

ELISA assay was used as first screen method to test the binding of hybridoma supernatants to human or mouse PD-<NUM> protein. Briefly, plates (Nunc) were coated with human or mouse PD-<NUM> ECD at <NUM>µg/ml overnight at <NUM>. After blocking and washing, the hybridoma supernatants were loaded to the coated plates and incubated at room temperature for <NUM>. The plates were then washed and subsequently incubated with secondary antibody goat anti rat IgG Fc HRP (Bethyl) for <NUM>. After washing, TMB substrate was added and the reaction was stopped by <NUM> HCl. The absorbance at <NUM> was read using a microplate reader (Molecular Device).

In order to confirm the native binding of anti-PD-<NUM> antibodies on conformational PD-<NUM> molecules expressed on cell membrane, FACS analysis was performed using PD-<NUM> transfected cell lines as second screening. CHO-S cells expressing human PD-<NUM> or 293F cells expressing mouse PD-<NUM> were transferred into <NUM>-well U-bottom plates (Corning) at a density of <NUM>×<NUM><NUM> cells/well. The hybridoma supernatants were then added and incubated with the cells for <NUM> at <NUM>. After washing with 1xPBS/<NUM>%BSA, the secondary antibody goat anti rat FITC (Jackson ImmunoResearch Lab) was applied and incubated with cells at <NUM> in the dark for <NUM>. The cells were then washed and resuspended in <NUM>×PBS/<NUM>%BSA or fixed with <NUM>% paraformldehyde, and analyzed by flow cytometery (BD) and FlowJo software. Antibody binding to parental CHO-S or 293F cell line was used as negative control, respectively.

To select potential antagonistic hits, selected antibodies were tested for their ability to block the binding of the ligand PD-L1 to PD-<NUM> transfected cells by FACS analysis. CHO-S cells expressing human PD-<NUM> or 293F cells expressing mouse PD-<NUM> were transferred into <NUM>-well U-bottom plates (BD) at a density of <NUM>×<NUM><NUM> cells/well. Hybridoma supernatants were added and incubated with the cells at <NUM> for <NUM>. After washing, mouse Fc fusion-human PD-L1 protein or mouse Fc fusion-mouse PD-L1 protein was added and incubated at <NUM> for <NUM>. The secondary antibody goat anti mouse IgG Fc FITC antibody (no cross-reactivity to rat IgG Fc, Jackson ImmunoResearch Lab) was incubated with cells at <NUM> in the dark for <NUM>. The cells were then washed and resuspended in <NUM>×PBS/<NUM>%BSA or fixed with <NUM>% paraformldehyde, and analyzed by flow cytometery (BD) and FlowJo software.

<FIG> shows graphs of <NUM> hybridoma antibodies binding to cell surface human and mouse PD-<NUM>. <FIG> shows binding to human PD-<NUM>. <FIG> shows binding to mouse PD-<NUM>.

Once specific binding and blocking activity were verified through first and confirmation screening, the positive hybridoma cell lines were used for subcloning. Briefly, for each hybridoma cell line, cells were counted and diluted to give <NUM> cells, <NUM> cell or <NUM> cell per <NUM>µL cloning medium. The cell suspension was plated <NUM>µL/well into <NUM>-well plates, one plate at <NUM> cells/well, one plate at <NUM> cell/well and four plates at <NUM> cell/well. Plates were cultured at <NUM>, <NUM>% CO<NUM>, till they were ready to be screened by binding ELISA or FACS as described above. The ESN of selected single clones were collected, and the antibodies were purified for further characterization.

<NUM>µL of goat anti-rat IgG1, IgG2a, IgG2b, IgG2c, IgG or IgM antibodies (<NUM>µg/mL) were coated in microtiter plates (Nunc) per well overnight. After blocking, <NUM>µL of hybridoma supernatant samples were added to each well, incubated for <NUM> hours at room temperature. Goat anti-rat IgG kappa or HRP labeled lambda light chain secondary antibody (Bethyl) is a detection antibody. Using TMB substrate for color, the reaction was then quenched with <NUM> HCl. The value of absorbs light at <NUM> is read using a microplate reader (Molecular Device).

Table <NUM> shows the subtype results of <NUM> hybridoma antibodies. <NUM> antibodies are polyclonal antibodies, and <NUM> antibodies are IgG2a kappa subtype. Considering the needs of anti-PD-<NUM> antibody to avoid the role of ADCC and CDC in vivo, the humanized antibody will be built as human IgG4 kappa subtype.

RNAs were isolated from monoclonal hybridoma cells with Trizol reagent. The VH and VL of PD-<NUM> chimeric antibodies were amplified as follows: RNA is first reverse transcribed into cDNA using a reverse transcriptase as described here,.

The resulting cDNA was used as templates for subsequent PCR amplification using primers specific for interested genes. The PCR reaction was done as follows:.

The resulting PCR product (<NUM>µL) was ligated with pMD18-T vector. Top <NUM> competent cells were transformed with <NUM>µL of the ligation product. Positive clones were checked by PCR using M13-<NUM> and M13-<NUM> primers followed by sequencing.

The rat anti-PD-<NUM> antibody from hybridomas were selected and humanized according to the high affinity and specificity of anti-PD-<NUM> antibody binding to PD-<NUM>, improving the homology with human antibody sequence. The said humanized usage is called as CDR-grafting technique. The variable region gene of antibody such as FR regions and CDR regions were divided by KABAT system and IMGT system. In antibody database, based on the alignments of binding sequence homology and structural similarity, the gene of murine region FR1-<NUM> was replaced by humanized variable region FR1-<NUM>, region FR4 of the murine gene was replaced by humanized FR4 region derived from JH and JK genes which had the most similar structures. After verifying the template sequence and codon optimization, the heavy chain variable region and light chain variable region were synthesized and cloned into the expression vector, and then expressing the humanized antibody.

According to the binding ability to cell surface human and mouse PD-<NUM>, W3052_r16. <NUM> and W3052_r16. <NUM> was selected for humanization. <NUM> shows the analysis of humanization scores. The clones W3052-<NUM>-z9-IgG4 (<NUM>) was selected for affinity maturation considering all these factors such as better affinity and humanization scores (Table.

Each amino acid of three complementary-determining regions (VH CDR3, VK CDR1, and VK CDR3) of parental clone was individually mutated to other <NUM> amino acids using a hybridization mutagenesis method. DNA primers containing a NNS codon encoding twenty amino acids were used to introduce mutation to each targeted CDR position. The individual degenerate primers were used in hybridization mutagenesis reactions. Briefly, each degenerate primer was phosphorylated, and then used in a <NUM>:<NUM> ratio with uridinylated ssDNA. The mixture was heated to <NUM> for <NUM> minutes then cooled down to <NUM> over <NUM> hour. Thereafter, T4 ligase and T4 DNA polymerase were added and mix was incubated for <NUM> hours at <NUM>. Synthesis products for VH and VL CDRs were pooled respectively. Typically, <NUM> ng of the pooled library DNA was electroporated into BL21 for plaque formation on BL21 bacterial lawn or for production of scFv fragments.

The primary screen consisted of a single point ELISA (SPE) assay which was carried out using periplasmic extract (PE) of bacteria grown in <NUM>-well plates (deep well). Briefly, this capture ELISA involved coating individual wells of a <NUM>-well Maxisorp Immunoplate with anti-c-myc antibody in coating buffer (<NUM> Na<NUM>CO<NUM>/NaHCO<NUM>) at pH <NUM> overnight at <NUM>. The next day, the plate was blocked with Casein for <NUM> at room temperature. scFv PE was then added to the plate and incubated at room temperature for <NUM> hr. After washing, biotinylated antigen protein was added to the well and the mixture was incubated for <NUM> at room temperature. This was followed by incubation with Streptavidin-HRP conjugate for <NUM> at room temperature. HRP activity was detected with TMB substrate and the reaction was quenched with <NUM> HCl. Plates were read at <NUM>. Clones exhibiting an optical density (OD) signal at <NUM> greater than the parental clone were picked and re-assayed by ELISA (as described above) in duplicate to confirm positive results. Clones that repeatedly exhibited a signal greater than that of the parental antibody were sequenced. The scFv protein concentration of each clone that had a CDR change was then determined by a quantitative scFv ELISA, where a scFv with known concentration was used as a reference. The scFv protein concentration was determined by comparing the ELISA signals with signals generated by the reference scFv. The binding assay was repeated once more for all positive variants under normalized scFv concentration in order to determine the relative binding affinity of the mutant scFv and the parental antibody.

The point mutations in VH and VL determined to be beneficial for binding to antigen were further combined to gain additional binding synergy. The combinatorial mutants were expressed as scFv and screened using the capture ELISA. Clones exhibiting an OD signal at <NUM> greater than the parental clone were sequenced and further confirmed by binding ELISA as described above.

After affinity maturation, a total of <NUM> humanized antibodies (2E5, 2G4, 1G10, 2C2, 2B1, 8C10, 1H6, 5C4, A6W and L1I) were obtained. <FIG> showed the result from first round mutagenesis library screen. Sequence and affinity data of <NUM> humanized antibodies in human, cynomolgus monkeys and mice were shown in Table <NUM>.

<NUM> showed the result from second round mutagenesis library screen. The clones 1H6, 2E5, 2G4 and 2C2 were selected for further analysis.

The vector containing affinity matured humanized antibody were transfected into 293F cells for antibody production and expression. Antibodies in the supernatant of 293F cells were purified using Protein A affinity chromatography.

Cross-reactivity was measured by FACS and ELISA. For FACS, the anti-PD-<NUM> antibodies were tested binding to cell surface human, mouse and cynomolgus PD-<NUM> as described in Example <NUM>.

<FIG> showed the results of cross-species test by FACS. <FIG> showed binding to human PD-<NUM> transfected CHO-S cells. The antibodies can bind specifically to the human PD-<NUM> with EC50 of <NUM>-<NUM>. <FIG> showed binding to mouse PD-<NUM> transfected 293F cells. The antibodies can bind specifically to the mouse PD-<NUM> with EC50 of <NUM>-<NUM>. <FIG> showed binding to activated cynomolgus PBMC in a dose dependent way. The isotype was human IgG4 kappa. The same below.

For ELISA, plates (Nunc) were coated with human, cynomolgus or mouse PD-<NUM> (Sino Biological) at <NUM>µg/ml overnight at <NUM>. After blocking and washing, antibodies were serially diluted in blocking buffer and added to the plates and incubated at room temperature for <NUM>. The plates were then washed and subsequently incubated with secondary antibody goat anti human IgG HRP (Bethyl) for <NUM>. After washing, TMB substrate was added and the reaction was stopped by <NUM> HCl. The absorbance at <NUM> was read using a microplate reader (Molecular Device).

<FIG> showed the result of cross-species test by ELISA. Figure 4A showed binding to human PD-<NUM>. <FIG> showed binding to mouse PD-<NUM>. <FIG> showed binding to cynomolgus PD-<NUM>.

Constructed cell lines that respectively express human PD-<NUM>, CD28, CTLA-<NUM> or ICOS were transferred in to <NUM>-well U-bottom plates (BD) at a density of <NUM>×<NUM><NUM> cells/well. Testing antibodies were diluted in wash buffer (<NUM>×PBS/<NUM>%BSA) and incubated with cells at <NUM> for <NUM>. After washing, the secondary antibody goat anti-human IgG Fc FITC (Jackson ImmunoResearch Lab) was added and incubated at <NUM> in the dark for <NUM>. The cells were then washed once and resuspended in <NUM>×PBS/<NUM>%BSA, and analyzed by flow cytometery (BD) and FlowJo software.

<FIG> showed the result of cross-family test. The anti-PD-<NUM> antibodies can bind specifically to human PD-<NUM>, but not to CD28 and CTLA-<NUM>.

<NUM> The ability of anti-PD-<NUM> antibodies to block PD-L1 binding to PD-<NUM> was tested by FACS as described in Example <NUM>.

<NUM> The ability of anti-PD-<NUM> antibodies to block PD-L2 binding to PD-<NUM> was tested by ELISA. Briefly, plates (Nunc) were coated with human PD-<NUM> at <NUM>µg/ml overnight at <NUM>. Antibodies were serially diluted in blocking buffer and mixed with his tag conjugated PD-L2. After blocking and washing the coated plates, the antibody/PD-L2 mixture were added to the plates, then incubated at room temperature for <NUM>. The plates were then washed and subsequently incubated with secondary antibody goat anti-his HRP (GenScript) for <NUM>. After washing, TMB substrate was added and the reaction was stopped by <NUM> HCl. The absorbance at <NUM> was read using a microplate reader (Molecular Device).

<FIG> showed the result of anti-PD-<NUM> antibodies blocking human PD-L1 binding to PD-<NUM> transfected CHO-S cells. <FIG> shows the result of anti-PD-<NUM> antibodies blocking mouse PD-L1 binding to PD-<NUM> transfected 293F cells. <FIG> showed that the anti-PD-<NUM> antibodies could block human PD-L2 binding to PD-<NUM> in a dose-dependent manner.

Antibodies were characterized for affinity and binding kinetics to PD-<NUM> by SPR assay using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma) was immobilized to a GLM sensor chip (Bio-Rad) through amine coupling. Purified antibodies were flowed over the sensor chip and captured by the Protein A. The chip was rotated <NUM>° and washed with running buffer (<NUM>×PBS/<NUM>% Tween20, Bio-Rad) until the baseline was stable. Seven concentrations of human PD-<NUM> and running buffer were flowed through the sensor chip at a flow rate of <NUM>µL/min for an association phase of <NUM>, followed by <NUM> dissociation. After regeneration, seven concentration of mouse PD-<NUM> and running buffer were flowed through the sensor chip at a flow rate of <NUM>µL/min for an association phase of <NUM>, followed by <NUM> dissociation. The chip was regenerated with pH <NUM><NUM>PO<NUM> after each run. The association and dissociation curve was fit by <NUM>:<NUM> Langmuir binding model using ProteOn software.

6A-6B showed the results of full kinetic binding affinity to human and mouse PD-<NUM> by SPR. WBP305BMK1 was synthesized according to the clone of 5C4 from BMS patent <CIT>. Keytruda was the anti-PD-<NUM> drug from Merck. The same below. The results showed that the affinity ability to human PD-<NUM> by SPR assay was from <NUM>. 43E-<NUM> to <NUM>. 64E-<NUM> mol/L. Comparing WBP305BMK1 with Keytruda, the KD value of antibody 2E5, 2G4 or 2C2 was much smaller, illustrating that 2E5, 2G4 or 2C2 had better binding ability to human PD-<NUM>. In addition, the affinity ability to mouse PD-<NUM> was from <NUM>. 37E-<NUM> to <NUM>. 89E-<NUM> mol/L.

CHO-S cells expressing human PD-<NUM> or 293F cells expressing mouse PD-<NUM> were transferred in to <NUM>-well U-bottom plates (BD) at a density of <NUM>×<NUM><NUM> cells/well. Testing antibodies were <NUM>:<NUM> serially diluted in wash buffer (<NUM>×PBS/<NUM>%BSA) and incubated with cells at <NUM> for <NUM>. The secondary antibody goat anti-human IgG Fc FITC (<NUM> moles FITC per mole IgG, (Jackson Immunoresearch Lab) was added and incubated at <NUM> in the dark for <NUM>. The cells were then washed once and resuspended in 1XPBS/<NUM>%BSA, and analyzed by flow cytometery (BD). Fluorescence intensity was converted to bound molecules/cell based on the quantitative beads (Quantum™ MESF Kits, Bangs Laboratories, Inc. KD was calculated using Graphpad PrismS.

7A-7B show the results of binding affinity of anti-PD-<NUM> antibodies to cell surface human and mouse PD-<NUM> molecules tested by flow cytometry. The results showed that the affinity ability to human PD-<NUM> by FACS assay was from <NUM>. 80E-<NUM> to <NUM>. 15E-<NUM> mol/L. In addition, the affinity ability to mouse PD-<NUM> was from <NUM>. 39E-<NUM> to <NUM>. 74E-<NUM> mol/L.

The binding epitope of anti-PD-<NUM> antibodies was compared with benchmark antibody A and B by FACS. CHO-S cells expressing human PD-<NUM> on the cell surface were incubated with mixture of biotinylated benchmark antibody A or B (1µg/ml) and testing antibodies (serially diluted in wash buffer) at <NUM> for <NUM>. The cells were washed and the second antibody Streptavidin-PE were added and incubated for <NUM> at <NUM>. The cells were then washed once and resuspended in <NUM>×PBS/<NUM>%BSA, and analyzed by flow cytometery (BD).

<FIG> showed the results of epitope binning assay suggesting that the anti-PD-<NUM> antibodies are in the same or close epitope bin as benchmark antibodies. <FIG> showed binning against WBP305BMK1 (<CIT>). <FIG> showed binning against Keytruda (<CIT>).

Furthermore, alanine scanning experiments on hPD-<NUM> were conducted and their effect to antibody binding was evaluated. Alanine residues on hPD-<NUM> were mutated to glycine codons, and all other residues were mutated to alanine codons. For each residue of the hPD-<NUM> extracellular domain (ECD), point amino acid substitutions were made using two sequential PCR steps. <NUM>-hPD-1_ECD. His plasmid that encodes ECD of human PD-<NUM> and a C-terminal His-tag was used as template, and a set of mutagenic primer was used for first step PCR using the QuikChange lightning multisite-directed mutagenesis kit (Agilent technologies, Palo Alto, CA). Dpn I endonuclease was used to digest the parental template after mutant strand synthesis reaction. In the second-step PCR, linear DNA expression cassette which composed of a CMV promoter, an extracellular domain (ECD) of PD-<NUM>, a His-tag and a herpes simplex virus thymidine kinase (TK) polyadenylation was amplified and transiently expressed in HEK293F cells (Life Technologies, Gaithersburg, MD).

Monoclonal antibodies W3052_r16. <NUM> and Keytruda were coated in plates for ELISA binding assay. After interacting with the supernatant that contains quantified PD-<NUM> mutant or human/mouse PD-1_ECD. His protein (Sino Biological, China), HRP conjugated anti-His antibody was added as detection antibody. Absorbance was normalized according to the average of control mutants. After setting an additional cutoff to the binding fold change (<<NUM>), the final determined epitope residues were identified.

The binding activities of the antibodies W3052_r16. <NUM> and Keytruda to both human and murine PD-<NUM> were conducted (<FIG>). <NUM> was found binding to both hPD-<NUM> and mPD-<NUM> while Keytruda only bound to the human one (<FIG>). This unique functional cross-reactivity of W3052_r16. <NUM> can help provide more animal model options in preclinical studies when evaluating the drug safety. To explore the origin of the observed binding behaviors, epitope mapping of both antibodies were conducted.

Top <NUM> point-substituted hPD-<NUM> mutants that significantly reduced antibody binding were shown in Table <NUM>. Checking the positions of all these residues on the hPD-<NUM> crystal structures (PDB code 3RRQ and 4ZQK) revealed that some amino acids (e.g. Val144, Leu142, Val110, Met108, Cys123 etc.) were fully buried in the protein, and were unlikely to directly contact any antibodies. The observed binding reductions most probably resulted from the instability or even collapse of hPD-<NUM> structure after alanine substitutions. According to the antigen structure analysis, some of the residues don't involve binding activity, but are expected to respond to the stability of the hPD-<NUM> structure, e.g. V144 and L142. Mutants that affect both antibodies were treated as false hot spots and were removed from the list. After setting an additional cutoff to the binding fold change (<<NUM>), the final determined epitope residues were listed in Table <NUM>. They are <NUM> positions to W3052_r16. <NUM> and <NUM> positions to Keytruda.

Comparing the epitope residues of W3052_r16. <NUM> and Keytruda in Table <NUM> only revealed two overlapped hot spot residues. The rest looked quite diverse, which indicated that two antibodies might have adopted very different mechanisms in terms of hPD-<NUM> binding and hPD-L1 blocking. Reading the residue IDs in Table <NUM> is not straightforward to interpret the mechanisms. All data in Table <NUM>, as well as the hPD-L1 binding site, were therefore mapped on the crystal structure of hPD-<NUM> to make a better visualization and comparison.

Two investigated antibodies W3052_r16. <NUM> and Keytruda, although both are functional in binding hPD-<NUM> and blocking hPD-L1, have obviously different epitopes (<FIG>). The epitope of Keytruda were mainly contributed by the residues on the C'D loop (corresponding to the C" strand on mPD-<NUM>), which didn't intersect the PD-L1 binding site at all. This suggested the hPD-L1 blocking function of Keytruda relied more on its steric hindrance effects provided by the size of the antibody. In contrast, the epitope mapping results show that the epitope of antibody W3052_r16. <NUM> was composed of hot spots distributed across multiple locations, and have direct overlap with the hPD-L1 binding site (<FIG>). <NUM> blocked hPD-L1 by means of competing with hPD-L1 in reacting to their common binding site. What's more, W3052_r16. <NUM> had no interactions with the flexible C'D loop (or the corresponding C" strand on mPD-<NUM>), where human and murine PD-<NUM> show big structural deviations (<FIG>). Its binding site is mostly located on the FG loop (<NPL>). That explains why W3052_r16. <NUM> can bind to both PD-<NUM> species while Keytruda only binds to the human one (<FIG>). Because of this unique functional cross-reactivity, the preclinical safety evaluations of W3052_r16. <NUM> could be conducted in mouse model, which will greatly simplify and accelerate the development. Overall, antibody W3052_r16. <NUM> is expected to be more functional and developable than Keytruda.

Human DCs, CD4+ T, CD8+ T and total T cells isolation: Human PBMCs were freshly isolated from healthy donors using Ficoll-Paque PLUS (GE) gradient centrifugation. Monocytes were isolated using Human Monocyte Enrichment Kit (StemCell) according to the manufacturer's instructions. Cells were cultured in medium containing rhGM-CSF and rhIL-<NUM> for <NUM> to <NUM> days to generate dendritic cells. <NUM> to <NUM> hours before MLR, <NUM>µg/mL LPS was added to the culture to induce the maturation of the DCs. Human CD4+ T cells were isolated using Human CD4+ T Cell Enrichment Kit (StemCell) according to the manufacturer's protocol. Mouse CD4+ T cells were obtained from the spleen of Balb/c mouse using Mouse CD4+ T Cell Isolation Kit (StemCell) according to the manufacturer's protocol. Mouse DCs were induced from bone marrow cells of C57BL/<NUM> mouse in medium containing rmGM-CSF and rmIL-<NUM> for <NUM> to <NUM> days. <NUM> to <NUM> hours before MLR, <NUM>µg/mL LPS was added to the culture to induce the maturation of the DCs.

Briefly, primary dendritic cell (DC)-stimulated MLR was conducted in <NUM>-well, U-bottom tissue culture plates in <NUM>µL of RPMI <NUM> containing <NUM>% FCS and <NUM>% antibiotics. DCs were mixed with <NUM>×<NUM><NUM> CD4+ T cells at a ratio between <NUM>:<NUM> and <NUM>:<NUM> DC: T cells in the presence or absence of testing antibodies or benchmark antibodies (form <NUM> down to <NUM>, generally total six concentrations). To determine the effect of anti-PD-<NUM> antibodies on T cell function, the cytokine production and T cell proliferation were determined. Results shown are representative of a minimum of five experiments performed.

Cytokine detection: Human IFN-γ and IL-<NUM> were measured by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs. The plates were pre-coated with capture antibody specific for human IFN-γ (cat# Pierce-M700A) or IL-<NUM> (cat# R&D-MAB602), respectively. The biotin-conjugated anti-IFN-γ antibody (cat# Pierce-M701B) or anti-IL-<NUM> antibody (cat# R&D-BAF202) was used as detecting antibody.

<FIG> showed anti-PD-<NUM> antibodies increased IL-<NUM> secretion in a dose-dependent manner. <FIG> shows anti-PD-<NUM> antibodies increase IFN-γ secretion in a dose-dependent manner.

Proliferation assay: <NUM>-thymidine (cat# PerkinElmer- NET027001MC) was diluted <NUM>:<NUM> in <NUM>% NaCl solution, and added to the cell culture plates at <NUM> uCi/well. The plates were cultured in <NUM>% CO<NUM> at <NUM> for <NUM> to <NUM> hours, before the incorporation of <NUM>-thymidine into the proliferating cells was determined. <FIG> shows anti-PD-<NUM> antibodies increase CD4+ T cells proliferation in a dose-dependent manner.

To determine the effect of anti-PD-<NUM> antibodies on mouse T cell function, the cytokine production and mouse T cell proliferation were determined similarly. <FIG> showed the results of mouse allo-MLR demonstrating that the anti-PD-<NUM> antibodies can enhance the function of mouse CD4+ T cell. <FIG> showed anti-PD-<NUM> antibodies increased IL-<NUM> secretion in a dose-dependent manner. <FIG> showed anti-PD-<NUM> antibodies increased IFN-γ secretion in a dose-dependent manner. <FIG> showed anti-PD-<NUM> antibodies increased CD4+ T cells proliferation in a dose-dependent manner.

In this assay, the CD4+ T cells and DCs were from a same donor. Briefly, CD4+ T cells were purified from PBMC and cultured in the presence of CMV pp65 peptide and low dose of IL-<NUM> (<NUM> U/mL), at the meanwhile, DCs were generated by culturing monocytes from the same donor's PBMC in GM-CSF and IL-<NUM>. After <NUM> days, the CMV pp65 peptide treated CD4+ T cells were co-cultured with DCs pulsed with CMV pp65 peptide in the absence or presence of human anti-PD-<NUM> antibodies or benchmark antibodies (as control). On day <NUM>, <NUM>µL of supernatants were taken from each of cultures for IFN-γ measurement by ELISA as described above. The proliferation of CMV pp65-specific T cells was assessed by <NUM>-thymidine incorporation as described above.

<FIG> showed the results of human auto-MLR demonstrating the anti-PD-<NUM> antibodies can enhance the function of human CD4+ T cell. <FIG> showed anti-PD-<NUM> antibodies increase IFN-γ secretion in a dose-dependent manner. <FIG> showed anti-PD-<NUM> antibodies increase CD4+ T cells proliferation in a dose-dependent manner.

Tregs, a subpopulation of T cells, are a key immune modulator and play critical roles in maintaining self-tolerance. Increased numbers of CD4+CD25+ Tregs were found in patients with multiple cancers and associated with a poorer prognosis. To determine whether the anti-PD-<NUM> antibodies affect the immune suppressive role of Tregs, we compared the T cell function in the presence of Tregs with or without anti-PD-<NUM> antibody treatment. CD4+CD25+ and CD4+CD25-T cells were separated using specific anti-CD25 microbeads (StemCell) per manufacture's instruction. Two thousand mature DCs, <NUM>×<NUM><NUM> CD4+CD25- T cells, <NUM>×<NUM><NUM> Treg cells and PD-<NUM> antibodies were incubated in <NUM>-well plates. The plates were kept at <NUM> in a <NUM>% CO<NUM> incubator for <NUM> days. IFN-γ production and CD4+CD25- cells proliferation were tested as described above.

<FIG> demonstrates that the anti-PD-<NUM> antibodies can reverse the suppressive function of Tregs. <FIG> showed anti-PD-<NUM> antibodies can restore the IFN-γ secretion. <FIG> showed anti-PD-<NUM> antibodies can restore the T-cell proliferation.

PD-<NUM> is expressed on variety of cell types. In order to minimize potential toxicity to healthy PD-<NUM> positive cells, the anti-PD-<NUM> antibodies were evaluated for their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Human activated CD4+ T cells and various concentrations of PD-<NUM> antibodies were pre-incubated in <NUM>-well plate for <NUM> minutes, and then PBMCs were added at the effector/target ratio of <NUM>:<NUM>. The plate was kept at <NUM> in a <NUM>% CO<NUM> incubator for <NUM> hours. Target cell lysis was determined by LDH-based cytotoxicity detection kit (cat# Roche-<NUM>). The absorbance at <NUM> was read using a microplate reader (Molecular Device). Herceptin-induced SK-Br-<NUM> cell lysis was used as positive control.

<FIG> showed the result of ADCC test demonstrating the anti-PD-<NUM> antibodies did not mediate ADCC activity on activated CD4+ T cells.

Human activated CD4+ T cells and various concentrations of PD-<NUM> antibodies were mixed in <NUM>-well plate. Human complement (Quidel-A112) was added at the dilution ratio of <NUM>:<NUM>. The plate was kept at <NUM> in a <NUM>% CO<NUM> incubator for <NUM> hours. Target cell lysis was determined by CellTiter-Glo. Rituxan®-induced Raji cell lysis was used as positive control. The luminescence was read using a microplate reader (Molecular Device).

<FIG> showed the result of CDC test demonstrating the anti-PD-<NUM> antibodies did not mediate CDC activity on activated CD4+ T cells.

Murine melanoma cell CloudmanS91 cell (ATCC-CCL-<NUM>) was cultured in vitro as monolayer, and the culture condition was F-<NUM> medium plus <NUM>% FBS and <NUM>% horse serum, <NUM> U/mL penicillin, and <NUM>µg/mL streptomycin, incubate at <NUM> and <NUM>% CO<NUM>. The cells were digested using trypsin-EDTA and passaged twice a week routinely. Cells were harvested, counted, and then inoculated when approximately <NUM>%-<NUM>% confluent and the number is as required.

<NUM> (<NUM>×<NUM><NUM> cells) CloudmanS91 cells were inoculated subcutaneously in the right backside of each animal. When the mean of tumor volume had reached approximately <NUM><NUM>, the administration started in groups. Grouping and dosing regimens were shown in Table <NUM>.

Experimental index is to investigate whether the tumor growth was inhibited, delayed or cured. Tumor diameters were measured with a caliper three times a week. Tumor volume is calculated using V=<NUM>a×b<NUM>, wherein a and b represents long and short diameters of the tumor, respectively.

Antitumor efficacy of the antibody was assessed by tumor growth inhibition TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflected the rate of tumor growth inhibition. TGI (%) was calculated as follows: TGI (%) = [(<NUM>-(average tumor volume at the end of administration in the treatment group - average tumor volume at the start of administration in the treatment group)) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × <NUM>%.

Relative tumor proliferation rate T/C (%) was calculated as follows: T/C% = TRTV / CRTV × <NUM>% (TRTV: treatment group RTV; CRTV: negative control group RTV). The relative tumor volume (RTV) was calculated according to the results of tumor measurements using RTV=Vt/V<NUM>, wherein V<NUM> was average tumor volume at the time of grouping (i.e., d<NUM>), Vt was average tumor volume of a certain measurement; the data of TRTV and CRTV were taken on the same day.

T-C (days) reflected tumor growth delay index, T represented average days passed when the tumor had reached a predetermined volume in the treatment group (eg. <NUM><NUM>), C represented the average days when tumors in the control group had reached the same volume.

Survival curves were plotted; animal survival time was defined as the time from the administration to animal deaths or the time when tumor volume had reached <NUM><NUM>. The median survival time (days) was calculated in each group. Increased life span (ILS) was calculated by comparison of the median survival times between the treated group and model control group and represented as a percentage over the lifetime of the model control group.

The data including the average tumor volume at each time point in each group and standard error (SEM) were analyzed statistically (refer to Table <NUM> for specific data). The experiment was completed on day <NUM> after the administration; on day <NUM> after the administration, start sacrificing animals successively; and therefore the statistical analysis and evaluation for inter-group differences were based on the tumor volume on day <NUM> after initiation of administration. For comparisons between the two groups, data were analyzed using T-test; for comparisons among three or more groups, data were analyzed using one-way ANOVA. If statistically significant difference was found for F value, data were analyzed using Games-Howell test. If no statistically significant difference was found for F value, Dunnet (<NUM>-sided) test was then used for analysis. SPSS <NUM> was used for all data analysis. p<<NUM> was considered as significant difference. Survival time was analyzed using Kaplan-Meier method with the Log-rank test.

Animal's weight is as an indirect reference for measurement of drug toxicity. The impact of 2E5 on the weight of CloudmanS91 subcutaneous syngeneic xenograft female DBA/<NUM> mice model was as shown in <FIG>. In this model, all administration groups showed no significant weight loss (<FIG>). Thus, 2E5 had no obvious toxicity in a mouse model of melanoma CloudmanS91.

Tumor volume in CloudmanS91 subcutaneously syngeneic xenograft female DBA/<NUM> mouse model after 2E5 treatment was as shown in Table <NUM>.

Survival curves in each group were shown in <FIG>.

In this study, we have evaluated the in vivo efficacy of 2E5 in CloudmanS91 syngeneic tumor model. Tumor volume in each group at different time points were shown in Table <NUM>, Table <NUM> and <FIG>, survival time were shown in <FIG> and Table <NUM>. On day <NUM> after administration, tumor volume of tumor-bearing mice in the solvent control group reached <NUM>,<NUM><NUM>. A weak inhibitory effect was observed in <NUM>/kg 2E5 group compared with the control group, and the tumor volume was <NUM>,<NUM><NUM> (T/C=<NUM>%, TGI=<NUM>%, p=<NUM>), tumor growth delay was <NUM> days. A significant anti-tumor effect was observed in <NUM>/kg 2E5 group compared with the solvent control group, and the tumor volume was <NUM><NUM> (T/C=<NUM>%, TGI=<NUM>%, p=<NUM>), tumor growth delay was <NUM> days. A significant anti-tumor effect was also observed in <NUM>/kg 2E5 group compared with the solvent control group, the tumor volume was <NUM><NUM> (T/C=<NUM>%, TGI=<NUM>%, p=<NUM>), tumor growth delay was <NUM> days.

In the experiment, the median survival time of tumor-bearing mice in solvent control group was <NUM> days. Compared with the vehicle control group, the median survival time of tumor-bearing mice in <NUM>/kg 2E5 group was <NUM> days, survival was prolonged <NUM>% (p=<NUM>); survival rate of tumor-bearing mice in <NUM>/kg 2E5 group was <NUM>% (p=<NUM>). The median survival time of tumor-bearing mice in <NUM>/kg 2E5 group was <NUM> days, survival was prolonged <NUM>% (p=<NUM>).

The changes in body weight of nude mice were shown in <FIG>. Good tolerability of drug 2E5 has been found in all tumor-bearing mice, and no significant weight loss was observed in all treatment groups. In summary, in this experiment, significant anti-tumor effects were shown in both <NUM>/kg group and <NUM>/kg group for CloudmanS91 subcutaneous synergistic tumor model, which is not dose-dependent. Anti-tumor effect in <NUM>/kg group is better than that in <NUM>/kg group.

Claim 1:
An antibody or antigen binding fragment thereof that specifically binds to PD-<NUM>, wherein the antibody or antigen binding fragment thereof comprises:
a) a variable region of a heavy chain having the amino acid sequence of SEQ ID NO: <NUM> and a variable region of a light chain having the amino acid sequence of SEQ ID NO: <NUM>;
b) a variable region of a heavy chain having the amino acid sequence of SEQ ID NO: <NUM> and a variable region of a light chain having the amino acid sequence of SEQ ID NO: <NUM>;
c) variable region of a heavy chain having the amino acid sequence of SEQ ID NO: <NUM> and a variable region of a light chain having the amino acid sequence of SEQ ID NO: <NUM>; or
d) a variable region of a heavy chain having the amino acid sequence of SEQ ID NO: <NUM> and a variable region of a light chain having the amino acid sequence of SEQ ID NO: <NUM>.