Canine PD-L1 antibodies, antigen binding fragments thereof and methods of use thereof

Abstract

Canine-derived antibodies and antigen-binding fragments thereof that bind to Programmed Death Ligand 1 in a canine subject are provided. Also provided are methods of treating cancer in a canine subject using the canylated antibodies and / or antigen-binding fragments, as well as methods of predicting and modeling the anti-cancer activity of a test compound in a subject.

Description

[0001] Priority

[0002] This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 383,909, filed on November 15, 2022. The content of the foregoing application is hereby incorporated by reference in its entirety into this disclosure. Technical Field

[0003] This disclosure relates to anti-canine programmed death ligand 1 (PD-L1) antibodies, antigen-binding fragments thereof, complementarity-determining regions (CDRs) thereof, and canineized antibodies against canine PD-L1. Also provided are methods of predicting and modeling the anti-cancer activity of test compounds in a subject using the antibodies, CDRs, and / or antigen-binding fragments thereof. Background Art

[0004] In a normal healthy system, immune checkpoints are surface proteins that are present to check and prevent any overstimulation of the immune response. In other words, their role is to prevent the immune response from becoming so strong that it destroys healthy cells in the body. In the case of cancer, when a T cell checkpoint protein (e.g., programmed cell death protein 1 (PD-1)) binds to a binding protein (programmed death ligand 1 (PD-L1)) on a tumor cell, it sends a "shut off" signal to the T cell and inhibits the anti-tumor immune response. In this way, tumor-killing cells within the immune system cannot kill the tumor because their method of attack is blocked by those checkpoints on the tumor itself.

[0005] Immune checkpoint blockade therapy employs immune checkpoint inhibitors and works by inhibiting the binding of checkpoint proteins to their ligands on tumor cells. PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation 3 (LAG3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell activation V-domain Ig suppressor (VISTA) are some examples of checkpoint proteins. Immune checkpoint blockade therapy is one of the most promising forms of cancer immunotherapy and has been successfully used in a variety of cancer types, including invasive urothelial carcinoma. In particular, blockade of the PD-1 / PD-L1 pathway using anti-PD-1 or anti-PD-L1 antibodies has elicited durable clinical responses in cancer patients, e.g., by normalizing the imbalanced anti-tumor immunity.

[0006] Given the promising and durable clinical responses, the U.S. Food and Drug Administration (FDA) has approved three PD-1 antibodies (pembrolizumab and nivolumab Nivolumab and pembrolizumab) and three PD-L1 antibodies (atezolizumab, avelumab, and durvalumab) are used for multiple types of human cancer. While this milestone demonstrates the promise of cancer immunotherapy treatment, current PD-1 / PD-L1 blockade therapies in cancer are not satisfactory due to limited response rates (10 - 40%). Thus, there is an urgent need for new immunotherapy strategies to improve the therapeutic efficacy of current PD-1 / PD-L1 blockade.

[0007] Strategies to improve PD-1 / PD-L1 blockade therapies in bladder and other cancers can include: 1) identifying host factors, including genetics, immune status, and molecular subtypes that drive responses, 2) evaluating biomarkers and combinations of biomarkers to predict responses and personalize therapies, 3) developing better tools to monitor immune effects, and 4) selecting combination drug approaches / protocols to address multiple "defects" in immune responses beyond PD-1 / PD-L1 blockade. Relevant preclinical animal models are commonly used to develop these strategies and test combination methods. Factors that can affect the PD-1 / PD-L1 axis, and thus can be manifested in animal models, include aggressive and metastatic cancer behavior, tumor heterogeneity, mutational landscape, genetic and epigenetic interactions, cancer molecular subtypes, immune cell responsiveness, and innate and acquired resistance mechanisms. While rodent models, including carcinogen-induced, transplanted, and genetically engineered models, are useful in cancer research, rodent models do not possess collective characteristics that are crucial for studying emerging therapies within and across molecular subtypes in bladder cancer and predicting the success or failure of treatment in humans. What is needed is an animal model that can be used to optimize checkpoint inhibitor treatment in humans.

[0008] In addition, much rodent data is not predictive for dogs, especially with regard to immune checkpoint blockade therapy. Many cancers remain untreatable in dogs, and treatments effective for one cancer often fail in another. Thus, there is also a need to treat certain forms of cancer in dogs. SUMMARY OF THE INVENTION

[0009] Provided is a canine antibody or antigen-binding fragment thereof that binds to programmed death ligand 1 (PD-L1) in a canine subject. In certain embodiments, the antibody or antigen-binding fragment is encoded by a nucleotide sequence comprising at least 80% sequence identity to SEQ ID NO: 2 and / or 4 or SEQ ID NO: 6 and / or 8 (e.g., comprising SEQ ID NO: 2 and / or 4 or SEQ ID NO: 6 and / or 8).

[0010] An antibody or antigen-binding fragment can include one or more complementarity determining regions (CDRs), the one or more complementarity determining regions including AAS, SEQ ID NO:9, and / or SEQ ID NO:10. The antibody or antigen-binding fragment can include CDRs, each CDR including at least 80% sequence identity with SEQ ID NO:17, WTS, and / or SEQ ID NO:12 (e.g., including SEQ ID NO:17, WTS, and / or SEQ ID NO:12). The antibody or antigen-binding fragment can include CDRs independently including at least 80% sequence identity with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11 (e.g., including SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11). The antibody or antigen-binding fragment can include CDRs independently including at least 80% sequence identity with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13 (e.g., including SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13).

[0011] In certain embodiments, the antibody or antigen-binding fragment is fully or partially canine-derived.

[0012] The antibody or antigen-binding fragment can be a chimeric form of a canine-derived antibody or antigen-binding fragment. In certain embodiments, the antibody or its antigen-binding fragment can specifically bind to canine PD-L1. In certain embodiments, the antibody or antigen-binding fragment includes a canine-derived murine PD-1 antibody or its antigen-binding fragment.

[0013] Also provided is a method of treating cancer in a canine subject. Such method can include administering to the canine subject a therapeutically effective amount of a canine-derived antibody or its antigen-binding fragment that binds PD-L1 and inhibits PD-L1 / PD-1 interaction in the subject.

[0014] The chimeric antibodies or antigen-binding fragments thereof of the method may include any chimeric antibodies or antigen-binding fragments thereof described herein. For example, in certain embodiments, the chimeric antibody or antigen-binding fragment thereof is encoded by a nucleotide sequence comprising at least 80% sequence identity to SEQ ID NO: 2 and / or 4 or SEQ ID NO: 6 and / or 8. The chimeric antibody or antigen-binding fragment may include one or more CDRs, wherein the one or more CDRs independently comprise at least 80% sequence identity to AAS, SEQ ID NO: 9 and / or SEQ ID NO: 10. The chimeric antibody or antigen-binding fragment may include one or more CDRs, wherein the one or more CDRs independently comprise at least 80% sequence identity to SEQ ID NO: 18, WTS and / or SEQ ID NO: 12. The chimeric antibody or antigen-binding fragment may include one or more CDRs, wherein the one or more CDRs independently comprise at least 80% sequence identity to SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 13.

[0015] In certain embodiments of the method, the chimeric antibody or antigen-binding fragment is encoded by a nucleotide sequence comprising SEQ ID NO: 2 and / or 4 or SEQ ID NO: 6 and / or 8. In certain embodiments of the method, the chimeric antibody or antigen-binding fragment includes one or more CDRs, wherein the one or more CDRs include AAS, SEQ ID NO: 9 and / or SEQ ID NO: 10. In certain embodiments of the method, the chimeric antibody or antigen-binding fragment includes one or more CDRs, wherein the one or more CDRs include SEQ ID NO: 17, WTS and / or SEQ ID NO: 12. In certain embodiments of the method, the chimeric antibody or antigen-binding fragment includes one or more CDRs, wherein the one or more CDRs include SEQ ID NO: 15, SEQ ID NO: 16 and / or SEQ ID NO: 11. The chimeric antibody or antigen-binding fragment of the method may include one or more CDRs, wherein the one or more CDRs include SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO: 13.

[0016] In certain embodiments, a therapeutically effective amount is from about 2 mg / kg of subject body weight (such as, for example, 2 mg / kg of subject body weight) to about 5 mg / kg of subject body weight (such as, for example, 5 mg / kg of subject body weight). The therapeutically effective amount can be 2 mg / kg of subject body weight. The therapeutically effective amount can be 5 mg / kg of subject body weight. The therapeutically effective amount can be from about 2.5 mg / kg of subject body weight (such as, for example, 2.5 mg / kg of subject body weight) to about 4.5 mg / kg of subject body weight (such as, for example, 4.5 mg / kg of subject body weight). The therapeutically effective amount can be from about 3.0 mg / kg of subject body weight (such as, for example, 3.0 mg / kg of subject body weight) to about 4.0 mg / kg of subject body weight (such as, for example, 4.0 mg / kg of subject body weight). The therapeutically effective amount can be from about 2.5 mg / kg of subject body weight (such as, for example, 3.0 mg / kg of subject body weight) to about 3.5 mg / kg of subject body weight (such as, for example, 3.5 mg / kg of subject body weight). The therapeutically effective amount can be 2 mg / kg of subject body weight. The therapeutically effective amount can be 5 mg / kg of subject body weight. The therapeutically effective amount can be from about 5.0 mg / kg of subject body weight (such as, for example, 5.0 mg / kg of subject body weight) to about 10.0 mg / kg of subject body weight (such as, for example, 10.0 mg / kg of subject body weight). The therapeutically effective amount can be from about 5.5 mg / kg of subject body weight (such as, for example, 5.5 mg / kg of subject body weight) to about 9.5 mg / kg of subject body weight (such as, for example, 9.5 mg / kg of subject body weight). The therapeutically effective amount can be from about 6.0 mg / kg of subject body weight (such as, for example, 6.0 mg / kg of subject body weight) to about 9.0 mg / kg of subject body weight (such as, for example, 9.0 mg / kg of subject body weight). The therapeutically effective amount can be from about 6.5 mg / kg of subject body weight (such as, for example, 6.5 mg / kg of subject body weight) to about 8.5 mg / kg of subject body weight (such as, for example, 8.5 mg / kg of subject body weight). The therapeutically effective amount can be from about 7.0 mg / kg of subject body weight (such as, for example, 7.0 mg / kg of subject body weight) to about 8.0 mg / kg of subject body weight (such as, for example, 8.0 mg / kg of subject body weight). The therapeutically effective amount can be from about 7.5 mg / kg of subject body weight (such as, for example, 7.5 mg / kg of subject body weight) to about 7.8 mg / kg of subject body weight (such as, for example, 7.8 mg / kg of subject body weight). The ranges specified in this paragraph include the stated endpoints and all 0.1 mg / kg increments contained within the specified ranges.

[0017] The cancer can be invasive urothelial carcinoma.

[0018] In certain embodiments of the method, the antibody or antigen-binding fragment thereof is formulated into a pharmaceutical composition.

[0019] Also provided is a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises any of the humanized antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable excipient.

[0020] Also provided is a method for predicting and modeling the anti-cancer activity of a test compound in a subject (e.g., having cancer). In certain embodiments, such method comprises: generating a population of humanized PD-L1 mice expressing humanized PD-L1 on the cell surface; evaluating the toxicity risk and / or efficacy of a set of test compounds in treating cancer in the population of humanized PD-L1 mice; and selecting one or more test compounds of the set that meet established toxicity risk and / or efficacy criteria.

[0021] Generating the population of humanized PD-L1 mice can further comprise replacing the murine cd274 gene in the mouse population with the canine PD-L1 gene using CRISPR.

[0022] The method can further comprise evaluating the toxicity risk and / or efficacy of one or more selected compounds in treating cancer in a human or canine cohort. In certain embodiments, the method can further comprise evaluating the oral bioavailability, absorption, distribution, metabolism, and excretion (ADME) values of the set of test compounds in the population of humanized PD-L1 mice. In certain embodiments, the method further comprises evaluating the oral bioavailability and ADME values of one or more selected test compounds in the population of humanized PD-L1 mice.

[0023] In certain embodiments, the set of test compounds comprises at least one compound that inhibits PD-L1 / PD-1 interaction in the subject.

[0024] Also provided are the CDRs of the antibody or antigen-binding fragment. The CDR can include at least 80% sequence identity with AAS, SEQ ID NO:9, and / or SEQ ID NO:10. The CDR of the antibody or antigen-binding fragment can include at least 80% sequence identity with SEQ ID NO:18, WTS, and / or SEQ ID NO:12. The CDR of the antibody or antigen-binding fragment can include at least 80% sequence identity with SEQ ID NO:16, SEQ ID NO:17, and / or SEQ ID NO:11. The CDR can include at least 80% sequence identity with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13. The CDR can include at least 80% sequence identity with SEQ ID NO:17, WTS, and / or SEQ ID NO:12. The CDR of the antibody or antigen-binding fragment can include at least 80% sequence identity with SEQ ID NO:16, SEQ ID NO:17, and / or SEQ ID NO:11. The CDR of the antibody or antigen-binding fragment can include at least 80% sequence identity with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13.

[0025] In certain embodiments, provided is the use of the humanized antibody or antigen-binding fragment of the present disclosure, the pharmaceutical composition described herein, or one or more of the CDRs described herein, such as in the preparation of a medicament for treating cancer in a canine subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description of various exemplary embodiments according to the present disclosure, the disclosed embodiments and other features, advantages, and aspects included herein, and the problems of achieving them will become apparent. Such detailed description will be better understood when combined with the drawings, wherein:

[0027] Figure 1Shows a schematic flow chart representing the generation and validation of the canine PD-L1 antibody used herein, where (A) immunization with antigen, injection of canine PD-L1 protein (cPD-L1), (B) establishment of hybridomas (more than 2,000 clones), (C) selection of cPD-L1-specific antibodies (Abs) by live cell-based Ab binding assay, (D) selection of therapeutic Abs by cPD-1 / PD-L1 blocking assay, (E) in vivo validation of the therapeutic efficacy of the cPD-L1 antibody in mice having only canine PD-L1 and PD-1 (i.e., canine PD-L1 humanized mice), (F) generation of cPD-L1 chimeric Ab (mouse / canine), (G) validation of the cPD-L1 chimeric Ab, and (H) initial safety profile and PK assay in dogs.

[0028] Figure 2A Is a schematic diagram of a canine programmed death ligand 1 (cPD-L1) antibody binding assay.

[0029] Figure 2B Is a schematic diagram of a cPD-L1 / canine programmed death protein 1 (cPD-1) blocking assay.

[0030] Figure 2C Is a kinetic graph showing the quantitative binding of a programmed death ligand 1 (PD-L1) antibody at each 3-hour time point to BT549 cells expressing cPD-L1, where positive clones are highlighted in red.

[0031] Figure 2D Is a representative image of cPD-L1 antibody binding (at 18 hours), showing a merged green fluorescence image of cPD-L1-expressing cells.

[0032] Figure 2E Is a kinetic graph showing the quantitative binding of PD-1 protein at each 3-hour time point to BT549 cells expressing cPD-L1 after addition of the cPD-L1 antibody. Positive clones blocking the PD-L1 / PD-1 protein interaction are highlighted in red.

[0033] Figure 2F Is a representative image of cPD-L1 blocking (at 18 hours), showing a merged green fluorescence image of cPD-L1-expressing cells. Note that there is no fluorescence because the antibody binds to PD-L1 and blocks the interaction with cPD-1.

[0034] Figure 3A Is a schematic diagram depicting the knock-in strategy for mice (c57BL / c background) containing canine PD-L1 and PD-1 molecules.

[0035] Figure 3B Is in MB49cPD-L1 Verification of cPD-L1 protein expression in cells, showing flow cytometry analysis of mPD-L1 and cPD-L1 proteins located on the membrane in MB49 cells expressing cPD-L1 (MB49 cPD-L1 ) or MB49 parental cells.

[0036] Figure 3C Showing immunofluorescence staining and protein expression patterns of mPD-L1 and cPD-L1 in MB49 or MB49 tumor masses from humanized PD-L1 mice (DAPI for nuclear counterstaining; scale bar 100 μM). cPD-L1 Showing immunofluorescence staining and protein expression patterns of mPD-L1 and cPD-L1 in MB49 or MB49 tumor masses from humanized PD-L1 mice (DAPI for nuclear counterstaining; scale bar 100 μM).

[0037] Figure 3D Showing graphs indicating the interaction of canine PD-1 (cPD-1) or murine PD-1 (mPD-1) proteins with cPD-L1 or murine PD-L1 (mPD-L1) proteins in the presence or absence of canine PD-L1 antibody (12C). His-tagged canine or mPD-L1 proteins were immobilized on nickel-nitrilotriacetic acid (Ni-NTA) 96-well plates, and horseradish peroxidase (HRP)-conjugated anti-human IgG Fc-specific secondary antibody and mPD-1-hFc or cPD-1-hFc proteins were added. OD was measured 450 to quantify the amount of bound PD-1 protein.

[0038] Figure 3E Showing the binding of cPD-L1 antibodies 12C and 3C to human PD-L1 (hPD-L1), mPD-L1, and cPD-L1 proteins. His-tagged human PD-L1, mPD-L1, or cPD-L1 proteins were immobilized on Ni-NTA 96-well plates, anti-cPD-L1 Ab (12C or 3C) was added, and HRP-conjugated anti-canine IgG specific secondary antibody. OD was measured 450 to quantify the amount of bound PD-L1 antibody (Ab = antibody).

[0039] Figure 3F Showing data related to tumor growth of MB49 in humanized PD-L1 mice treated with cPD-L1 antibodies 12C or 3C. The IgG isotype of 12C and 3C antibodies is murine IgG1, which is equivalent to human IgG4. Tumors were measured at the indicated time points and dissection analysis was performed at the end point (n = 8 per group). cPD-L1 Showing data related to tumor growth of MB49 in humanized PD-L1 mice treated with cPD-L1 antibodies 12C or 3C. The IgG isotype of 12C and 3C antibodies is murine IgG1, which is equivalent to human IgG4. Tumors were measured at the indicated time points and dissection analysis was performed at the end point (n = 8 per group).

[0040] Figure 3G Showing representative images of immunofluorescence staining of protein expression patterns of CD8 and granzyme B in MB49 tumor masses from mice treated with IgG, 12C, or 3C. DAPI was used for nuclear counterstaining. Scale bar, 100 μm.

[0041] Figure 3H and 3I shows the quantification of CD8( Figure 3H ) and granzyme B( Figure 3I ) in immunofluorescence staining and protein expression patterns using Gen5 software (BioTek, Winooski, VT). n = 10.

[0042] Figure 3J and 3K are graphs representing the treatment efficacy on PD-L1 mice, measuring the functions of mouse kidneys( Figure 3J ) and livers( Figure 3K ) at the end of the experiment. ALT, alanine aminotransferase. Treatment with the PD-L1 antibody does not affect renal function (serum creatinine) or hepatic enzyme activity (ALT = alanine aminotransferase), which was measured in the blood collected at the end of the experiment.

[0043] Figures 4A - 4F shows the quality attributes of the purified anti-cPD-L1, 1210E4 (12C) chimeric antibody, where Figure 4A shows a schematic diagram of the cPD-L1, 12C10E4 chimeric antibody expression construct pTRIOZ-cIgG2-cPD-L1 12C10E4; Figure 4B shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purity of the 12C chimeric antibody under non-reducing and reducing (2-mercaptoethanol) conditions (HC, heavy chain; LC, light chain; SM, protein size marker); Figure 4C shows the isoelectric focusing (IEF) analysis of the 12C chimeric antibody (standard, pI standard); Figure 4D shows the peptide mapping analysis of the cPD-L1 12C chimeric antibody (SEQ ID NO:14); after reduction and alkylation, the 12C chimeric antibody was enzymatically digested with trypsin on an S-trap microcolumn from Protifi. The peptides were then separated and analyzed by reversed-phase liquid chromatography tandem mass spectrometry (RP-LC / MS-MS). The resulting mass spectrometry data was analyzed using the PEAK PTM workflow in the PEAKS X PRO Studio 10.6 software package from Bioinformatics Solutions Inc. to map the detected MS1 and MS2 ions to the amino acid sequence of the antibody. The sequence coverages of the heavy chain and light chain were 100% (453 out of 453 amino acids) and 98.2% (223 out of 227 amino acids), respectively; Figure 4EShows the size exclusion chromatography (SEC) analysis (standard, SEC standard) of the 12C chimeric antibody; and Figure 4F Shows the matrix-assisted laser desorption / ionization (MALDI)-mass spectrometry (MS) profile of the fully methylated N-glycans released from the PNGase F-treated 12C chimeric antibody. The mass of the indicated glycan species represents the [M+Na+] value.

[0044] Figure 5A Shows the 12C antibody cPD-L1 bound to BT549

[0045] Figure 5B Shows the flow cytometry analysis of 12C chimeric antibody in BT549 cPD-L1 cells, where cIgG was used as a negative control.

[0046] Figure 5C Shows the 12C chimeric antibody that binds to cPD-L1 and cPD-L2.

[0047] Figure 5D Shows the binding affinity (K D ) analysis of the 12C chimeric antibody by Octet.

[0048] Figure 5E Shows the EC 50 of the 12C chimeric antibody 12C10E4. 50 EC = 0.419 μg / ml, measured by quantifying the bound cPD-1 protein by measuring green fluorescence in the S3 live cell analysis system (Sartorius AG, Gottingen, Germany).

[0049] Figure 5F and 5G Shows an image related to data from a canine IO assay panel (NanoString) analysis used to query changes in gene expression after activation of canine peripheral blood mononuclear cells (cPBMCs) from three healthy pet dogs. RNA from resting and activated PBMCs was used for NanoString work. The canine IO assay panel was used to query changes in approximately 700 genes. Groupwise analysis was performed using "Rosalind". When comparing control PBMCs with activated PBMCs, 65 genes were differentially expressed (P<0.05, FC>1.5), including 30 upregulated and 35 downregulated genes. In the heatmap, each column consists of data from one sample.

[0050] Figure 5H and 5IData related to the concentrations of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in activated cPBMCs are shown.

[0051] Figure 5J and 5K are flow cytometry analysis data of cPD-L1 protein expression on K9TCC or K9TCC cells expressing nuclear-restricted RFP (K9TCC nRFP ). Endogenous PD-L1 expression was stimulated with 50 ng / mL canine IFNγ for 12 hours. cIgG was used as a negative control.

[0052] Figure 5L Data from quantitative reverse transcription-polymerase chain reaction (RT-PCR) of cPD-L1 (CD274) mRNA expression in K9TCC or K9TCC nRFP cells are shown.

[0053] Figure 5M are image and graphical data related to K9TCC cells co-cultured with cPBMCs activated with CD3 antibody (100 ng / mL) and interleukin-2 (IL-2) (10 ng / mL) at a ratio of 1 tumor cell:15 cPBMCs. The viable tumor cell count at 72 hours is shown in the bar graph (right). The 12C chimeric antibody enhanced tumor cell killing.

[0054] Figure 5N Data related to the analysis of IFNγ concentration in the co-culture medium of K9TCC cells and activated cPBMCs with and without 12C chimeric antibody treatment are shown.

[0055] Figure 6A A schematic diagram of an enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic analysis is shown.

[0056] Figure 6B is a graph of the 12C chimeric antibody concentration measured in the serum of dogs treated with 2 mg / kg of the 12C antibody.

[0057] Figure 6C is a graph of the 12C chimeric antibody concentration measured in the serum of dogs treated with 5 mg / kg of the 12C antibody.

[0058] Although the present disclosure is susceptible to various modifications and alternative forms, its exemplary embodiments are shown by way of example in the drawings and are described in detail herein.

[0059] Sequence Listing

[0060] The sequences of this article (SEQ ID NO: 1-26) are also provided in computer-readable form, encoded in the file submitted herewith, and incorporated herein by reference. According to 37 C.F.R.§1.821(f), the information recorded in computer-readable form is the same as the written sequence listing provided herein.

[0061] SEQ ID NO: 1 is the amino acid sequence (inferred from the DNA sequence) of the cPD-L1_12C10E4_dK light chain of the cPD-L1 12C10E4 antibody:

[0062] MESDTLLLWVLLLWVPGSAG DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWYQQKPGQPPKLLIFAASNVKSGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSGKVPHTFGGGTKLEIKR SMEIKRTDAQPA VYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHEL YSCEITHKSLPSTLIKSFQRSECQRVD *,

[0063] wherein, Underline , signal sequence; italic, FR; bold, CDR; Bold and Underline , CL; * stop; CDR1: ESVEYYGTSL (SEQ ID NO: 9); CDR2: AAS; and CDR3: CQQSGKVPHTF (SEQ ID NO: 10).

[0064] SEQ ID NO:2 is the nucleotide sequence of the cPD-L1_12C10E4_dK light chain of the cPD-L1 12C10E4 antibody (and encodes SEQ ID NO:1) (codons optimized for CHO cells): ATGGAGAGCGACACCCTCCTGCTGTGGGTGCTGCTACTGTGGGTTCCTGGGAGCGCGGGAGACATCGTGCTGACACAGAGCCCTGCAAGCCTGGCCGTGAGCCTGGGACAGAGAGCCACCATCAGCTGCAGAGCAAGCGAGAGCGTGGAGTACTACGGCACAAGCCTGATGCAGTGGTATCAGCAGAAGCCTGGACAGCCTCCTAAGCTGCTGATCTTCGCCGCAAGCAACGTGAAGAGCGGCGTGCCTGCTAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCAGCCTGAACATCCACCCTGTGGAGGAGGACGACATCGCCATGTACTTCTGTCAGCAGAGCGGCAAGGTGCCTCACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGAtCCATGGAAATCAAACGTACGGATGCCCAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGTTGTGTGTTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTGTCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGCCTCAGCAGCACCCTGACGATGTCCAGTACTGAGTACCTAAGTCATGAGTTGTACTCCTGTGAGATCACTCACAAGAGCCTGCCCTCCACCCTCATCAAGAGCTTCCAAAGGAGCGAGTGTCAGAGAGTGGACTAA。

[0065] SEQ ID NO:3 is the amino acid sequence of the cPD-L1_12C10E4_dIgG2 heavy chain of the cPD-L1 12C10E4 antibody (deduced from the DNA sequence): Wherein, Underline , the signal sequence; FR; CDR; CH; *, stop; CDR1: (SEQ ID NO:15); CDR2: (SEQ ID NO:16); and CDR3: (SEQ ID NO:11).

[0066]

[0067] SEQ ID NO:5 is the amino acid sequence of the cPD-L1_3C8D3_dK light chain of the cPD-L1 3C chimeric antibody clone: wherein, Underline , signal sequence; FR; CDR; CL; *, stop; CDR1: (SEQ ID NO:17); CDR2: and CDR3: (SEQ ID NO:12).

[0068] SEQ ID NO:6 is the nucleotide sequence of the cPD-L1_3C8D3_dK light chain of the cPD-L1 3C chimeric antibody clone (encoding SEQ ID NO:5): ATGGAGACACATTCTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGAAGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGTTAGTCAGGATGTGGGTACCGCTGTAGCCTGGTATCAACAGAAACCAGGGCAATGTCCCAAAAGACTGATTTACTGGACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAGTCTGAAGACTTGGTAGATTATTTCTGTCAGCAATATAGCAGTTATCCTCTCACGTTCGGTGGTGGGACCAAGCTGGAGCTGAAACGGTCCATGGAAATCAAACGTACGGATGCCCAGCCAGCCGTCTATTTGTTCCAACCATCTCCAGACCAGTTACACACAGGAAGTGCCTCTGTTGTGTGTTTGCTGAATAGCTTCTACCCCAAAGACATCAATGTCAAGTGGAAAGTGGATGGTGTCATCCAAGACACAGGCATCCAGGAAAGTGTCACAGAGCAGGACAAGGACAGTACCTACAGCCTCAGCAGCACCCTGACGATGTCCAGTACTGAGTACCTAAGTCATGAGTTGTACTCCTGTGAGATCACTCACAAGAGCCTGCCCTCCACCCTCATCAAGAGCTTCCAAAGGAGCGAGTGTCAGAGAGTGGACTAA。

[0069] SEQ ID NO:7 is the amino acid sequence of the cPD-L1_3C8D3_dIgG2 heavy chain of the cPD-L1 3C chimeric antibody clone: Among them, Underline , the signal sequence; FR; CDR; CL; *, stop; CDR1: (SEQ ID NO:18); CDR2: (SEQ ID NO:19); and CDR3: (SEQ ID NO:13).

[0070]

[0071] SEQ ID NO:9 is the amino acid sequence of CDR1: ESVEYYGTSL.

[0072] SEQ ID NO:10 is the amino acid sequence of CDR3: CQQSGKVPHTF.

[0073] SEQ ID NO:11 is the amino acid sequence of CDR3: ARGGGPDWYFDV.

[0074] SEQ ID NO:12 is the amino acid sequence of CDR3: CQQYSSYPLTF.

[0075] SEQ ID NO:13 is the amino acid sequence of CDR3: CARSDYSNYVGFAYW.

[0076] SEQ ID NO:14 is part of the amino acid sequence of the cPD-L1 12C chi antibody clone: GDTFICAVMHEALHNHYTQK.

[0077] SEQ ID NO:15 is the amino acid sequence of CDR1: GFSLTSFG.

[0078] SEQ ID NO:16 is the amino acid sequence of CDR2: IWSGGST.

[0079] SEQ ID NO:17 is the amino acid sequence of CDR1: QDVGTA.

[0080] SEQ ID NO:18 is the amino acid sequence of CDR1: GYTFTDYV.

[0081] SEQ ID NO:19 is the amino acid sequence of CDR2: INPSNGDT.

[0082] SEQ ID NO:20 is the nucleotide sequence of the pre-assembled guide RNA related to the full length of canine CD274 cDNA (NM_001291972): CAGCAAATATCCTCATGTTTTGG.

[0083] SEQ ID NO:21 is the nucleotide sequence of the forward primer of primer set 1 described herein: CCACTTGGTTCTACATGGCT.

[0084] SEQ ID NO:22 is the nucleotide sequence of the reverse primer of primer set 1 described herein: CCTCAGCCTGACACATTAGTT.

[0085] SEQ ID NO:23 is the nucleotide sequence of the forward primer of primer set 2 described herein: CCTGTCACCTCTGAACATGAA.

[0086] SEQ ID NO:24 is the nucleotide sequence of the reverse primer of primer set 2 described herein: GGACTAAGCTCTAGGTTGTCC.

[0087] SEQ ID NO:25 is the nucleotide sequence of the forward primer of primer set 3 described herein: GACTGGCTTTTAGGGCTTATGT.

[0088] SEQ ID NO:26 is the nucleotide sequence of the reverse primer of primer set 3 described herein: ACACCCCACAAATTACTTCCATT. Detailed Description

[0089] To facilitate understanding of the principles of the present disclosure, embodiments shown in the accompanying drawings will now be referred to, and these embodiments will be described using specific language. However, it should be understood that the description of these embodiments is not intended to limit the scope. On the contrary, the present disclosure aims to cover substitutions, modifications, and equivalents that may be included within the spirit and scope of the present application as defined by the appended claims. As previously mentioned, although such technology may be illustrated and described in one or more preferred embodiments, its compositions, compounds, and methods may include many different configurations, forms, materials, and accessories.

[0090] Novel canine programmed death ligand 1 (cPD-L1) antibodies and cPD-L1 antigen-binding fragments are provided. Unless otherwise specified, the term "canine" includes all domestic dogs, Canis lupus familiaris, or Canis familiaris. For example, these antibodies and antigen-binding fragments can be used as immuno-oncology drugs. When administered, these antibodies and antigen-binding fragments can increase tumor cell killing activity in a subject. In certain embodiments, the antibodies and their antigen-binding fragments can inhibit the immunosuppressive function of cPD-L1 in the presence of cancer cells. Given that the expression of PD-L1 on T cells and natural killer (NK) cells has been reported, and that PD-L1 on cancer cells is blocked by antibodies and / or their antigen-binding fragments, the direct effect of PD-L1 on T cells or other immune cells, in addition to targeting PD-L1 to cancer cells, can enhance the efficacy of the antibodies and their antigen-binding fragments when administered to a subject.

[0091] Antibodies and Complementary Determining Regions (CDRs)

[0092] Canine programmed death protein 1 (PD-1) / PD-L1 blocking antibodies cannot be widely used in dogs with invasive urothelial carcinoma (InvUC). Tumor regression in dogs with oral melanoma and soft tissue sarcoma has been reported to respond to a canine chimeric monoclonal antibody targeting PD-L1. However, the anti-tumor effect of this antibody is still uncertain because the role of combination drugs in tumor regression is not clear (Maekawa et al., A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma, Sci Rep 7:8951 (2017); Knapp et al., Phase I trial of piroxicam in 62 dogs bearing naturally occurring tumors, Cancer Chemother Pharmacol 29, 214-218 (1992); and Nemoto et al., Development and characterization of monoclonal antibodies against canine PD-1 and PD-L1, Vet Immunol Immunopathol 198:19-25 (2018)). In other work, anti-canine PD-1 and PD-L1 antibodies have been developed for diagnostic applications but have not been therapeutically tested (Choi et al., Development of canine PD-1 / PD-L1 specific monoclonal antibodies and amplification of canine T cell function, PLoS One 15:e0235518 (2020)). Therefore, canine immune checkpoint blocking antibodies or anti-canine PD-L1 antibodies generally cannot be used in translational research or treatment of dogs.

[0093] Provided is an immunotherapeutic PD-L1 antibody (or antigen-binding fragment thereof). As used herein, the term "antibody" refers to any form of immunoglobulin that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and single-domain antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen prior to modifying the antibody for the intended use, such as humanizing the antibody for use as a human therapeutic antibody.

[0094] The variable regions of each light / heavy chain pair form the antibody binding site. Thus, generally, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are usually identical. Generally, the variable domains of the heavy and light chains include three hypervariable regions, also known as CDRs, which are located between relatively conserved variable framework regions (FRs). The CDRs are typically flanked by FRs, enabling binding to a specific epitope. Generally, from the N-terminus to the C-terminus, the light and heavy chain variable domains both include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0095] As used herein, unless otherwise specified, an "antibody fragment" or "antibody-binding fragment" refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to bind antigen specifically with the same antigen specificity as the full-length antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., sc-Fv); nanobodies; and multispecific antibodies formed from antibody fragments. A "Fab fragment" consists of a light chain constant region, a light chain variable region, a CH1 domain of one heavy chain, and a variable domain. A "Fab fragment" can be the product of papain cleavage of an antibody. The "fragment crystallizable" (Fc) region is the tail region of an antibody that interacts with cell surface receptors and allows the antibody to deactivate the immune system. The Fc region contains at least two heavy chain fragments including the CH2 and CH3 domains of the antibody (i.e., two identical polypeptides). The two heavy chain fragments can be held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0096] A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain that includes the variable heavy (VH) domain and the CH1 domain, and also includes the region between the CH1 and CH2 domains such that interchain disulfide bonds can form between the two heavy chains and the two Fab' fragments to form an F(ab')2 molecule.

[0097] In certain embodiments, the PD-L1 antibody (or antigen-binding fragment thereof) is a chimeric form of a canine antibody (e.g., monoclonal) or antigen-binding fragment thereof that specifically binds to PD-L1 in a canine subject (e.g., has a high binding affinity for PD-L1 in a canine subject). As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, wherein the first and second antibodies are from different species. For example, the gene variable segments (e.g., framework and CDR portions) from, e.g., a murine antibody (e.g., monoclonal or polyclonal antibody) can be used in combination with a canine or human constant segment to produce a chimeric antibody. Thus, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a murine antibody (e.g., one or more CDRs of a murine antibody) and the constant or effector domain from a human or canine antibody, although other mammalian species can also be used. A chimeric antibody can also include amino acid sequences obtained from protein sources other than antibodies.

[0098] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to canine PD-L1. As used herein, an antibody or antigen-binding fragment thereof "specifically" binds to a polypeptide comprising a portion of the amino acid sequence of a canine antigen, e.g., cPD-L1, if it binds to the polypeptide but does not bind to other canine proteins lacking the sequence portion of the canine antigen. For example, an antibody that specifically binds to a polypeptide comprising canine PD-L1 can bind to a FLAG-tagged form of canine PD-L1 but will not specifically bind to other FLAG-tagged canine proteins. An antibody or binding compound derived from the antigen-binding site of an antibody "specifically" binds to its canine antigen (or variant or mutant protein thereof) when its affinity for its canine antigen or variant or mutant protein is at least ten-fold, at least fifteen-fold, at least twenty-fold, or at least one hundred-fold higher than its affinity for any other tested canine antigen.

[0099] The cPD-L1 antibody can include the amino acid sequence of SEQ ID NO:1 and / or 3, or SEQ ID NO:5 and / or 7. cPD-L1 is encoded by a nucleotide sequence comprising SEQ ID NO:2 and / or 4, or SEQ ID NO:6 and / or 8.

[0100] In certain embodiments, the antibody (or antigen-binding fragment thereof) is chimerized. In certain embodiments, the antibody (or antigen-binding fragment thereof) is fully chimerized. In some embodiments, the antibody (or antigen-binding fragment thereof) is partially chimerized. As used herein, the term "chimeric antibody" refers to an antibody form that contains sequences from both canine and non-canine (e.g., murine or rat) antibodies. Generally, a chimeric antibody may include substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-canine immunoglobulin (e.g., including 6 murine anti-canine PD-L1 CDRs, as illustrated below), and all or substantially all of the parental frameworks. A chimeric antibody may also refer to an antibody in which a point mutation has changed one amino acid to reflect the amino acid typically found at that position in a dog, rather than the amino acid typically found at that position in a mouse or rat species.

[0101] Chimerized regions are amino acids where the amino acid identity at selected positions changes from the original (i.e., rat or mouse) amino acid to an amino acid that more closely reflects the common canine amino acid at those positions. These changes are made to prevent or reduce the host anti-antibody response during or after treatment. These unwanted anti-antibody responses are typically found when the host (in this case, the dog) detects an unusual amino acid identity at critical positions. As a non-limiting example, a dog may have an unwanted immune response to lysine (Lys29) found at position 29 because dog antibodies rarely, if ever, have lysine at position 29. The chimerization process can then result in changing lysine 29 to alanine (Lys29Ala) in an attempt to avoid a strong immune response to the antibody, which may have severe side effects. The most common chimerization changes are located in the variable domains of therapeutic antibodies, although chimerization changes in one or more other antibody regions may also be required. The anti-PD-L1 antibody (or antigen-binding fragment thereof) may include a chimerized murine PD-L1 antibody or antigen-binding fragment thereof.

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof includes a chimeric antibody, wherein all or a portion of the murine (or other mammalian) CDR sequences are replaced with one or more corresponding canine CDR sequences or portions thereof. The antibody or antigen-binding fragment thereof may include a fusion of one or more variable regions of murine DNA with canine DNA constant regions. In certain embodiments, the chimeric anti-canine PD-L1 antibody is a chimerized mammalian (e.g., murine) anti-canine PD-L1 antibody.

[0103] CDRs are those portions of the variable regions of immunoglobulins (i.e., antibodies) and T cell receptors that are involved in the binding of specific antigens, epitopes, or peptides. For example, there are three non - contiguous CDRs (CDR1, CDR2, and CDR3) on the amino acid sequence of the variable domain of a complete antigen receptor. Since an antigen receptor typically consists of two variable domains (on two different chains - heavy and light chains), each antigen receptor has six CDRs that can together contact the antigen.

[0104] An antibody (or an antigen - binding fragment thereof) can include one or more genetically modified CDRs. In certain embodiments, a CDR (e.g., a CDR of an antibody or an antigen - binding fragment thereof) includes AAS, SEQ ID NO:9 and / or SEQ ID NO:10, or has at least 80% or about 80% - 100% (such as about 80% to 100%, 80% to about 100%, or 80% - 100%) sequence identity with AAS, SEQ ID NO:9 and / or SEQ ID NO:10. In certain embodiments, a CDR includes at least 80% or about 85% - 95% (such as about 85% to 95%, 85% to about 95%, or 85% - 95%) sequence identity with AAS, SEQ ID NO:9 and / or SEQ ID NO:10. In certain embodiments, a CDR includes about 87% - 93% (such as about 87% to 93%, 87% to about 93%, or 87% - 93%) sequence identity with AAS, SEQ ID NO:9 and / or SEQ ID NO:10. In certain embodiments, a CDR includes about 90% (such as 90%) sequence identity with AAS, SEQ ID NO:9 and / or SEQ ID NO:10. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, a CDR (e.g., a CDR of an antibody or an antigen - binding fragment thereof) includes AAS, SEQ ID NO:9 and / or SEQ ID NO:10 or a sequence 90%, 95%, 98%, or 99% identical to the sequence of AAS, SEQ ID NO:9 and / or SEQ ID NO:10. In certain embodiments, a CDR includes a sequence substantially identical to AAS, SEQ ID NO:9 and / or SEQ ID NO:10.

[0105] In certain embodiments, the CDR comprises SEQ ID NO:17, WTS, and / or SEQ ID NO:12, or has at least about 80%, or about 80%-100% (such as about 80% to 100%, 80% to about 100%, or 80%-100%) sequence identity (or is substantially the same) with SEQ ID NO:17, WTS, and / or SEQ ID NO:12. In certain embodiments, the CDR comprises a sequence identity between about 85%-95% (such as about 85% to 95%, 85% to about 95%, or 85%-95%) with SEQ ID NO:17, WTS, and / or SEQ ID NO:12. In certain embodiments, the CDR comprises a sequence identity between about 87%-93% (such as about 87% to 93%, 87% to about 93%, or 87%-93%) with SEQ ID NO:17, WTS, and / or SEQ ID NO:12. In certain embodiments, the CDR comprises a sequence identity of about 90% (such as 90%) with SEQ ID NO:17, WTS, and / or SEQ ID NO:12. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the CDR (e.g., the CDR of an antibody or an antigen-binding fragment thereof) comprises SEQ ID NO:17, WTS, and / or SEQ ID NO:12 or a sequence 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:17, WTS, and / or SEQ ID NO:12.

[0106] In certain embodiments, the CDR comprises SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11, or has at least about 80%, or has a sequence identity (or is substantially identical) of about 80% - 100% (such as about 80% to 100%, 80% to about 100%, or 80% - 100%) with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11. In certain embodiments, the CDR comprises a sequence identity between about 85% - 95% (such as about 85% to 95%, 85% to about 95%, or 85% - 95%) with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11. In certain embodiments, the CDR comprises a sequence identity between about 87% - 93% (such as about 87% to 93%, 87% to about 93%, or 87% - 93%) with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11. In certain embodiments, the CDR comprises a sequence identity of about 90% (such as 90%) with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the CDR (e.g., the CDR of an antibody or an antigen-binding fragment thereof) comprises SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11 or a sequence that is 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11.

[0107] In certain embodiments, the CDR comprises SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13, or has at least about 80%, or has a sequence identity (or is substantially identical) of about 80% - 100% (such as about 80% to 100%, 80% to about 100%, or 80% - 100%) with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13. In certain embodiments, the CDR has a sequence identity between about 85% - 95% (such as about 85% to 95%, 85% to about 95%, or 85% - 95%) with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13. In certain embodiments, the CDR has a sequence identity between about 87% - 93% (such as about 87% to 93%, 87% to about 93%, or 87% - 93%) with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13. In certain embodiments, the CDR has a sequence identity of about 90% (such as 90%) with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the CDR (e.g., the CDR of an antibody or an antigen-binding fragment thereof) comprises SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13 or a sequence that is 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13.

[0108] In certain embodiments, the anti-cPD-L1 antibody or antigen-binding fragment can include one or more CDRs that independently include at least 80% sequence identity to AAS, SEQ ID NO:9, and / or SEQ ID NO:10. In certain embodiments, the anti-cPD-L1 antibody or antigen-binding fragment can include one or more CDRs that independently include at least about 80% sequence identity to SEQ ID NO:17, WTS, and / or SEQ ID NO:12. In certain embodiments, the cPD-L1 antibody or antigen-binding fragment can include one or more CDRs that independently include at least about 80% sequence identity to SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:11. In certain embodiments, the cPD-L1 antibody or antigen-binding fragment can include one or more CDRs that independently include at least about 80% sequence identity to SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:13.

[0109] In certain embodiments, the antibodies (or antigen-binding fragments thereof) herein include two or more of the CDRs described herein.

[0110] In certain embodiments, the antibodies herein are monoclonal antibodies. In certain embodiments, the monoclonal antibody is a murine antibody. In certain embodiments, the monoclonal antibody is a chimeric antibody. In certain embodiments, the monoclonal antibodies herein are chimeric murine antibodies. The antibodies (and / or their antigen-binding fragments) can be isolated antibodies (or isolated antigen-binding fragments). An "isolated antibody" or "isolated antigen-binding fragment" refers to a purified state and in this context means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium. Generally, unless otherwise stated, the term "isolated" does not mean the complete absence of such substances or the absence of water, buffer, or salt, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compounds described herein.

[0111] In certain embodiments, the antibody is a recombinant antibody or antigen-binding fragment. In certain embodiments, the heavy chain variable domain and the light chain variable domain are linked by a flexible linker to form a single-chain antibody.

[0112] The antibody or antigen-binding fragment can be a Fab fragment. The antibody or antigen-binding fragment can be a Fab' fragment. The antibody or antigen-binding fragment can be an F(ab')2 molecule. In certain embodiments, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody.

[0113] Also provided are nucleic acids (including isolated nucleic acids) encoding any antibody, fragment, and / or portion thereof (including CDRs). In certain embodiments, nucleic acids encoding any light chain or humanized antibody or portion thereof are provided. Similarly, nucleic acids encoding any heavy chain or humanized antibody or portion thereof are provided.

[0114] The nucleic acid sequence encoding the antibody herein can include SEQ ID NO:2. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes at least about 80% sequence identity with SEQ ID NO:2, or has a sequence identity of about 80% - 100% (such as about 80% to 100%, 80% to about 100%, or 80% - 100%) (or is substantially the same) with SEQ ID NO:2. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity between about 85% - 95% (such as about 85% to 95%, 85% to about 95%, or 85% - 95%) with SEQ ID NO:2. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity of 87% - 93% (such as about 87% to 93%, 87% to about 93%, or 87% - 93%) with SEQ ID NO:2. The nucleic acid sequence encoding the antibody herein includes a sequence identity of about 90% (such as 90%) with SEQ ID NO:2. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:2 or a sequence 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:2.

[0115] The nucleic acid sequence encoding the antibody herein may include SEQ ID NO:4. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes at least about 80% sequence identity with SEQ ID NO:4, or has about 80%-100% (such as about 80% to 100%, 80% to about 100%, or 80%-100%) sequence identity (or is substantially the same) with SEQ ID NO:4. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity between about 85%-95% (such as about 85% to 95%, 85% to about 95%, or 85%-95%) with SEQ ID NO:4. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity of 87%-93% (such as about 87% to 93%, 87% to about 93%, or 87%-93%) with SEQ ID NO:4. The nucleic acid sequence encoding the antibody herein includes a sequence identity of about 90% (such as 90%) with SEQ ID NO:4. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:4 or a sequence 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO:4.

[0116] In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:2 and 4.

[0117] In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:6, has at least 80% sequence identity with SEQ ID NO:6, or has about 80%-100% (such as about 80% to 100%, 80% to about 100%, or 80%-100%) sequence identity (or is substantially the same) with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity between about 85%-95% (such as about 85% to 95%, 85% to about 95%, or 85%-95%) with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity of 87%-93% (such as about 87% to 93%, 87% to about 93%, or 87%-93%) with SEQ ID NO:6. The nucleic acid sequence encoding the antibody herein includes a sequence identity of about 90% (such as 90%) with SEQ ID NO:6. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ IDNO:6 or a sequence 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO:6.

[0118] In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:8, has at least 80% sequence identity with SEQ ID NO:8, or has a sequence identity of about 80% - 100% (such as about 80% to 100%, 80% to about 100%, or 80% - 100%) (or is substantially the same). In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity between about 85% - 95% (such as about 85% to 95%, 85% to about 95%, or 85% - 95%) with SEQ ID NO:8. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes a sequence identity of 87% - 93% (such as about 87% to 93%, 87% to about 93%, or 87% - 93%) with SEQ ID NO:8. The nucleic acid sequence encoding the antibody herein includes a sequence identity of about 90% (such as 90%) with SEQ ID NO:8. The ranges specified in this paragraph include the stated endpoints and all 1% increments therebetween. In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:8 or a sequence 90%, 95%, 98% or 99% identical to the sequence of SEQ ID NO:8.

[0119] In certain embodiments, the nucleic acid sequence encoding the antibody herein includes SEQ ID NO:6 and 8.

[0120] In certain embodiments, an antibody (or an antigen-binding fragment thereof) of canine PD-L1 includes one or more CDRs described herein and / or binds to the amino acid sequence of PD-L1. In certain embodiments, the dissociation constant (K D ) of the canine antibody - canine PD-L1 binding is between about 4.0 nmol / L and 10.0 nmol / L (such as 4.0 nmol / L to about 10.0 nmol / L, about 4.0 nmol / L to about 10.0 nmol / L, or about 4.0 nmol / L to 10.0 nmol / L). In certain embodiments, the antibody (e.g., canine antibody) or their antigen-binding fragments have a K D for binding to canine PD-L1 between about 4.2 nmol / L and 9.8 nmol / L (such as 4.2 nmol / L to about 9.8 nmol / L, about 4.2 nmol / L to about 9.8 nmol / L, or about 4.2 nmol / L to 9.8 nmol / L) or therebetween. In certain embodiments, the antibody (e.g., canine antibody) or their antigen-binding fragments have a K DBinds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of from about 4.6 nmol / L to 9.4 nmol / L (such as from 4.6 nmol / L to about 9.4 nmol / L, from about 4.6 nmol / L to about 9.4 nmol / L, or from about 4.6 nmol / L to 9.4 nmol / L) or between them D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of from about 4.8 nmol / L to 9.2 nmol / L (such as from 4.8 nmol / L to about 9.2 nmol / L, from about 4.8 nmol / L to about 9.2 nmol / L, or from about 4.8 nmol / L to 9.2 nmol / L) or between them D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of from about 5.0 nmol / L to 9.0 nmol / L (such as from 5.0 nmol / L to about 9.0 nmol / L, from about 5.0 nmol / L to about 9.0 nmol / L, or from about 5.0 nmol / L to 9.0 nmol / L) or between them D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of from about 5.2 nmol / L to 8.8 nmol / L (such as from 5.2 nmol / L to about 8.8 nmol / L, from about 5.2 nmol / L to about 8.8 nmol / L, or from about 5.2 nmol / L to 8.8 nmol / L) or between them D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of from about 5.4 nmol / L to 8.6 nmol / L (such as from 5.4 nmol / L to about 8.6 nmol / L, from about 5.4 nmol / L to about 8.6 nmol / L, or from about 5.4 nmol / L to 8.6 nmol / L) or between them D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of 8.6 nmol / L D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine - humanized antibody) or an antigen - binding fragment thereof has a K of from about 5.6 nmol / L to 8.4 nmol / L (such as from 5.6 nmol / L to about 8.4 nmol / L, from about 5.6 nmol / L to about 8.4 nmol / L, or from about 5.6 nmol / L to 8.4 nmol / L) or between them DBinds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine antibody) or an antigen-binding fragment thereof has a K between about 5.8 nmol / L and 8.2 nmol / L (such as 5.8 nmol / L to about 8.2 nmol / L, about 5.8 nmol / L to about 8.2 nmol / L, or about 5.8 nmol / L to 8.2 nmol / L) or thereabouts D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine antibody) or an antigen-binding fragment thereof has a K between about 6.0 nmol / L and 8.0 nmol / L (such as 6.0 nmol / L to about 8.0 nmol / L, about 6.0 nmol / L to about 8.0 nmol / L, or about 6.0 nmol / L to 8.0 nmol / L) or thereabouts D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine antibody) or an antigen-binding fragment thereof has a K between about 6.2 nmol / L and 7.8 nmol / L (such as 6.2 nmol / L to about 7.8 nmol / L, about 6.2 nmol / L to about 7.8 nmol / L, or about 6.2 nmol / L to 7.8 nmol / L) or thereabouts D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine antibody) or an antigen-binding fragment thereof has a K between about 6.4 nmol / L and 7.6 nmol / L (such as 6.4 nmol / L to about 7.6 nmol / L, about 6.4 nmol / L to about 7.6 nmol / L, or about 6.4 nmol / L to 7.6 nmol / L) or thereabouts D Binds to canine PD-L1. In certain embodiments, the antibody (e.g., a canine antibody) or an antigen-binding fragment thereof has a K between about 6.6 nmol / L and 7.4 nmol / L (such as 6.6 nmol / L to about 7.4 nmol / L, about 6.6 nmol / L to about 7.4 nmol / L, or about 6.6 nmol / L to 7.4 nmol / L) or thereabouts D Binds to canine PD-L1. The ranges specified in this paragraph include the stated endpoints and all 0.1 increments therebetween

[0121] Antibodies or their antigen-binding fragments can stimulate an antigen-specific memory response against tumors or pathogens. In certain embodiments, the antibodies or their antigen-binding fragments can stimulate an antibody response in a subject. In certain embodiments, the antibodies or their antigen-binding fragments can stimulate an immune response in an animal subject (e.g., a canine animal subject). The term "immune response" refers to, for example, the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which can lead to the selective damage, destruction, or clearance of cancer cells, cells or tissues infected with an invading pathogen, or pathogens in a mammalian body (e.g., a canine body).

[0122] In certain embodiments, the antibodies or their antigen-binding fragments can bind to canine PD-L1 and also block the binding of canine PD-L1 to PD-1. In certain embodiments, the canine antibodies and their antigen-binding fragments herein can bind to canine PD-L1 and block the binding of canine PD-L1 to PD-1.

[0123] Antibodies or antigen-binding fragments can be used to prepare a medicament for treating cancer in a canine subject. The antibody (or its antigen-binding fragment) can be administered in the form of a unit dosage form and / or composition containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles and combinations thereof. The term "administer" and its derivatives generally refer to any and all means of introducing the compounds described herein into a host subject, including but not limited to administration routes such as oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, oral, ocular, sublingual, vaginal, rectal, etc.

[0124] Alternatively, or in combination, any antibody or antibody fragment used herein is provided for diagnostic use. In certain embodiments, an expression vector comprising a isolated nucleic acid encoding any of the canine anti-canine PD-L1 antibodies or antigen-binding fragments herein is provided. A host cell comprising one or more of the expression vectors described herein is also provided. In certain embodiments, these nucleic acids, expression vectors, or polypeptides can be used in a method for preparing an antibody.

[0125] A pharmaceutical composition is also provided. The pharmaceutical composition can include one or more of the antibodies, antigen-binding fragments, antigenic peptides of canine PD-L1 (including isolated antigenic peptides), fusion proteins comprising canine PD-L1 antigenic peptides, nucleic acids encoding the antigenic fragments and / or fusion proteins herein, expression vectors comprising such nucleic acids, or any combination thereof, and a pharmaceutically acceptable carrier or diluent. The term "composition" generally refers to any product comprising more than one ingredient, including but not limited to an antibody or its antigen-binding fragment. In certain embodiments, the pharmaceutical composition includes a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient.

[0126] The composition can be prepared from an isolated antibody or its antigen-binding fragment or from salts, solutions, hydrates, solvates, and other forms of the antibody or its antigen-binding fragment. The composition can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms of the antibody or its antigen-binding fragment, and the composition can be prepared from various hydrates and / or solvates of the compound.

[0127] The antibody or its antigen-binding fragment can be formulated into a pharmaceutical composition and administered to a mammalian host, such as a human or canine subject, in a variety of forms suitable for the selected route of administration. For example, the pharmaceutical composition can be formulated for and administered by oral or parenteral, intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, topical, inhaled, and / or subcutaneous routes. In fact, in at least one embodiment, the compound and / or composition can be administered directly into the bloodstream, muscle, or internal organ.

[0128] For example, in at least one embodiment, the antibody or its antigen-binding fragment can be systemically administered (e.g., orally) in combination with a pharmaceutically acceptable negative carrier, such as an inert diluent or an absorbable edible carrier. For oral therapeutic administration, the antibody or its antigen-binding fragment can be combined with one or more excipients and used in the form of ingestible tablets, lozenges, troches, capsules, elixirs, suspensions, syrups, wafers, etc. The percentages of the composition and formulation can vary and can be one or more active ingredients and binders, excipients, disintegrants, lubricants, and / or sweeteners (as known in the art) between about 1 to about 99% by weight. In such therapeutically useful compositions, the amount of the antibody or its antigen-binding fragment is an amount capable of achieving an effective dosage level.

[0129] Using standard pharmaceutical techniques well known to those of skill in the art, the preparation of parenteral compounds / compositions under sterile conditions can be readily accomplished, for example, by lyophilization. In at least one embodiment, the solubility of the compound used to prepare the parenteral composition can be increased by using suitable formulation techniques, such as incorporating solubilizing agents.

[0130] As described above, the antibody or antigen-binding fragment thereof can also be administered by infusion or injection (e.g., using a needle (including a microneedle) syringe and / or a needleless syringe). The solution of the active composition can be aqueous, optionally mixed with a non-toxic surfactant, and / or can contain carriers or excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9). However, for certain applications, they may be more suitably formulated as a sterile non-aqueous solution or in a dry form, to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or phosphate-buffered saline (PBS). For example, dispersions can be prepared in glycerol, liquid PEG, triacetin, and their mixtures and oils. Under ordinary storage and use conditions, these formulations can further contain preservatives to prevent microbial growth.

[0131] Therapeutic Methods

[0132] A method of treating animal cancer, particularly canine invasive urothelial carcinoma (InvUC), which comprises more than about 90% of dog bladder cancers, using the antibodies or antigen-binding fragments herein is provided. In at least one embodiment, the method comprises administering to a canine subject a therapeutically effective amount of any antibody or antigen-binding fragment thereof (e.g., for treating cancer). The cancer can be, for example, any canine cancer, including but not limited to InvUC. In certain embodiments, administering a therapeutically effective amount of a canine antibody, chimeric antibody, or antigen-binding fragment thereof inhibits PD-L1 / PD-1 interaction in the subject. In certain embodiments, administering a therapeutically effective amount of a canine antibody, chimeric antibody, or antigen-binding fragment thereof increases the activity of immune cells in the subject.

[0133] The subject can be a mammal. The subject can be a dog. The subject can be a companion dog.

[0134] "Effective amount" or "therapeutically effective amount" means an amount of a therapeutic agent (e.g., an antibody or an antigen-binding fragment thereof) or a composition comprising the same that elicits a desired biological or medical response in a subject (i.e., a tissue, organ, or organism, such as a vertebrate, e.g., a mammal, such as a human or a dog), which is sought by a researcher, veterinarian, physician, or other clinician, and the biological or medical response includes, but is not limited to, imaging and / or alleviating the signs and / or symptoms of the disease or disorder being treated. In one aspect, an effective amount is an amount of an active agent that is capable of treating or alleviating the signs and / or symptoms of a disease with a reasonable benefit / risk ratio applicable to any pharmaceutical treatment. The "effective amount" or "therapeutically effective amount" with respect to therapeutic use means the amount of the active agent / antibody in a formulation that, when administered as part of a desired dosing regimen (e.g., to a mammal, such as a human or a dog), alleviates symptoms, improves the condition, or slows the onset of the disease according to clinically acceptable criteria for the disorder or condition to be treated or for cosmetic purposes, e.g., with a reasonable benefit / risk ratio applicable to any medical treatment.

[0135] In combination with the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be designed by selecting among various active agents and trade-off factors such as potency, relative bioavailability, subject body weight, severity of adverse side effects, and mode of administration, which will not cause significant adverse toxicity and will be effective for treating a particular subject.

[0136] A wide range of tolerated doses are contemplated herein depending on the type of cancer, route of administration, and / or whether the antibody or an antigen-binding fragment thereof is administered locally or systemically. The amount of the composition required for therapeutic use (e.g., a therapeutically or prophylactically effective amount or dose) will vary not only with the particular application and dosing regime, but also with the salt selected (if applicable) and the characteristics of the subject (such as, for example, species, breed, age, condition, sex, body surface area and / or mass of the subject, tolerance to the drug), and will ultimately be determined by the attending physician, veterinarian, clinician, or other person.

[0137] The range of therapeutically effective amounts or doses can be, for example, from about 0.05 mg / kg of the subject's body weight to about 30.0 mg / kg of the subject's body weight, or from about 0.01 mg / kg of the subject's body weight to about 5.0 mg / kg of the subject's body weight, including but not limited to 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg, all of which are per kg of the subject's body weight. The total therapeutically effective amount of the compound can be administered as a single dose or in divided doses and may fall outside of the typical ranges given herein according to the judgment of the practitioner.

[0138] Parenteral administration may require higher doses. The effective amount for any particular application can vary depending on factors such as the disease or disorder being treated, the particular antibody being administered, the size of the subject, and / or the severity of the disease or disorder. A person of ordinary skill in the art can empirically determine the effective amount of a particular antibody, its antigen-binding fragment, and / or other therapeutic agent without undue experimentation. The maximum dose, i.e., the highest safe dose according to some medical judgment, can be used. Multiple daily administrations can be used to achieve a suitable systemic level of the compound. A suitable systemic level can be determined, for example, by measurement of the peak or sustained plasma levels of the drug in the patient. "Dose" and "dosage" are used interchangeably herein.

[0139] Adjusting the dose to achieve maximum efficacy based on the methods described herein and other methods known in the art is well within the capabilities of a person of ordinary skill. Depending on the mode of administration, the dose can be appropriately adjusted to achieve the desired local or systemic drug level. For example, the dose for intravenous administration can vary from one order of magnitude lower to several orders of magnitude per day. If the response of the subject is insufficient at such a dose, even higher doses (or higher effective doses obtained by different, more localized delivery routes) can be used within the tolerance of the subject. Multiple daily administrations are contemplated to achieve a suitable systemic level of the compound.

[0140] Any effective regimen for administering an antibody or an antigen-binding fragment thereof can be used. The dose can be a single dose or divided doses, and can be administered according to a variety of regimens, including q.d., b.i.d., t.i.d., or even every other day, every two weeks (b.i.w.), once a week, once a month, once a quarter, etc. In each of these cases, it should be understood that the effective amount described herein corresponds to the administered case, or alternatively corresponds to the total daily, weekly, monthly, or quarterly dose, as determined by the dosing regimen.

[0141] For example, the antibody can be administered as a single dose, or the dose can be divided and administered in a multiple-dose daily regimen. In addition, a staggered regimen, such as Monday through Friday each week, can be used as an alternative to daily treatment. Such intermittent or staggered daily regimens are considered equivalent to daily treatment. A subject being treated can be treated for cancer by multiple injections of an antibody or an antigen-binding fragment thereof. The subject can be injected with an antibody or an antigen-binding fragment thereof multiple times (e.g., approximately 2 - 50x), for example, at intervals of 12 - 72 hours or at intervals of 48 - 72 hours. After one or more initial injections, additional injections of an antibody or an antigen-binding fragment thereof can be administered to the subject at intervals of days or months, and such additional injections can prevent recurrence of cancer.

[0142] These methods can be used in combination with one or more additional therapies and / or active agents. Such additional therapies include, but are not limited to, additional immunotherapy, administration of DNA damage response pathway inhibitors, chemotherapy, radiotherapy, and / or surgery.

[0143] Methods for Predicting and Modeling the Anticancer Activity of Test Compounds

[0144] Companion dogs naturally develop several types of cancer, which are in many ways similar to clinical cancers in human patients. Although mouse models are the most commonly used animal models in cancer research, they do not possess collective characteristics such as tumor heterogeneity, mutational landscape, cancer molecular subtypes, and immune cell responsiveness that mimic the conditions required for the best animal model. Studies in mouse models can be complemented by other models, such as specific forms of cancer that occur naturally in pet dogs. This can be particularly beneficial when developing or studying new immuno-oncology drugs, such as novel immune checkpoint inhibitors (ICIs) or their combination regimens, where companion dogs can provide naturally occurring cancers in the context of a complete immune system and invasive heterogeneous cancers. The development of canine ICIs can expand comparative oncology approaches to improve the current therapeutic efficacy of human cancer immunotherapy. Thus, canine studies of immuno-oncology drugs can be translated into knowledge to inform and prioritize new immuno-oncology treatments for humans. However, traditionally, the challenge has been the inability to obtain ICIs that target canine immune checkpoint molecules such as cPD-1 and cPD-L1.

[0145] In the development of immuno-oncology drugs, particularly immuno-therapeutic antibodies, the translation of discoveries from mouse models to clinical trials is hampered by numerous biological differences between mice and humans, such as lack of cross-reactivity between species and / or the response of the mouse physiological system to cancer and other pathological insults being different from that of the human or canine system. For example, if an anti-human or canine PD-L1 antibody does not recognize the mouse PD-L1 protein, the therapeutic efficacy of the human or canine antibody cannot be evaluated in syngeneic mouse models. Mice transplanted with the human immune system have been developed for translational research to overcome this limitation. Indeed, the anti-human PD-1 antibody pembrolizumab has shown tumor growth inhibition and CD8+ T cell activation in humanized NSG (HuNSG) mice receiving tumor grafts from patient-derived xenografts (PDX) (see, e.g., Wang et al., Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy, FASEB J 32:1537-1549 (2018)). Although mice transplanted with the human immune system bearing human tumors are important models for preclinical immuno-oncology research, there are significant obstacles in these humanized mouse models, such as limited sources of human cells and tissues, immune rejection, and high costs (see, e.g., Yong et al., Humanized mice as unique tools for human-specific studies, Arch Immunol Ther Exp (Warsz) 66:245-266 (2018)).

[0146] As an alternative mouse model for the development of immuno-therapeutic antibodies, mice humanized for immune checkpoint molecules are commercially available. For example, using the clustered regularly interspaced short palindromic repeats (CRISPR) / CAS9 strategy, humanized PD-L1 mice were generated by replacing the mouse PD-L1 gene (cd274) with the human PD-L1 gene (CD274). Humanized PD-L1 mice can be used to evaluate the therapeutic efficacy of anti-human PD-L1 antibodies in vivo; however, a canine PD-L1 gene knock-in mouse model that can be used to evaluate the therapeutic efficacy of anti-canine PD-L1 antibodies has not been available to date, which has posed a major obstacle to the development of canine immune checkpoint inhibitors such as PD-1 / PD-L1 blocking antibodies. The PD-L1 mouse model of the present invention can be used not only for preclinical immuno-oncology research, but also as a translational research tool to bridge the gap between dogs and humans, thereby increasing the success rate of human immuno-therapy applications.

[0147] Also provided are methods of predicting and modeling (e.g., optimizing) the anti-cancer activity of test compounds in a secondary subject using the antibodies or antigen-binding fragments of the present disclosure. In certain embodiments, the antibody or antigen-binding fragment thereof is humanized and evaluated in a dog (e.g., the primary subject), and those test compounds that are most successful are advanced to human trials (e.g., where the secondary subject is human).

[0148] In certain embodiments, methods of predicting and modeling the anti-cancer activity of test compounds in a subject include generating a population of mice that express canine PD-L1 on the cell surface; evaluating the toxicity risk and / or efficacy of a group of test compounds in treating cancer in the PD-L1 mouse population; and selecting one or more test compounds of the group that meet established toxicity risk and / or efficacy criteria. The group of test compounds can include, for example, at least one compound that inhibits (or is reasonably expected to inhibit) the PD-L1 / PD-1 interaction in the subject.

[0149] In certain embodiments, the method further includes evaluating the oral bioavailability, absorption, distribution, metabolism, and excretion (ADME) values of the group of test compounds in a mouse population that contains canine PD-L1 and PD-1. In certain embodiments, the method further includes evaluating the oral bioavailability and ADME values of one or more test compounds selected in the PD-L1 mouse population.

[0150] The method can further include evaluating the toxicity risk and / or efficacy of one or more selected compounds in treating cancer in a human or canine cohort.

[0151] The toxicity risk and / or efficacy criteria can be established according to known protocols.

[0152] Generating the PD-L1 mouse population can further include replacing the murine cd274 gene in the mouse population with the canine PD-L1 gene using CRISPR or other known techniques.

[0153] The canine model of the present invention can be used for the study of InvUC-specific therapies. This concept is supported by data that human InvUC can be replicated in dogs and the similarities between human InvUC and canine InvUC. Canine InvUC is similar to human InvUC in terms of manifestation, pathology, local invasion, distant metastasis (lungs, etc., in more than 50% of cases), and chemotherapy response (see, for example, Cekanova et al., Molecular imaging of cyclooxygenase-2 in canine transitional cell carcinomas in vitro and in vivo, Cancer Prev Res (Phila) 6:466-76 (2013); Fulkerson et al., Naturally occurring canine invasive urinary bladder cancer: A complementary animal model to improve the success rate in human clinical trials of new cancer drugs, Int J Genomics, 6589529 (2017); Knapp et al., Cisplatin versus cisplatin combined with piroxicam in a canine model of human invasive urinary bladder cancer, Cancer Chemother Pharmacol 46:221-226 (2000); Knapp et al., Urinary bladder cancer in dogs, a naturally occurring model for cancer biology and drug development, ILAR J 55:100-118 (2014); Lin et al., Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand, Mol Cancer 10:9 (2011); Patrick et al.,Classification of canine urinary bladder urothelial tumours based on the World Health Organization / International Society of Urological Pathology consensus classification, J CompPathol 135:190 - 199(2006); Sommer et al., Naturally-occurring canine invasive urothelial carcinoma: A model for emerging therapies, Bladder Cancer 4:149 - 159(2018); and Suarez-Bonnet et al., Expression of cell cycle regulators, 14-3-3sigma and p53 proteins, and vimentin in canine transitional cell carcinoma of the urinary bladder, Urol Oncol 33:332e1-7(2015)). Since InvUC accounts for two percent (2%) of the approximately six million new canine cancer cases in the United States each year, there is a large pool of dogs available for translational research (see, e.g., Davis and Ostrander, Domestic dogs and cancer research: a breed-based genomics approach, ILAR J 55:59 - 68(2014)). In canine clinical trials, the test subjects continue to live as pets after the study, which is a win-win for each dog, and the knowledge gained can help both people and pet dogs. Thus, dogs provide an excellent opportunity to advance PD-1 / PD-L1 blockade therapies in humans. Successful therapies in rodents can be evaluated in dogs, and those that are most successful can move on to human trials.

[0154] Example

[0155] The present disclosure will be better understood by reference to the following examples, which are provided by way of illustration and not limitation.

[0156] Materials

[0157] The BT549 human breast cancer and MB49 mouse bladder cancer cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA) and Sigma-Aldrich (St. Louis, MO), respectively. The canine bladder cancer cell line K9TCC was generated in the Knapp laboratory (see Igase et al. (2020), supra). The human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (Waltham, MA). The cell lines were verified by short tandem repeat DNA fingerprinting using the AmpFISTR Identifier PCT amplification kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions.

[0158] The cells were tested for mycoplasma using the Mycoplasma PCR Detection Kit (ABM).

[0159] The cells were grown for no more than 15 passages in Dulbecco's Modified Eagle Medium (DMEM) or DMEM / Nutrient Mixture F-12 (DMEM / F-12) medium supplemented with 10% fetal bovine serum (FBS).

[0160] To stably express PD-L1, the cDNA of canine PD-L1 (SinoBiological, Wayne, PA) was inserted into the pGIPZ vector (Horizen Discovery, Waterbeach, UK) as described by Lim et al. (2016), supra. The endogenous human PD-L1 was knocked out and Flag-cPD-L1 was simultaneously reconstituted using the pGIPZ-shPD-L1 / Flag-cPD-L1 double-expression construct to establish the endogenous PD-L1 knockout and Flag-cPD-L1-expressing BT549 cell line. See, e.g., Lim et al., EGFR signaling enhances aerobic glycolysis in triple negative breast cancer cells to promote tumor growth and immune escape, Cancer Res (2016).

[0161] Lentiviruses were packaged by co - transfecting the transfer plasmid with plasmids pMD2.G (Addgene #12259) and pCMV dR8.2 (Addgene #12263) into HEK293FT cells using X - tremeGENE HP (Roche Diagnostics, Indianapolis, IN). The supernatant was harvested for lentiviral transduction. Selection was routinely performed using 1 μg / mL puromycin (InvivoGen, San Diego, CA) to maintain ectopic gene expression.

[0162] For mouse PD - L1 knockout, the mouse PD - L1 double - incision enzyme plasmid (Santa Cruz Biotechnology, Dallas, TX) was transfected into MB49 cells using the X - tremeGENE transfection reagent.

[0163] For canine PD - L1 - overexpressing MB49 cells (MB49 cPDL1 ), mouse PD - L1 KO MB49 cells were infected with lentivirus carrying pGIPZ - Flag - cPD - L1 and then selected with puromycin.

[0164] All animal experiments were approved by the Purdue Animal Care and Use Committee (PACUC) at Purdue University.

[0165] For flow cytometry analysis, MB49 or BT549 cells were washed twice with ice - cold cell staining buffer (Biolegend, San Diego, CA) and stained with cIgG control or 12C10E4 cIgG at 4 °C for 1 hour. After washing three times with the staining buffer, the cell samples were stained with Alexa Fluor 488 - conjugated anti - canine IgG - specific secondary antibody at 4 °C for 30 minutes. The cell samples were loaded onto a BD LSRFortessa (BD, Franklin Lakes, NJ) for analysis. Data analysis was performed on FlowJo v9 software (BD). Every hour, the green fluorescence signal was measured and quantified by IncuCyte S3 (Sartorius, Goettingen, Germany). Image analysis was performed according to the manufacturer's protocol.

[0166] Example 1

[0167] Preparation and screening of anti - canine PD - L1 monoclonal antibody

[0168] At the Vanderbilt University Antibody and Protein Resource Core Facility, monoclonal antibodies 3C8D3 (mAb 3C) and 12C10E4 (mAb 12C) were generated by a conventional hybridoma procedure using A / J mice immunized with the extracellular domain of canine programmed death ligand 1 (cPD-L1) (attached to a human Fc tag). Briefly, splenocytes were isolated from the immunized mice and fused with SP2 / 0 myeloma cells (see Figure 1 ). Supernatants from the isolated clones were screened for the ability to block the canine programmed cell death protein 1 (cPD-1) / cPD-L1 interaction by an enzyme-linked immunosorbent assay (ELISA) based on cPD-L1-expressing cells, and mAb 3C and 12C were selected for further study. The cloned antibodies were purified from the supernatants, and the same assay was repeated. Anti-cPD-L1 mAbs were successfully generated using these conventional hybridoma procedures.

[0169] To screen antibodies capable of blocking the cPD-1 / cPD-L1 interaction for use in a therapeutic setting, live-cell-based antibody-binding and PD-1 / PD-L1 blockade assays were developed (see Figure 2A and 2B ), similar to those previously published. See, e.g., Li et al., Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1, Cancer Cell 33:187-201e10 (2018); Lim et al., Deubiquitination and stabilization of PD-L1 by SCN5, Cancer Cell (2016); Li et al., Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity, Nat Commun 7:12632 (2016).

[0170] Human cancer cell line BT549 cells expressing cPD-L1 were seeded in 96-well plates or 384-well plates. cPD-L1 antibodies (from the hybridoma) and Alexa 488-conjugated anti-mouse IgG fc-specific secondary antibody were added, and by The S3 live cell analysis system (Sartorius AG, Göttingen, Germany) measures the green fluorescence signal to quantify the amount of bound PD-L1 antibody ( Figure 2A ).

[0171] BT549 cells expressing cPD-L1 were seeded in 96-well or 384-well plates. cPD-1-human Fc (hFc) protein, Alexa 488-conjugated anti-human IgG Fc specific secondary antibody and / or cPD-L1 antibody were added, and then the green fluorescence signal was measured by the S3 live cell analysis system (Sartorius AG, Göttingen, Germany) to quantify the amount of bound PD-1 protein ( Figure 2B ).

[0172] Among more than 2,000 hybridomas, 154 clones were screened for membrane-localized cPD-L1 protein by live cell-based antibody binding assays. The cloned antibodies were purified from the supernatant and the same assay was repeated. Figure 2C A kinetic plot of the quantitative binding of PD-L1 antibody to BT549 cells expressing cPD-L1 at each 3-hour time point is shown. Positive clones are highlighted in bold boxes. Figure 2D A representative image of cPD-L1 antibody binding (acquired at 18 hours) is shown, which shows the green-fluorescence merged image of cPD-L1-expressing cells.

[0173] Ten of these clones have the ability to block the cPD-L1 / cPD-1 interaction. Figure 2E A kinetic plot of the quantitative binding of PD-1 protein to BT549 cells expressing cPD-L1 at each 3-hour time point after addition of cPD-L1 antibody is shown. Positive clones that block the PD-L1 / PD-1 protein interaction are highlighted in bold boxes. Figure 2F A representative image of cPD-L1 blockade (acquired at 18 hours) is shown, which shows the green-fluorescence merged image of cPD-L1-expressing cells. Note that there is no fluorescence (in the square highlighted by the white box) because the antibody binds to PD-L1 and blocks the interaction with cPD-1.

[0174] Representative positive clones are as Figures 2B - 2F shown. Based on specificity, binding affinity, and PD-1 / PD-L1 blockade efficacy, mAb 3C and mAb 12C were selected for further analysis.

[0175] Example 2

[0176] Generation of canine PD-L1 knock-in mice as a preclinical model

[0177] To further evaluate the clinical application of the cPD-L1 antibody as an immunotherapeutic agent, its therapeutic efficacy was evaluated in an in vivo model. To this end, mice (C57BL / 6 background) expressing canine PD-L1 were generated. This was achieved by generating cPD-L1 mice in which the mouse cd274 (PD-L1) gene was replaced with cPD-L1 using the CRISPR knock-in mouse strategy, a long single-stranded DNA (ssDNA) donor, and CRISPR ribonucleoproteins ( Figure 3A ).

[0178] More specifically, by replacing murine CD274 with canine CD274, a highly efficient addition method using ssDNA insertion-CRISPR (Easi-CRISPR) for human CD247 knock-in mouse generation was employed to create mice expressing PD-L1 on the cell surface (C57BL / 6 background). See Quadros et al., Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long single-stranded DNA (ssDNA) donors and CRISPR ribonucleoproteins, Genome Biol 18:92 (2017). Easi-CRISPR is a targeting strategy in which a long ssDNA donor is co-injected with a pre-assembled crRNA+tracrRNA+Cas9 ribonucleoprotein (ctRNP) complex into mouse fertilized eggs to generate targeted insertion alleles in the resulting live offspring (here replacing the murine cd274 (PD-L1) gene with the canine PD-L1 gene). A long ssDNA (full-length canine CD274 cDNA; NM_001291972) was co-injected with a pre-assembled guide RNA (gRNA, CAGCAAATATCCTCATGTTTTGG (SEQ ID NO:20)) and Cas9 ribonucleoprotein (ctRNP) complex into mouse fertilized eggs. The ssDNA and sgRNA were synthesized at Integrated DNA Technologies (IDT, Coralville, IA, USA). All animal experiments for knock-in mouse generation were approved by the Purdue Animal Care and Use Committee (PACUC) at Purdue University (West Lafayette, IN). Four-week-old C57BL / 6N female mice (Envigo, Indianapolis, IN, USA) were superovulated and then mouse fertilized eggs were obtained by mating superovulated females with C57BL / 6N males.

[0179] Inject 20 ng / μl Cas9 protein, 10 ng / μl sgRNA, and 5 ng / μl ssDNA into the pronucleus of single-cell stage fertilized mouse embryos. Microinjection and mouse transgenesis were performed as described (40). Mouse genomic DNA was extracted from the tail tip and then used for genotyping (primer set 1 forward, 5’-CCACTTGGTTCTACATGGCT-3’ (SEQ ID NO:21); primer set 1 reverse, 5’-CCTCAGCCTGACACATTAGTT-3’ (SEQ ID NO:22); primer set 2 forward, 5’-CCTGTCACCTCTGAACATGAA-3’ (SEQ ID NO:23); primer set 2 reverse, 5’-GACTAAGCTCTAGGTTGTCC-3’ (SEQ ID NO:24); primer set 3 forward, 5’-GACTGGCTTTTAGGGCTTATGT-3’ (SEQ ID NO:25); primer set 3 reverse, 5’-ACACCCCACAAATTACTTCCATT-3’ (SEQ ID NO:26)) and sequencing (primer set 3 forward, 5’-GACTGGCTTTTAGGGCTTATGT-3’ (SEQ ID NO:25); primer set 3 reverse, 5’-ACACCCCACAAATTACTTCCATT-3’ (SEQ ID NO:26)) to verify the insertion site and DNA sequence of the canine CD274 gene.

[0180] To evaluate the therapeutic efficacy of the cPD-L1 antibody in a syngeneic animal model, a mouse bladder cancer MB49 cell line expressing cPD-L1 was generated by knocking out mPD-L1 and re-expressing cPD-L1 (MB49 cPD-L1 )( Figure 3B and 3C ). Figure 3B Flow cytometry analysis of cPD-L1 protein located on the membrane in MB49 cells expressing cPD-L1 (MB49 cPD-L1 ) is shown.

[0181] Example 3

[0182] In vitro interaction of cPD-1 or mPD-1 protein with cPD-L1 or mPD-L1 with or without treatment with the cPD-L1 antibody (mAb 12C)

[0183] Although MB49 cPD-L1The canine PD-L1 mice express the cPD-L1 protein but not the mPD-L1 protein, while the canine PD-L1 mice express the murine PD-1 protein. Therefore, before evaluating the therapeutic efficacy of a cPD-L1 antibody in canine PD-L1 mice, it is first checked whether the cPD-L1 protein interacts with the mPD-1 protein.

[0184] To measure the interaction of immune receptors and ligands, His-tagged canine or murine PD-L1 proteins (cPD-L1-His or mPD-L1-His, respectively) were incubated in nickel-nitrilotriacetic acid (Ni-NTA)-coated 96-well plates. The plates were then incubated with the recombinant Fc-tagged protein for 1 hour, and the secondary antibody used was an anti-human or canine IgG Fc-specific Alexa488 dye conjugate (Jackson ImmunoResearch Inc., West Grove, PA). The fluorescence intensity of the Alexa fluor 488 dye was measured by a microplate reader (Synergy Neo2; BioTek Instruments, Inc., Winooski, VT).

[0185] The binding of cPD-L1 and mPD-1 was similar to that of the homologous cPD-L1 and cPD-1 pair (OD450 was measured to quantify the amount of bound PD-1 protein ( Figure 3D ))). Consistently, the cPD-L1 antibody (mAb 12C) effectively blocked the cPD-L1 / mPD-1 interaction as well as the cPD-L1 / cPD-1 interaction, but did not block the mPD-L1 / mPD-1 or mPD-L1 / cPD-1 interaction (because the current cPD-L1 antibody does not recognize mPD-L1) ( Figure 3D and 3E ).

[0186] Example 4

[0187] Evaluation of the therapeutic efficacy of a cPD-L1 antibody in canine PD-L1 mice

[0188] All procedures performed on B mice (C57BL / c background strain; 6 to 8 weeks old) with canine PD-L1 were carried out in accordance with the guidelines approved by the PACUC of Purdue University. The mice were grouped according to the average tumor volume of each group. MB49 cPD-L1 (2×10 5 cells in 25 μL of medium mixed with 25 μL of Matrigel basement membrane matrix (BD Biosciences, San Jose, CA)) were injected into the flanks of the humanized PD-L1 mice.

[0189] After establishing MB49 tumors in cPD-L1 mice cPD-L1 the mice were divided into a control group, an mAb12C treatment group, and an mAb3C treatment group, and the mice in each subgroup were divided according to the average tumor volume in each group.

[0190] For antibody treatment, on days 4, 6, 8, 10, and 12 after tumor cell inoculation, when the tumor size was approximately 30 to 40 mm 3 100 μg of cPD-L1 antibody (mAb 12C or mAb 3C clone) or control mouse IgG (BioXCell) was injected intraperitoneally. Tumors were measured every other day with calipers, and tumor volume was calculated using the following formula:

[0191] π / 6 × length × width 2 .

[0192] Tumors were dissected at the end point for analysis (n = 8 per group)( Figure 3F ).

[0193] Immunofluorescence staining was performed on the protein expression patterns of CD8 and granzyme B in MB49 tumor masses from IgG-treated mice (control group), 12C-treated mice, and 3C-treated mice( Figures 3G - 3I ). Tumor masses were immediately frozen in optimal cutting temperature (OTC) blocks after excision. 5-μm thick cryostat sections were attached to saline-coated slides. The cryostat sections were fixed with 4% paraformaldehyde at room temperature for 30 minutes and blocked with a blocking solution (1% bovine serum albumin, 2% donkey and / or chicken serum, and 0.1 M phosphate buffered saline (PBS)) at room temperature for 30 minutes. Samples were stained overnight at 4 °C with primary antibodies against CD8 and granzyme B, and then stained for 1 hour at room temperature with secondary antibodies. Nuclear staining was performed with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA). Stained sections were observed by automated microscopy (Lionheart LX; BioTek Instruments, Inc., Winooski, VT). The granzyme B-positive area per high power field (200X) and the number of CD8-positive cytotoxic T lymphocytes (CTLs) were evaluated. The number of CD8-positive CTLs and the granzyme B-positive area of each tissue were examined in fourteen microscopic fields randomly selected from four consecutive sections of each tissue block.

[0194] CD8 was also analyzed using BioTek Gen5 data analysis software (Agilent Technologies, Santa Clara, CA)( Figure 3H) and Granzyme B ( Figure 3I ) were quantified (n = 10). Treatment with mAb 12C or mAb 3C significantly reduced tumor size ( Figure 3F ) and increased the number of infiltrating cytotoxic T cells, as measured by CD8+ and Granzyme B expression, compared to mice treated with control IgG ( Figures 3G - 3I ).

[0195] In addition, at the end of treatment, the therapeutic effect on PD-L1 mice was evaluated by measuring the function of the mouse kidneys ( Figure 3J ) and livers ( Figure 3K ). The data supported both that the cPD-L1 antibody enhanced anti-tumor immunity in the PD-L1 syngeneic mouse model and demonstrated good safety characteristics, as the mice maintained their body weight; and that renal function was not altered by serum creatinine or liver enzyme activity. In vitro and in vivo validation results showed that the cPD-L1 antibody recognizing canine PD-L1 could effectively inhibit the PD-1 / PD-L1 pathway and enhance anti-tumor immunity in mice.

[0196] Example 5

[0197] Characterization of Canine PD-L1 Chimeric Antibody

[0198] For the clinical application of the canine PD-L1 antibody in dogs, mAb 12C was humanized by replacing the murine constant domain with the canine IgG2 (equivalent to human IgG1) constant domain. Briefly, full-length variable heavy (VH) and variable light (VL) RNA transcripts obtained from hybridoma clones were sequenced by 5' / 3' rapid amplification of cDNA ends (RACE) and codon-optimized for CHO VL, and the VH chain was cloned into the pTRIOZ-cIgGB-ck vector (InvivoGen, San Diego, CA, USA), which is designed to produce full monoclonal antibodies with high yield using a single plasmid. Then the constant light and heavy chains were replaced with the canine κ light constant chain and canine IgG2 heavy constant chain: pTRIOZ-cIgG2-12C10E4. Figure 4A A schematic diagram of the chimeric antibody expression construct (pTRIOZ-cIgG2-cPD-L1 mAb12C) of cPD-L1 (mAb 12C) is shown. The chimeric cPD-L1 antibody retained the cPD-L1-binding VH and VL chains of the murine hybridoma.

[0199] According to the transfection kit instructions (GIBCO, A29133), the plasmid encoding the 12C chimeric antibody pTRIOZ cIgG212C10E4 was transfected into ExpiCHO-S cells. The ExpiCHO-S cells were cultured with ExpiCHO expression medium (ThermoFisher Scientific, Waltham, MA) in a shaking incubator set at 120 rpm, 37 °C, and 8.0% CO2. Ten days after transfection, the cells were collected by centrifugation at 4,000 x g for 20 minutes at 4 °C. The antibody supernatant was passed through a 0.22-μm filter and neutralized with 10X PBS buffer (Lonza TM BioWhittaker TM Phosphate Buffered Saline (10X), BW17-517Q). The antibody supernatant was pre-incubated with Protein A Sepharose for 2 hours. The agarose A-conjugated antibody was applied to a column (BioRad poly-prep chromatography column, #731-1550). The column was washed with low endotoxin PBS (Lonza TM BioWhittaker TM Dulbecco's Phosphate Buffered Saline (1X) w / o calcium and magnesium, BW17512F24). The bound antibody was eluted with elution buffer (ThermoFisher Scientific, Waltham, MA; elution buffer, 0.1 M glycine-HCl, pH 2.8, #21004) into neutralization buffer (Tris HCl, 1 M, BP1757-500). The purified antibody was concentrated and buffer-exchanged with PBS at pH 7.0. The antibody concentration was determined by UV absorbance at 280 nm.

[0200] To monitor the batches during the antibody production process, the properties of the purified chimeric antibody, such as purity, isoelectric point (pI) value, amino acid sequence, and N-glycomics profile ( Figures 4B - 4F ) were evaluated. Briefly, the purity and pI value were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and isoelectric focusing (IEF), respectively. SDS-PAGE or IEF gels were purchased from Bio-Rad Laboratories (Hercules, CA) or ThermoFisher Scientific (Waltham, MA). The purity of the 12C chimeric antibody was analyzed by SDS-PAGE under non-reducing and reducing (2-mercaptoethanol) conditions according to the manufacturer's protocol ( Figure 4B)。IEF and Coomassie blue staining were also performed according to the manufacturer's protocol. SDS-PAGE, image acquisition, and quantification of band intensities were performed using an Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE). Figure 4B shows the SDS-PAGE analysis of the 12C chimeric antibody, and Figure 4C shows its IEF analysis.

[0201] Peptide mapping comparisons of the cPD-L1 chimeric antibody (per mAb 12C batch) were also performed to evaluate the amino acid sequence. Briefly, after reduction and alkylation, the antibody was enzymatically digested with trypsin on an S-trap microcolumn from Protifi (Farmingdale, NY). Peptides were then separated and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry (RP-LC-MS / MS) using a Q Exactive HF hybrid quadrupole-orbitrap MS equipped with a Nanospray Flex ion source and coupled to a Dionex UltiMate 3000 RSLC nano system (ThermoFisher Scientific, Waltham, MA). The resulting mass spectrometry data were analyzed using the PEAKPTM workflow in the PEAKS X PRO Studio 10.6 software package from Bioinformatics Solutions Incorporated to map the detected MS1 and MS2 ions to the amino acid sequence of the antibody. See Ma et al., PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry, Rapid Commun Mass Spectrom 17:2337-2342 (2003). Peptide mapping analysis was performed at the Proteomics Core Facility at Purdue University (West Lafayette, IN). LC / MS-MS data were used to map the glycosylation (0.98 Da) of asparagine (N) and glutamine (Q) residues of the mapped antibody sequences.

[0202] The sequence coverages of the heavy and light chains were 100% (453 out of 453 amino acids) and 98.2% (223 out of 227 amino acids), respectively, which is a strong positive indicator of the presence of SEQ ID NO:14 ( Figure 4D )。The chimeric cPD-L1 antibody retained the cPD-L1 binding VH and VL chains of the murine hybridoma.

[0203] Size-exclusion chromatography (SEC) analysis of the 12C chimeric antibody was performed to detect antibody aggregates and monomers. The antibody was analyzed for 135 minutes at a flow rate of 0.3 ml / min using an AKTA Pure 150M (Cytiva, Marlborough, MA) and a Superdex 200 Increase 10 / 300 GL column (Cytiva, Marlborough, MA). Elution was monitored using UV absorption at 280 nm, and the data were processed by Unicorn 7 software (Cytiva, Marlborough, MA). The SEC analysis was performed at the Molecular Evolution, Protein Engineering, and Production Core Facility at Purdue University (West Lafayette, IN). Figure 4E The SEC analysis results are shown.

[0204] In addition, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the pre-methylated N-glycans released from PNGase F-treated 12C chimeric antibody was performed by the method described in Shajahan et al., Glycomic and glycoproteomic analysis of glycoproteins - a tutorial, Anal Bioanal Chem 409:4483 - 4505 (2017). Briefly, the N-glycans of the 12C10E4 antibody were released by treating the reduced and alkylated protein with PNGase F. The released N-glycan fraction was then fully methylated. The fully methylated N-glycans were evaluated by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) using an AB SCIEX TOF / TOF 5800 mass spectrometer (Applied Biosystem / MDS Analytical Technologies, Sunnyvale, CA). The structural assignment of the N-glycans was based on the molecular weight and followed the principles of the N-glycan biosynthesis pathway. Carbohydrate analysis was performed at the Complex Carbohydrate Research Center at the University of Georgia (funded by NIH R24GM137782). Figure 4F The MALD-MS analysis results are shown.

[0205] Example 6

[0206] Canine cPD-L1 chimeric antibody binding and blocking assay

[0207] According to known protocols, a cell-free cPD-L1 / cPD-1 blockade assay was used to evaluate the canine cPD-L1 chimeric antibody. See Li et al. (2018), supra.

[0208] Antibody binding and blockade assays were performed as described in Li et al. (2018), supra. Briefly, to measure the interaction between PD-L1 protein and PD-L1 antibody, 1 x 10 4 BT549 cPD-L1 cells per well were seeded in a 96-well plate. The plate was then incubated with cIgG control (Rockland Immunochemicals, Pottstown, PA) or 12C10E4 antibody and anti-canine Alexa Fluor 488 dye conjugate (Southern Biotech, Birmingham, AL). Every hour, the green fluorescence signal ( Figure 5A ) was measured and quantified by IncuCyte S3 (Sartorius, Göttingen, Germany).

[0209] To measure PD-1 protein on cells, 1 x 10 4 BT549 cPD-L1 cells per well were seeded in a 96-well plate. The plate was then incubated with cIgG control (Rockland Immunochemicals, Pottstown, PA) or 12C10E4 antibody, cPD-1-hFc protein (human Fc protein conjugated; Sino Biological US, Wayne, PA) and / or anti-human Alexa Fluor 488 dye conjugate (Thermo Fisher Scientific, Waltham, MA). Every 3 hours, the green fluorescence signal ( Figure 5B ) was measured and quantified by IncuCyte S3 (Sartorius, Göttingen, Germany). Image analysis was performed according to the manufacturer's protocol.

[0210] ELISA-based assays were also performed to compare receptor / ligand and receptor / antibody binding. The 6XHis-tagged extracellular domain of cPD-L1 protein was expressed in the ExpiCHO cell system (Thermo Fisher Scientific, Waltham, MA) and purified by Ni-NTA agarose (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.

[0211] For the cPD-L1 / cPD-L1 blocking assay, Pierce Ni-NTA coated 96-well plates (ThermoFisher Scientific, Waltham, MA) were coated with canine PD-L1-His protein and PD-1-Fc protein (human Fc-protein conjugated) (SinoBiological US, Wayne, PA). An anti-human IgG Fc specific horseradish peroxidase (HRP) conjugated secondary antibody (SouthernBiotech, Birmingham, AL) was added, followed by addition of anti-canine PD-L1, 12C10E4 antibody. Bound PD-1-Fc protein was quantified by measuring OD 450 values with a Synergy LX multimode reader.

[0212] The chimeric antibody 12C10E4 binds to membrane-localized cPD-L1 protein ( Figure 5A and 5B ), but does not recognize cPD-L2 protein ( Figure 5C ).

[0213] Example 7

[0214] Binding affinity (K D ) determination of the canine cPD-L1 chimeric antibody

[0215] The binding affinity (K D ) of the chimeric cPD-L1 / cPD-L1 antibody (12C10E4) for canine PD-L1 was determined by Octet Biolayer Interferometry (BLI) using an Octet RED384 system (Sartorius, Bohemia, NY). Briefly, His-tagged cPD-L1 protein was loaded onto an Octet NTA biosensor at a concentration of 200 nM. The association step was performed by immersing the sensor in three concentrations of 12C10E4 antibody (50, 100, 200 nmol / L) in kinetic buffer. Dissociation was performed in fresh kinetic buffer and monitored. Data were analyzed with Octet Analysis HT software (Sartorius, Bohemia, NY), and bound cPD-1 protein was quantified by measuring green fluorescence in an IncuCyte S3.

[0216] The K D of the chimeric antibody determined by Octet was 8.6 nmol / L ( Figure 5D ). Similar to the murine 12C antibody obtained from a hybridoma, the 12C chimeric antibody blocked the cPD-L1 / cPD-1 interaction (EC 50 = 0.419 μg / ml;Figure 5E )。

[0217] Example 8

[0218] Activation and cytokine measurement of peripheral blood mononuclear cells (PBMCs)

[0219] Primary canine PBMCs (cPBMCs) were isolated from dog blood using SepMate PBMC isolation tubes (Stemcell Technologies Inc., Vancouver, B.C., Canada) and Histopaque-1077 (Millipore Sigma, Burlington, MA) according to the manufacturer's protocol. The activation of canine T cells by anti-canine CD3 and CD28 antibodies has been well established in previous studies.

[0220] Briefly, canine T cells in PBMCs were activated with 10 ng / mL canine interleukin-2 (IL-2) (10 ng / mL) (Novus Biologicals, Centennial, CO) and co-treated with and without 1 μg / mL anti-canine CD3ε antibody (clone CA17.2A12, coated; ThermoFisher Scientific, Waltham, MA) and 3 μg / mL (in medium) anti-canine CD28 antibody (clone 1C6; ThermoFisher Scientific, Waltham, MA) for 48 hours. Following the manufacturer's protocol, multiplex measurements of IFNγ, IL-10, and TNFα in these activated canine PBMCs were performed using the MILLIPLEX canine cytokine / chemokine magnetic bead assay kit (Sigma-Aldrich, St. Louis, MO).

[0221] Samples were incubated with the cytokine magnetic beads on a shaker for 2 hours and then with the secondary detection antibody provided in the kit. The plate was read on an Attune flow cytometer, using the FL2 channel for reporting (PE channel) and the FL4 (APC) channel for gating. For each cytokine, 300 beads were measured, and data were collected for forward and side scatter and log-scale FL2 vs FL4. The concentration of cytokines was quantified as ng / ml using cytokine multiplex analysis software (MPLEX, Cytomic Analtyical LLC). Data showed that treatment with anti-CD3 / CD28 and IL2 significantly induced the expression of IFNγ, IL-10, and TNFα compared to treatment with IL-2 alone, and this activation protocol was used for the remaining studies.

[0222] Example 9

[0223] Gene Expression Analysis of Canine-Derived cPD-L1 Chimeric Antibody

[0224] In Example 8, a canine IO assay panel (NanoString Technologies, Inc., Seattle, WA) analysis was used to query changes in gene expression from activated PBMCs of three healthy pet dogs. Activation of cPBMCs was performed as previously described in Example 8.

[0225] RNA was isolated from resting and activated cPBMCs (RNeasy kit, Qiagen, Germantown, MD) and submitted to the Stark Neurosciences Research Institute Biomarker Core, Indiana University School of Medicine (Indiana University, Indianapolis, IN) for use with the canine IO assay panel (NanoString Technologies, Inc., Seattle, WA) to detect regulation of genes after activation.

[0226] The canine IO assay panel was used to query changes in approximately 700 genes. Groupwise analysis of the data was performed using Rosalind (Rosalind, San Diego, CA) with control cPBMCs (n = 3) and compared to activated cells (n = 3). When comparing control PBMCs to activated PBMCs, 65 genes were differentially expressed (FC ≥ 1.5; P < 0.05), including 30 upregulated and 35 downregulated genes.

[0227] Data was visualized using heatmaps, volcano plots, and histograms for specific genes. In the heatmap, each column consists of data from one sample. IFNγ ( Figure 5H ) and TNFα ( Figure 5I ) concentrations were also analyzed in activated canine PBMCs.

[0228] Canine immuno-oncology assay panel analysis ( Figure 5F and 5G ; Table 1) and analysis of secreted cytokines (IFNγ and TNFα; Figure 5H and 5I ) demonstrated activation of canine PBMCs by anti-canine CD3 and CD28 antibodies and canine IL2 treatment.

[0229] Table 1. Gene alterations in activated cPBMCs from the nCounter Canine Immuno-Oncology Panel Assay.

[0230]

[0231]

[0232]

[0233]

[0234] Example 10

[0235] Canineized cPD-L1 Chimeric Antibody Enhances T Cell-Mediated Tumor Cell Killing

[0236] To demonstrate immune checkpoint inhibition by the 12C chimeric antibody and to analyze the killing of tumor cells by inactivated T cells, a tumor cell killing assay was performed on an ex vivo canine system according to a known protocol (see Li et al. (2016), supra), in which cPD-L1-positive canine bladder cells (K9TCC) were co-cultured with activated canine PBMCs. To quantify the number of viable or dead tumor cells in the tumor cell killing assay, K9TCC cells expressing nuclear-restricted red fluorescent protein (RFP) were established. nRFP Cells. The expression of endogenous cPD-L1 protein and mRNA in K9TCC parental cells and K9TCC nRFP cells after IFNγ treatment was confirmed ([ Figures 5J - 5L ). These activated canine PBMCs and K9TCC nRFP cells were then used to perform the tumor cell killing assay.

[0237] Briefly, K9TCC nRFP cells were co-cultured with activated cPBMCs in DMEM / F12 containing 10% FBS. The cPBMCs were activated by incubation with 100 ng anti-canine CD3ε antibody (see Example 8) (clone CA17.2A12, ThermoFisher Scientific, Waltham, MA) and 10 ng / mL canine interleukin-2 (IL-2) (NovusBiologicals, Littleton, CO) in DMEM / F12 containing 10% FBS. Primary cPBMCs were isolated from dog blood using SepMate PBMC isolation tubes (Stemcell Technologies, Cambridge, MA) and Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's protocol.

[0238] Viable tumor cell counts at 72 hours are shown in the bar graph of Figure 5J . After 96 hours, RFP signal was measured as viable tumor cells, and the expression of IFNγ, IL10, and TNFα in the supernatant of co-cultured cells was measured by the MILLIPLEX canine cytokine / chemokine magnetic bead assay kit according to the manufacturer's protocol.

[0239] Although PBMCs and tumor cells were from different dogs, and thus the dog lymphocyte antigen (DLA) was mismatched between cPBMCs and K9TCC cells, the 12C chimeric antibody enhanced tumor cell killing activity and IFNγ secretion ( Figure 5M and 5N ).

[0240] Example 11

[0241] Treatment of laboratory dogs with the 12C10E4 chimeric antibody

[0242] A single-dose pilot study was conducted in six laboratory beagle dogs (including male and female dogs) approximately 12 - 15 months old to evaluate initial safety and pharmacokinetic parameters. These dogs were housed and evaluated in the preclinical research laboratory of the College of Veterinary Medicine at Purdue University in West Lafayette, IN. The 12C10E4 cPD-L1 chimeric antibody used in the laboratory dog study was produced in the Molecular Evolution, Protein Engineering, and Production Facility at Purdue University (West Lafayette, IN) as described above. The antibody solution was mycoplasma-free and contained less than 0.5 EU endotoxin / mg antibody (consistent with the endotoxin limit for human PD-L1 antibody solution).

[0243] After acclimation to the facility, the dogs were treated with the 12C10E4 cPD-L1 chimeric antibody diluted in sterile water, which was used for intravenous (IV) administration (total volume of 6 ml / kg body weight) and administered via an IV catheter over 1 hour. Six dogs were treated and received either 2 mg / kg or 5 mg / kg of the antibody.

[0244] Blood was collected at 1, 6, 24, 48, and 72 hours after initiation of antibody administration for pharmacokinetic analysis, then once a week for 4 weeks. Monitoring for adverse events included physical examinations before and during treatment, then twice a day for 7 days, then once a week for 4 weeks; daily observations for 4 weeks; and a complete blood count (CBC), serum biochemistry panel (including but not limited to measurement of the concentration of the 12C10E4 chimeric antibody in the serum of dogs treated with the 12C10E4 antibody ( Figure 6A), and pre-treatment and weekly urinalysis were performed for 4 weeks. Additional testing was planned for any adverse events observed. Adverse events were classified using the Veterinary Cooperative Oncology Group (VCOG) criteria. See VCOG, Common terminology criteria for adverse events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.1, Vet Comp Oncol 14:417-446 (2016).

[0245] The 12C10E4 chimeric antibody was well tolerated and had a half-life of approximately 3 days ( Figure 6B and 6C ; Table 2). The half-life of the cPD-L1 antibody was shorter than that of human checkpoint inhibitors, indicating that weekly dosing may be suitable for dogs. A possible infusion reaction in one dog resolved without intervention. In a single-dose study in laboratory dogs, the antibody had good antibody tolerance and body weight remained unchanged (Table 2). Nonspecific changes such as a slight decrease in monocyte count and slight increases in CO2 and gamma-glutamyl transferase (GGT) were transient and resolved without intervention (Table 2).

[0246] Table 2. Summary of potential adverse events following initial administration of cPD-L1 antibody to laboratory dogs.

[0247]

[0248]

[0249] General and Specific Definitions

[0250] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification represent the state of the art of those skilled in the art to which the present disclosure pertains. All such publications are hereby incorporated by reference to the same extent as if each individual publication had been specifically and individually indicated to be incorporated by reference.

[0251] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. Certain embodiments may be practiced without some or all of these specific details, and it should be understood that the present disclosure is not limited to specific biological systems, specific cancers, or specific organs or tissues, which may of course vary, but which are still applicable in light of the data provided herein.

[0252] Various techniques and mechanisms of the present disclosure will sometimes describe a connection or link between two components. Terms such as attached, linked, coupled, connected, and the like, as well as their inflectional morphemes, may be used interchangeably, unless a difference is noted in the context or otherwise made clear. These words and expressions do not necessarily denote a direct connection, but include a connection through intermediate components. It should be noted that the connection between two components does not necessarily imply a direct and unobstructed connection, as various other components may be located between these two components. Thus, unless otherwise stated, a connection does not necessarily imply a direct and unobstructed connection.

[0253] In addition, whenever feasible and convenient, the same reference numerals are used in the drawings and the specification to refer to the same or similar parts or steps. These drawings are in simplified form and are not to exact scale. It should be understood that the disclosure presented in this manner is for purposes of explanation only, and the principles and embodiments described herein can be applied to antibodies and / or composition components having configurations different from those specifically described herein. In fact, it is explicitly contemplated that the components of the compositions and compounds of the present disclosure can be customized to facilitate their desired applications.

[0254] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the chemical and biological arts. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this application, the preferred methods and materials are described herein. In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, when a compound / composition is "substituted by an" alkyl or aryl, the compound / composition is optionally substituted by at least one alkyl and / or at least one aryl.

[0255] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and sub - combinations of ranges and specific embodiments therein are intended to be included. When referring to a number or numerical range, the term "about" means that the number or numerical range involved is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise", "comprises", "having", or "including") is not intended to exclude that in certain other embodiments, for example, an embodiment of any substance composition, composition of matter, method, or process described herein may "consist of the recited features" or "consist essentially of the recited features".

[0256] "Essentially" may allow a degree of variability of a value or range, for example, within 90%, 95%, or 99% of the limits of the value or range.

[0257] In the case of two or more polypeptide sequences, the terms "sequence identity" or "percent identity" refer to two or more such sequences or subsequences that are identical or have a specified percentage of identical peptides (i.e., having about 60% identity within a particular region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity within a particular region when compared and aligned in a comparison window or specified region, such as a targeted gene, to achieve maximum correspondence), as measured using sequence comparison algorithms known in the art or by manual alignment and visual inspection. Such sequences are then referred to as "substantially identical". In other words, there is identity in one or more regions of the overall sequence, provided that the general shape and structure of the molecule and one or more appropriate hydrogen bonds are maintained such that it is substantially suitable for the targeted binding site and functions as its agonist.

[0258] The term "fully canine antibody" refers to an antibody that includes only the protein sequence of canine immunoglobulin. If a fully canine antibody is produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, it may contain murine carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that includes only the mouse immunoglobulin sequence. Alternatively, if a fully canine antibody is produced in a rat, rat cell, or hybridoma derived from a rat cell, it may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that includes only the rat immunoglobulin sequence.

[0259] In certain embodiments, the compounds, compositions, and methods of the present disclosure can be used for the prevention and / or treatment of cancer. In certain embodiments, the provided compounds and / or compositions can also be used for the treatment of cancer. In certain embodiments, the compounds provided herein are provided or used alone, in combination with targeting agents, and / or in combination therapies with other interventions such as cytokine-based and other immunotherapies.

Claims

1. A caninized antibody or an antigen-binding fragment thereof that specifically binds to programmed death ligand 1 (PD-L1) in a canine subject.

2. The antibody or antigen-binding fragment according to claim 1, encoded by a nucleotide sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and / or 4 or SEQ ID NO: 6 and / or 8.

3. The antibody or antigen-binding fragment according to claim 1, comprising one or more complementarity-determining regions (CDRs), each CDR comprising at least 80% sequence identity with AAS, SEQ ID NO: 9 and / or SEQ ID NO:

10.

4. The antibody or antigen-binding fragment according to claim 1, comprising CDRs that independently comprise at least 80% sequence identity with SEQ ID NO: 17, WTS and / or SEQ ID NO:

12.

5. The antibody or antigen-binding fragment according to claim 1, comprising CDRs that independently comprise at least 80% sequence identity with SEQ ID NO: 15, SEQ ID NO: 16 and / or SEQ ID NO:

11.

6. The antibody or antigen-binding fragment according to claim 1, comprising CDRs that independently comprise at least 80% sequence identity with SEQ ID NO: 18, SEQ ID NO: 19 and / or SEQ ID NO:

13.

7. The antibody or antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment is fully or partially caninized.

8. The antibody or antigen-binding fragment according to any one of claims 1-7, wherein, The antibody or antigen-binding fragment is a chimeric form of a caninized antibody or antigen-binding fragment.

9. The antibody or its antigen-binding fragment according to claim 1, comprising a caninized murine PD-1 antibody or an antigen-binding fragment thereof.

10. A method for treating cancer in a canine subject, comprising administering to the canine subject a therapeutically effective amount of the antibody or antigen-binding fragment according to any one of claims 1-9 or the pharmaceutical composition according to claim 22.

11. The method according to claim 10, wherein, The caninized antibody or its antigen-binding fragment is encoded by a nucleotide sequence comprising at least 80% sequence identity with SEQ ID NO: 2 and / or 4 or SEQ ID NO: 6 and / or 8.

12. The method according to claim 10, wherein, The caninized antibody or antigen-binding fragment comprises one or more complementarity-determining regions (CDRs), and the one or more complementarity-determining regions independently comprise at least 80% sequence identity with AAS, SEQ ID NO: 9 and / or SEQ ID NO:

10.

13. The method according to claim 10, wherein, The caninized antibody or antigen-binding fragment comprises one or more CDRs, and the one or more CDRs independently comprise at least 80% sequence identity with SEQ ID NO: 18, WTS and / or SEQ ID NO:

12.

14. The method according to claim 10, wherein, The caninized antibody or antigen-binding fragment comprises one or more CDRs, and the one or more CDRs comprise at least 80% sequence identity with SEQ ID NO: 15, SEQ ID NO: 16 and / or SEQ ID NO:

11.

15. The method according to claim 10, wherein The canine - derived antibody or antigen - binding fragment comprises one or more complementarity - determining regions (CDRs), and the one or more complementarity - determining regions comprise at least 80% sequence identity with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:

13.

16. The method according to claim 10, wherein, The antibody or its antigen - binding fragment is formulated into a pharmaceutical composition.

17. The method according to claim 10, wherein The therapeutically effective amount is from about 2 mg / kg of the subject's body weight (such as, for example, 2 mg / kg of the subject's body weight) to about 5 mg / kg of the subject's body weight (such as, for example, 5 mg / kg of the subject's body weight).

18. The method according to claim 10, wherein The therapeutically effective amount is 2 mg / kg to 5 mg / kg of the subject's body weight.

19. The method according to claim 10, wherein, The therapeutically effective amount is 2 mg / kg of the subject's body weight.

20. The method according to claim 10, wherein, The therapeutically effective amount is 5 mg / kg of the subject's body weight.

21. The method according to claim 10, wherein, The cancer is invasive urothelial carcinoma.

22. A pharmaceutical composition comprising the canine - derived antibody or its antigen - binding fragment according to any one of claims 1 - 9 and a pharmaceutically acceptable excipient.

23. A method for predicting and modeling the anti-cancer activity of a test compound in a subject with cancer, wherein, The method comprises: Generating a population of canine - PD - L1 - humanized mice that express canine PD - L1 on the cell surface; Evaluating the toxicity risk and / or efficacy of a set of test compounds in treating cancer in the population of canine - PD - L1 - humanized mice; and Selecting one or more test compounds of the set that meet established toxicity risk and / or efficacy criteria.

24. The method according to claim 23, further comprising evaluating the toxicity risk and / or efficacy of the one or more selected compounds in treating cancer in a human or canine cohort.

25. The method according to claim 23, wherein Generating the population of canine - PD - L1 - humanized mice further comprises replacing the murine cd274 gene in the mouse population with the canine PD - L1 gene using CRISPR.

26. The method according to claim 23, further comprising evaluating the oral bioavailability, absorption, distribution, metabolism, and excretion (ADME) values of the test compounds of the set in the population of canine - PD - L1 - humanized mice.

27. The method according to claim 26, further comprising evaluating the oral bioavailability and ADME values of one or more selected test compounds in the population of canine - PD - L1 - humanized mice.

28. The method according to claim 23, wherein, The set of test compounds comprises at least one compound that inhibits the PD - L1 / PD - 1 interaction in a subject.

29. A complementarity - determining region (CDR) of an antibody or antigen - binding fragment, comprising at least 80% sequence identity with AAS, SEQ ID NO:9, and / or SEQ ID NO:

10.

30. A complementarity - determining region (CDR) of an antibody or antigen - binding fragment, comprising at least 80% sequence identity with SEQ ID NO:18, WTS, and / or SEQ ID NO:

12.

31. A complementarity - determining region (CDR) of an antibody or antigen - binding fragment, comprising at least 80% sequence identity with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:

11.

32. A complementarity-determining region (CDR) of an antibody or antigen-binding fragment, comprising at least 80% sequence identity with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:

13.

33. A complementarity-determining region (CDR) of an antibody or antigen-binding fragment, comprising at least 80% sequence identity with SEQ ID NO:17, WTS, and / or SEQ ID NO:

12.

34. A complementarity-determining region (CDR) of an antibody or antigen-binding fragment, comprising at least 80% sequence identity with SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:

11.

35. A complementarity-determining region (CDR) of an antibody or antigen-binding fragment, comprising at least 80% sequence identity with SEQ ID NO:18, SEQ ID NO:19, and / or SEQ ID NO:

13.

36. Use of a humanized antibody or antigen-binding fragment according to any one of claims 1-9, a pharmaceutical composition according to claim 22, or one or more CDRs according to any one of claims 29-35 in the preparation of a medicament for treating cancer in a canine subject.

Images