Anti-canine PD-1 antibodies, compositions containing, and therapeutic or diagnostic use thereof

Abstract

This invention relates to a novel antibody (HugPet9) which recognizes canine PD-1. The invention also relates to the use thereof for treating and / or detecting canine cancer. In some embodiments the antibody is administered in conjunction with CPMV particles and / or the treatment regimen includes cancer surgery. In some embodiments the HugPet9 antibody and / or CPMV particles are administered intratumorally. In some embodiments the treated canine has mammary cancer, optionally inflammatory mammary cancer.

Description

PATENT ATTY. DOCKET NO.1143252.007213 ANTI-CANINE PD-1 ANTIBODIES, COMPOSITIONS CONTAINING, AND THERAPEUTIC OR DIAGNOSTIC USE THEREOF RELATED APPLICATIONS The present application claims benefit of priority to US Provisional Application Nos.: 63 / 598,756, filed on November 14, 2023, and 63 / 672,987 filed July 18, 2024, the contents of each of which are incorporated by reference in their entirety. SEQUENCE LISTING DISCLOSURE The contents of the electronic sequence listing (1143252_007213_SL.xml; Size: 74,782 bytes; and Date of Creation: September 23, 2024) is herein incorporated by reference in its entirety. FIELD The present application generally relates to novel antibodies and antigen binding fragments thereof which recognize canine Programmed cell death protein 1 (also known as PD-1 and CD279). These antibodies and antibody fragments may be used as a monotherapy or in combination therapies for treating cancer in canines, e.g., another anti-cancer agent or cancer regime such as another anti-tumor antibody, checkpoint inhibitor fusion protein, chemotherapeutic, toxin, checkpoint inhibitor antibody or cytokine. In particular the invention includes combination therapies comprising the administration of an anti-PD-1 antibody and Cowpea Mosaic Virus (CPMV)-based neoantigens. BACKGROUND Approximately 1 in 4 dogs will, at some stage in their life, develop neoplasia. Almost half of dogs over the age of 10 will develop cancer. Dogs get cancer at roughly the same rate as humans, while there is less information about the rate of cancer in cats. Some cancers, such as lymphoma, are more common in cats than in dogs. Cancer is the most common cause of death in adult dogs. Many features of spontaneously developing tumors in pet dogs contribute to their potential utility as a human disease model. These include similar environmental exposures, similar clonal evolution as it applies to important factors such as immune avoidance, a favorable body size for imagingPATENT ATTY. DOCKET NO.1143252.007213 and serial biopsy, and a relatively contracted time course of disease progression, which makes evaluation of temporal endpoints such as progression free or overall survival feasible in a comparatively short time frame. These criteria have been leveraged to evaluate novel local therapies, demonstrate proof of tumor target inhibition or tumor localization, evaluate potential antimetastatic approaches, and assess the efficacy, safety and immune effects of a variety of immune-based therapeutics. Some of these canine proof of concept studies have been instrumental in informing subsequent human clinical trials. In particular the addition of immunotherapy to other pillars of oncology (surgical, radiation, and systemic therapy) has been pivotal in transforming the therapeutic landscape for treating many different types of human tumors (Adams S et al., (2019) “Pembrolizumab monotherapy for previously treated metastatic triple-negative breast cancer: cohort A of the phase II KEYNOTE-086 study”, Ann Oncol 30:397-404. doi: 10.1093 / annonc / mdy517; Adams S et al., (2019) “Pembrolizumab monotherapy for previously untreated, PD-L1- positive, metastatic triple-negative breast cancer: cohort B of the phase II KEYNOTE-086 study”, Ann Oncol 30:405-411. doi: 10.1093 / annonc / mdy518; Cortes J et al., (2020) KEYNOTE-355: “Randomized, double-blind, phase III study of pembrolizumab + chemotherapy versus placebo + chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer”, J Clin Oncol 38:1000-1000. doi: 10.1200 / JCO.2020.38.15_suppl.1000; Torres ETR et al., (2022) “Emerging combination immunotherapy strategies for breast cancer: dual immune checkpoint modulation, antibody–drug conjugates and bispecific antibodies”, Breast Cancer Res Treat 191:291-302. doi: 10.1007 / s10549-021-06423-0; Debien V et al., (2023) “Immunotherapy in breast cancer: an overview of current strategies and perspectives”, NPJ Breast Cancer 9:7. doi: 10.1038 / s41523-023-00508-3). In light of the good antitumor responses elicited by immune checkpoint inhibitors against many human tumors, the search for equivalent monoclonal antibodies to treat canine cancer patients is slowly gaining traction Klingemann H (2021) “Immunotherapy for Dogs: Still Running Behind Humans”, Front Immunol 12. doi: 10.3389 / fimmu.2021.665784. Also, the use of cowpea mosaic virus (CPMV) as a neoadjuvant in canines is known. Particularly, in situ vaccination (ISV) by introducing CPMV particles (empty or RNAPATENT ATTY. DOCKET NO.1143252.007213 containing) directly into a tumor to stimulate antitumor immunity both against the treated tumor and systemically against untreated tumors (Mao C, et al. “In situ vaccination with cowpea mosaic virus elicits systemic antitumor immunity and potentiates immune checkpoint blockade”, Journal for Immunotherapy of Cancer, 2022;10:e005834. doi:10.1136 / jitc-2022-005834) has been reported. Specifically, it has been reported that cowpea mosaic virus (CPMV) is a potent multi-toll-like receptor (TLR) agonist with potent efficacy for treating tumors in mice and dogs by ISV. Also, combination therapies wherein CPMV is combined with CD40 agonist antibodies and / or anti-PD-1 antibodies have been reported and allegedly the results demonstrate that the combination of local CPMV / aCD40 and systemic aPD-1 eliminates local and distant tumors after repeated administration. (Mao C, et al., Id.) Notwithstanding the foregoing, it would be beneficial to provide novel and improved anti-canine PD-1 antibodies, particularly those which possess greater specificity, enhanced antigen binding affinity (lower KD), and / or which elicit greater antitumor potency, alone or in association with other antitumor agents compared to existing anti-canine PD-1 antibodies. Further, it would be beneficial to novel therapeutic regimens involving the administration of anti-canine PD-1 antibodies. Also, it would be beneficial to provide combination therapies using these novel and improved anti-canine PD-1 antibodies. The present invention achieves these objectives. SUMMARY The inventors initially identified five antibodies using hybridoma technology targeting the canine Programmed cell death protein 1 (also known as PD-1 and CD279). From these five antibodies, an anti-PD-1 antibody referred to as “HugPet9” was identified as possessing properties making it well suited for use as a diagnostic and / or a therapeutic agent for detecting / treating canine cancers alone or in combination with other actives, e.g., cowpea mosaic virus (CPMV) particles (empty and non-empty CPMV particles). The HugPet9 antibody is also referred to as “77A6H9” herein (see the Sequence Listing). Also, a closely related variant of the HugPet9 antibody, i.e., “77A6H7”, and having the same CDRs as HugPet9 and comprising the sequences disclosed in the Sequence Listing is also provided. As is disclosed in more detail infra, HugPet9 is a mouse anti-canine PD-1 antibody (acPD-1) which was demonstrated using flow cytometry to avidly bind to canine peripheralPATENT ATTY. DOCKET NO.1143252.007213 blood mononuclear cells (PBMCs). Also, HugPet9 was demonstrated to elicit IFN-γ production in PBMCs activated with Concanavalin A. Further, HugPet9 was demonstrated to bind to different canine tumor tissues that express PD-1 protein. Further, in a proof-of- concept safety trial when HugPet9 was used as an intratumoral (IT) immunotherapy in an open-labelled phase I preclinical study in canine mammary cancer (CMC) patients alone or in combination with CPMV neoadjuvant, this antibody was shown to be safe and effective, i.e., it enhanced patient survival, reduced tumor size and inhibited metastasis. OBJECTS OF THE INVENTION Accordingly, it is an object of the invention to provide novel monoclonal antibodies and antigen binding fragments thereof which bind to canine PD-1 having the sequences disclosed herein. Particularly, it is an object of the invention to provide monoclonal antibodies and fragments thereof which bind to canine PD-1 having the same CDRs as HugPet9 or 77A6H9 or 77A6H7. Also, it is a specific object of the invention to provide an antibody or antibody fragment which comprises the same CDRs as HugPet9 or 77A6H9 or 77A6H7 and which comprises (i) a VH polypeptide possessing an amino acid sequence which possesses at least 90% sequence identity to the VH polypeptide of HugPet9 or 77A6H9 or 77A6H7, and (ii) a VL polypeptide possessing an amino acid sequence which possesses at least 90% sequence identity to the VL polypeptide of HugPet9 or 77A6H9 or 77A6H7. Also, it is a specific object of the invention to provide an antibody or antibody fragment as above described, which comprises the same CDRs as HugPet9 or 77A6H9 or 77A6H7 and which comprises (i) a VH polypeptide possessing an amino acid sequence which possesses at least 95% sequence identity to the VH polypeptide of HugPet9 or 77A6H9 or 77A6H7, and (ii) a VL polypeptide possessing an amino acid sequence which possesses at least 95% sequence identity to the VL polypeptide of HugPet9 or 77A6H9 or 77A6H7. Also, it is a specific object of the invention to provide an antibody or antibody fragment as above described, which comprises which comprises the same CDRs as HugPet9 or 77A6H9 or 77A6H7 and which comprises (i) a VH polypeptide possessing an amino acid sequence which possesses at least 98-99% sequence identity to the VH polypeptide ofPATENT ATTY. DOCKET NO.1143252.007213 HugPet9 or 77A6H9 or 77A6H7, and (ii) a VL polypeptide possessing an amino acid sequence which possesses at least 98-99% sequence identity to the VL polypeptide of HugPet9 or 77A6H9 or 77A6H7. Also, it is a specific object of the invention to provide an antibody or antibody fragment as above described, which comprises (i) a VH polypeptide possessing an amino acid sequence which is identical to the VH polypeptide of HugPet9 or 77A6H9 or 77A6H7, and (ii) a VL polypeptide which is identical to the VL polypeptide of HugPet9 or 77A6H9 or 77A6H7. Also, it is a specific object of the invention to provide an antibody or antibody fragment as above described, which comprises an Fc or constant region, optionally a murine, human or canine Fc or constant region. Also, it is a specific object of the invention to provide an antibody or antibody fragment as above described, which comprises a canine Fc region selected from canine IgGA, canine IgGB, canine IgGC and canine IgGD, which optionally comprises at least one modification that enhances or inhibits at least one antibody effector function, further optionally at least one modification that enhances or inhibits at least one antibody effector function selected from glycosylation, FcR binding, FcRN binding, phagocytosis, antibody dependent cellular cytotoxicity (ADCC), and / or complement dependent cytotoxicity (CDC). Also, it is a specific object of the invention to provide an antibody or antibody fragment as above described, optionally a Fab, Fab2or scFv. Also, it is an object of the invention to provide an immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising an antibody or antibody fragment according to any of the foregoing. Also, it is a specific object of the invention to provide an antibody or antibody fragment according to any of the foregoing, or immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising same, which is expressed in a recombinant host cell, optionally a mammalian, yeast, fungal, plant, insect or bacterial cell. Also, it is a specific object of the invention to provide an antibody or antibody fragment according to any of the foregoing, or immunoconjugate, antibody drug conjugatePATENT ATTY. DOCKET NO.1143252.007213 (ADC) or chimeric antigen receptor (CAR) comprising same, which is expressed in a CHO, BHK, COS, Hela, HEK or insect cell. Also, it is a specific object of the invention to provide a nucleic acid or nucleic acids which encode for an antibody, antibody fragment, immunoconjugate, ADC, or CAR according to any of foregoing. Also, it is a specific object of the invention to provide an expression vector comprising the nucleic acid or nucleic acids of the foregoing. Also, it is a specific object of the invention to provide a recombinant cell which comprises an expression vector according to the foregoing, optionally a mammalian, yeast, fungal, plant, insect or bacterial cell, further optionally a CHO, BHK, COS, Hela, HEK or insect cell. Also, it is a specific object of the invention to provide a diagnostic or therapeutic composition comprising an antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of the foregoing and a therapeutically or diagnostically acceptable carrier. Also, it is a specific object of the invention to provide a method of treating canine cancer comprising the administration of an antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of the foregoing. Also, it is a specific object of the invention to provide a method of treating canine cancer comprising the administration of an antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of the foregoing, wherein the administration thereof is intratumoral. Also, it is a specific object of the invention to provide a method of treating canine cancer comprising the administration of an antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of the foregoing, wherein the canine cancer comprises an adenoma, adenocarcinoma, anal sac adenocarcinoma, basal tumor, bladder cancer, bone cancer (e.g., osteosarcoma), brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, hemangiosarcoma, histiocytic sarcoma, leukemia, lipoma, liver cancer, lung cancerPATENT ATTY. DOCKET NO.1143252.007213 e.g., canine primary lung cancer, lymphoma, lymphosarcoma, mammary cancers or mastocytoma, mast cell tumor, melanoma, oral cancers such as melanoma, squamous cell carcinoma or fibrosarcoma; ovarian cancer, nasal cancer or nasal tumor, pancreatic cancer, prostate cancer, skin cancers (e.g., melanoma), soft tissue sarcoma, splenic hemangiosarcoma (HSA), squamous cell carcinoma, testicular cancer, transitional cell carcinoma, uterine cancers, among others, and preferably for use in treating mammary cancers or inflammatory mammary cancers. Also, it is a specific object of the invention to provide a method of treating canine cancer comprising the intratumoral administration of an antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of the foregoing, and at least one other therapeutic agent or therapeutic treatment, optionally wherein said other therapeutic agent comprises an anti- cancer agent, further optionally empty or non-empty CPMV particles and / or a CD40 agonist antibody and / or optionally the other cancer treatment includes cancer surgery or radiation. Also, it is a specific object of the invention to provide a therapeutic composition or therapeutic method according to the foregoing, wherein said other therapeutic agent comprises another checkpoint inhibitor antibody or checkpoint inhibitor fusion protein, a hormone, a cytokine, a growth factor, a chemotherapeutic, another anti-cancer antibody, or a combination of any of the foregoing. Also, it is a specific object of the invention to provide an antibody or antibody fragment, immunoconjugate, ADC, or CAR composition, or method according to any one of the foregoing, wherein the antibody is chimeric. Also, it is a specific object of the invention to provide an antibody or antibody fragment, immunoconjugate, ADC, or CAR composition, or method according to any one of the foregoing, wherein the antibody or antibody fragment is caninized. Also, it is a specific object of the invention to provide a pharmaceutical composition comprising the antibody or antibody fragment, immunoconjugate, ADC, or CAR, according to any one of the foregoing, and a pharmaceutically acceptable carrier.PATENT ATTY. DOCKET NO.1143252.007213 Also, it is a specific object of the invention to provide a kit comprising the antibody or antibody fragment, immunoconjugate, ADC, or CAR according to any one of the foregoing. Also, it is a specific object of the invention to provide method of treatment, comprising administering an antibody or antibody fragment, immunoconjugate, ADC, or CAR, or composition containing, or a cell expressing according to any of the foregoing to a canine subject in need thereof. Also, it is a specific object of the invention to provide a treatment method of any one of the foregoing, wherein said anti-PD-1 antibody and said second therapeutic agent, optionally empty or non-empty CPMV particles and / or a CD40 agonist antibody are administered to said subject, optionally via intratumoral delivery, separately and / or in combination. Also, it is a specific object of the invention to provide a method of detecting a PD-1 antigen and / or cells which express PD-1 in a canine, which detection method is effected in vivo and / or in vitro, which optionally is used to assess the disease status of a canine subject or optionally to assess the treatment status of a canine subject, optionally after administration of an antibody or antibody fragment, immunoconjugate, ADC, or CAR, or composition according to any one of the foregoing or a cell which expresses antibody or antibody fragment, immunoconjugate, ADC, or CAR according to any one of the foregoing. Also, it is an object of the invention to provide therapeutic compositions comprising an anti-PD-1 antibody having the sequences disclosed herein and a pharmaceutically acceptable carrier or excipient and optionally another therapeutic, e.g., another anticancer agent, e.g., CPMV particles. Also, it is an object of the invention to provide therapeutic methods comprising the use of the subject anti-PD-1 antibodies or a PD-1 binding fragment thereof disclosed infra for treating canine cancers optionally characterized by the expression or increased expression of PD-1, e.g., an adenoma, adenocarcinoma, anal sac adenocarcinoma, basal tumor, bladder cancer, bone cancer (e.g., osteosarcoma), brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, hemangiosarcoma, histiocytic sarcoma, leukemia, lipoma, liver cancer, lung cancer e.g., canine primary lung cancer,PATENT ATTY. DOCKET NO.1143252.007213 lymphoma, lymphosarcoma, mammary cancers or mastocytoma, mast cell tumor, melanoma, oral cancers such as melanoma, squamous cell carcinoma or fibrosarcoma; ovarian cancer, nasal cancer or nasal tumor, pancreatic cancer, prostate cancer, skin cancers (e.g., melanoma), soft tissue sarcoma, splenic hemangiosarcoma (HSA), squamous cell carcinoma, testicular cancer, transitional cell carcinoma, uterine cancers, among others, and preferably for use in treating mammary cancers or inflammatory mammary cancers, wherein the antibody or fragment thereof is administered as a monotherapy or in combination with other therapies, e.g., other antitumor agents, CPMV particles and / or an anti-CD40 agonist antibody. Also, it is an object of the invention to provide therapeutic methods comprising the use of HugPet9 and other cancer treatment methods, such as surgery, radiotherapy, endocrine therapy, biologic response modifiers (interferons, interleukins, antibodies, aptamers, siRNAs, oligonucleotides, enzymes, ion channel and receptor inhibitors or activators et seq.), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g., antiemetics), chemotherapeutic drugs, including, but not limited to, alkylating drugs (e.g., mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites (e.g., Methotrexate), purine antagonists and pyrimidine antagonists (e.g., 6- Mercaptopurine, 5-Fluorouracil, Cytarabine, Gemcitabine), spindle poisons (e.g., Vinblastine, Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (e.g., Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (e.g., Carmustine, Lomustine), inorganic ions (e.g., Cisplatin, Carboplatin), enzymes (e.g., Asparaginase), and hormones (e.g., Tamoxifen, Leuprolide, Flutamide, and Megestrol), among others. Also, it is a specific object of the invention to provide pharmaceutical compositions which comprise a therapeutically effective amount of HugPet9 or a variant thereof formulated together with one or more pharmaceutically acceptable carriers and optionally one or more other active agents such as one or more of those above-identified. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type which optionally can be administered to canines and other human or non-human animals orally, intratumorally, rectally, parenterally, intracisternally, intravaginally,PATENT ATTY. DOCKET NO.1143252.007213 intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray. Also, it is a specific object of the invention to provide methods of using HugPet9 and fragments thereof to detect PD-1 in vitro in canine patient samples and / or in vivo, e.g., to assess disease status. Further, it is a specific object of the invention to provide methods of using HugPet9 and fragments thereof to stage cancer prognosis, design specific treatment regimens, and / or to establish the efficacy of a specific cancer treatments in canines. DESCRIPTION OF THE DRAWINGS FIG.1. This Figure depicts the use of HugPet9 for detecting PD-1 expression on Hek293T cells. The Hek293T cells were transfected with canine PD-1, expanded and used to detect PD-1 levels in these cells by immunocytochemistry (A) or embedded in paraffin by standard IHC with HugPet9 (B). The formats reflect tissues and archival paraffin-embedded samples respectively. FIG.2A-C show the effect of HugPet9 on IFNγ expression in PMBCs of heathy and cancer dogs. In FIG.2A-B canine PMBCs from healthy (A) and cancer-bearing dogs (B) were activated with an isotype IgG1 antibody, Staphylococcus enterotoxin B (SEB, 50 ng / ml) or SEB plus HugPet9 at the doses indicated in the X-axis. The IFN-γ levels are indicated on the Y axis. FIG.2C how the effect of HugPet9 on IFNγ expression in PMBCs of heathy and cancer dogs activated with Con A (2.5 μg / ml; Figure 2, right panel) and at the same time incubated with HugPet9 antibody. As shown therein there was a dose-dependent increase in IFN-γ release compared with an isotype-matched, irrelevant antibody. Fig 3A-F shows the detection of PD-1 levels in cancer samples using HugPet9. PD- 1 protein is detected in jejunum (A), melanoma (B), lymphoma (C-D), E is a negative control, no HugPet9 was added, and canine tonsils (F). Fig 4A-B show the results of an intratumoral (IT) monotherapy using HugPet9. The effect of IT HugPet9 in injected tumors is shown in (A), and the tumor response evaluated by tumor growth inhibition (% TGI relative to D0 on the X-axis is presented in (B)PATENT ATTY. DOCKET NO.1143252.007213 where the dotted lines (20% and ~30%) represent the stable disease area with values up or down representing progressive disease and partial response, respectively. Fig 5A-F shows the staining of canine normal lymph node and intestinal lymphoma samples with HugPet9. The HugPet9 antibody shows membranous and cytoplasmic positivity. Reactivity mostly within germinal centers in the superficial cortex and very few positive cells in T lymphocytes areas, surrounding germinal centers, paracortex, deep cortex (A). A commercial comparator antibody (clone JC053) presents a membranous and cytoplasmic positivity in abundant cells within germinal centers (pro-B lymphocytes, some T lymphocytes), surrounding germinal centers, paracortex, deep cortex (T lymphocytes). Also, some stromal cells show cytoplasmic staining in fibroblasts and endothelial cells (B). In the canine intestinal lymphoma sample (D-E), HugPet9 is negative, showing no reactivity (D). The commercial comparator antibody (clone JC053) presents membranous and cytoplasmic reactivity in approximately 25% of neoplastic lymphocytes (suspected to be T lymphocytes not confirmed by IHC). In addition, there is a non-specific reactivity (membranous and cytoplasmic) in epithelial cells, endothelial cells and stromal cells (E). In the assay, canine lymph node (C) and canine lymphoma tissues (F) were used as controls (no antibody was used in the assay). Figure 6 schematically describes the clinical trial treatment regimen. As is shown in the Figure 6 one canine treatment group received HugPet9 alone, i.e., 0.125 mg of HugPet9 on day 0, 0.25 mg of HugPet9 on day 7, 0.5 mg of HugPet9 on day 14 and 1.0 mg of HugPet9 on day 21. As is further shown in the Figure 6, a second canine group received HugPet9 and CPMV VLPS on different days, i.e., 0.2 mg of CPMV on days 0, 7, 14 and 21 and 0.2 mg of HugPet9 on days 2, 9, 16 and 23. Finally, the schematic in Figure 6 shows that on day 29 patients were administered a combination of CPMV VLPs and HugPet9, respectively at dosages of 0.4 mg and 0.5mg. Figure 7A-F contain data evidencing that acPD-1 and CPMV / acPD-1 treatments are associated with tumor control. Weekly acPD-1 as monotherapy (patients P1-P3; purple arrows; A) or CPMV (light green arrows in C) plus acPD-1 (purple arrows in C) was given for four weeks to patients P4-P6; in P5, tumors P5.1 and P5.2 were treated. Long-term weekly CPMV / acPD-1 (E; dark green arrows) was given to patients P1, P5 (P5.1 and P52 werePATENT ATTY. DOCKET NO.1143252.007213 treated) and P6. The %TGI (relative to D0) in B, D, and F indicates SD in the dotted areas (-30 to 20), above 20% indicates PD, and below -30%, partial response. Figure 8A-L contain data evidencing that CPMV / acPD-1 treatments have a variable abscopal effect on established lung metastases. Of the five established lung metastatic nodules in P5, M1, M2, M4, and M5 nodules were responsive to CPMV / acPD-1 treatments, while M3 was not and had a steady tumor growth (A). During treatment, we observe CR in M2 and M4, PR in M1 and M5, and PD in M3 (B). After surgery on D113, we observed CR in M5, PR in M1, and PD in M3 (B). In P6, M1 and M3 nodules showed transient responses, and M2 slowly grew during treatment and after surgery on D79 (C). In this patient, we observed transient SD in the largest M1, and a rapid transition from SD to PD in M2 and M3 (D). The vertical dotted line in the x-axis indicates palliative surgery (mastectomy or tumor resection) of the primary tumors. Representative radiographs illustrate tumor changes in the left (E-H) and right (I-L) lung metastatic nodules in P5 before (D-2), during (up to D94), and after surgery (D254-D315). Note changes in tumor volume in M1 and M5 as well as the absence of M2, M4 and M5. Figure 9A-C contain data evidencing that acPD-1 and combination CPMV / acPD-1 treatments induce transcriptomic changes in the tumor microenvironment. During the four- week treatment period acPD-1 and CPMV / acPD-1 treatments affects gene expression in injected tumors (A). Extended CPMV / acPD-1 treatments affects gene expression in injected tumors, noninjected lung metastases (B), and noninjected tumors and inguinal nodes (C). Details in text. Legends: Each row indicates a gene; a column, an individual patient. Patient characteristics are indicated on the right side of the graphs. Patients are identified by a number (P3-P6), followed by the number of the treated or untreated tumor or metastatic lesion, and the day the sample was collected. P3 and P6 had a single treated tumor and are represented as P3.0 and P6.0 while P4 and P5 have the injected tumor (P4.1, P5.1 and P5.2), noninjected tumors (P5.3.113 and P5.4.113) and inguinal nodes in P5 (P5.5) and P6 (P6.2). The numbers at the end of each sample represent the day when the sample was collected. Metastatic lesions are represented with the letter M. Gene expression levels are represented by z-scores. Figure 10A-B contain data evidencing that CPMV / acPD-1 treatments induce changes in immune cell contents in injected tumors and noninjected, establishedPATENT ATTY. DOCKET NO.1143252.007213 metastases. CPMV / acPD-1 treatments increased immune cell content in both P5 (A) and P6 (B) patients with variations observed in the type of immune cells and the intensity of the increase with some immune cells having high content from D0 up to surgery day in P5 (D113), but not in P6 (D79)(compare CD8 T cells, mast cells, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDC) between P5 and P6) or an increase from D0 to D29 and then a drop in the content (compare plasma cells between P5 and P6). It is also noticeable the decrease in cell contents from surgery to metastases in P5 while an increase was observed in most of the immune cells in P6 metastases. Figure 11A-C contain data evidencing that acPD-1 or CPMV / acPD-1 treatments induced transient changes in blood cells. As shown therein treatment elicited a decrease in erythrocytes (A), hemoglobin (B), and hematocrit (C) in P1 and P5. Dotted areas indicate the normal range values for each variable. Figure 12A-C contain data evidencing that acPD-1 or CPMV / acPD-1 treatments induced transient changes in blood biochemistry. In the figure (A) shows total protein levels (g / dl) days after treatment initiation; (B) shows globulin levels (g / dl) days after treatment initiation; and (C) shows albumin levels (g / dl) days after treatment initiation. As can be seen treatment elicited a decrease in total proteins in some dogs, and slight hyperglobulinemia and hypoalbuminemia was observed in P1 and P5 patients. Dotted areas indicate the normal range values for each variable. Figure 13A-D contain data evidencing that acPD-1 or CPMV / acPD-1 treatments induced differential effects on treated patients. As can be seen high ALT levels decreased with treatment in P1, P5 and P6 and remain normal in other dogs (A). AST levels remained low in P1 and P5 (B); creatinine (C); and Urea (D) changes were observed in P1, P3 and P5 patients. Dotted areas indicate the normal range values for each variable. Figure 14 contains data evidencing that acPD-1 or CPMV / acPD-1 treatments induced different effects on noninjected tumors. As can be seen tumor reduction was observed in all three noninjected tumors in P2 and tumor control in one noninjected tumor in P4 (P4.3) and no response in another noninjected tumor (P4.2). Figure 15A-D contain data evidencing that acPD-1 or CPMV / acPD-1 treatment induced tumor changes in target injected and noninjected lesions in CMC patients byPATENT ATTY. DOCKET NO.1143252.007213 itRECIST criteria. The figure shows the percent change from baseline in diameters of target injected lesions in CMC cases treated with IT acPD-1 as monotherapy (A) or IT CPMV / acPD-1 combined therapy (B) during the first 4 weeks of treatment, and during the long-term IT CPMV / acPD-1 treatment (C), and percent change from baseline in diameters of noninjected lesions in P2 and P4 patients (D). The broken lines indicated SD (-30 to 20); PD, progressive disease; PR, partial response. Figure 16A-B contain data in Swimmer Plots showing the effects of acPD-1 or CPMV / acPD-1 treatment in target injected lesions and established lung metastases in CMC patients based on itRECIST criteria. In the figure (A) shows the response to IT treatment during follow-up in the target injected lesions in CMC cases treated with acPD-1 IT as monotherapy (blue lanes) or IT CPMV / acPD-1 combined therapy (green lanes); note that P1 received acPD-1 monotherapy until D29, and then continued with the combined therapy. Note, death of P5 is presented at D250 (*) though she died at D386 (A and B). (B) The abscopal effect of IT CPMV / acPD-1 treatment on established lung metastases in P5 (gray) and P6 (light orange) patients. *Per itRECIST guidelines, unconfirmed PD means the presence of PD, followed by SD, PR or CR; two consecutive evaluations showing PD are required for a confirmed PD. Figure 17A-B contain data evidencing that acPD-1 or CPMV / acPD-1 treatment induced Tumor changes in non-injected lung metastases in patients P5 and P6 by itRECIST criteria. The figure shows the percent change from baseline in diameters of noninjected lung lesions in P5 and P6. Surgery was performed at D113 (A) and 79 (B). SD, stable disease (-30% to 20%), PR, partial response (<-30%), PD, progressive disease (>20%), and complete response (-100%). Vertical dotted lines indicate the start of therapy (D0) and surgery day (D113 in A and D79 in B). Figure 18A-C contains data showing that CPMV / acPD-1 treatment has a variable effect on the reactome pathways in injected tumors. The top 10 immune-related reactome pathways are highlighted for clusters 1 (A), 2 (B), and 3 (C). Prior to providing the detailed description of the invention, the following definitions are provided. DEFINITIONSPATENT ATTY. DOCKET NO.1143252.007213 Unless defined otherwise presents membranous and cytoplasmic reactivity in approximately 25% of neoplastic lymphocytes (suspected to be e, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the invention or testing of the present invention, suitable methods and materials are described herein. The materials, methods and examples are illustrative only, and are not intended to be limiting. As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. "Adjuvant," as used herein, refers broadly to any substance which is incorporated into or administered simultaneously with HugPet9 and fragments thereof of the invention which potentiates the immune response in the subject. Adjuvants include but are not limited to CPMV particles (empty or non-empty), aluminum compounds, e.g., gels, aluminum hydroxide and aluminum phosphate, and Freund's complete or incomplete adjuvant (e.g., in which the PS / A antigen is incorporated in the aqueous phase of a stabilized water in paraffin oil emulsion). The paraffin oil may be replaced with different types of oils, e.g., squalene or peanut oil. Other materials with adjuvant properties, include BCG (attenuated Mycobacterium tuberculosis), calcium phosphate, levamisole, Isoprinosine, polyanions (e.g., poly A:U), lentinan, pertussis toxin, lipid A, saponins, QS-21 and peptides, e.g., muramyl dipeptide. Rare earth salts, e.g., lanthanum and cerium, may also be used as adjuvants. The amount of adjuvants depends on the subject and the particular antigen used and can be readily determined by one skilled in the art without undue experimentation. "Amino acid," as used herein, refers broadly to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.PATENT ATTY. DOCKET NO.1143252.007213 Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. "Antibody," as used herein, refers broadly to any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, from all sources, e.g., human, rodent, rabbit, cow, sheep, pig, dog, chicken, are considered to be "antibodies." Antibodies include but are not limited to chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (scFvs), camelbodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small-modular immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fabs, Fab', F(ab')2.) Numerous antibody coding sequences have been described; and others may be raised by methods well-known in the art. See Streltsov, et al. (2005) Protein Sci.14(11): 2901-9; Greenberg, et al. (1995) Nature 374(6518): 168-173; Nuttall, et al. (2001) Mol Immunol. 38(4): 313-26; Hamers-Casterman, et al. (193) Nature 363(6428): 446-8; Gill, et al. (2006) Curr Opin Biotechnol.17(6): 653-8. The term "antibody" is used in the broadest sense and specifically covers, for example, single monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), antibody compositions with poly-epitopic specificity, polyclonal antibodies, single chain antibodies, and fragments of antibodies as long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeable with antibody herein. An "isolated antibody" is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) toPATENT ATTY. DOCKET NO.1143252.007213 greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the alpha and gamma chains and four CH domains for mu and epsilon isotypes. Each L chain has at the N- terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chainsPATENT ATTY. DOCKET NO.1143252.007213 designated alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by thePATENT ATTY. DOCKET NO.1143252.007213 hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol Biol., 222:581-597 (1991), for example. The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc), and human constant region sequences. An "intact" antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions. "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a singlePATENT ATTY. DOCKET NO.1143252.007213 antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen- binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells. "Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in “The Pharmacology of Monoclonal Antibodies”, Vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994). The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having twoPATENT ATTY. DOCKET NO.1143252.007213 antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). "Caninized" forms of non-canine (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from the non-canine antibody. For example, caninized antibodies may comprise canine immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient antibody are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the canine immunoglobulin are replaced by corresponding non-human residues. Furthermore, caninized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the caninized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a canine immunoglobulin sequence. The caninized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a canine immunoglobulin, optionally one of canine IgGA, canine IgGB, canine IgGC and canine IgGD. Antibody caninization may be performed using a conventional CDR grafting method followed by construction and screening of a caninized phage display antibody library with rationally designed back mutations. A "species-dependent antibody," e.g., a mammalian anti-human IgE antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody "bind specifically" to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 10-7M, preferably no more than about 10-8M and most preferably no more than about 10-9M) but has a binding affinity for a homologue of the antigen from a second non-human mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its bindingPATENT ATTY. DOCKET NO.1143252.007213 affinity for the human antigen. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody. The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Additionally, when referring to a modified Fc domain or “Fc variant”, the terms “Kabat numbering system,” “Kabat position,” “Kabat residue,” “Kabat number” or the like, or in any instance in which an Fc modification is identified by number without reference to a specific numbering system (e.g., “position” followed by a number), refer to positions numbered according to the EU index or EU numbering scheme (Kabat et al., 1991, “Sequences of Proteins of Immunological Interest”, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, incorporated by reference). The terms “EU index” or “EU index as in Kabat” and the like refer to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated by reference). Additionally, except where stated otherwise, when referring to an Fc variant relative terms (such as “increased” or “decreased”) refer to the change in that attribute relative to the same Fc variant (or polypeptide containing said Fc variant) without said modification. Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation ofPATENT ATTY. DOCKET NO.1143252.007213 cell surface receptors (e.g., B cell receptor); and B cell activation. Exemplary antibodies of the present disclosure may include one or more modifications that decrease one or more effector functions, such as alterations in the amino acid sequence, or alterations in the location, extent, or type of glycosylation. "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express Fc gamma RIII only, whereas monocytes express Fc gamma RI, Fc gamma RII and Fc gamma RIII. FcR expression on hematopoietic cells is summarized on page 464 of Ravetch and Kinet, Annu Rev. Immunol.9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No.5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci., USA 95:652-656 (1998). "Antigen," as used herein, refers broadly to a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen may have one epitope, or have more than one epitope. The specific reaction referred to herein indicates that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by otherPATENT ATTY. DOCKET NO.1143252.007213 antigens. Antigens may be tumor specific (e.g., expressed by neoplastic cells of pancreatic and colon carcinoma.) "Antigenic composition," as used herein, refers broadly to a composition that elicits an immune response. "Cancer," as used herein, refers broadly to any neoplastic disease (whether invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor. "Chimeric antibody," as used herein, refers broadly to an antibody molecule in which the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug; or the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. "Conservatively modified variants," as used herein, applies to both amino acid and nucleic acid sequences, and with respect to particular nucleic acid sequences, refers broadly to conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) may be modified to yield a functionally identical molecule. "Complementarity determining region," "hypervariable region," or "CDR," as used herein, refers broadly to one or more of the hyper-variable or complementarily determining regions (CDRs) found in the variable regions of light or heavy chains of an antibody. See Kabat, et al. (1987) "Sequences of Proteins of Immunological Interest"PATENT ATTY. DOCKET NO.1143252.007213 National Institutes of Health, Bethesda, MD. These expressions include the hypervariable regions as defined by Kabat, et al (1991) "Sequences of Proteins of Immunological Interest" U.S. Dept. of Health and Human Services, or the hypervariable loops in 3-dimensionaI structures of antibodies. Chothia and Lesk (1987) J Mol. Biol.196: 901- 17. The CDRs in each chain are held in close proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids that have been described as the selectivity determining regions (SDRs) which represent the critical contact residues used by the CDR in the antibody-antigen interaction. Kashmiri (2005) Methods 36: 25-34. "Control amount," as used herein, refers broadly to a marker can be any amount or a range of amounts to be compared against a test amount of a marker. For example, a control amount of a marker may be the amount of a marker in a patient with a particular disease or condition or a person without such a disease or condition. A control amount can be either in absolute amount (e.g., microgram / ml) or a relative amount (e.g., relative intensity of signals). "Differentially present," as used herein, refers broadly to differences in the quantity or quality of a marker present in a sample taken from patients having a disease or condition as compared to a comparable sample taken from patients who do not have one of the diseases or conditions. For example, a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and / or NAT-based assays. A polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker may be considered to be differentially present. Optionally, a relatively low amount of up-regulation may serve as the marker. "Diagnostic," as used herein, refers broadly to identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "falsePATENT ATTY. DOCKET NO.1143252.007213 negatives." Subjects who are not diseased and who test negative in the assay are termed "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. "Diagnosing," as used herein, refers broadly to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and / or prospects of recovery. The term "detecting" may also optionally encompass any of the foregoing. Diagnosis of a disease according to the present invention may, in some embodiments, be affected by determining a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease. It should be noted that a "biological sample obtained from the subject" may also optionally comprise a sample that has not been physically removed from the subject. An "effective amount" of a composition such as a polypeptide, drug, siRNA or analog thereof, antibody, pharmaceutical, small molecule, or other compound, or an agonist or antagonist thereof, is an amount sufficient to carry out a specifically stated purpose. An "effective amount" may be determined empirically and in a routine manner, in relation to the stated purpose. "Expression vector," as used herein, refers broadly to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell. The term includes recombinant expression cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.PATENT ATTY. DOCKET NO.1143252.007213 "Framework region" or "FR," as used herein, refers broadly to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody. See Kabat, et al (1987) "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, MD. These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody. "Heterologous," as used herein, refers broadly to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). "High affinity," as used herein, refers broadly to an antibody having a dissociation constant of about or less than 10-8M, more preferably about or less than 10-9 M and even more preferably about or less than 10-10M for a target antigen. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a dissociation constant of about or less than 10-7M and even more preferably about or less than 10-8M for a target antigen. "Homology," as used herein, refers broadly to a degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between a polypeptide sequence and a reference polypeptide sequence. Homology may be partial or complete. Complete homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is one that is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" may be used interchangeably with "homology." "Host cell," as used herein, refers broadly to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect (e.g., SF9),PATENT ATTY. DOCKET NO.1143252.007213 amphibian, or mammalian cells such as CHO, HeLa, HEK-293, e.g., cultured cells, explants, and cells in vitro. Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. "K-assoc" or "Ka", as used herein, refers broadly to the association rate of a particular antibody-antigen interaction, whereas the term "Kdiss" or "Kd," as used herein, refers to the dissociation rate of a particular antibody-antigen interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. , Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. "Immunoassay," as used herein, refers broadly to an assay that uses an antibody to specifically bind an antigen. The immunoassay may be characterized by the use of specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen. "Isolated," as used herein, refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or anyPATENT ATTY. DOCKET NO.1143252.007213 material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated antibody”). "Label" or a "detectable moiety" as used herein, refers broadly to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. "Low stringency," "medium stringency," "high stringency," or "very high stringency conditions," as used herein, refers broadly to conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel, et al. (2002) “Short Protocols in Molecular Biology”, (5th Ed.) John Wiley & Sons, NY. Exemplary specific hybridization conditions include but are not limited to: (1) low stringency hybridization conditions in 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2XSSC, 0.1 % SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); (2) medium stringency hybridization conditions in 6XSSC at about 45°C, followed by one or more washes in 0.2XSSC, 0.1 % SDS at 60°C; (3) high stringency hybridization conditions in 6XSSC at about 45°C, followed by one or more washes in 0.2XSSC, 0.1 % SDS at 65°C; and (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2XSSC, 1 % SDS at 65°C. "Mammal," as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, gerbils, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington DC. "Nucleic acid" or "nucleic acid sequence," as used herein, refers broadly to a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogsPATENT ATTY. DOCKET NO.1143252.007213 of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. "Patient," as used herein, refers broadly to any animal who is in need of treatment either to alleviate a disease state or to prevent the occurrence or reoccurrence of a disease state. Also, "Patient" as used herein, refers broadly to any animal who has risk factors, a history of disease, susceptibility, symptoms, signs, was previously diagnosed, is at risk for, or is a member of a patient population for a disease. The patient may be a clinical patient such as a human or a veterinary patient such as a companion, domesticated, livestock, exotic, or zoo animal. The term "subject" may be used interchangeably with the term "patient". Generally herein this term refers to a canine subject. "Promoter," as used herein, refers broadly to an array of nucleic acid sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that isPATENT ATTY. DOCKET NO.1143252.007213 active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. "Prophylactically effective amount," as used herein, refers broadly to the amount of a compound that, when administered to a patient for prophylaxis of a disease or prevention of the reoccurrence of a disease, is sufficient to effect such prophylaxis for the disease or reoccurrence. The prophylactically effective amount may be an amount effective to prevent the incidence of signs and / or symptoms. The "prophylactically effective amount" may vary depending on the disease and its severity and the age, weight, medical history, predisposition to conditions, preexisting conditions, of the patient to be treated. "Prophylaxis," as used herein, refers broadly to a course of therapy where signs and / or symptoms are not present in the patient, are in remission, or were previously present in a patient. Prophylaxis includes preventing disease occurring subsequent to treatment of a disease in a patient. Further, prevention includes treating patients who may potentially develop the disease, especially patients who are susceptible to the disease (e.g.., members of a patent population, those with risk factors, or at risk for developing the disease). "Recombinant" as used herein, refers broadly with reference to a product, e.g.., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. "Specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," or "specifically interacts or binds," as used herein, refers broadly to a protein or peptide (or other epitope), refers, in some embodiments, to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. For example, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample. Typically a specific or selective reaction will be at leastPATENT ATTY. DOCKET NO.1143252.007213 twice background signal or noise and more typically more than about 10 to 100 times background. "Signs" of disease, as used herein, refers broadly to any abnormality indicative of disease, discoverable on examination of the patient; an objective indication of disease, in contrast to a symptom, which is a subjective indication of disease. "Solid support," "support," "substrate," or "solid phase" as used herein, refer to a non-aqueous matrix to which an antibody or other molecule of the present invention can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149. The definition broadly includes any material that provides a solid or semi-solid structure with which another material can be attached including but not limited to smooth supports (e.g., metal, glass, plastic, silicon, and ceramic surfaces) as well as textured and porous materials. "Subjects" as used herein, refers broadly to anyone suitable to be treated according to the present invention include, but are not limited to, avian and mammalian subjects, and are preferably mammalian. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, primates, and humans. Generally it will refer to a canine subject. "Subjects" is used interchangeably with "patients." "Symptoms" of disease as used herein, refers broadly to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease, typically cancer. "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those inPATENT ATTY. DOCKET NO.1143252.007213 whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a cancer if, after receiving a therapeutic amount of an antibody (or other drug), the patient shows observable and / or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and / or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the antibody may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient. The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating". To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. The term “synergistic effect” refers to the result achieved using a combination being greater than the sum of the results that would be achieved using the individual components of the combination. For example, in the context of a treatment method (e.g., treatment of cancer), a synergistic effect indicates that the effect of two or more treatments in combination (e.g., two or more of administration of an antibody, a chemotherapeutic agent, another anti-cancer agent, surgery, or radiation) have an effect that is more than just the additive effect of the individual agents. In an exemplary embodiment, the synergistic effect can be measured with respect to tumor burden, mean survival, or another endpoint as known in the art.PATENT ATTY. DOCKET NO.1143252.007213 "Variable region" or "VR," as used herein, refers broadly to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. "Vector," as used herein, refers broadly to a plasmid, cosmid, phagemid, phage DNA, or other DNA molecule which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of restriction endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vector, and into which DNA may be inserted in order to bring about its replication and cloning. The vector may further contain a marker suitable for use in the identification of cells transformed with the vector. DETAILED DESCRIPTION OF THE INVENTION the inventors have identified a panel of antibodies using hybridoma technology targeting PD-1. Of these antibodies, one anti-canine PD-1 antibody referred to as “HugPet9” or alternatively “77A6H9” or “acPD-1”was identified as possessing properties making it well suited for use as a diagnostic and / or a therapeutic agent for detecting / treating canine cancers alone or in combination with other actives, e.g., cowpea mosaic virus (CPMV) particles (empty and non- empty CPMV particles). HugPet9 (also referred to as “77A6H9” or “acPD-1”) is a mouse anti-canine PD-1 antibody (acPD-1) which was demonstrated using flow cytometry to bind to canine peripheral blood mononuclear cells (PBMCs). Also, HugPet9 was demonstrated to elicit IFN- γ production in PBMCs activated with Concanavalin A. Further, HugPet9 was demonstrated to bind to different canine tumor tissues that comprise PD-1 protein. Moreover, in a proof- of-concept safety trial, i.e., when HugPet9 was used as an intratumoral (IT) immunotherapy in an open-labelled phase I preclinical study in canine mammary cancer (CMC) patientsPATENT ATTY. DOCKET NO.1143252.007213 alone or in combination with CPMV neoadjuvant this antibody was shown to be safe and effective, i.e., it enhanced patient survival, reduced tumor size, and inhibited metastasis. We also show herein the effects of neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV / acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that weekly acPD-1 (n=3 dogs) or CPMV / acPD-1 (n=3 dogs) treatments for four weeks, and extended CPMV / acPD-1 treatments (9-11 weeks)(tree dogs not candidates for surgery) were safe without immune-related adverse events. We found that acPD-1 and CPMV / acPD-1 injections resulted in tumor control and reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of the treated dogs. CPMV / acPD-1 treatments resulted in control and reduction of established lung metastases in two metastatic CMC patients. CPMV / acPD-1 treatments altered gene expression with treatment-specific and temporal gene expression changes related to TLR1-4 signaling and complement. These novel therapies could be effective for CMC patients. Because of extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC. Breast cancer (BC) remains the most common cancer in US women with ~298,000 estimated new cases in 2023. BC was the second leading cause of cancer deaths in the US with ~43,200 estimated deaths in 2023 despite multidisciplinary treatments including chemotherapy, surgery, radiation, and, when appropiate, targeted therapy [1]. Anti-PD-1 immunotherapy for solid tumors has a wide range of efficacy from modest for most tumors, including BC [2-5], to practice-changing results in mismatch repair–deficient, locally advanced rectal cancer [6]. This low efficacy highlights the urgent need for strategies that can alter the immune-suppressive tumor microenvironment (TME) and increase efficacy of anti-PD-1 therapy in BC patients. Developing immunotherapies for BC is hindered by the lack of optimal preclinical models with biological and immunological features close to humans, which if available would enable better evaluation of novel treatments. We, and others, have demonstrated that companion dogs with spontaneous canine mammary cancer (CMC) share clinicopathologic, genomic, and immunologic features with human BC patients [7-18]. ThesePATENT ATTY. DOCKET NO.1143252.007213 characteristics in animals with large tumors makes them a uniquely valuable heterogeneous animal population to evaluate the clinical efficacy of new immunotherapeutic agents and their combinations with high confidence that findings will predict human clinical trial outcomes. We previously demonstrated that intratumoral (IT) therapy with cowpea mosaic virus nanoparticles (CPMV), a plant virus that does not infect animals, deliver strong immunostimulatory signals through toll-like receptor (TLR) 2, 4, and 7. When applied IT, CPMV converts cold tumors into hot tumors by activating innate immune cells which release pro-inflammatory cytokines that stimulate local and systemic T cell anti-tumor immunity [19, 20]. Our previous studies in ovarian, colorectal and melanoma mouse models demonstrated that IT CPMV significantly increased PD-1 levels in Foxp3-CD4+effector T cells and CD44+CD8+effector T cells

[0021] , implying that the strong immunostimulatory properties of CPMV could synergize with anti-PD-1 therapy. Indeed, combining IT CPMV with systemic anti-PD-1 increased the total number of CD4+and CD8+T cells and their effector memory subsets (CD44+CD62L-), the CD8+ / regulatory T cell ratio and proportion of NK cells, and elicited a long-term immune response by generating systemic tumor specific T cells and increased survival in these murine models

[0021] . It should be noted that human patients receive systemic (intravenous; IV) anti-PD-1 therapy. However, in this study we propose IT anti-PD-1 therapy for the following reasons: 1. In both human and canine cancer patients, systemic anti-PD-1 therapy is linked to irAEs, including therapy-related death [22-24] while IT treatment has not been associated with serious irAEs in humans cancer patients [25, 26] or in canine patients treated with anti- PD-1 (our preliminary data), other IT treatments like IL2 / IL12 in canine soft tissue sarcoma and melanoma patients [27, 28], or anti-OX40 combined with a toll-like receptor (TLR3 / 8) agonist in various canine solid tumors

[0029] .2. The IV route uses a large amount of anti-PD-1 for a canine patient, which can lead to both biological toxicities, including death

[0022] , and economic toxicities associated with the high cost of the large doses needed to systemically treat dogs. The IT route requires low amounts of drugs, significantly decreasing the high cost linked to IV immunotherapies

[0030] , and will reduce the risk of irAEs by lowering systemic exposure to the immunotherapy while keeping their potential effect on injected lesions.3. Of note, IT therapy, including anti-PD1, has been applied to human BC demonstratingPATENT ATTY. DOCKET NO.1143252.007213 feasibility, safety, good clinical responses, and low irAEs [25, 26, 31].4. Lastly, it should be highlighted that the IT procedure for accessible superficial tumor masses is a simple and straightforward, minimally invasive procedure, requiring minimal equipment, and can be performed in the outpatient setting by a physician or veterinarian with minimum training [26, 32]. In our hands, it takes 3-5 min to carefully apply the IT drugs in CMC patients. We have demonstrated that IT CPMV treatments are safe and effective in CMC patients independent of clinical stage, tumor size, histopathologic grade, and tumor subtypes [33, 34]. To model current PD-1-based immunotherapy in BC [4, 35, 36] and accelerate the implementation of human clinical trials with IT CPMV, we developed a monoclonal mouse antibody against canine PD-1 (acPD-1), and applied IT acPD-1 as a monotherapy and as combined therapy with IT CPMV (IT CPMV / acPD-1) in six female CMC patients. Our study demonstrates that acPD-1 monotherapy and CPMV / acPD-1 combined therapy are safe, well-tolerated and do not cause immune-related adverse events (irAEs), have a positive effect on controlling tumor burden in injected and noninjected tumors, and CPMV / acPD-1 controlled established lung metastases. COMBINATION THERAPIES The present disclosure also specifically describes therapeutic methods and compositions for use in such methods comprising the HugPet9 antibody or one comprising the same CDRs and / or VH and VL sequences as HugPet9, utilized in combination with another therapeutic agent, e.g., CPMV particles, wherein the HugPet9 antibody or one comprising the same CDRs and / or VH and VL sequences as HugPet9, and the other agent may be in the same or different compositions; or may be used in combination with another cancer treatment regimen for treating cancers, e.g., canine cancers optionally characterizedby the expression or overexpression of PD-1 lymphomas, lymphosarcomas, bonetumors (e.g., osteosarcoma), mast cell tumors, soft tissue sarcomas, leukemias, colorectal cancers, pancreatic cancers, oral cancers such as melanoma, squamous cell carcinoma and fibrosarcoma, esophageal cancers, nasal tumors, liver cancer, adenoma, lung cancers, breast cancers, prostate cancers, skin cancers (e.g., melanoma), ovarian cancers, cervical cancers, uterine cancers, among others, optionally wherein the combination provides for enhanced therapeutic efficacy, e.g., a synergistic enhancement of antitumor efficacy. In somePATENT ATTY. DOCKET NO.1143252.007213 exemplary embodiments the canine cancer comprises canine mammary cancer or canine inflammatory mammary cancer. The present disclosure also specifically describes methods for selecting canine patients for treatment in a therapeutic regimen involving the use of HugPet9. Said patient may be a canine patient with a cancer (lymphomas, lymphosarcomas, bone tumors (e.g., osteosarcoma), mast cell tumors, soft tissue sarcomas, leukemias, colorectal cancers, pancreatic cancers, oral cancers such as melanoma, squamous cell carcinoma and fibrosarcoma, esophageal cancers, nasal tumors, liver cancer, adenoma, lung cancers, breast cancers, prostate cancers, skin cancers (e.g., melanoma), ovarian cancers, cervical cancers, uterine cancers, among others). The patient may be selected for treatment based upon the presence of a cancer at a specified stage, such as pre-cancer and Stage I, II, II and IV cancers including metastatic cancers. Said cancer may express PD-1), e.g., lymphomas, lymphosarcomas, bone tumors (e.g., osteosarcoma), mast cell tumors, soft tissue sarcomas, leukemias, colorectal cancers, pancreatic cancers, oral cancers such as melanoma, squamous cell carcinoma and fibrosarcoma, esophageal cancers, nasal tumors, liver cancer, adenoma, lung cancers, breast cancers, prostate cancers, skin cancers (e.g., melanoma), ovarian cancers, cervical cancers, uterine cancers, among others, or metastatic cancer cells originating from said tissue or organ. Again, in some exemplary embodiments the canine cancer comprises canine mammary cancer or canine inflammatory mammary cancer. The present disclosure also specifically describes therapeutic methods and compositions for use in such methods comprising the use of HugPet9 in combination with another therapeutic agent, wherein HugPet9 and the other agent may be in the same or different compositions; for treating cancers optionally characterized by the expression ofPD-1 lymphomas, lymphosarcomas, bone tumors (e.g., osteosarcoma), mast celltumors, soft tissue sarcomas, leukemias, colorectal cancers, pancreatic cancers, oral cancers such as melanoma, squamous cell carcinoma and fibrosarcoma, esophageal cancers, nasal tumors, liver cancer, adenoma, lung cancers, breast cancers, prostate cancers, skin cancers(e.g., melanoma), ovarian cancers, cervical cancers, uterine cancers, among others whereinthe other agent is selected from CPMV particles, other therapeutic antibodies, other checkpoint inhibitors, chemotherapeutics, and the like, which optionally result in enhanced therapeutic efficacy relative to the individual therapeutic agents, optionally by triggeringPATENT ATTY. DOCKET NO.1143252.007213 apoptotic pathways, enhancing ADCC, enhancing CDC, and thereby promote tumor regression, enhanced cell killing, or increased patient survival. Again, in some exemplary embodiments the canine cancer comprises canine mammary cancer or canine inflammatory mammary cancer. The present disclosure also specifically describes the use of HugPet9 for detecting PD-1 expressing cells in vivo or in patient samples and based thereon monitoring the disease status of cancers characterized by PD-1 expression. The present disclosure also specifically describes therapeutic methods and compositions for use in such methods comprising the use HugPet9 in combination with another therapeutic agent, for treating cancers characterized by the expression of PD-1, wherein the other agent is selected from antimetabolites, alkylators, corticosteroids, radiation, monoclonal antibodies, platins and PARP inhibitors. Exemplary combinations include use of HugPet9, together with epirubicin, cisplatin, dacarbazine, fludarabine / cyclophosphamide, dexamethasone, doxorubicin, or other anti-cancer agents such as FTS, CMH, TMS, and estradiol (E2). The present disclosure also specifically describes therapeutic methods and compositions for use in such methods comprising the use HugPet9 in combination with another therapeutic agent, for treating cancers characterized by the expression of PD-1, wherein the other agent is selected from chemotherapy agents, e.g., alkylating agents, antimetabolites, plant alkaloids, and anti-cancer antibiotics, further optionally one or more selected from cyclophosphamide, cisplatin, carboplatin, oxaliplatin, etoposide, irinotecan, lurbinectedin, paclitaxel, docetaxel, cabazitaxel, altretamine, capecitabine, gemcitabine, ifosfamide, melphalan, pemetrexed, topotecan, vinorelbine, mitoxantrone, ixabepilone, eribulin, estramustine, vinblastine, vincristine, 5-fluorouracil (5-FU), doxorubicin, epirubicin, dactinomycin, or a derivative thereof. In certain embodiments, chemotherapy agents may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP). The present disclosure also specifically describes therapeutic methods and compositions for use in such methods comprising the use HugPet9 in combination with another therapeutic agent, for treating cancers characterized by the expression of PD-1, wherein the other agent is selected from other cancer treatment methods, such as surgery, radiotherapy, hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g.,PATENT ATTY. DOCKET NO.1143252.007213 antiemetics), and other approved chemotherapeutic drugs, including, but not limited to, alkylating drugs (e.g., mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites (e.g., Methotrexate), purine antagonists and pyrimidine antagonists (e.g., 6-Mercaptopurine, 5-Fluorouracil, Cytarabine, Gemcitabine), spindle poisons (e.g., Vinblastine, Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (e.g., Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (e.g., Carmustine, Lomustine), inorganic ions (e.g., Cisplatin, Carboplatin), enzymes (e.g., Asparaginase), and hormones (e.g., Tamoxifen, Leuprolide, Flutamide, and Megestrol),among others. The present disclosure also specifically describes a kit comprising HugPet9, and one of said other agents. Typically HugPet9 and / or said other agent are provided at therapeutically effective dosages for the treatment of a disease or condition, e.g., canine PD-1 expressing cancers. The present disclosure also specifically describes pharmaceutical compositions which comprise a therapeutically effective amount of HugPet9, formulated together with one or more pharmaceutically acceptable carriers and optionally one or more other active agents such as those above-identified. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of this invention can be administered to canines orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray. In a preferred embodiment administration is intratumoral and / or systemic, e.g., intravenous or subcutaneous administration. Dosage forms In another aspect, the disclosure provides dosage forms comprising HugPet9 or a fragment thereof, and optionally another active suitable for in vivo use, e.g., liquid dosage forms. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylPATENT ATTY. DOCKET NO.1143252.007213 benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying agents, suspending agents, sweetening, flavoring, and perfuming agents. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In order to prolong the effect of a drug, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.PATENT ATTY. DOCKET NO.1143252.007213 The active can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons. Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. According to the methods of treatment of the present invention, disorders are treated or prevented in a subject, such as a canine, by administering to the subject a therapeutically effective amount of HugPet9, in such amounts and for such time as isPATENT ATTY. DOCKET NO.1143252.007213 necessary to achieve the desired result. The term "therapeutically effective amount" of a compound of the invention, as used herein, means a sufficient amount of the compound so as to decrease the symptoms of a disorder in a subject, generally a cancer characterized by expression of the canine PD-1 antigen bound by HugPet9. As is well understood in the medical arts a therapeutically effective amount of a compound of this invention will be at a reasonable benefit / risk ratio applicable to any medical treatment. In general, HugPet9, will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents, optionally CPMV VLPs and / or a CD40 agonist. In some exemplary embodiments one or more of the actives are administered via intratumoral administration. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg / kg per body weight (0.05 to 4.5 mg / m2). An indicated daily dosage in a canine, may range from about 0.5 mg to about 100 mg. Suitable unit dosage forms for oral administration comprise from 0.1 to 100 mg active ingredient. In general, treatment regimens according to the present invention comprise administration to a canine patient in need of such treatment from about .10 mg to about 100 mg of the compound(s) of this invention per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration (e.g., systemic, optionally via intravenous, subcutaneous or intratumoral injection), the size of the canine, the condition of the canine patient (e.g., comorbidities) as well as the possibility of co-usage with other agents. The invention also provides for a pharmaceutical combinations, e.g., a kit, comprising a) HugPet9 as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit can comprise instructions for its administration to a subject suffering from or susceptible to a disease or disorder. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected therapeutic agents toPATENT ATTY. DOCKET NO.1143252.007213 a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound of the invention and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound of the invention and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidantsPATENT ATTY. DOCKET NO.1143252.007213 can also be present in the composition, according to the judgment of the formulator. The agents or salts thereof may be formulated into pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions, which comprise an amount of the protein inhibitor effective to treat or prevent a cancer and a pharmaceutically acceptable carrier, are another embodiment of the present invention. These pharmaceutical compositions may be used in combination with other cancer treatments such as radiation therapy (also referred to as radiotherapy). This radiation can have an electromagnetic form, such as a high-energy photon, or a particulate form, such as an electron, proton, neutron, or alpha particle. A common form of radiation used in practice today is high-energy photons. Photon absorption in human tissue is determined by the energy of the radiation, as well as the atomic structure of the tissue in question. The basic unit of energy used in radiation oncology is the electron volt (eV); 10^3 eV=1 keV, 10^6 eV=1 MeV. Three interactions can be involved in photon absorption in tissue: the photoelectric effect, Compton effect, and pair production. Additionally provided are diagnostic methods comprising administering said HugPet9 to a canine patient and detecting the binding of said antibody to tumor cells in said patient. Exemplary embodiments of the invention provide compositions comprising HugPet9, (such as therapeutic compositions or diagnostic compositions), which compositions may comprise a pharmaceutically acceptable carrier, and additionally may comprise one or more additional therapeutic agents, such as another anti-cancer agent. Further exemplary embodiments of the invention provide nucleic acids encoding HugPet9. Additionally provided are cells (such as mammalian, prokaryotic, yeast, or other eukaryotic cells) or vectors comprising a nucleic acid encoding HugPet9. Additional exemplary embodiments of the invention provide methods of making HugPet9 by expressing a nucleic acid encoding HugPet9 in a suitable cell. In another aspect, this disclosure provides methods of detecting a cancer cell that expresses PD-1 using HugPet9. Detecting the expression of cancer-associated antigen using HugPet9 may be used for diagnosis and staging of cancers (e.g., in radioimaging). For example, the level or extent of expression of PD-1 antigens may indicate the stage of cancer,PATENT ATTY. DOCKET NO.1143252.007213 may be correlated with patient outcome, or may be predictive of the outcome of different treatment options. Generally, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Detection of cancer cells using HugPet9 can be used in conjunction with one or more therapies, typically cancer surgery. Therapy may be targeted to the cancer cells by intratumoral administration thereby promoting effective treatment and / or reducing the effect on normal non-cancerous tissue. The therapeutic course (e.g., regimen and dosages of radiotherapy, surgical plan, or course of cryotherapy) that are therapeutically effective will depend on the particular canine cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician. Expression of a PD-1 antigens may be evaluated using an in vivo diagnostic assay, e.g., by administering HugPet9, which binds the PD-1 antigens to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody. Aside from the above assays, various in vivo and in vitro assays for detecting the presence of a cancer-associated antigen are available to the skilled practitioner. The PD-1 antigen may be present on the cell surface. Alternatively or in addition the PD-1 antigen may be produced and secreted at detectable levels. For example, PD-1 antigens may be detected in a biological fluid such as serum, e.g., using antibody-based assays (see also, e.g., U.S. Pat. No.4,933,294 issued Jun.12, 1990; WO91 / 05264 published Apr.18, 1991; U.S. Pat. No.5,401,638 issued Mar.28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). HugPet9 may also be used for purification or immunoprecipitation of the cancer- associated antigen from cells or other samples, for detection and quantitation of cancer- associated antigen in vitro, e.g., in an ELISA or a Western blot, to kill and eliminate cancer-PATENT ATTY. DOCKET NO.1143252.007213 associated antigen-expressing cells from a population of mixed cells, e.g., as a step in the purification of other cells. In another aspect, the invention provides a diagnostic kit comprising an HugPet9. In one embodiment, the antibody may be directly or indirectly fixed to a solid phase support, such as a bead, plate, matrix, polymer, test tube, sheet, culture dish, or test strip. In another embodiment, the solid support may be an array. HugPet9 may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. HugPet9 may optionally be produced in mammalian cells (such as CHO cells), bacterial cells, yeast cells, or other cells or using cell-free methods as known in the art. For diagnostic purposes, HugPet9 optionally may be detectably labeled, attached to a solid support, or the like. For example, the antibody may be labeled by conjugation to a radiolabel such as 111In or 86Y. A further embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a canine, wherein the method comprises detecting the level of expression of PD-1 (a) in a test sample of tissue cells obtained from said canine, and (b) in a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of the PD-1 antigen in the test sample, as compared to the control sample, is indicative of the presence of tumor in the canine from which the test sample was obtained. Another embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a canine wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the canine with HugPet9 and (b) detecting the formation of a complex between the HugPet9 antibody and PD-1 in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the HugPet9 antibody employed is detectably labeled, attached to a solid support, or the like, and / or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.PATENT ATTY. DOCKET NO.1143252.007213 Yet another embodiment of the present invention is directed to a method of binding an HugPet9 antibody to a cell that expresses PD-1 antigen, wherein the method comprises contacting a cell that expresses PD-1 antigen with said HugPet9 antibody under conditions which are suitable for binding of the antibody to said PD-1 antigen and allowing binding therebetween. In preferred embodiments, the HugPet9antibody is labeled with a molecule or compound that is useful for qualitatively and / or quantitatively determining the location and / or amount of binding of the antibody to the cell. Except where otherwise provided the techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook, et al (2001) “Molecular Cloning: A Laboratory Manual [3rd Ed] Cold Spring Harbor Laboratory Press”. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. The subject HugPet9 antibodies, and antigen-binding fragments thereof may be used in diagnostic methods for detecting the presence or absence of PD-1 antigen. HugPet9 and antigen-binding fragments thereof, may be used in methods comprising (a) contacting a test sample with HugPet9, or fragment thereof, and (b) assaying for antibody- PD-1 antigen complexes, wherein the presence thereof is indicative of a carcinoma. Further, HugPet9, may be used in a method for detecting the presence of PD-1, comprising (a) administering to said patient HugPet9 or fragment thereof, that binds canine PD-1, and (b) detecting the presence of canine PD-1; wherein the presence thereof is indicative of a carcinoma. The antibody-antigen complexes may be detected by Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay,PATENT ATTY. DOCKET NO.1143252.007213 immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement-fixation assay, immunohistochemical assay, fluorescent immunoassay, and protein A immunoassay. The sample may be sample is a tissue biopsy, lymph, urine, cerebrospinal fluid, amniotic fluid, inflammatory exudate, blood, serum, stool, or liquid collected from the colorectal tract. HugPet9, and fragments thereof which selectively bind to canine PD-1 may be used in diagnostic methods for detecting the presence or absence of canine PD-1 antigen, wherein the presence of the antigen is indicative of cancer including but not limited to canine cancers optionally characterized by the expression of PD-1, e.g., lymphomas, lymphosarcomas, bone tumors (e.g., osteosarcoma), mast cell tumors, soft tissue sarcomas, leukemias, colorectal cancers, pancreatic cancers, oral cancers such as melanoma, squamous cell carcinoma and fibrosarcoma, esophageal cancers, nasal tumors, liver cancer, adenoma, lung cancers, breast cancers, prostate cancers, skin cancers (e.g., melanoma), ovarian cancers, cervical cancers, uterine cancers, among others. The diagnostic methods may be used with canine patients at risk of cancer (e.g., dog breeds that are very cancer prone) or canine patients without symptoms. In exemplary embodiments the canine cancer comprises canine mammary cancer or canine inflammatory mammary cancer. The PD-1 antigen bound by HugPet9 may be used as a cancer biomarker. Detection of PD-1 in a biological sample, such as a subject's serum, biopsied neoplastic cells or fecal sample, may be performed by means using HugPet9. For example, a biological sample (e.g., a tumor, serum or fecal sample) is obtained from a canine subject, then PD-1 antigen is measured (e.g., by ELISA or PCR), and compared with corresponding samples from normal subjects. Measuring methods include any method of nucleic acid detection, for example in situ hybridization using antisense DNA or cRNA oligonucleotide probes, ultra- high throughput sequencing, nanostring technology, microarrays, rolling circle amplification, proximity-mediated ligation, PCR, qRT-PCR ChIP, ChIP-qPCR, or PD-1 antigen-binding antibodies. Comparatively high levels of the PD-1 antigen bound by HugPet9 indicate the presence and / or severity of a cancer characterized by expression of PD-1, and may indicate metastasis or poor cancer prognosis. HugPet9 which selectively binds to canine PD-1, and antigen-binding fragments thereof, may be used in SQUID (Superconducting Quantum Interference Device) techniquesPATENT ATTY. DOCKET NO.1143252.007213 for diagnostic methods. The SQUID technique comprises attaching nanoparticles of iron oxide to antibodies, which are then injected into the patient. If a tumor is present, the antibodies with conjugated nanoparticles recognize and bind to PD-1 antigen expressed on tumor cells. See, e.g., Hao, et al. (2010) Journal of Physics 43: 474004. In a SQUID method, the patient is then surrounded with sensitive magnetic coils in a superconducting quantum interference device (SQUID). A magnetic field is generated and all of the metal nanoparticles align in one direction. When the magnetic field is broken, the nanoparticles emit an electromagnetic signal as they relax back into their original state. By measuring the strength of the signal, one may tell how many metal particles, and therefore how many tumor cells, may be present, and where in the patient the tumor cells are located. See, e.g., Shao, et al. (2010) Beilstein, Journal of Nanotechnology 1: 142-154. Samples and Procurement of Samples The samples used in the methods described herein may be taken from a canine subject (patient) include but are not limited to a body fluid or secretion including but not limited to blood, serum, urine, plasma, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, secretions of the breast ductal system (and / or lavage thereof), broncho alveolar lavage, lavage of the reproductive system and lavage of any other part of the body or system in the body; samples of any organ including isolated cell(s) or tissue(s), wherein the cell or tissue can be obtained from an organ selected from, but not limited to lung, colon, ovarian, uterine, cervical, and / or breast tissue; stool or a tissue sample, or any combination thereof. In some embodiments, the term encompasses samples of in vivo cell culture constituents. Prior to be subjected to the diagnostic assay, the sample can optionally be diluted with a suitable diluent. Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and / or polypeptide of the marker of interest in the subject. Examples of tissue or fluid collection methods include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy / sample is obtained the level of the marker may be determined and a diagnosis can thus be made.PATENT ATTY. DOCKET NO.1143252.007213 Immunoassays HugPet9 and binding fragments thereof, may be used in immunoassays to qualitatively or quantitatively detect and analyze markers in a sample. This method comprises providing an antibody specifically binds to canine PD-1; contacting a sample with the antibody; and detecting the presence of a complex of the antibody bound to the marker in the sample. The PD-1 antigen bound by HugPet9 may be detected and / or quantified using any of a number of well recognized immunological binding assays. Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a Western blot assay, or a slot blot assay. See, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168. Optionally, HugPet9 can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include but are not limited to glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead. Antibodies may be attached to a solid support. After incubating the sample with antibodies, the mixture is washed and the antibody-marker complex formed may be detected. This can be accomplished by incubating the washed mixture with a detection reagent. Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and / or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture. Throughout the assays, incubation and / or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, marker, volume of solution, concentrations. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures (e.g., 10 degrees C.-40 degrees C.).PATENT ATTY. DOCKET NO.1143252.007213 The immunoassay can be used to determine a test amount of a marker in a sample from a subject. First, a test amount of a marker in a sample may be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody-marker complex with an antibody specifically binds the marker under suitable incubation conditions described above. The amount of an antibody-marker complex can optionally be determined by comparing to a standard. As noted above, the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control amount and / or signal. Such immunoassays are known in the art and include but not limited to radio-immunoassay (RIA), enzyme linked immunosorbent assay (ELISA), magnetic immunoassay, immunoblot, Western blot, immunoprecipitation assays, immunohistochemical analysis, and fluorescence activated cell sorting (FACS). See Wild, (2008) [Ed.] The Immunoassay Handbook [3rd Ed.] Elsevier. Radio-Imaging Methods The PD-1 antigen bound by t HugPet9 and antigen-binding fragments thereof, may be used in radio-imaging methods to diagnosis PD-1 expressing cancers, including e.g., lymphomas, prostate cancers, skin cancers (e.g., melanoma), ovarian cancers, cervical cancers, uterine cancers, , or to monitor the progression of PD-1 antigen expressing tumors. In exemplary embodiments the canine cancer comprises canine mammary cancer or canine inflammatory mammary cancer. These methods include but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPECT). Both of these techniques are non-invasive, and can be used to detect and / or measure a wide variety of tissue events and / or functions, such as detecting cancerous cells for example. SPECT may optionally be used with two labels simultaneously. See U.S. Pat. No.6,696,686. In order that the invention herein described may be fully understood, the above detailed description is set forth. Various embodiments of the invention are described in detail and may be further illustrated by the provided examples. The examples provided are intended to be illustrative, rather than limiting, on the scope of the invention, which is limited only by the scope of the claims provided after the examples.PATENT ATTY. DOCKET NO.1143252.007213 EXAMPLES EXAMPLE 1: Identification of Anti-Canine PD-1 Antibodies A panel of 5 monoclonal antibodies (mAbs) specific for canine PD-1 were generated by immunizing mice with purified recombinant canine PD-1 antigen and screening for binding of secreted antibodies from hybridomas to plate bound canine rPD-1 (data not shown). The individual PD-1 mAbs were isotyped and prepared as purified IgG for binding and functional studies. Control antibodies included irrelevant isotype-matched antibodies (feline Immunodeficiency virus) (Sigma-Aldrich, St Louis, MO, USA and BioXCell, West Lebanon, NH, USA). Specifically, total RNA was isolated from the hybridoma cells following the manufacturer’s instructions. Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers following the technical manual of SMARTScribe Reverse Transcriptase. Antibody fragments of heavy chain and light chain were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. From these five monoclonal antibodies HugPet9 was selected as the lead antibody. HugPet9 is a chimeric mouse anti-canine PD-1 antibody. The variable heavy and light sequences of the HugPet9 antibody (alternatively referred to as 77A6H9) and the CDRs thereof are contained in the Sequence Listing which precedes the claims. Also, the variable heavy and light sequences of a closely related variant referred to as 77A6H7 and the CDRs thereof are contained in the Sequence Listing which precedes the claims. EXAMPLE 2: Binding of Lead anti-Canine PD-1 Antibody to Recombinant Canine PD-1 Recombinant canine PD-1 was expressed in 293 T cells by transfection of these cells with a DNA encoding the full-length canine cDNA for PD-1, coupled to the human Fc molecule. The resultant secreted recombinant canine PD-1 was then purified using anti- human Fc columns, to generate a PD-1 fusion protein of the appropriate molecular weight, resulting in a semi-GMP quality of HugPet9; endotoxin purification.PATENT ATTY. DOCKET NO.1143252.007213 As is shown in Figure 1A-B, the lead antibody, HugPet9, detects PD-1 protein expressed in 293T cells used in two formats: fresh frozen and fixed cells, and formalin-fixed and embedded in paraffin. Therefore, HugPet9 may be used to detect PD-1 in fresh-frozen and archival paraffin canine tissues, respectively. EXAMPLE 3: Functional Studies using Lead anti-Canine PD-1 Antibody and Canine PMBCs Whole blood was obtained from healthy donor dogs for isolation of PBMCs to examine binding of mouse anti-canine PD-1 antibodies and commercial anti-canine PD- 1 / PD-L1 antibodies to primary canine lymphocytes and monocytes after stimulation with various activation agents. Blood was processed for PBMCs by density centrifugation and used for evaluation of INF-g production and flow cytometry. The isolated healthy canine donor PBMCs were stimulated with Con A (2.5 μg / ml for 3 days at 37˚C to evaluate interferon-gamma (IFN-γ) production after PD-1 / PD-L1 blockade. Canine PBMCs (5x10e5) were stimulated with 1 μg / ml Con A and incubated in 0.2 ml DMEM complete medium in a U-bottom 96-well plate for 96 hours with or without PD-1 or PD-L1 antibodies. After incubation, supernatant was collected and IFN-γ production was evaluated using an IFN-γ Quantikine ELISA Kit (R&D Systems, Inc., Minneapolis, MN). Particularly, to address the functional effects of PD-1 blockade using HugPet9, when PBMCs from either cancer-bearing dogs or healthy dogs were incubated with HugPet9 antibody, it was observed that there was no spontaneous T-cell activation, as assessed by IFN-g release. As is shown in FIG 2A-C HugPet9 elicited gamma interferon production in PMBCs from healthy dogs. When PBMCs were activated with Staphylococcal endotoxin B (SEB; Figure 2A, left panel) or Con A (2,5 μg / ml; Figure 2C, right panel) and at the same time incubated with HugPet9 antibody, there was a dose-dependent increase in IFN- γ release compared with an isotype-matched, irrelevant antibody (unstimulated in Figure 2C). EXAMPLE 4: Immunohistochemistry (IHC) Studies using Lead anti-Canine PD-1 Antibody Immunohistochemistry (IHC) To demonstrate that HugPet9 is a good and specific antibody for the detection of PD-1 protein levels in archival canine tumor samples, we applied standard IHC assays inPATENT ATTY. DOCKET NO.1143252.007213 several canine tumor samples. As is illustrated in Figure 3A-F, PD-1 protein is detected in jejunum T cell lymphomas (A), metastatic melanomas (B), lymphomas (C-E), and canine tonsils (F). We detected PD-1 levels in a wide range of canine tissues, including mammary tumors, histiocytomas, synovial carcinomas, squamous cell carcinomas and B and T cell lymphomas (data not shown). Immunostaining with HugPet9 demonstrated a strong membranous localization of PD-1 protein in the samples with minor to no cytoplasmic staining. Based on the foregoing, HugPet9 may be used for the detection of PD-1 protein levels in canine tumor samples, and potentially may be used for staging canine cancer and / or for assessing the efficacy of cancer treatments. EXAMPLE 5: Phase 1 In Vivo Studies Validating Efficacy of Lead anti-Canine PD-1 Antibody for Immunotherapy The safety and efficacy of intratumoral (IT) HugPet9 immunotherapy was effected in an open-labelled phase I preclinical study in canine mammary cancer (CMC) patients, using a modified version of the accelerated titration design (Simon R et al, (1997) “Accelerated titration designs for phase I clinical trials in oncology”, J Natl Cancer Inst 89:1138-47. doi: 10.1093 / jnci / 89.15.1138) wherein single patient cohorts with double-dose escalation steps are treated. It is noted that this treatment approach finds known usage for systemic therapies in humans, but to the best of Applicant’s knowledge, this clinical regimen has not been used in teating cancer in dogs. Notwitrhtanding, it is an appropriate approach to obtain persuasive clinical information. To track any abnormal change related to IT HugPet9 immunotherapy, a hemogram (hemoglobin content, hematocrit, total leucocytes, neutrophils, monocytes, lymphocytes, erythrocytes, and platelets) and biochemical analyses (total protein, albumin, globulin, alanine aminotransferase (ALT), aspartate-aminotransferase (AST), urea, creatinine, and cholesterol) were performed before any intratumoral HugPet9 injection. After IT injections, dogs were kept for a 4- to 6-h observation period in the veterinary clinic and a follow-up three days later in the clinic. Owners were in direct contact with the attending veterinary doctor and had a designated questionnaire to document the physical status of the patient daily during the whole week. In this study, we randomly enrolled three dogs for IT HugPet9 immunotherapy (P1, P2, and P3). The enrolled patients reflect what we see in human BC patients: middle-PATENT ATTY. DOCKET NO.1143252.007213 advanced age, different breeds, and human-related comorbidities like chronic kidney disease, cardiopathies, increase in the hepatic enzyme activity, cholesterolemia, and blood variations associated with a chronic and / or inflammatory disease, such as mild anemia, increase in plasmatic globulins and in the number of leukocytes in peripheral blood. The largest tumor in each CMC patient was the target tumor (injected) for IT HugPet9 immunotherapy. The inclusion criteria has been previously described (Adams S, et al., (2019) “Pembrolizumab monotherapy for previously untreated, PD-L1-positive, metastatic triple- negative breast cancer: cohort B of the phase II KEYNOTE-086 study”, Ann Oncol 30:405- 411. doi: 10.1093 / annonc / mdy518. In the enrolled patients, a pretreatment fine needle aspiration (FNA) was taken (BARD Mission™ Disposable Core Biopsy Instrument; needle, 14g x 10 cm, 10- and 20-mm throw; 9.4- and 18.8-mm sample notch; BD, Tempe, AZ), followed by an IT HugPet9 injection immediately after obtaining the biopsy sample. IT HugPet9 doses were given once a week; the dose was escalated in each dog starting with 125 mg, and doubling the dose to 250 mg, 500 mg and 1,000 mg per injection once a week (Figure 4A-B). The IT HugPet9 dose was diluted in ~0.5 ml of sterile phosphate buffered saline (PBS) and injected using a 22G needle. Depending on the tumor size, the total PBS solution ranged from 0.3 ml to 0.8 ml. The injected PBS volume was equally distributed in three locations within a treated tumor. Before the fourth injection an FNA was taken, followed by surgery one-two weeks later when the injected tumor was resected and a surgical biopsy was collected for histopathology and research studies. Surgical procedures were performed per institutional standard of care protocol. The IT HugPet9 immunotherapy was provided as a neoadjuvant therapy and after surgery, adjuvant therapy was provided per standard institutional procedures. The adjuvant therapy is maintained for two years unless local recurrence or metastases are observed. After surgery, follow-up is performed every three months until two years or until death or euthanasia due to tumor progression or any other cause. Thoracic radiographs and abdominal ultrasound are performed every three months to search for metastases. During the four weeks we treated the dogs with IT HugPet9 immunotherapy, the hemogram and biochemical analyses showed fluctuations within the normal range without significant changes suggestive of immune-related adverse events (irAEs) which could bePATENT ATTY. DOCKET NO.1143252.007213 associated with the therapy (data not shown). Furthermore, the owners reported a positive change in the behavior of the dogs like being more alert, active, energetic, and eating better. Hence, the IT HugPet9 immunotherapy is safe. More specifically, while the main focus of this small clinical trial was safety, as illustrated in Fig.4A, intratumoral administration of HugPet9 was demonstrated to control one large tumor in one patient (P2) and one small one in another patient (P3), and a mixed response, pseudoprogression, and antitumor response in another patient (P1) by day 29 after antibody administration. The clinical effect tumor growth is illustrated in Fig.4B indicating that patients P2 and P3 had stable disease (SD), and P1, partial response (PR). This translates into a clinical benefit (SD+PR) of 100%. Moreover, this Phase I trial demonstrated safety at doses up to 1 mg per IT injection without any serious irAEs, and it demonstrated clinical benefit in the treated CMC dogs. For comparison, in the trial of Igase’s group, the rat-canine chimeric anti- PD-1 antibody was given to 21 dogs and the caninized anti-PD-1 to nine dogs. The dogs received intravenous anti-PD-1 at 3 mg / kg every two weeks in a 10-week treatment cycle. Most of the 30 dogs (21 oral melanomas, two CMC (stage III and IV), two skin melanomas, two squamous cell carcinomas, and lymphoma, renal carcinoma, and lung adenocarcinoma, one each) had been previously treated with radiation therapy, surgery, and chemotherapy. They demonstrated that their anti-PD-1 antibodies resulted in PR in 17%, SD in 17%, and progressive disease (PD) in 67% of the dogs (Igase M, et al., (2020) “A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs”, Sci Rep 10:18311. doi: 10.1038 / s41598-020-75533-4.) Merck has recently received conditional approval of gilvetmab (a caninized anti- PD-1 monoclonal antibody) for the treatment of dogs with mast cell tumors or melanomas.

[0014] . When comparing HugPet9 to gilvetmab, we have demonstrated both safety and efficacy in cancer patients. When comparing safety and efficacy of HugPet9 vs. Igase’s study with acPD-1 antibodies, HugPet9 immunotherapy elicited no irAE in the three CMC patients, whereas in Igase’s study, there were irAEs grade ≥3 in ~7% (2 out of 30) of dogs, including one treatment-related death (pneumonitis) observed in a 14-year-old Chihuahua with stage IV oral melanoma who received three doses of the chimeric antibody (Merck AH (2023) Gilvetmab for veterinary professionals. available at https: / / www.merck-animal-health-PATENT ATTY. DOCKET NO.1143252.007213 usa.com / gilvetmab). Also, HugPet9 immunotherapy resulted in PR (33%) and SD (67%) while Igase’s group reported mostly PD (67%) followed by SD and PR (17%, each one).Therefore, we have shown that HugPet9 provides an effective immunotherapy for CMC patients with a good safety profile and clinical benefit in treated patients. When compared to similar antibodies, it appears to yield better responses without irAEs and better quality of life. EXAMPLE 6: Comparison of Immunostaining of Canine Normal Lymph Nodes and an Intestinal Lymphoma Sample with HugPet9 versus Comparator Anti-Canine PD-1 Antibody for Immunodiagnosis and immunodetection As evidenced by the immunostaining results in Figure 5A-F, HugPet9 antibody may be used as a research tool, especially to detect PD-1 levels in canine fresh-frozen tissues, and archival canine paraffin-embedded tissues. One commercially available antibody, clone JC053 from QED Bioscience (San Diego, CA, USA), has been proposed as being suitable for research studies, including use in the detection of PD-1 in archival tissues. However, as shown in Figure 5A-G, while HugPet9 clearly and with negligible background stains T lymphocytes in canine tonsils (A), JC053 instead stains in the same canine tonsil sample several types of cells, including fibroblast and endothelial cells, indicating that it is not specific (B). The nonspecific staining is further demonstrated in an intestinal lymphoma where HugPet9 had no reactivity in this tissue (D), while JC05 shows reactivity in epithelial cells, endothelial cells, and stromal cells (E). In Figure 5, canine lymphocytes (C) and intestinal lymphoma (F) (without antibody) were used as controls. Given the high demand for PD-1 antibody to determine PD-1 expression levels in canine tumors, and the lack of suitable specific anti-PD-1 antibodies for such assays, HugPet9 meets a significant need in the art as it provides a new tool for diagnosing cancer in canines and / or for determining the efficacy and / or prognosis of cancer in a canine patient based on detected PD-1 levels on different tissues and tumor samples. EXAMPLE 7: In Vivo Immunotherapy bi Intratumoral HugPet9 Administration with Neoadjuvant and CPMV / ac-PD1 injections Materials and Methods Canine patient recruitment and selection criteriaPATENT ATTY. DOCKET NO.1143252.007213 CMC is rare in the US because female dogs are generally spayed when young. This is not the case in many other countries which do not aggressively spay young female dogs. This prospective proof-of-concept, open label study was performed at Centro Veterinario Valles, Zapopan, Jalisco, Mexico, and DIAGSA, Naucalpan, Mexico. All patient’s owners signed an informed consent. This study is approved by the Internal Committee for the Care and Use of Animals, Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico (Protocol #153). Client-owned dogs with histologically confirmed diagnosis of mammary gland cancer with a tumor mass of at least 1.5 cm in any length, with or without metastatic disease, were eligible for enrollment. Prior chemotherapy, radiation therapy, or another investigational drug was not allowable. The characteristics of six individual dogs are described in Table 1 and Figure 11A-C. The clinical staging system, histopathological classification of tumors and the histological grade of malignancy were evaluated as described elsewhere [37-39]. Table 1. Clinicopathologic characteristics of enrolled CMC patients. Age, Weight, Clinical Histo. Receptor Adj. OS, Patient Vol.; cm Histo. Type y. kg stage Grade status Ther. days ¥¥tumor is triple-negative (TN), and the right tumor is ER+PR+. OS, overall survival counted from first treatment day up to 05 / 31 / 2024. ¥, dogs are alive. Safety evaluation Hemograms and biochemistry analyses were performed weekly to evaluate systemic changes and track irAEs (additional details are described in supplemental file). After IT injections, each canine patient was closely observed by the attending veterinarian for ~ four hours in the veterinary clinic with a follow-up three days later in the clinic. InPATENT ATTY. DOCKET NO.1143252.007213 addition, dogs were observed daily by owners to detect possible irAEs using a preestablished quality of life (QOL) survey

[0040] , which was reviewed by the attending veterinarian prior to planned weekly treatment. The evaluation of hematological, biochemical and other adverse events related to immunotherapy was conducted per the Veterinary Cooperative Oncology Group criteria (Version 2)

[0041] . Study design and treatment The primary objective of this open-labelled study in CMC patients was to determine the safety profile, tolerability, and dosage of neoadjuvant IT acPD-1 as monotherapy or in combination with IT CPMV (CPMV / acPD-1 ITIT) in CMC patients. Secondary endpoints evaluated overall response rate (ORR), immune-related adverse events (irAEs) and quality of life (QOL). The largest tumor mass in each patient was selected as the target tumor (injected) for IT injections. Other mammary nodules present in the same and contralateral chains were observed to evaluate systemic impact on noninjected nodules in the same canine patient. Similarly, thoracic radiographs were used to track the abscopal effect on lung metastases in patients P5 and P6, who had metastatic lung disease at diagnosis. Dogs were randomly enrolled in the acPD-1 arm (P1-P3) and CPMV / acPD-1 arm (P4-P6). CPMV nanoparticles were produced in plants as described previously

[0042] , and the IT CPMV dose 0.2 mg was used as in previous CMC studies [33, 34]. Depending on the tumor volume, the CPMV dose was dissolved in 0.3 to 0.8 ml of sterile phosphate buffered saline (PBS) and injected in two to four different sites of the selected target tumor using a 25G needle. The acPD-1 mouse monoclonal antibody is a proprietary antibody, which will be described separately (Noelle and Arias-Pulido, manuscript in preparation). For the acPD-1 dosage, we used a modified version of the accelerated titration design

[0043] wherein single patient cohorts with double-dose escalation steps are treated. Based on published clinical trials in canine cancer patients using systemic anti-PD-1 (3 mg / kg)

[0022] , we allometrically scaled dosing to dogs of 5 kg to 30 kg body weight, and used 1 / 100thof the systemic dose which gave a range of 0.15 to 0.90 mg per single injection. The starting IT acPD-1 dose was 0.125 mg and increased weekly to 0.25 mg, 0.50 mg, and 1.00 mg. For the IT CPMV / anti-PD-1 dose, the same acPD-1 dose escalation was done with a fixed CPMV dose of 0.2 mg. The acPD-1 dose in PBS was applied as described above for the CPMV dose. For the combinedPATENT ATTY. DOCKET NO.1143252.007213 treatment, CPMV dose was mixed with the acPD-1 dose and given as a single IT injection as described above. P5 had two target tumors (P5.1 and P5.2), each treated with an individual IT dose. No attempts were made to avoid necrotic tumor regions. The scheme of the trial is illustrated in Figure 6. As shown therein IT acPD-1 was provided weekly for four weeks (purple arrow in Figure 6 left section). As we previously demonstrated, combining IT CPMV plus anti-PD-1 elicited a long-term immune system activation by generating systemic tumor specific T cells and increasing survival in these murine models

[0021] . Based on this rationale, we first applied IT CPMV on the target tumor (green day numbers in Figure 6; middle section) and two days later (D2), we applied IT acPD- 1 (purple day numbers in Figure 6). This weekly treatment was administered for four weeks. After the four weeks of treatment, patients P2, P3, and P4 underwent planned surgery with adjuvant therapy as recommended (described in supplemental file). Surgical procedures were performed per institutional standard of care protocol (described in supplemental file). Patients P1, P5, and P6 continued on the combination therapy at D29 because they were not candidates for surgery (Figure 6; right section): P1 suffered from spondylosis deformans and other comorbidities, limiting surgery for her; P5 and P6, two metastatic patients, were in good health at D29 and the combined immunotherapy was having a possitive effect on their tumors and lung metastases. Hence, they continued with weekly IT CPMV / anti-PD-1 treatments. Based on hemogram and biochemistry data and tumor response at D29, the IT dose for the combination therapy was 0.4 mg of CPMV and 0.5 mg of acPD-1 from this day onwards. Before treatment, patient evaluation included collection of a blood sample, thoracic radiographs, tumor measurements, quality of life (QOL) evaluation, collection of a fine needle aspirate (FNA). During a four-week treatment period, three dogs (P1-P3) received IT acPD-1 weekly (left section) and three dogs (P4-P6) received IT CPMV (green days) and two days later, IT acPD-1 (purple days). Three dogs (P1, P5 and P6) were treated further after D29 with CPMV / acPD-1 as a single weekly IT injection (right section). Tumor response evaluation The tumor response to the IT treatment was evaluated once or twice a week during the treatment period by measuring the tumor volume (Tv) using the formula Tv = 0.5*long axis*(short axis)2. The injected tumor and any uninjected mammary nodules in the same or contralateral chains were evaluated in a similar manner. The percentage of tumorPATENT ATTY. DOCKET NO.1143252.007213 growth inhibition (%TGI) was estimated as %TGI= 100*(final Tv - initial Tv) / initial Tv. All measurements are in cubic centimeters (cm3). Taking D0 as the reference, responses were defined as complete response (CR) when there was disappearance of all target lesions; partial response (PR), when at least a 30% decrease of target lesions occurred; progressive disease (PD), when at least a 20% increase in the target lesions occurred or one or more new lesions appeared; and stable disease (SD), when neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD. ORR was defined as CR+PR. In addition, for exploratory analyses, we applied the itRECIST criteria

[0044] . The clinical benefit (CB) in humans is defined as CR+PR+SD for at least 6 months from the best response date. For the CB in dogs, and considering the dog’s age, size, and weight

[0045] , the 6 months life period in humans translates to roughly one month in a dog’s life. In dogs, three-view thoracic radiography, consisting of the right and left lateral and ventrodorsal or dorsoventral views, is standard for detecting pulmonary metastases

[0046] and each patient had 3-view radiographs. Radiographs were digital with a direct system (Xmaru model DR, Rayence, Gyeonggi-do, Korea). A grid (>10 cm in thickness) was used in all patients. All radiographs were assessed as being of adequate diagnostic quality. The interpreters (Dr. DLD, a veterinary expert on canine radiographs, and Dr. PJL, an expert on human breast radiographs) were blinded to the patient’s information. All three radiographs were interpreted separately with the final call made by Dr. DLD. The location (lung lobe affected) and size of the pulmonary nodules were recorded using images in Digital Imaging and Communications in Medicine format and analyzed with the HOROS software (https: / / horosproject.org / ). Histopathology and Immunohistochemistry (IHC) assays Single 4 mm tumor tissue sections were used for histopathology and IHC assays for estrogen receptor, progesterone receptor, and human epidermal factor receptor-2 (HER2). Transcriptome analysis of the TME Changes in the TME induced by injecƟons in treated tumors were analyzed with the nCounter canine IO panel (NanoString, Seattle, WA) using RNA isolated from FNA samples collected during treatment and paraffin-embedded tissues (FFPET) from surgeriesPATENT ATTY. DOCKET NO.1143252.007213 and necropsies. The RNA quality of P1 and P2 was poor and samples were excluded from the transcriptomic analyses. Details of gene expression analyses are provided in the supplemental file. Statistical analyses Linear regression analysis was performed for evaluation of individual IT-induced changes in tumor size between treatment points, follow-up points and start of treatment. To evaluate potential toxic and immunological effects of IT therapy in dogs, a two-tailed Student’s t-test or, as appropriate, Wilcoxon test was performed to compare therapy- induced changes in blood cell numbers and biochemistry variables. Two-tailed p-values less than 0.05 were considered statistically significant. Statistical analyses were carried out using IBM SPSS Statistics program (version v.25; Armonk, NY, USA) and GraphPad Prism (version 7.02; GraphPad San Diego, CA, USA) software. Additional details described in the supplemental file. RESULTS Clinico-pathological characteristics of CMC patients. The enrolled canine patients reflect the heterogeneity seen in human BC patients: middle-advanced age (9 to 13 years old), different breeds, and human-related comorbidities like cholesterolemia, chronic kidney disease, increase in hepatic enzyme activity, and blood variations associated with a chronic and / or inflammatory disease, such as mild anemia, increase in plasmatic globulins and in the number of peripheral blood leukocytes; different tumor subtypes (ER, PR, and TN), low (I), intermediate (II) and high histological tumor grade (III), and clinical stage I to V (Table 1). In addition to the target injected tumor, P2 had 3 tumors on the left mammary glands (P2.2-P2.4); P4, two tumors, one on the right (P4.2) and one on the left (P4.3) mammary chains; P5, had one tumor on the left (P5.3) and one on the right (P5.4) mammary chain; these two tumors were initially tracked, but during treatment, tumors became too small to correctly measure, and their measurement was discontinued. At the time of diagnosis, thoracic radiographs indicated that patient P5 had five lung metastatic nodules (three left, P5.M1-M3, and two right, P5.M4-M5 nodules). Patient P6 previously underwent a mastectomy on the right mammary chain and, at the time ofPATENT ATTY. DOCKET NO.1143252.007213 enrollment, she had a primary tumor on the 5thright mammary gland not resected when the mastectomy was performed (this tumor was treated), and also had two left (P6.M1-M2) and one right (P6.M3) lung metastatic nodules. acPD-1 monotherapy or acPD-1 / CPMV combined therapy is safe. To facilitate data presentation, instead of reporting safety data for acPD-1 and combined CPMV / acPD-1 treatments separately, it is presented together. No skin reactions in the tumor injection site or changes in the physical status of the dogs were observed in the treated dogs during the ~4-h observation period in the veterinary clinic. Per QOL questionnaire, during the four-week treatment period, all owners reported all treated dogs being more alert, more active, and some having an increased appetite, and in good physical status. This status remained the same for the three dogs during the long-term treatment. The hemogram analysis indicated no significant changes in leukocytes, lymphocytes, monocytes, neutrophils, and platelets numbers in any dogs (data not shown). However, a decrease in the erythrocyte numbers, hemoglobin, and hematocrit was observed in P1 and P5 during the first week (D2-D7) compared to basal levels at D0 (Figure 11A-C). Although these three parameters were below the normal range at the beginning of the combined therapy in P5, at D7 we observed a sustained drop in those three markers. We never observed any CPMV-related drop in these parameters in previous CMC studies [33, 34], and those changes could be related to either the disease in this dog and / or IT acPD- 1 therapy. Hence, to avoid potential adverse events, P5 did not receive the planned acPD1 dose (0.25 mg) on D9 but did receive that dose when she was clear to continue IT treatments on the next week. Once P5 continued with the combined therapy, those variables rose a bit, but remained below the normal range up to D113. A similar trend was also observed in P1, but she received the planned acPD-1 up to 1 mg. P5 ended up with only 0.5 mg injected in each of her two tumors (or 1 mg total), instead of the planned 1 mg per tumor or 2 mg total. Changes in blood and biochemistry variables were not of concern in the other treated dogs (Figure 11A-C). While total serum proteins were high before treatment in most of the dogs, except P2, there was a trend to decrease towards the normal range during treatment (Figure 12A). Similarly, the total globulin levels were a bit higher than the normal range in P1, P3 and P6, but levels dropped during treatment (Figure 12B). The albumin levels werePATENT ATTY. DOCKET NO.1143252.007213 within the normal levels during treatment with P1 and P5 remaining in the low range or below the normal range during treatment (Figure 12C). The albumin to globulin range remained below the normal range as it was before treatment, and no significant changes were observed in total proteins or in cholesterol levels during treatment when compared to basal levels (data not shown). For the liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), at D0, P1, P5, and P6 had high ALT levels, which decreased to normal range during treatment (Figure 13A). AST levels remained in the normal range in most of the dogs, except in P1 and P5 which were low (P1) or below (P5) the normal range at D0 and remained below during the treatment period (Figure 13B). Kidney function measured by creatinine levels showed slight fluctuations in P3 and P5; it was high in P1 at D0 and remained high during the treatment up to D73 (Figure 13C). Urea levels were high in P1 and P3 and remained higher during the whole treatment; some fluctuations were seen in P5 but remained in the normal range by the end of treatment (Figure 13D). It should be noted that abnormal renal function was also a limitation for surgery in P1 and she died of renal failure ~10 weeks after the last CPMV / acPD-1 ITI treatment. Collectively, the results demonstrate that neoadjuvant IT acPD-1 and IT CPMV / acPD-1 therapy is safe without serious irAEs or local reactions requiring medical intervention in treated patients. acPD-1 or acPD-1 / CPMV therapy controls tumor burden in injected and noninjected CMC tumors Figure 7A-F provides data showing that acPD-1 and CPMV / acPD-1 treatments are associated with tumor control. In the experiments therein, weekly acPD-1 as monotherapy (patients P1-P3; purple arrows; A) or CPMV (light green arrows in C) plus acPD-1 (purple arrows in C) was given for four weeks to patients P4-P6; in P5, tumors P5.1 and P5.2 were treated. Long-term weekly CPMV / acPD-1 (E; dark green arrows) was given to patients P1, P5 (P5.1 and P52 were treated) and P6. The %TGI (relative to D0) in B, D, and F indicates SD in the dotted areas (-30 to 20), above 20% indicates PD, and below -30%, partial response. During the four-week treatment period in the IT acPD-1 arm (P1-P3), we observed a tumor reduction in P1 followed by a subsequent tumor growth and decrease,PATENT ATTY. DOCKET NO.1143252.007213 while tumors did not grow in P2, and P3 tumors (Figure 7A). The %TGI indicates that SD was observed in P3 and PR in P1 and P2 by D29 (Figure 7B). In the IT CPMV / acPD-1 arm (P4-P6), there was an initial small tumor reduction in both P4 and P6 tumors with subsequent tumor growth and reduction in P4 but not in P6, where the tumor grew from D10 to D17 and remained controlled up to D29 (Figure 7C). Patient P5 presented with two large tumors in her fourth right (77.3 cm3; P5.1) and left (115.7 cm3; P5.2) mammary glands, which were treated as individual tumor masses. As illustrated in Figure 7C a higher tumor reduction was observed in the right tumor than the left tumor up to D29. Regression analysis indicates that tumor reduction in P5 (P5.1 and P5.2) and tumor growth in P6 tumors were significant (P<0.03; data not shown). The %TGI indicates that CPMV / acPD-1 treatment resulted in PR in P5 in both tumors and SD in P4 and P6 (See Figure 7D). The ORR (CR+PR) during the 4-week treatments was 67% (two PR out of three patients) in acPD-1, and 33% (one PR out of three patients) in CPMV / acPD-1. The CB is 100% for both arms (acPD-1, two PR and one SD; and CPMV / acPD-1, one PR and two SD). During long-term IT CPMV / acPD-1 treatment, we observed tumor growth in both P1 and P6 injected tumors starting on D29 with a sharp increase in tumor growth in P6 at D36, while we observed continued response in P5.1 and, to a lesser extent, in P5.2 (Figure 7E). Regression analysis indicates significant tumor reduction in P5.1 tumor (P<0.001), and tumor growth in P1 (P=0.024) and P6 (P<0.001) (data not shown). The %TGI indicates SD in P1, PD in P6, and PR in both P5 tumors (Figure 7F). The ORR is 33% (PR in one out of 3 patients); the CB is 67% (one PR and one SD out of 3 patients). The systemic effect of the IT treatment in noninjected malignant tumors were tracked in patients P2 and P4. P2 had three noninjected tumors in the left mammary chain with no tumor growth observed in two small tumors (<1.0 cm3; P2.2 and P2.3), and tumor reduction observed in the larger (~3.0 cm3, P2.4) tumor. The larger right tumor in P4 (3.5 cm3, P4.2) grew, while the smaller left tumor (~0.2 cm3, P4.3) did not grow during the four- week treatment period (See Figure 14). The exploratory itRECIST evaluation indicates that SD was observed in the acPD-1 (See Figure 15A) and CPMV / acPD-1 arms (See Figure 15B) during the four-week treatment,PATENT ATTY. DOCKET NO.1143252.007213 as well as during the long-term with CPMV / acPD-1 (See Figure 15C). While in the noninjected tumors P2.2 and P2.3 we observed SD and PR in P2.4, P4.2 had PD and P4.3 had SD (See Figure 15D). The Swimmers plot illustrating the response to CPMV / acPD-1 in target injected lesions in CMC patients based on itRECIST criteria is presented in Figure 16A. Further, as shown in Figure 8A-C, CPMV / acPD-1 treatments elicited a variable abscopal effect on established lung metastases. Of the five established lung metastatic nodules in P5, M1, M2, M4, and M5 nodules were responsive to CPMV / acPD-1 treatments, while M3 was not and had a steady tumor growth (A). During treatment, we observe CR in M2 and M4, PR in M1 and M5, and PD in M3 (B). After surgery on D113, we observed CR in M5, PR in M1, and PD in M3 (B). In P6, M1 and M3 nodules showed transient responses, and M2 slowly grew during treatment and after surgery on D79 (C). In this patient, we observed transient SD in the largest M1, and a rapid transition from SD to PD in M2 and M3 (D). The vertical dotted line in the x-axis indicates palliative surgery (mastectomy or tumor resection) of the primary tumors. Representative radiographs illustrate tumor changes in the left (E-H) and right (I-L) lung metastatic nodules in P5 before (D-2), during (up to D94), and after surgery (D254-D315). Note changes in tumor volume in M1 and M5 as well as the absence of M2, M4 and M5. The abscopal effect of CPMV / acPD-1 treatments on established lung metastases We were able to track real time changes in the lung metastatic nodules with thoracic radiographs during and after treatment in patients P5 and P6. Patient P5 had three left nodules: M1 (27.80 cm3), M2 (0.24 cm3), and M3 (0.46 cm3) and two right nodules: M.4 (3.61 cm3) and M5 (10.21 cm3)( Figure 8A). During treatment, the five metastatic nodules had a variable response to CPMV / acPD-1 with M2 and M4 nodules not detectable by D65 and D94 (Figure 8A), M1 and M5 were responsive, but M3 was not, grew steadily up to D315 (A). After surgery on D113, M5 tumor reduction was observed and by D254, it was no longer observed by radiograph, while tumor reduction was seen in M1 (Figure 8A) with subsequent tumor growth observed by D315. In P5, during treatment we observed CR in M2 and M4, PR in M1 and M5, and PD in M3 (Figure 8B). After surgery, we observed CR in M5, PR in M1, and PD in M3 (Figure 8B). Representative radiographs illustrate the tumor changes in the left (Figure 8E-H) and right (Figure 8I-L) lung nodules in patient P5 before (D- 2), during (up to D94) and after treatment surgery on D113 (D254-D315).PATENT ATTY. DOCKET NO.1143252.007213 In P6, the M1 (12.0 cm3) and M3 (3.5 cm3) nodules showed transient response, and M2 (2.3 cm3) slowly grew during treatment and after surgery on D79 (Figure 8C). In this patient, we observed SD in the largest M1 nodule and a rapid transition from SD to PD in M2 and M3 (Figure 8D). The exploratory itRECIST evaluation for the lung metastases indicates that during treatment, P5 had CR in M2 and M4, SD in M1, PD in M3 and M5 (FIG 17A). After treatment, we observed CR in M5, SD in M1, and PD in M3 (FIG 17A). Patient P6 had SD in M1 and M2, and PD in M3 (Figure 17B). The Swimmers plot illustrating the response to IT CPMV / acPD-1 in noninjected distant lung metastases in P5 and P6 patients based on itRECIST evaluation is presented in Figure 16B. While survival outcomes were not within the objectives of the study, the outcome for the acPD-1 (P1-P3) and CPMV / acPD-1 (P4-P6) arms was as follows: After four weeks of treatments, P1 was in good health and was treated further. P2, P3, and P4 underwent planned surgery with adjuvant therapy given to P3. P2 and P4 are alive and in good health (472 days after last treatment); P3, died of a cardiopathy issue 97 days after last treatment. During long-term CPMV / acPD-1 treatments, P1 received weekly treatment up to D94, and remained without treatment up to D162 when she died of renal failure (68 days after last treatment); P6 underwent palliative surgery at D79, followed by adjuvant chemotherapy, and died at D219 of lung metastasis (139 days after last treatment); P5 received treatments up to D108 and palliative surgery at D113, followed by adjuvant chemotherapy, and was euthanized at D386 (278 days after last treatment). Overall survival is illustrated in Table 1. acPD-1 and CPMV / acPD-1 treatments induced changes in the TME Transcriptomic analysis indicated the presence of altered gene expression in injected and noninjected samples. During the four-week treatment course (D0 and D29), hierarchical clustering showed the presence of three gene clusters (Figure 9A). Pathways associated with TLR1 to TLR4 cascades were enriched in clusters 1 and 3 (Figure 9A, pink and black boxes, respectively; and Figure 18), whereas pathways associated with complement, TNF, interleukins, and chemokine pathways were enriched in cluster 2 (Figure 9A, red box; and Figure 18A-C). P5.1 and P5.2 tumors showed a strong upregulation of genes in cluster 2 on D29 compared to all other samples, potentially reflecting a patient-PATENT ATTY. DOCKET NO.1143252.007213 specific response. Genes in cluster 1 seemed to be associated with a treatment response to CPMV / acPD-1 as all D29 tumors treated with CPMV / acPD-1 showed a modest upregulation of genes in this cluster. This maybe related to the known effect of CPMV on TLR2,4,7

[0019] . Genes in cluster 3 seemed associated with treatment response as these genes were modestly downregulated in D29 samples. P3 was treated with acPD-1 only and had very few altered genes compared to all other samples in both D0 and D29 samples. Expanding the hierarchical clustering analysis to samples treated after D29 in both patients P5 and P6, the patients with the largest number of biopsies, we observed a range of changes in gene expression. The 2 gene clusters (cluster 1 and 2, Figure 9A) upregulated in D29 P5.1 and P5.2 tumors were again reflected in this clustering analysis. Interestingly, genes in these 2 clusters were also upregulated in metastatic samples in P6 collected on D212 (Figure 9B, pink and red boxes). Genes in P5 noninjected metastases (collected at D386) clustered together with only a small number of genes showing upregulation in the three responsive nodules (P5.M1, P5.M2.4, and P5.M5) but not in the nonresponsive metastatic nodule (P5.M3; Figure 9B, blue box). Lastly, we noted that genes in clusters 1 and 2 (Figure 9B, red and pink boxes) were lowly expressed in P6 D0 samples and their expression gradually increased as time progressed; P6 D79 showed a slight increase and metastatic P6 D113 showed a clear increase in gene expression. The abscopal effect on gene expression was also observed in the two P5 untreated tumors we stopped tracking when their tumor growth reduction occurred and measurements were not reliable (P5.3.113 and P5.4.113 in Figure 9C). While gene expression in P5.3.113 (5thleft mammary gland) shares similarities with its treated neighbor P5.2.113 tumor (4thleft mammary gland), gene expression in P5.4.113 (5thright mammary gland) differs completely from the treated P5.1.113 (4thsample from the right)(Figure 9C). It is interesting that the inguinal nodes in P5 (P5.5.113) and P6 (P6.2.79) showed similar gene expression patterns having small common clusters (Figure 9C). More specifically, as shown in Figure 9A-C, acPD-1 and CPMV / acPD-1 treatments induces transcriptomic changes in the tumor microenvironment. During the four-week treatment period acPD-1 and CPMV / acPD-1 treatments affects gene expression in injected tumors (A). Extended CPMV / acPD-1 treatments affects gene expression in injected tumors, noninjected lung metastases (B), and noninjected tumors and inguinal nodes (C). Details inPATENT ATTY. DOCKET NO.1143252.007213 text. Legends: Each row indicates a gene; a column, an individual patient. Patient characteristics are indicated on the right side of the graphs. Patients are identified by a number (P3-P6), followed by the number of the treated or untreated tumor or metastatic lesion, and the day the sample was collected. P3 and P6 had a single treated tumor and are represented as P3.0 and P6.0 while P4 and P5 have the injected tumor (P4.1, P5.1 and P5.2), noninjected tumors (P5.3.113 and P5.4.113) and inguinal nodes in P5 (P5.5) and P6 (P6.2). The numbers at the end of each sample represent the day when the sample was collected. Metastatic lesions are represented with the letter M. Gene expression levels are represented by z-scores. Transcriptome analysis of immune cell changes within the TME in CPMV / acPD-1 injected tumors and noninjected metastases show variable changes in immune cells in both P5 and P6 tumor samples (See Figure 10A-B). Most of the immune cell contents increased by D29 in both P5 and P6 tumors. However, CD8 T cells, plasmacytoid and myeloid dendritic cells, neutrophils, monocyte and B cells remained high, while B memory cells, T reg, CD8 T memory, and plasma cells decreased in P5 by surgery day (D113) (See Figure 10A). In P6 tumor, most of the immune cells decreased by surgery day (D79), except plasma cells, monocytes, and neutrophils, which went up (See Figure 10B). Of note, compared to injected tumors, immune cell contents went down in P5 lung metastases while most of them went up in P6, except fibroblast, endothelial cells, and mast cells, which slightly decreased by D212 (See Figure 10A-B). Discussion Current use of anti-PD-1 immunotherapy in BC has been minimally effective [4, 5]. We have demonstrated good efficacy of IT CPMV in CMC patients [33, 34], supporting the potential to implement this safe and effective immunotherapy in CMC, and potentially in human BC. Given the increasing number of PD-1-focused clinical trials in BC [4, 35, 36], we sought to evaluate the feasibility of combining IT CPMV with IT anti-PD-1 using an optimal animal model like dogs with spontaneous mammary tumors to support the implementation of clinical trials of IT CPMV with approved anti-PD-1 in human BC patients. While IT anti-PD- 1 therapy will be less toxic clinically and economically than systemic anti-PD-1 for humans, systemic anti-PD-1 is commonly used clinically and could be combined with IT CPMV as many other combinatorial immunotherapies, including IT therapies, are now performed [4,PATENT ATTY. DOCKET NO.1143252.007213 35, 36]. The immunologic rationale for combining IT with systemic anti-PD-1 is that IT injection of an immunostimulatory reagent like CPMV disrupts local immune suppression and expands antitumor effector T cells which then can improve clinical responses to systemic anti-PD-1. Particularly, the results in Figure 10A-B show that CPMV / acPD-1 treatments induce changes in immune cell contents in injected tumors and noninjected, established metastases. CPMV / acPD-1 treatments increased immune cell content in both P5 (A) and P6 (B) patients with variations observed in the type of immune cells and the intensity of the increase with some immune cells having high content from D0 up to surgery day in P5 (D113), but not in P6 (D79)(compare CD8 T cells, mast cells, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDC) between P5 and P6) or an increase from D0 to D29 and then a drop in the content (compare plasma cells between P5 and P6). It is also noticeable the decrease in cell contents from surgery to metastases in P5 while an increase was observed in most of the immune cells in P6 metastases. Two groups have reported clinical studies targeting PD-1 in canine patients [22, 47-49]. One group reported safety and efficacy of systemically administered rat-canine- chimeric and caninized anti-PD-1 antibodies in 30 dogs with oral malignant melanoma (OMM), and other spontaneous tumors, including two CMC cases [22, 47], and in 37 non- OMM patients

[0048] , and another case report of a canine salivary adenocarcinoma treated with systemic caninized anti-PD-1

[0049] . Although most of the dogs previously received various treatments, clinical responses were observed along with irAEs as observed in humans

[0024] . While some fluctuations in hemogram and biochemistry variables were observed in our CMC treated patients, none were concerning or suggestive of irAEs that require medical intervention during the four-week period as well as during the long-term combined CPMV / acPD-1 treatments. Hence, our feasibility study demonstrated that neoadjuvant IT acPD-1 as monotherapy or combined with IT CPMV is safe, tolerable, and without irAEs; and they could represent a potential treatment option for CMC patients. We observed efficacy with both acPD-1 monotherapy and combined CPMV / acPD-1 treatments with reduction of tumor burden in the injected and noninjected primary tumors as well in established lung metastases. We have previously documentedPATENT ATTY. DOCKET NO.1143252.007213 that neoadjuvant IT CPMV injections resulted in tumor reduction in both the injected and noninjected tumors (abscopal effect) in CMC patients [33, 34]. Because IT CPMV was the only therapy provided to CMC patients, the observed systemic response was a bona fide abscopal effect. Moreover, we observed abscopal effect in noninjected tumors. However, the more striking tumor reduction was observed in noninjected lung metastatic nodules in P5 and P6 patients treated with IT CPMV / acPD-1, suggestive of a potent systemic immune response. The abscopal effect in noninjected tumors and lung node metastases observed manually and with radiographs was confirmed by transcriptomic analysis where we demonstrated the systemic changes of gene expression in (i) noninjected tumors and inguinal nodes with some small gene clusters seen in the samples analyzed; (ii) the three lung metastases, which had tumor changes observed in the radiographs, and three additional lung nodes not seen in P6 radiographs; (iii) variable gene expression in P5 metastases, which was different from the injected and noninjected P5 primary tumors, and P6 metastases; and (iv) a wide range of immune cell activation in injected tumors, noninjected tumors and metastases as a result of the CPMV / acPD-1-induced systemic response. The decrease in immune cell contents observed in P5 lung metastases compared to an increase in P6 metastases could be related to adjuvant therapy. P5 received toceranib phosphate (a receptor tyrosine kinase inhibitor)

[0050] , and P6, doxorubicin, which has a positive effect in immune cells

[0051] . Furthermore, despite being a relapsed mCMC patient, P6 had responses in the established metastatic nodules. While this could suggest that a good responding patient like P5, may have improved patient outcomes from immune- activating adjuvant therapy like doxorubicin than drugs that do not activate the immune system. However, additional studies are warranted to substantiate this observation as well as to determine whether specific genes in cluster 2 are associated with the constant response observed in P5 tumors, but not in other tumors. In relation to the abscopal effect on metastases, our findings are significant because once metastatic disease has occurred, there is no cure in canine or human patients. Metastatic breast cancer (mBC), whether present at diagnosis (de novo) or occurring later (relapse), is responsible for 90% of BC deaths [52, 53]. Of note, women with de novo mBC have superior outcomes compared with women with relapsed mBC

[0052] . Similarly to human mBC, P5 is a de novo and P6 is a relapse metastatic CMC patient (mCMC), and we observedPATENT ATTY. DOCKET NO.1143252.007213 better responses in the primary tumors and metastatic lung nodules in P5 than in P6. Adding additional treatments to CPMV / acPD-1 could improve the observed reduction of lung metastases and, therefore, survival outcomes. It should be noted that the median survival time for mCMC patients treated with surgery alone or with chemotherapy is generally from ~50-200 days [54-57]. While survival outcomes are out of the scope of this small feasibility study, P5 overall survival was 386 days (9.1 months after last treatment) with good QOL. The lack of a strong response in P6 could be related to the fact that the treated tumor was originally not resected when P6 underwent mastectomy a long time before entering the trial, and P6 underwent adjuvant chemotherapy. With time, the small tumor evolved into a more aggressive, chemo-resistant tumor. However, CPMV / acPD-1 resulted in SD in the treated tumor as well as in controlling lung metastases. Hence, CPMV / acPD-1 could be a good treatment option for de novo and relapse metastatic patients, but studies with a larger number of cases are needed. CONCLUSIONS Collectively, our findings demonstrated that IT acPD-1, by itself or with IT CPMV is safe and well tolerated; acPD-1 controlled tumor burden in injected and noninjected tumors, and CPMV / acPD-1 was effective against lung metastases. These could be effective therapy for CMC patients, with CPMV / acPD-1 representing a novel therapy against mCMC. Given the striking similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy against mBC. We also have shown that the inventive anti-PD-1 antibody, HugPet9, appears to be superior to a commercially known antibody, gilvetmab, which has been conditionally approved for treating cancer in canines, in both safety and efficacy in canine cancer patients. When comparing safety and efficacy of our HugPet9 antibody to a clinical study reported by Igase with acPD-1 antibodies, HugPet9 immunotherapy elicited no irAE in the three CMC patients, whereas in Igase’s study, there were irAEs grade ≥3 in ~7% (2 out of 30) of dogs, including one treatment-related death (pneumonitis) observed in a 14-year-old Chihuahua with stage IV oral melanoma who received three doses of their chimeric antibody (Igase M et al., (2020) “A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs”, Sci Rep 10:18311. doi: 10.1038 / s41598-020-75533-4).PATENT ATTY. DOCKET NO.1143252.007213 Regarding efficacy, HugPet9 immunotherapy resulted in a PR of (33%) and an SD of (67%) while the canine anti-PD-1 antibody therapy reported by Igase achieved a PD of (67%) and a SD and PR (17%, each one). We also note that in our study, patients P1 and P2 underwent planned surgery and adjuvant chemotherapy, and patient P3 underwent no additional treatments. Our clinical approach differs from Igase in that we utilize intratumoral administration to induce an immune response directly in the tumor microenvironment instead of the intravenous or systemic injection mode of administration used by Igase which, in part, may explain the irAE associated with a large amount of systemic acPD-1 which was required during the Igase treatment. A second technical advantage of HugPet9 is that our IT approach, aside from achieving enhanced clinical safety as described above, should be more cost effective because less anti-PD-1 antibody is needed for treatment. For comparisons and assuming that we will treat one cancer patient with four doses (one a week for four weeks), we will inject 0.5 mg of HugPet9 per injection or 2 mg total. If we assume the same dose, but at 0.5 mg / kg (if given systemically), for a small dog of 2 kg, or a large one of 30 kg, we will need 4 mg or 60 mg, respectively. As the cost of anti-PD-1 antibody treatment is largely driven by antibody cost this would substantially increase the cost of treatment. In addition, we note that gilvetmab has only been approved only for mast cell tumors (stages I, II, and III) or dogs with melanomas (stage II and III) melanomas; whereas Igase’s antibodies were analyzed in oral melanomas and other tumor types. By contrast, we expect based on our results to date that HugPet9 will likely be efficacious in other tumor types beyond CMC where we have demonstrated safety and efficacy. Therefore, HugPet9 represents a new immunotherapy for CMC patients with a good safety profile and clinical benefit in treated patients. When compared to similar antibodies, we observed better responses without irAEs and better quality of life. HugPet9 is a chimeric mouse anti-canine PD-1 antibody. In the future, HugPet9 will be engineered into canine antibodies or “caninized” using known methods. However, using our IT approach, the current chimeric antibody is suitable for IT immunotherapy as we have not seen any serious irAEs, and have attained good antitumor responses in treated canine patients.PATENT ATTY. DOCKET NO.1143252.007213 In addition, and similar to what we are observing in human cancer patients, monotherapies with most of the immunotherapeutic agents result in good outcomes in a small number of patients (about one third of patients or less benefit from single agents). 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Front Vet Sci 10:1144869. doi: 10.3389 / fvets.2023.1144869 London CA, Hannah AL, Zadovoskaya R, Chien MB, Kollias-Baker C, Rosenberg M, Downing S, Post G, Boucher J, Shenoy N, Mendel DB, McMahon G and Cherrington JM (2003) Phase I dose-escalating study of SU11654, a small molecule receptor tyrosine kinase inhibitor, in dogs with spontaneous malignancies. Clin Cancer Res 9:2755-68. Galluzzi L, Humeau J, Buqué A, Zitvogel L and Kroemer G (2020) Immunostimulation with chemotherapy in the era of immune checkpoint inhibitors. Nat Rev Clin Oncol 17:725-741. doi: 10.1038 / s41571-020-0413-z Dawood S, Broglio K, Ensor J, Hortobagyi GN and Giordano SH (2010) Survival differences among women with de novo stage IV and relapsed breast cancer. Ann Oncol 21:2169-2174. doi: 10.1093 / annonc / mdq220 Tevaarwerk AJ, Gray RJ, Schneider BP, Smith ML, Wagner LI, Fetting JH, Davidson N, Goldstein LJ, Miller KD and Sparano JA (2013) Survival in patients with metastaticPATENT ATTY. DOCKET NO.1143252.007213 recurrent breast cancer after adjuvant chemotherapy. Cancer 119:1140-1148. doi: doi:10.1002 / cncr.27819 Marconato L, Lorenzo RM, Abramo F, Ratto A and Zini E (2008) Adjuvant gemcitabine after surgical removal of aggressive malignant mammary tumours in dogs. Vet Comp Oncol 6:90-101. doi: 10.1111 / j.1476-5829.2007.00143.x CB DEC, Lavalle GE, Monteiro LN, Pêgas GRA, Fialho SL, Balabram D and Cassali GD (2018) Adjuvant Thalidomide and Metronomic Chemotherapy for the Treatment of Canine Malignant Mammary Gland Neoplasms. In Vivo 32:1659-1666. doi: 10.21873 / invivo.11429 Damasceno KA, Santos-Conceição AMD, Silva LP, Cardoso TMS, Vieira-Filho C, Figuerêdo SHS, Martins-Filho E, Faria BGO, Costa-Neto JMD, Cassali GD and Estrela- Lima A (2022) Factors related to the suppression of the antitumour immune response in female dogs with inflammatory mammary carcinoma. PLoS One 17:e0267648. doi: 10.1371 / journal.pone.0267648 Philibert JC, Snyder PW, Glickman N, Glickman LT, Knapp DW and Waters DJ (2003) Influence of host factors on survival in dogs with malignant mammary gland tumors. J Vet Intern Med 17:102-6. doi: 10.1892 / 0891- 6640(2003)017<0102:iohfos>2.3.co;2 1. Adams S, Schmid P, Rugo HS, Winer EP, Loirat D, Awada A, Cescon DW, Iwata H, Campone M, Nanda R, Hui R, Curigliano G, Toppmeyer D, O'Shaughnessy J, Loi S, Paluch-Shimon S, Tan AR, Card D, Zhao J, Karantza V and Cortés J (2019) Pembrolizumab monotherapy for previously treated metastatic triple-negative breast cancer: cohort A of the phase II KEYNOTE-086 study. Ann Oncol 30:397-404. doi: 10.1093 / annonc / mdy517 Adams S, Loi S, Toppmeyer D, Cescon DW, De Laurentiis M, Nanda R, Winer EP, Mukai H, Tamura K, Armstrong A, Liu MC, Iwata H, Ryvo L, Wimberger P, Rugo HS, Tan AR, Jia L, Ding Y, Karantza V and Schmid P (2019) Pembrolizumab monotherapy for previously untreated, PD-L1-positive, metastatic triple-negative breast cancer: cohort B of the phase II KEYNOTE-086 study. Ann Oncol 30:405-411. doi: 10.1093 / annonc / mdy518PATENT ATTY. DOCKET NO.1143252.007213 Cortes J, Cescon DW, Rugo HS, Nowecki Z, Im S-A, Yusof MM, Gallardo C, Lipatov O, Barrios CH, Holgado E, Iwata H, Masuda N, Otero MT, Gokmen E, Loi S, Guo Z, Zhao J, Aktan G, Karantza V and Schmid P (2020) KEYNOTE-355: Randomized, double-blind, phase III study of pembrolizumab + chemotherapy versus placebo + chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer. J Clin Oncol 38:1000-1000. doi: 10.1200 / JCO.2020.38.15_suppl.1000 Torres ETR and Emens LA (2022) Emerging combination immunotherapy strategies for breast cancer: dual immune checkpoint modulation, antibody–drug conjugates and bispecific antibodies. Breast Cancer Res Treat 191:291-302. doi: 10.1007 / s10549-021-06423-0 Debien V, De Caluwé A, Wang X, Piccart-Gebhart M, Tuohy VK, Romano E and Buisseret L (2023) Immunotherapy in breast cancer: an overview of current strategies and perspectives. npj Breast Cancer 9:7. doi: 10.1038 / s41523-023-00508-3 Klingemann H (2021) Immunotherapy for Dogs: Still Running Behind Humans. Front Immunol 12. doi: 10.3389 / fimmu.2021.665784 Ceeraz S, Nowak EC and Noelle RJ (2013) B7 family checkpoint regulators in immune regulation and disease. Trends Immunol 34:556-63. doi: 10.1016 / j.it.2013.07.003 Simon R, Freidlin B, Rubinstein L, Arbuck SG, Collins J and Christian MC (1997) Accelerated titration designs for phase I clinical trials in oncology. J Natl Cancer Inst 89:1138-47. doi: 10.1093 / jnci / 89.15.1138 Coy J, Caldwell A, Chow L, Guth A and Dow S (2017) PD-1 expression by canine T cells and functional effects of PD-1 blockade. Vet Comp Oncol 15:1487-1502. doi: 10.1111 / vco.12294 Maekawa N, Konnai S, Takagi S, Kagawa Y, Okagawa T, Nishimori A, Ikebuchi R, Izumi Y, Deguchi T, Nakajima C, Kato Y, Yamamoto K, Uemura H, Suzuki Y, Murata S and Ohashi K (2017) A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma. Sci Rep 7:8951. doi: 10.1038 / s41598-017-09444-2PATENT ATTY. DOCKET NO.1143252.007213 68. Nemoto Y, Shosu K, Okuda M, Noguchi S and Mizuno T (2018) Development and characterization of monoclonal antibodies against canine PD-1 and PD-L1. Vet Immunol Immunopathol 198:19-25. doi: 10.1016 / j.vetimm.2018.02.007 69. Igase M, Nemoto Y, Itamoto K, Tani K, Nakaichi M, Sakurai M, Sakai Y, Noguchi S, Kato M, Tsukui T and Mizuno T (2020) A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs. Sci Rep 10:18311. doi: 10.1038 / s41598-020-75533-4 70. Choi JW, Withers SS, Chang H, Spanier JA, De La Trinidad VL, Panesar H, Fife BT, Sciammas R, Sparger EE, Moore PF, Kent MS, Rebhun RB and McSorley SJ (2020) Development of canine PD-1 / PD-L1 specific monoclonal antibodies and amplification of canine T cell function. PLoS One 15:e0235518. doi: 10.1371 / journal.pone.0235518 71. Merck AH (2023) Gilvetmab for veterinary professionals. available at https: / / www.merck-animal-health-usa.com / gilvetmab. Accessed on August 15, 202 72. Ni JJ, Zhang ZZ, Ge MJ, Chen JY and Zhuo W (2022) Immune-based combination therapy to convert immunologically cold tumors into hot tumors: an update and new insights. Acta Pharmacol Sin. doi: 10.1038 / s41401-022-00953-z 73. Yap TA, Parkes EE, Peng W, Moyers JT, Curran MA and Tawbi HA (2021) Development of Immunotherapy Combination Strategies in Cancer. Cancer Discov 11:368-1397. doi: 10.1158 / 2159-8290.Cd-20-1209 Each document cited herein, including all patent and non-patent literature, published or unpublished patent applications, abstracts, and any other document cited, is hereby incorporated by reference in its entirety.PATENT ATTY. DOCKET NO.1143252.007213 INFORMAL SEQUENCE LISTING ANTIBODY SEQUENCES OF ANTI-CANINE PD-1 ANTIBODY 77A6H9: 77A6H9 Heavy chain: DNA sequence (414 bp) Signal sequence (Bold)-FR1 (thick line)-CDR1 (broken underline)-FR2 (thick line)-- CDR2 (broken underline)-FR3 (thick line)-CDR3 (broken underline)--FR4 (thick line) ATGGCTGTCCTGGTGCTGCTCCTCTGCCTGGTGACATTCCCAAGCTGTGTCCTGTCCCA GGTGCAACTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCATAACCTGCACA GTCTCTGGTTTCTCATTAACTAAGTATGGTGTACACTGGGTTCGCCAGTCTCCAGAAAAGGGTCTGG AGTGGCTGGGAGTGATATGGACAGGTGGAAACACAGACTACAATGCAGCTTTCATGTCCAGACTGA GCATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTGATGACAC TGCCATATATTACTGTGCCAAAAATGGGGCATCCGATGATAACGGGGGGGACTCCTGGGGTCAAGG AACCTCAGTCACCGTCTCATCA (SEQ ID NO: 1) 77A6H9 Heavy chain: Amino acid sequence (138 aa) Signal sequence (Bold)-FR1 (thick line)-CDR1 (broken underline)-FR2 (thick line)-- CDR2 (broken underline)-FR3 (thick line)-CDR3 (broken underline)--FR4 (thick line) MAVLVLLLCLVTFPSCVLSQVQLKQSGPSLVQPSQSLSITCTVSGFSLTKYGVHWVRQSPEK GLEWLGVIWTGGNTDYNAAFMSRLSITRDNSKSQVFFKMNSLQADDTAIYYCAKNGASDDNGGDSW GQGTSVTVSS (SEQ ID NO: 2) 77A6H9 Heavy chain CDR1: Nucleic acid sequence (15 nt) AAGTATGGTGTACAC (SEQ ID NO: 3) 77A6H9 Heavy chain CDR1: Amino acid sequence (5 aa) KYGVH (SEQ ID NO: 4) 77A6H9 Heavy chain CDR2: Nucleic acid sequence (48 nt) GTGATATGGACAGGTGGAAACACAGACTACAATGCAGCTTTCATGTCC (SEQ ID NO: 5) 77A6H9 Heavy chain CDR2: Amino acid sequence (16 aa) VIWTGGNTDYNAAFMS (SEQ ID NO: 6)PATENT ATTY. DOCKET NO.1143252.007213 77A6H9 Heavy chain CDR3: Nucleic acid sequence (33 nt) AATGGGGCATCCGATGATAACGGGGGGGACTCC (SEQ ID NO: 7) 77A6H9 Heavy chain CDR3: Amino acid sequence (11 aa) NGASDDNGGDS (SEQ ID NO: 8) 77A6H9 Heavy chain FR1 Sequence (DNA Sequence) ( 90 nt) CAGGTGCAACTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCA TAACCTGCACAGTCTCTGGTTTCTCATTAACT (SEQ ID NO: 9) 77A6H9 Heavy chain FR1 Sequence (Amino Acid Sequence) (30 aa) QVQLKQSGPSLVQPSQSLSITCTVSGFSLT (SEQ ID NO: 10) 77A6H9 Heavy chain FR2 Sequence (DNA Sequence) (42 nt) TGGGTTCGCCAGTCTCCAGAAAAGGGTCTGGAGTGGCTGGGA (SEQ ID NO: 11) 77A6H9 Heavy chain FR2 Sequence (Amino Acid Sequence) WVRQSPEKGLEWLG (SEQ ID NO: 12) 77A6H9 Heavy chain FR3 Sequence (DNA Sequence) (96 nt) AGACTGAGCATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCT GCAAGCTGATGACACTGCCATATATTACTGTGCCAAA (SEQ ID NO: 13) 77A6H9 Heavy chain FR3 Sequence (Amino Acid Sequence) (32 aa) RLSITRDNSKSQVFFKMNSLQADDTAIYYCAK (SEQ ID NO: 14) 77A6H9 Heavy chain FR4 Sequence (DNA Sequence) (33 nt) TGGGGTCAAGGAACCTCAGTCACCGTCTCATCA (SEQ ID NO: 15) 77A6H9 Heavy chain FR4 Sequence (Amino acid sequence) (11 aa) WGQGTSVTVSS (SEQ ID NO: 16) 77A6H9 Heavy chain Signal Sequence (DNA Sequence) (57 nt) ATGGCTGTCCTGGTGCTGCTCCTCTGCCTGGTGACATTCCCAAGCTGTGTCCTGTCC (SEQ ID NO: 17) 77A6H9 Heavy chain Signal Sequence (Amino Acid Sequence) (19 aa)PATENT ATTY. DOCKET NO.1143252.007213 MAVLVLLLCLVTFPSCVLS VH SIGNAL SEQUENCE AA (SEQ ID NO: 18) 77A6H9 Heavy chain Coding Sequence (DNA Sequence) (357 nt) CAGGTGCAACTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCA TAACCTGCACAGTCTCTGGTTTCTCATTAACTAAGTATGGTGTACACTGGGTTCGCCAGTCTCCAGAA AAGGGTCTGGAGTGGCTGGGAGTGATATGGACAGGTGGAAACACAGACTACAATGCAGCTTTCAT GTCCAGACTGAGCATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAA GCTGATGACACTGCCATATATTACTGTGCCAAAAATGGGGCATCCGATGATAACGGGGGGGACTCC TGGGGTCAAGGAACCTCAGTCACCGTCTCATCA (SEQ ID NO: 19) 77A6H9 Heavy chain Polypeptide (Amino Acid Sequence) (119 aa) QVQLKQSGPSLVQPSQSLSITCTVSGFSLTKYGVHWVRQSPEKGLEWLGVIWTGGNTDYN AAFMSRLSITRDNSKSQVFFKMNSLQADDTAIYYCAKNGASDDNGGDSWGQGTSVTVSS VH POLYPEPTIDE SEQUENCE AA (SEQ ID NO: 20) 77A6H9 Light chain: DNA sequence (393 bp) Signal sequence (Bold)-FR1 (thick line)-CDR1 (broken underline)-FR2 (thick line)-- CDR2 (broken underline)-FR3 (thick line)-CDR3 (broken underline)--FR4 (thick line) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT AGATCTAGT CAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTC CAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGG ATCAGGGACAGATTTCACACTCAAGATCGGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGSignal sequence (Bold)-FR1 (thick line)-CDR1 (broken underline)-FR2 (thick line)-- CDR2 (broken underline)-FR3 (thick line)-CDR3 (broken underline)--FR4 (thick line) MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYL QKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIGRVEAEDLGVYYCFQGSHIPLTFGAGTKLELK (SEQ ID NO: 22) 77A6H9 Light chain CDR1: Nucleic acid sequence (15 nt) GATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAA (SEQ ID NO: 23)PATENT ATTY. DOCKET NO.1143252.007213 77A6H9 Light chain CDR1: Amino acid sequence (5 aa) RSSQNIVHSNGNTYLE (SEQ ID NO: 24) 77A6H9 Light chain CDR2: Nucleic acid sequence (48 nt) AAAGTTTCCAACCGATTTTCT (SEQ ID NO: 25) 77A6H9 Light chain CDR2: Amino acid sequence (16 aa) KVSNRFS (SEQ ID NO: 26) 77A6H9 Light chain CDR3: Nucleic acid sequence (27 nt) TTTCAAGGTTCACATATTCCTCTCACG (SEQ ID NO: 27) 77A6H9 Light chain CDR3: Amino acid sequence (11 aa) FQGSHIPLT (SEQ ID NO: 28) 77A6H9 Light chain FR1 Sequence (DNA Sequence) (69 nt) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC ATCTCTTGC (SEQ ID NO: 29) 77A6H9 Light chain FR1 Sequence (Amino Acid Sequence) (23 aa) DVLMTQTPLSLPVSLGDQASISC (SEQ ID NO: 30) 77A6H9 Heavy chain FR2 Sequence (DNA Sequence) (42 nt) TGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTAC (SEQ ID NO: 31) 77A6H9 Light chain FR2 Sequence (Amino Acid Sequence) (15 aa) WYLQKPGQSPKLLIY (SEQ ID NO: 32) 77A6H9 Heavy chain FR3 Sequence (DNA Sequence) (96 nt) AGACTGAGCATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCT GCAAGCTGATGACACTGCCATATATTACTGTGCCAAA (SEQ ID NO: 33) 77A6H9 Light chain FR3 Sequence (Amino Acid Sequence) (31 aa) VPDRFSGSGSGTDFTLKIGRVEAEDLGVYYC (SEQ ID NO: 34) 77A6H9 Light chain FR4 Sequence (DNA Sequence) (30 nt)PATENT ATTY. DOCKET NO.1143252.007213 TTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 35) 77A6H9 Light chain FR4 Sequence (Amino acid sequence) (10 aa) FGAGTKLELK (SEQ ID NO: 36) 77A6H9 Light chain Signal Sequence (DNA Sequence) (57 nt) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT VL SIGNAL SEQUENCE NA (SEQ ID NO: 37) 77A6H9 Light chain Signal Sequence (Amino Acid Sequence) (19 aa) MKLPVRLLVLMFWIPASSS VL SIGNAL SEQUENCE AA (SEQ ID NO: 38) 77A6H9 Variable Light chain Coding Sequence (DNA Sequence) (XX nt) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC CATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCT GCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCC AGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCGGCAGAGTGGAGGCT GAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATATTCCTCTCACGTTCGGTGCTGGGACCA AGCTGGAGCTGAAA (SEQ ID NO: 39) 77A6H9 Variable Light chain Polypeptide (Amino Acid Sequence) (112 aa) DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTDFTLKIGRVEAEDLGVYYCFQGSHIPLTFGAGTKLELK VL POLYPEPTIDE SEQUENCE AA (SEQ ID NO: 40) ANTIBODY SEQUENCES OF ANTI-CANINE PD-1 ANTIBODY 77A6H7: Heavy chain: DNA sequence (414 bp) Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGGCTGTCCTGGTGCTGCTCCTCTGCCTGGTGACATTCCCAAGCTGTGTCCTGTCCCAG GTGCAACTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCATAACCTGCACAGGTGGCTGGGAGTGATATGGACAGGTGGAAACACAGACTACAATGCAGCTTTCATGTCCAGACTGAGPATENT ATTY. DOCKET NO.1143252.007213 CATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACT GCCATATATTACTGTGCCAAAAATGGGGCATCCGATGATAACGGGGGGGACTCCTGGGGTCAAGGAHeavy chain: Amino acid sequence (138 aa) Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 MAVLVLLLCLVTFPSCVLSQVQLKQSGPSLVQPSQSLSITCTVSGFSLTKYGVHWVRQSPEK GLEWLGVIWTGGNTDYNAAFMSRLSITRDNSKSQVFFKMNSLQADDTAIYYCAKNGASDDNGGDSW GQGTSVTVSS (SEQ ID NO: 42) 77A6H7 Heavy chain CDR1: Nucleic acid sequence (15 nt) AAGTATGGTGTACAC (SEQ ID NO: 43) 77A6H7 Heavy chain CDR1: Amino acid sequence (5 aa) KYGVH (SEQ ID NO: 44) 77A6H7 Heavy chain CDR2: Nucleic acid sequence (48 nt) GTGATATGGACAGGTGGAAACACAGACTACAATGCAGCTTTCATGTCC (SEQ ID NO: 45) 77A6H7 Heavy chain CDR2: Amino acid sequence (16 aa) VIWTGGNTDYNAAFMS (SEQ ID NO: 46) 77A6H7 Heavy chain CDR3: Nucleic acid sequence (33 nt) AATGGGGCATCCGATGATAACGGGGGGGACTCC (SEQ ID NO: 47) 77A6H7 Heavy chain CDR3: Amino acid sequence (11 aa) NGASDDNGGDS (SEQ ID NO: 48) 77A6H7 Heavy chain FR1 Sequence (DNA Sequence) (90 nt) CAGGTGCAACTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCA TAACCTGCACAGTCTCTGGTTTCTCATTAACT (SEQ ID NO: 49) 77A6H7 Heavy chain FR1 Sequence (Amino Acid Sequence) (30 aa) QVQLKQSGPSLVQPSQSLSITCTVSGFSLT (SEQ ID NO: 50)PATENT ATTY. DOCKET NO.1143252.007213 77A6H7 Heavy chain FR2 Sequence (DNA Sequence) (42 nt) TGGGTTCGCCAGTCTCCAGAAAAGGGTCTGGAGTGGCTGGGA (SEQ ID NO: 51) 77A6H7 Heavy chain FR2 Sequence (Amino Acid Sequence) WVRQSPEKGLEWLG (SEQ ID NO: 52) 77A6H7 Heavy chain FR3 Sequence (DNA Sequence) (96 nt) AGACTGAGCATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCT GCAAGCTGATGACACTGCCATATATTACTGTGCCAAA (SEQ ID NO: 53) 77A6H7 Heavy chain FR3 Sequence (Amino Acid Sequence) (32 aa) RRLSITRDNSKSQVFFKMNSLQADDTAIYYCAK (SEQ ID NO: 54) 77A6H7 Heavy chain FR4 Sequence (DNA Sequence) (33 nt) TGGGGTCAAGGAACCTCAGTCACCGTCTCATCA (SEQ ID NO: 55) Heavy chain FR4 Sequence (Amino acid sequence) (11 aa) WGQGTSVTVSS (SEQ ID NO: 56) 77A6H7 Heavy chain signal sequence (Nucleic acid Sequence) ATGGCTGTCCTGGTGCTGCTCCTCTGCCTGGTGACATTCCCAAGCTGTGTCCTGTCC (SEQ ID NO: 57) 77A6H7 Heavy chain signal sequence (Amino acid Sequence) MAVLVLLLCLVTFPSCVLS (SEQ ID NO: 58) 77A6H7 Variable Heavy chain Polypeptide Coding Sequence (Nucleic Acid) (357 nt) CAGGTGCAACTGAAGCAGTCAGGACCTAGCCTAGTGCAGCCCTCACAGAGCCTGTCCA TAACCTGCACAGTCTCTGGTTTCTCATTAACTAAGTATGGTGTACACTGGGTTCGCCAGTCTCCAGAA AAGGGTCTGGAGTGGCTGGGAGTGATATGGACAGGTGGAAACACAGACTACAATGCAGCTTTCAT GTCCAGACTGAGCATCACCAGGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAA GCTGATGACACTGCCATATATTACTGTGCCAAAAATGGGGCATCCGATGATAACGGGGGGGACTCC TGGGGTCAAGGAACCTCAGTCACCGTCTCATCA (SEQ ID NO: 59) 77A6H7 Variable Heavy chain Polypeptide Amino Acid Sequence) (119 aa)PATENT ATTY. DOCKET NO.1143252.007213 QVQLKQSGPSLVQPSQSLSITCTVSGFSLTKYGVHWVRQSPEKGLEWLGVIWTGGNTDYN AAFMSRLSITRDNSKSQVFFKMNSLQADDTAIYYCAKNGASDDNGGDSWGQGTSVTVSS (SEQ ID NO: 60) Light chain: DNA sequence (393 bp) Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTG ATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA GATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGG(SEQ ID NO: 62) 77A6H7 Light chain CDR1: Nucleic acid sequence (48 nt) AGATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAA (SEQ ID NO: 63) 77A6H7 Light chain CDR1: Amino acid sequence (16 aa) RSSQNIVHSNGNTYLE (SEQ ID NO: 64) 77A6H7 Light chain CDR2: Nucleic acid sequence (21 nt) AAAGTTTCCAACCGATTTTCT (SEQ ID NO: 65) 77A6H7 Light chain CDR2: Amino acid sequence (7 aa) KVSNRFS (SEQ ID NO: 66)PATENT ATTY. DOCKET NO.1143252.007213 77A6H7 Light chain CDR3: Nucleic acid sequence (27 nt) TTTCAAGGTTCACATATTCCTCTCACG (SEQ ID NO: 67) 77A6H7 Light chain CDR3: Amino acid sequence (9 aa) FQGSHIPLT (SEQ ID NO: 68) 77A6H7 Light chain FR1 Sequence (DNA Sequence) (69 nt) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC ATCTCTTGC (SEQ ID NO: 69) 77A6H7 Light chain FR1 Sequence (Amino Acid Sequence) (23 aa) DVLMTQTPLSLPVSLGDQASISC (SEQ ID NO: 70) 77A6H7 Light chain FR2 Sequence (DNA Sequence) (42 nt) TGGTACCTGCAGAAACCAGGCCAGTCTCCAAAACTCCTGATCTAC (SEQ ID NO: 71) 77A6H7 Light chain FR2 Sequence (Amino Acid Sequence) WYLQKPGQSPKLLIY (SEQ ID NO: 72) 77A6H7 Light chain FR3 Sequence (DNA Sequence) (96 nt) GGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCG GCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC (SEQ ID NO: 73) 77A6H7 Light chain FR3 Sequence (Amino Acid Sequence) (32 aa) GVPDRFSGSGSGTDFTLKIGRVEAEDLGVYYC (SEQ ID NO: 74) 77A6H7 Light chain FR4 Sequence (DNA Sequence) (30 nt) TTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 75) Light chain FR4 Sequence (Amino acid sequence) (10 aa) FGAGTKLELK (SEQ ID NO: 76) 77A6H7 Light chain signal sequence (Nucleic acid Sequence) (57 nt) ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGT (SEQ ID NO: 77) 77A6H7 Light chain signal sequence (Amino acid Sequence) (19 aa)PATENT ATTY. DOCKET NO.1143252.007213 MKLPVRLLVLMFWIPASSS (SEQ ID NO: 78) 77A6H7 Variable Light chain Polypeptide Coding Sequence (Nucleic Acid Sequence) (336 nt) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC ATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCA GAAACCAGGCCAGTCTCCAAAACTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGAC AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCGGCAGAGTGGAGGCTGAGGA TCTGGGAGTTTATTACTGCTTTCAAGGTTCACATATTCCTCTCACGTTCGGTGCTGGGACCAAGCTGG AGCTGAAA (SEQ ID NO: 79) 77A6H7 Variable Light chain Polypeptide (Amino Acid Sequence) (112 aa) DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTDFTLKIGRVEAEDLGVYYCFQGSHIPLTFGAGTKLELK (SEQ ID NO: 80) Having fully described the invention, the following claims are now provided below.

Claims

PATENT ATTY. DOCKET NO.1143252.007213 What is claimed is:

1. An antibody or antibody fragment which binds to canine PD-1, wherein said antibody or antibody fragment comprises the same CDRs as 77A6H9 (HugPet9) or 77A6H7.

2. The antibody or antibody fragment of claim 1, which : (i) comprises a VH region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 4, 6 and 8 respectively; and a VL region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 24, 26 and 28 respectively; or (ii) comprises a VH region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 44, 46 and 48 respectively; and a VL region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 64, 66 and 68, respectively.

3. The antibody or antibody fragment of claim 1 or 2, which comprises a VH region which comprises an amino acid sequence which possesses at least 90, 95, 96, 97, 98, 99 or 100% sequence identity to that of 77A6H9 (HugPet9) or 77A6H7 or to that of SEQ ID NO: 20 or SEQ ID NO:

60.

4. The antibody or antibody fragment of claim 1 or 2, which comprises a VL region which comprises an amino acid sequence which possesses at least 90, 95, 96, 97, 98, 99 or 100% sequence identity to that of 77A6H9 (HugPet9) or 77A6H7 or to that of SEQ ID NO: 40 or SEQ ID NO:

80.

5. The antibody or antibody fragment of claim 1 or 2, which comprises a VH region which comprises an amino acid sequence which possesses at least 90, 95, 96, 97, 98, 99 or 100% sequence identity to that of 77A6H9 (HugPet9) or 77A6H7 or to that of SEQ ID NO: 20 or SEQ ID NO: 60; and further comprises a VL region which comprises an amino acid sequence which possesses at least 90, 95, 96, 97, 98, 99 or 100% sequence identity to that of 77A6H9 (HugPet9) or 77A6H7 or to that of SEQ ID NO: 40 or SEQ ID NO:

80.

6. The antibody or antibody fragment of claim 1 or 2, which comprises a VH region which comprises an amino acid sequence which is identical to that of 77A6H9 or 77A6H7 or to SEQ ID NO: 20 or SEQ ID NO: 60; and comprises a VL region which is identical to that of 77A6H9 or 77A6H7 or to SEQ ID NO: 40 or SEQ ID NO: 80.PATENT ATTY. DOCKET NO.1143252.007213 7. The antibody or antibody fragment of any one of the previous claims, which comprises an Fc region.

8. The antibody or antibody fragment of any one of the previous claims, which comprises a murine, human, feline or canine Fc region, optionally a canine IgGA, IgGB, IgGC or IgGD Fc region.

9. The antibody or antibody fragment of any one of the previous claims, which comprises canine or murine framework regions.

10. The antibody or antibody fragment of any one of the previous claims, which is caninized.

11. An antibody or antibody fragment according to any one of the previous claims, which comprises an Fc region which comprises at least one modification that enhances or inhibits at least one antibody effector function, optionally complement binding, FcR binding, ADCC, glycosylation, FcRN binding, and the like.

12. An antibody or antibody fragment according to any one of the previous claims, which is a Fab, Fab2or scFv.

13. An antibody or antibody fragment according to any one of the previous claims, which comprises an Fc or constant region, which comprises at least one modification that enhances at least one antibody effector function selected from glycosylation, FcR binding, FcRN binding, phagocytosis, antibody dependent cellular cytotoxicity (ADCC), or complement dependent cytotoxicity (CDC).

14. An antibody or antibody fragment according to any one of the previous claims, which comprises an Fc or constant region, which comprises at least one modification that inhibits at least one antibody effector function selected from glycosylation, FcR binding, FcRN binding, phagocytosis, antibody dependent cellular cytotoxicity (ADCC), or complement dependent cytotoxicity (CDC).

15. An immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) comprising an antibody or antibody fragment which: (i) comprises a VH region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 4, 6 and 8 respectively; and a VL region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 24, 26 and 28 respectively; or (ii) comprises a VH region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 44, 46 and 48 respectively; and aPATENT ATTY. DOCKET NO.1143252.007213 VL region comprising a CDR1, 2 and 3 comprising the amino acid sequence of SEQ ID NO: 64, 66 and 68, respectively.

16. An antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor according to any one of claims 1-15, which is expressed in a recombinant host cell, optionally a mammalian, yeast, fungal, plant, insect or bacterial cell.

17. An antibody or antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor according to any one of claims 1-15, which is expressed in a CHO, BHK, COS or Hela or HEK cell.

18. A nucleic acid or nucleic acids which encode for an antibody, antibody fragment, immunoconjugate, ADC, or CAR according to any one of claims 1-15.

19. An expression vector comprising the nucleic acid or nucleic acids of claim 18.

20. A recombinant cell which comprises an expression vector according to claim 19.

21. The recombinant cell of claim 20, which is mammalian, yeast, fungal, plant, insect or bacterial cell.

22. The recombinant cell of claim 21, which is a CHO, BHK, COS, Hela, or HEK (e.g., HEK- 293) cell.

23. A diagnostic or therapeutic composition comprising an antibody, antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of claims 1-15 and a therapeutically or diagnostically acceptable carrier.

24. A method of treating cancer in a canine comprising the administration of an antibody, antibody fragment, immunoconjugate, antibody drug conjugate (ADC) or chimeric antigen receptor (CAR) according to any one of claims 1-15.

25. The treatment method of claim 24 wherein the cancer comprises an adenoma, adenocarcinoma, anal sac adenocarcinoma, basal tumor, bladder cancer, bone cancer (e.g., osteosarcoma), brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, hemangiosarcoma, histiocytic sarcoma, leukemia, lipoma, liver cancer, lung cancer e.g., canine primary lung cancer, lymphoma, lymphosarcoma, mammary cancers or mastocytoma, mast cell tumor, melanoma, oral cancers such as melanoma, squamous cell carcinoma or fibrosarcoma; ovarian cancer, nasal cancer or nasal tumor, pancreatic cancer, prostate cancer, skin cancersPATENT ATTY. DOCKET NO.1143252.007213 (e.g., melanoma), soft tissue sarcoma, splenic hemangiosarcoma (HSA), squamous cell carcinoma, testicular cancer, transitional cell carcinoma, uterine cancers, among others, and preferably for use in treating mammary cancers or inflammatory mammary cancers.

26. The treatment method of claim 24 or 25 which includes the use of at least one other therapeutic agent or treatment regimen.

27. The treatment method of any one of claims 24-26 wherein the antibody or antibody fragment and / or optionally another active are administered to the canine intratumorally and / or systemically, optionally by intravenous or subcutaneous administration.

28. The treatment method of claim 26 or 27, wherein said other therapeutic agent comprises an anti-cancer agent.

29. The treatment method of claim 26 or 27, wherein said other therapeutic agent comprises Cowpea Mosaic Virus (CPMV) particles (empty or non-empty) and optionally is intratumorally administered.

30. The treatment method of claim 26 or 27, wherein said other therapeutic agent comprises a checkpoint inhibitor antibody or checkpoint inhibitor fusion protein, a hormone, a cytokine, a growth factor, a chemotherapeutic, another anti-cancer antibody, or a combination of any of the foregoing.

31. The treatment method of claim 26 or 27, wherein said other therapeutic agent targets the extrinsic apoptotic pathway or the intrinsic apoptotic pathway or the common apoptotic pathway.

32. The treatment method of claim 26 or 27, wherein said other therapeutic agent enhances ADCC of cancer cells.

33. The treatment method of claim 26 or 27, wherein the other therapeutic agent comprises gemcitabine and / or cisplatin.

34. The treatment method of claim 26 or 27, wherein the other therapeutic agent comprises necrosis factor alpha (TNF-alpha), tumor necrosis factor beta (TNF-beta, lymphotoxin alpha), lymphotoxin beta (LT-beta), TRAIL (Apo2L), CD95 (Fas, APO-I) ligand, TRAMP (DR3, Apo-3) ligand, DR4 ligand, DR6 ligand as well as fragments, variants, and derivatives thereof.PATENT ATTY. DOCKET NO.1143252.007213 35. The treatment method of claim 26 or 27, wherein said other therapeutic agent comprises an endocrine therapeutic, a biologic response modifier, interferon, interleukin, antibody, aptamer, siRNA, oligonucleotide, enzyme, ion channel and receptor inhibitor or activator, hyperthermia, cryotherapy, agent to attenuate any adverse effects, or antiemetic.

36. The treatment method of claim 26 or 27, wherein said other therapeutic agent comprises an alkylating drug, mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide, antimetabolite, Methotrexate, purine antagonist, pyrimidine antagonist, 6-Mercaptopurine, 5-Fluorouracil, Cytarabine, Gemcitabine, spindle poison, Vinblastine, Vincristine, Vinorelbine, Paclitaxel, podophyllotoxin, Etoposide, Irinotecan, Topotecan, antibiotic, doxorubicin, Bleomycin, Mitomycin, nitrosoureas, Carmustine, Lomustine, inorganic ion, Cisplatin, Carboplatin, enzyme, Asparaginase, hormone, Tamoxifen, Leuprolide, Flutamide, or Megestrol.

37. The antibody, antibody fragment, immunoconjugate, ADC, CAR, or composition, according to any one of claims 1-15, wherein the antibody or antibody fragment is chimeric.

38. The antibody, antibody fragment, immunoconjugate, ADC, CAR, or composition according to any one of claims 1-15, wherein the antibody or antibody fragment is caninized.

39. A pharmaceutical composition comprising the antibody, antibody fragment, immunoconjugate, ADC, or CAR, according to any one of claims 1-15, and a pharmaceutically acceptable carrier.

40. A kit comprising the antibody, antibody fragment, immunoconjugate, ADC, or CAR according to any one of claims 1-15.

41. A method of treatment, comprising administering an antibody, antibody fragment, immunoconjugate, ADC, or CAR, or composition containing according to any one of claims 1-15 to a canine subject in need thereof.

42. A method of treatment, comprising administering an antibody, antibody fragment, immunoconjugate, ADC, or CAR, or composition containing according to any one of claims 1-15 to a canine subject in need thereof and further comprising administering radiation therapy to a subject and / or effecting cancer surgery.PATENT ATTY. DOCKET NO.1143252.007213 43. A method of detecting canine PD-1 using an antibody or antibody fragment according to any one of claims 1-15.

44. The method of claim 43 which is effected in vivo.

45. The method of claim 43 which is effected in vitro.

46. The detection method of any one of claims 43-45, which is used to assess the disease status of a canine subject.

47. The detection method of any one of claims 43-45, which is used to assess the treatment status of a canine subject, optionally after administration of an antibody or antibody fragment, immunoconjugate, ADC, or CAR, or composition according to any one of the foregoing claims or a cell which expresses antibody or antibody fragment, immunoconjugate, ADC, or CAR according to any one of the foregoing claims.

48. The detection method of any one of claims 43-47, which includes any one of the following: Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement-fixation assay, immunohistochemical assay, fluorescent immunoassay, and protein A immunoassay.

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