Anti-canine PD-L1 antibody

Abstract

This invention relates to anti-canine PD-L1 antibodies. The antibodies are preferably complete canine antibodies. The invention further relates to canine PD-L1 epitopes that are conjugated by these antibodies and inhibit the binding of canine PD-1 to PD-L1. The invention further relates to the use of these antibodies in the treatment of dogs, including cancer treatment.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority from EP23164436.0, filed on 27 March 2023, which is incorporated in its entirety by reference.

[0002] This invention relates to anti-canine PD-L1 antibodies. The antibodies are preferably complete canine antibodies. The invention further relates to canine PD-L1 epitopes that are conjugated by these antibodies and inhibit the binding of canine PD-1 to PD-L1. The invention further relates to the use of these antibodies in the treatment of dogs, including cancer treatment. [Background technology]

[0003] In human medicine, the introduction of immune checkpoint inhibitors (ICIs) has led to many advances in the immunotherapeutic management of cancer, particularly malignant melanoma. Ipilimumab, an anti-CTLA-4 monoclonal antibody, was the first ICI approved to demonstrate efficacy in human advanced / metastatic melanoma. Subsequently, nivolumab and pembrolizumab, both anti-PD-1 antibodies, were approved for the treatment of advanced melanoma. In total, since May 2006, six anti-PD1 / PD-L1 specific ICIs for various cancer indications have been approved by the FDA and / or EMA: pembrolizumab (anti-PD1, IgG4κ), nivolumab (anti-PD1, IgG4), cemiprimab (anti-PD1, IgG4), atezolizumab (anti-PD-L1, IgG1, silent Fc format), avelumab (anti-PD-L1, IgG1, wild-type Fc format), and durvalumab (anti-PD-L1, IgG1, silent Fc format) (Ai et al. 2020).

[0004] ICIs represent a true breakthrough in the treatment of human melanoma, significantly improving the prognosis of responsive animals. Therefore, veterinary medicine is now shifting its focus to the use of ICIs as a potentially effective systemic therapy, also for tumor-bearing dogs. However, experience in dogs remains limited. The expression of cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1), and PD-1 ligand-1 (PD-L1) on canine immune cells and / or cancer cells has already been investigated and reported (Igase et al. 2020, Maekawa et al. 2017, Mason et al. 2021).

[0005] PD-1 is a 55 kDa type I transmembrane glycoprotein containing an extracellular Ig variable (type V) domain that binds to its ligand and a cytoplasmic tail that binds to signaling molecules. PD-1 is an inhibitor of both adaptive and innate immune responses and is expressed on activated T cells, natural killer (NK) cells, B lymphocytes, macrophages, dendritic cells (DCs), and monocytes. Importantly, PD-1 is highly expressed on tumor-specific T cells (Ahmadzadeh et al. 2009). PD-1 plays two opposing roles, both beneficial and harmful. It plays a crucial role in reducing the modulation of ineffective immune responses or damaging immune responses. Thus, PD-1 maintains immune tolerance by suppressing harmful responses to self-proteins. On the other hand, PD-1 causes the proliferation of malignant cells by impairing protective immune responses. Suppression of the immune response is achieved by the binding of PD-L1, a ligand for PD-1, to PD-1.

[0006] PD-L1 is a 33 kDa type I transmembrane glycoprotein containing both IgV and IgC-like domains in its extracellular domain, along with a short cytoplasmic region lacking known signaling motifs (Sanmamed und Chen 2014). PD-L1 is typically expressed by macrophages, some activated T and B cells, dendritic cells, and some epithelial cells, particularly under inflammatory conditions (Han et al. 2020). Furthermore, PD-L1 is overexpressed in a variety of tumors, where it binds to PD-1, inhibits the proliferation of PD-1-positive cells, and contributes to tumor immune evasion, leading to treatment failure (Ohaegbulam et al. 2015). Such tumor tissues include cancers of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, oral squamous epithelial cells, urothelial cells, and pancreas, as well as head and neck tumors.

[0007] PD-L1 on the surface of tumor cells can be upregulated by interferon-gamma (IFN-γ) produced by activated T cells (Tang et al. 2018) and NK cells (Bellucci et al. 2015). Binding of PD-L1 on tumor cells to PD-1 on immune cells may reduce T cell-mediated immune surveillance, leading to the absence of an immune response against the tumor and even T cell apoptosis (Iwai et al. 2017).

[0008] In summary, abnormal PD-L1 expression has been reported in many human cancers and is considered a mechanism of immune evasion in cancer. Importantly, PD-L1 expression has been demonstrated in a variety of canine cancers, particularly oral melanoma (OMM) (36 / 40 patients; (Maekawa et al. 2016)). Other PD-L1-positive cancers include osteosarcoma, angiosarcoma, mast cell tumors, mammary gland cancer, and prostate cancer (Takeuchi et al. 2020, Maekawa et al. 2014, Cascio et al. 2021, Ariyarathna et al. 2020, Hartley et al. 2017, Maekawa et al. 2016). Most recently, higher PD-L1 expression in neoplastic lymphocytes compared to normal B cells has been demonstrated in flow cytometry-mediated canine B-cell lymphoma (Hartley et al. 2018). Increased PD-L1 expression is associated with a higher risk of progression and lymphoma-related death, regardless of treatment (Aresu et al. (al. 2021). This result is consistent with recent evidence in humans and suggests that upregulation of PD-L1 in tumor cells allows tumors to evade the host immune system and increase chemoresistance. PD-1 expression was elevated in tumor-infiltrating lymphocytes obtained from oral melanoma, suggesting that lymphocytes in this type of cancer may have been functionally exhausted (Maekawa et al. 2016). Similar results were shown in another study of canine melanoma cell lines and tumor-infiltrating macrophages that upregulate PD-L1 expression upon exposure to interferon-γ, suggesting an important mechanism of tumor-mediated T cell suppression (Hartley et al. 2017).

[0009] Several attempts have been made to design candidate therapeutic antibodies against canine ICIs, but clinical experience is still in its early stages. Chimeric rat-canine anti-PD-L1 (Maekawa et al. 2017), "canine-like" anti-CTLA-4 (Mason et al. 2021), and anti-PD-1 (Igase et al. 2020) monoclonal antibodies (mAbs) have been developed.

[0010] Maekawa and colleagues demonstrated that a canine-chimeric PD-L1 monoclonal antibody (designated c4G12) improved cytokine production and proliferation of canine peripheral blood mononuclear cells (Maekawa et al. 2017). More importantly, in a pilot clinical study using c4G12, an antitumor response was observed in 1 out of 7 dogs with oral malignant melanoma and 1 out of 2 dogs with undifferentiated sarcoma (Maekawa et al. 2017). In a follow-up study using 29 dogs diagnosed with primary OMM with confirmed lung metastasis, an increase in antitumor response and overall survival after c4G12 treatment was also reported (Maekawa et al. 2021).

[0011] It is noteworthy that this canine-chimeric antibody features variable light (VL) and heavy (VH) chains of rat origin, representing an important source for the development of anti-drug antibodies (ADA). The authors did not mention investigating the presence of ADA in any of the treated dogs. However, ADA can reduce the pharmacological efficacy of a compound after repeated dosing. In addition, chimeric antibodies increase the risk of inducing allergic reactions with common side effects including fever, chills, headache, nausea, vomiting, diarrhea, rash, or fatigue after administration.

[0012] The generation of species-specific mAbs that are expected to have a better safety profile is technically difficult, and the experience in the development of antibodies for companion animals has only gradually evolved. Technical approaches for generating therapeutic antibodies for companion animals, such as dogs or cats, namely, the modification of existing compounds and the use of transgenic animals, are available in only a few cases. Methods for "dogging" or "catting" antibodies have been disclosed (Gearing et al. 2016, Gearing et al. 2013). However, even subtle changes in the protein sequence of an antibody can result in a significant loss of efficacy and altered biophysical properties, which has characterized such methods as time-consuming and prone to failure. More advanced techniques relate to transgenic rodents that express canine immunoglobulins (Wabl 5 / 23 / 2017). The disadvantages are the need to sacrifice animals for the initial antibody discovery process and the fact that the immunization process is hardly controllable. In this regard, in vitro selection methods, such as phage display, offer a great advantage as they enable an antibody selection process adapted to the purpose. Very recently, a synthetic phage display library containing full-length canine antibody fragments has been disclosed (Tiller et al. 6 / 21 / 2018).

[0013] The present invention relates to a full-length canine antibody against canine PD-L1 derived from a species-specific canine phage display library having high target specificity and favorable biophysical properties, which is therapeutically used in various canine malignancies.

[0014] The citation of any reference in this specification should not be construed as an admission that such reference is available as "prior art" for the present application. SUMMARY OF THE INVENTION

[0015] The present invention relates to an antibody or antibody fragment that binds to canine programmed death ligand 1 (canine PD-L1). The antibody or antibody fragment is characterized by its complementarity-determining region (CDR) or its light and / or heavy variable domains.

[0016] In a first aspect, the inventors identified an antibody or antibody fragment that may include the light chain CDR1 (LCDR1) region described in SEQ ID NO: 3, the light chain CDR2 (LCDR2) region described in SEQ ID NO: 4, and / or the light chain CDR3 (LCDR3) region described in SEQ ID NO: 18 or SEQ ID NO: 49. Furthermore, the antibody or antibody fragment may include the heavy chain CDR1 (HCDR1) region described in SEQ ID NO: 6 or SEQ ID NO: 44, the heavy chain CDR2 (HCDR2) region described in SEQ ID NO: 30 or SEQ ID NO: 60, and / or the heavy chain CDR3 (HCDR3) region described in SEQ ID NO: 8 or SEQ ID NO: 45.

[0017] By maturing an antibody having a light chain variable domain as described in SEQ ID NO: 1, which includes the LCDR3 described in SEQ ID NO: 5, combined with the heavy chain variable domain as described in SEQ ID NO: 7, which includes the HCDR2 described in SEQ ID NO: 7, several high-affinity antibodies were obtained, each containing an LCDR3 region having an amino acid sequence selected from any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, and 17, and an HCDR2 region having an amino acid sequence selected from any one of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29. Based on these LCDR3 regions, the inventors were able to identify a first anti-PD-L1 high-affinity LCDR3 consensus sequence described in SEQ ID NO: 18, and a first anti-PD-L1 high-affinity HCDR2 consensus sequence described in SEQ ID NO: 30.

[0018] In an alternative embodiment of the present invention, several high-affinity antibodies were obtained by maturing an antibody having a light-chain variable domain as described in SEQ ID NO: 41, which includes the LCDR3 described in SEQ ID NO: 43, combined with the heavy-chain variable domain described in SEQ ID NO: 42, which includes the HCDR2 described in SEQ ID NO: 45, thereby maturing an antibody having an LCDR3 region having an amino acid sequence selected from SEQ ID NO: 47 or 48, and an HCDR2 region having an amino acid sequence selected from SEQ ID NOs: 50, 51, 52, 53, 54, 55, 56, 57, 58, and 59, preferably one of SEQ ID NOs: 19 or 52. Based on these LCDR3 regions, the inventors were able to identify a second anti-PD-L1 high-affinity LCDR3 consensus sequence described in SEQ ID NO: 49, and a second anti-PD-L1 high-affinity HCDR2 consensus sequence described in SEQ ID NO: 60.

[0019] Therefore, the antibody or antibody fragment of the present invention may include the high-affinity LCDR3 consensus sequence described in SEQ ID NO: 18 or SEQ ID NO: 49, and / or the high-affinity HCDR2 consensus sequence described in SEQ ID NO: 30 or 60.

[0020] Preferably, the antibody or antibody fragment comprises a light chain containing various combinations of LCDR1, LCDR2, and LCDR3 as disclosed herein, and / or a heavy chain containing various combinations of HCDR1, HCDR2, and HCDR3 as disclosed herein. Most preferably, the antibody or antibody fragment comprises a light chain containing combinations of LCDR1, LCDR2, and LCDR3 as disclosed herein, along with a heavy chain containing HCDR1, HCDR2, and HCDR3 as disclosed herein.

[0021] In a preferred embodiment, the antibody or antibody fragment comprises a variable light chain containing the LCDR3 region described in SEQ ID NO: 18 combined with a variable heavy chain containing the HCDR2 region described in SEQ ID NO: 30, or a variable light chain containing the LCDR3 region described in SEQ ID NO: 49 combined with a variable heavy chain containing the HCDR2 region described in SEQ ID NO: 60.

[0022] In certain preferred embodiments, the present invention provides an anti-PD-L1 antibody with remarkably high affinity, comprising a light chain variable domain described in SEQ ID NO: 39 combined with a heavy chain variable domain described in SEQ ID NO: 40, or a light chain variable domain described in SEQ ID NO: 65 combined with a heavy chain variable domain described in SEQ ID NO: 66.

[0023] The antibody or antibody fragment is preferably a fully canine antibody or antibody fragment, optionally a recombinant canine antibody. The antibody or antibody fragment can be used in the treatment of a disease in a subject requiring treatment, preferably a canine subject. The disease may be, for example, cancer, or an inflammatory or autoimmune disease.

[0024] Notably, the fully canine antibodies of the present invention can overcome the immunogenicity burden associated with chimeric antibodies derived from non-canine species, such as rodents. Importantly, chimeric antibodies described in the literature exhibit only weak affinity, potentially severely limiting their use as therapeutic antibodies.

[0025] Furthermore, the present invention relates to a pharmaceutical composition comprising an antibody or antibody fragment as disclosed herein, a polynucleotide encoding the antibody or antibody fragment, a vector comprising the polynucleotide, or a vector comprising the polynucleotide or a plurality of polynucleotides. In a preferred embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of the antibody of the present invention, further comprising a pharmaceutically acceptable carrier.

[0026] In one or more embodiments, the antibody or antibody fragment of the present invention provides a method for treating PD-L1-related disorders. In one or more embodiments, the PD-L1-related disorder is cancer. In one or more embodiments, the type of cancer is selected from, but is not limited to, melanoma, lung cancer, bladder cancer, renal cell carcinoma, head and neck cancer, breast cancer, esophageal cancer, and lymphoma. In preferred embodiments, the PD-L1 disorder is melanoma.

[0027] In one or more embodiments, the present invention provides a method for treating a subject with PD-L1-related disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject.

[0028] In one or more embodiments, the present invention provides a host cell that produces one or more of the antigen-binding proteins of the present invention.

[0029] In one or more embodiments, the present invention provides a vector comprising one or more polynucleotides encoding the antibody or antibody fragment of the present invention.

[0030] In one or more embodiments, the present invention provides a host cell comprising one or more of the polynucleotides of the present invention.

[0031] In one or more embodiments, the present invention provides a host cell comprising a vector containing one or more of the nucleic acids of the present invention.

[0032] In one or more embodiments, the present invention provides a host cell comprising one or more of the nucleic acids of the present invention.

[0033] In one or more embodiments, the present invention provides a method for producing an antigen-binding protein by culturing host cells of the present invention under conditions that induce the production of an antigen-binding protein, and then isolating the antigen-binding protein from the host cells or the culture medium of the host cells.

[0034] In one or more embodiments, the present invention provides a kit comprising the antibody or antibody fragment of the present invention. [Brief explanation of the drawing]

[0035] [Figure 1] The consensus sequence based on the maturation of the LCDR3 (Figure 1A) and HCDR2 regions (Figure 1B) of CAN1005010. [Figure 2]Consensus sequence based on the mature LCDR3 (Figure 2A) and HCDR2 regions (Figure 2B) of CAN1005001. [Figure 3] cPD-L1 binding and neutralization of cPD-L1 / cPD-1 interactions by CAN1005001 and CAN1005010 [Figure 4-1] Concentration-dependent binding of CAN1005010L1 and CAN1005010H2 (Figure 4.A), CAN1005001L1, CAN1005001H3, and CAN1005001 (Figure 4.B) in a cPD-L1-biotin ELISA assay. [Figure 4-2] Derived IC50 values ​​in the cPD-L1-biotin ELISA assay (Figure 4.C). [Figure 5] Concentration-dependent binding of CAN1005016 (Figure 5.A) and CAN1005019 (Figure 5.B) in a cPD-L1-biotin ELISA assay. [Figure 6] Affinity measurement of CAN1005016 (Figure 6.A) and CAN1005019 (Figure 6.B) using GCI with 2BIND. [Figure 7] Candidate cPD-L1 binding was derived from cross-cloning of mature IgG. Serial titrations of biotinylated CAN1005016 (Figure 7.A) and CAN1005019 (Figure 7.B) were added to plate-bound cPD-L1_hFc. [Figure 8-1] FACS binding of CAN1005016 (Figure 8A) and CAN1005019 (Figure 8B) to HEK293c18 cells transfected with target proteins. [Figure 8-2] FACS binding of control IgG to HEK293c18 cells transfected with target protein. [Figure 9] ELISA-based ligand binding inhibition assays for CAN1005016 (Figure 9A) and CAN1005019 (Figure 9B). [Figure 10] Biolayer interference (BLI)-based PD-1 binding inhibition assay for CAN1005016 antibody candidate. [Figure 11A] CAN1005016 [Figure 11B] CAN1005019 [Figure 11C] Canine IgG control [Modes for carrying out the invention]

[0036] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0037] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise clearly indicated by the context.

[0038] In the context of a quantity, "approximately" refers to an average deviation of up to ±20%, preferably ±10%, and most preferably ±5%, based on the given value. For example, a quantity of approximately 20 mg / ml means 20 mg / ml ± 6 mg / ml, preferably 20 mg / ml ± 4 mg / ml, and most preferably 20 mg / ml ± 2 mg / ml. This also includes the value itself, which has no deviation whatsoever.

[0039] All ranges described herein in the summary and description of the present invention include all numbers or values ​​of approximately the number of a range, or all numbers or values ​​between the numbers of a range. The ranges of the present invention expressly represent and describe all integer, decimal, and fractional values ​​within the range. The term "approximately" may be used to describe a range.

[0040] As used herein, the terms “antibody” or “polypeptide binder” include the entire antibody, and any antigen-binding fragment (i.e., “antigen-binding portion”), or their individual chains. Naturally occurring “antibodies” are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region of IgG, IgA, or IgD antibodies consists of three domains: CH1, CH2, and CH3, while the heavy chain of IgM and IgE antibodies consists of four domains: CH1, CH2, CH3, and CH4. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FWs). Each VH and VL consists of three CDRs and four FWs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FW1, CDR1, FW2, FW2, CDR2, FW3, CDR3, FW4. Polypeptides containing the variable regions FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4 of the heavy and light chains may be referred to as "VH or VL polypeptides." The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), via the Fc receptor and the first component (C1q) of the classical complement system.

[0041] The framework region and CDR range of human antibodies are precisely defined (see Kabat, 1991, J.Immunol., 147, 915-920; Chothia & Lesk, 1987, J.Mol.Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883; Al-Lazikani et al., 1997, J.Mol.Biol. 273:927-948). The antibody framework region, i.e., the combined framework region of the constituent light and heavy chains, helps to position and align the CDR, which is primarily involved in binding to the antigen. However, while canine antibodies can be partially aligned with human antibodies, the above numbering scheme is not ideally suited to describing amino acid positions within the antibody heavy or light chain sequence. In this invention, the following numbering scheme is used.

[0042] The antibody heavy chain is defined as VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4. Framework 1 (FW1) consists of 30 amino acids (X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21, X22, X23, X24, X25, X26, X27, X28, X29, X30). HCDR1 is a length of 5 amino acids and is defined by positions X31-X35 (X31, X32, X33, X34, X35). Framework 2 (FW2) is defined by the length of 14 amino acids at positions X36-X49 (X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49). HCDR2 is defined by positions X50-X65 (X50, X51, X52, X52a, X53, X54, X55, X56, X57, X58, X59, X60, X61, X62, X63, X64, X65). Framework 3 (FW3) is defined by positions X66-X94 and is the length of 32 amino acids. HCDR3 is defined by positions X95–X102 (X98, X99, X100, X100a, X100b, X100c, X100d, X100e, X100f, X100g, X101, X102). This CDR is diverse in length, indicated by positions X100a–X100g, which may or may not contain amino acids. For clarification, if one of these positions is empty, the subsequent positions up to X101 are also empty. Framework 4 (FW4) is defined by positions X103–X113 (X103, X104, X105, X106, X107, X108, X109, X111, X112, X113). The general concept of the numbering scheme is also shown in Figure 9.

[0043] The antibody light chain is defined as VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4. Framework 1 (FW1) is defined at positions Y1-Y23, with one length variation at position Y10. For clarification, position Y10 may be empty or contain an amino acid (Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23). LCDR1 is a length of 11 amino acids and is defined at positions 24-34 (Y24, Y25, Y26, Y27, Y28, Y29, Y30, Y31, Y32, Y33, Y34). Framework 2 (FW2) is defined by positions 35-49 (Y35, Y36, Y37, Y38, Y39, Y40, Y41, Y42, Y43, Y44, Y45, Y46, Y47, Y48, Y49). LCDR2 is the length of seven amino acids and is defined by positions 50-56 (Y50, Y51, Y52, Y53, Y44, Y55, Y56). Framework 3 (FW3) is defined at positions Y57 to Y88 (Y57, Y58, Y59, Y60, Y61, Y62, Y63, Y64, Y65, Y66, Y67, Y68, Y69, Y70, Y71, Y72, Y73, Y74, Y75, Y76, Y77, Y78, Y79, Y80, Y81, Y82, Y83, Y84, Y85, Y86, Y87, Y88). LCDR3 is defined at positions Y89 to Y97 (Y89, Y90, Y91, Y92, Y93, Y94, Y95, Y95a, Y95b, Y95c, Y96, Y97). This CDR has length variation, which is indicated by positions Y95a to Y95c, which may or may not contain amino acids. For clarification, if one of these positions is empty, then the subsequent positions up to Y91 are also empty. Framework 4 (FW4) is defined by positions Y98 to Y107 (Y98, Y99, Y100, Y101, Y102, Y103, Y104, Y105, Y106, Y107).

[0044] The terms “antigen-binding moiety” or “fragment” of an antibody are used interchangeably in this application. These terms refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term “antigen-binding moiety” of an antibody include the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; the F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide crosslinks in a hinge region; the Fd fragment, consisting of VH and CH1 domains; the Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; the single-domain antibody (dAb) fragment, consisting of the VH domain (Ward et al., 1989 Nature 341:544-546); and isolated complementarity-determining regions (CDRs). The preferred antigen-binding moiety or fragment of an antibody is the Fab fragment. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic peptide linker using recombination, which allows them to be produced as a single protein chain, in which case the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242:423-426 and Huston et al., 1988 Proc.Natl.Acad.Sci.85:5879-5883). Such single-chain antibodies contain one or more "antigen-binding moieties" of the antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. The antigen-binding moiety can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136).One type of single-domain antibody is a heavy-chain variable domain, referred herein as "VHH" (heavy-chain variable domain of heavy-chain antibody), derived from HCAb (heavy-chain antibody) found in camelids (e.g., Camelus dromedarius, Camelus bactrianus, Vicugna pacos, or Lama glama). V-NAR is a heavy-chain-only binding factor derived from having heavy-chain antibodies (IgNAR, "immunoglobulin novel antigen receptor") from cartilaginous fish. The antigen-binding portion of the antibody can be implanted into scaffold bases and polypeptides, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199 describing fibronectin polypeptide monobodies). The antigen-binding moiety can be incorporated into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding domains together with a complementary light chain polypeptide (Zapata et al., 1995 Protein Eng. 8(10)1 057-1062, and U.S. Patent No. 5,641,870).

[0045] The term "isolated" refers to a compound that may be substantially free of other antibodies or antibody fragments having different antigen specificities, for example, an antibody or antibody fragment. Furthermore, an isolated antibody or antibody fragment may substantially free of other cellular material and / or chemicals. Thus, in some embodiments, the antibody provided is an isolated antibody separated from an antibody having different specificities. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant may cross-reactive to other related antigens, such as antigens from other species (e.g., species homologs).

[0046] As used herein, the term “complete canine antibody” refers to an antibody having a variable region in which both the framework and CDR region of the variable region are derived from a canine sequence. For example, both the framework and CDR region may be derived from a canine sequence. Furthermore, if the antibody contains a constant region, the constant region may also be derived from such a canine sequence, e.g., a canine germline sequence, or a variant version of a canine germline sequence. The canine antibodies of the present invention may contain amino acid residues not encoded by a canine sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo).

[0047] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to a preparation of an antibody molecule with a single-molecule composition. Monoclonal antibody compositions exhibit single-binding specificity and affinity to a particular epitope.

[0048] The term "germline" refers to a complete germline sequence, and in addition, to a modified or manipulated germline sequence having minor mutations in the amino acid sequence for purposes such as removing undesirable post-translational modification (PTM) sites, removing undesirable cysteine, optimizing an antibody (e.g., affinity, half-life), introducing a desired restriction site, or modifications resulting from errors in synthesis, amplification, or cloning.

[0049] As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding domain of the polypeptide interacts with its target at multiple sites through weak non-covalent forces, and the higher the interaction, the stronger the affinity.

[0050] When used herein, "K DThe term "KD" refers to the dissociation constant, which is derived from the Kd to Ka ratio (i.e., Kd / Ka) and expressed as a molar concentration (M). For example, the KD value of an antigen-binding moiety, such as a monoclonal antibody, can be determined using methods well established in the art. D Methods for determining this include SET (soluble equilibrium titration) or surface plasmon resonance using a biosensor system such as the Biacore® system.

[0051] The light chain variable domain described herein, "combined" with the heavy chain variable domain, relates to paired light and heavy chains that are paired with each other. The pairing may be between different domains of one polypeptide chain containing a VL polypeptide sequence and a VH polypeptide sequence, for example, in scFv, or between two polypeptide chains containing a VL polypeptide sequence and a VH polypeptide sequence, for example, in a full-length antibody or Fab fragment.

[0052] The term "expression vector" refers to a vector containing recombinant polynucleotides that include an expression control sequence operably ligated to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, and these include cosmids, plasmids (e.g., naked plasmids or plasmids contained within liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0053] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an alpha carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids. Amino acids are generally identified herein according to known one- or three-letter amino acid codes.

[0054] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly includes its conservatively modified variants.

[0055] In the context of two or more nucleic acid or polypeptide sequences, the terms “identical” or “percent “identical” refer to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if, when compared and aligned for the greatest match across a comparison frame or designated region, they have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity across a designated region, or, when not specified, across the entire sequence). Optionally, identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. Accordingly, the “substantially identical sequences” according to the present invention may include any of the disclosed sequences having one, two, three, four, or five amino acid exchanges, preferably one to three, more preferably one or two amino acid exchanges. Preferably, the exchanges may be conservative amino acid exchanges.

[0056] For sequence comparison, typically one sequence serves as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, sub-sequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence with respect to the reference sequence based on the programmed parameters. Alignment for the purpose of determining the sequence identity percentage within this invention can be performed in various ways well known to those skilled in the art using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINE™ (DNASTAR) software. Those skilled in the art can routinely determine appropriate parameters for measuring alignment, including any algorithm required to achieve the maximum alignment over the entire length of the sequences being compared. Unless otherwise defined, alignment is performed using the default settings of the alignment tool.

[0057] The term “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such offspring may not actually be identical to the parent cell, but as used herein, they still fall within the scope of the term “host cell.”

[0058] The term “vector” refers to a polynucleotide molecule capable of transporting another polynucleotide to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector to which an additional DNA segment can be ligated into a viral genome. Certain vectors can self-replicate in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial replication origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can direct the expression of a gene to which they are functionally ligated. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA techniques are often in plasmid form. Hereinafter, “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vectors. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0059] "Cross-competition" refers to the ability of an antibody, antibody fragment, or other antigen-binding moiety to interfere with the binding of another antibody, antibody fragment, or antigen-binding moiety to a particular antigen in a standard competitive binding assay. The ability or extent to which an antibody, antibody fragment, or other antigen-binding moiety can interfere with the binding of another antibody, antibody fragment, or antigen-binding moiety to a particular antigen, and therefore whether it can be called cross-competition according to the present invention, can be determined using a standard competitive binding assay. One preferred assay involves the use of Biacore technology (e.g., by using the BlAcore3000 instrument (Biacore, Uppsala, Sweden)), which can measure the degree of interaction using surface plasmon resonance technology.

[0060] (Detailed explanation) In general embodiments, the present invention relates to an antibody or antibody fragment comprising the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and / or the LCDR3 region described in SEQ ID NO: 18 or SEQ ID NO: 49. The antibody or antibody fragment may further comprise the HCDR1 region described in SEQ ID NO: 6 or SEQ ID NO: 44, the HCDR2 region described in SEQ ID NO: 30 or SEQ ID NO: 60, and / or the HCDR3 region described in SEQ ID NO: 8 or SEQ ID NO: 46.

[0061] The antibodies and antibody fragments described herein specifically bind to canine programmed cell death ligand 1 (PD-L1), which may be characterized, for example, by the amino acid sequence described in NCBI reference sequence: NP_001278901.1 or the sequence described in Sequence ID No. 67.

[0062] By maturing an antibody having a light chain CAN1005010_VL variable domain, described in SEQ ID NO: 1, which includes an LCDR3 (CAN1005010_VL_LCDR3) described in SEQ ID NO: 5 combined with a heavy chain variable domain (CAN1005010_VH) described in SEQ ID NO: 7, which includes an HCDR2 (CAN1005010_VH_HCDR2) described in SEQ ID NO: 7, several high-affinity antibodies were obtained, each containing an LCDR3 region having an amino acid sequence selected from any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, and 17, and an HCDR2 region having an amino acid sequence selected from any one of SEQ ID NOs: 19, 20, 21, 22, 23, 34, 25, 26, 27, 28, and 29. Based on these LCDR3 regions, the inventors were able to identify the first anti-PD-L1 high affinity LCDR3 consensus sequence described in SEQ ID NO: 18 and the first anti-PD-L1 high affinity HCDR2 consensus sequence described in SEQ ID NO: 30. The alignments resulting from the LCDR3 consensus sequence described in SEQ ID NO: 18 and the HCDR2 consensus sequence described in SEQ ID NO: 30 are shown in Figure 1.

[0063] In an alternative embodiment of the present invention, several high-affinity antibodies were obtained by maturing an antibody having a light chain variable domain (CAN1005001_VL) described in SEQ ID NO: 41, which includes an LCDR3 (CAN1005001_VL_LCDR3) described in SEQ ID NO: 43, combined with a heavy chain variable domain (CAN1005001_VH) described in SEQ ID NO: 42, which includes an HCDR2 (CAN1005001_VH_HCDR2) described in SEQ ID NO: 45, thereby obtaining an LCDR3 region having an amino acid sequence selected from either SEQ ID NOs. 47 or 48, and an HCDR2 region having an amino acid sequence selected from either SEQ ID NOs. 50, 51, 52, 53, 54, 55, 56, 57, 9, and 59, preferably SEQ ID NO: 19 or 52. Based on these LCDR3 and HCDR3 regions, the inventors were able to identify a second anti-PD-L1 high affinity LCDR3 consensus sequence described in SEQ ID NO: 49 and a second anti-PD-L1 high affinity HCDR2 consensus sequence described in SEQ ID NO: 60. The alignments resulting from the LCDR3 consensus sequence described in SEQ ID NO: 49 and the HCDR2 consensus sequence described in SEQ ID NO: 60 are shown in Figure 2.

[0064] According to one embodiment, the antibody or antibody fragment of the present invention may include an LCDR3 region having an amino acid sequence selected from any one of SEQ ID NOs: 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 43, 47, and 48. Preferably, the antibody includes the LCDR3 described in SEQ ID NO: 9 or SEQ ID NO: 47.

[0065] Furthermore, the antibody or antibody fragment of the present invention may include an HCDR2 region having an amino acid sequence selected from any one of SEQ ID NOs: 7, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 50, 51, 52, 53, 54, 55, 56, 57, 58, and 59. Preferably, the antibody contains the HCDR2 described in SEQ ID NO: 19 or 52.

[0066] In one embodiment, the variable light chain may include the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and the LCDR3 region, preferably SEQ ID NO: 5, selected from any one of SEQ ID NOs: 5, 9, 10, 11, 12, 13, 14, 15, 16, and 17. This light chain may be combined with, for example, another independent embodiment of the variable heavy chain HCDR1 region described in SEQ ID NO: 6, the HCDR2 region having the amino acid sequence of SEQ ID NO: 19, selected from any one of SEQ ID NOs: 7, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29, preferably SEQ ID NO: 19, and the HCDR3 region described in SEQ ID NO: 8.

[0067] Therefore, a very preferred embodiment of the present invention relates to an antibody or antibody fragment comprising a variable light chain including the LCDR1 region of SEQ ID NO: 3, the LCDR2 region of SEQ ID NO: 4, and the LCDR3 region of SEQ ID NO: 5, combined with the variable heavy chain HCDR1 region of SEQ ID NO: 6, the HCDR2 region of SEQ ID NO: 19, and the HCDR3 region of SEQ ID NO: 8.

[0068] In one embodiment, the light chain may include the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and the LCDR3 region described in SEQ ID NOs: 43, 47, 48, preferably SEQ ID NO: 47. This light chain may be combined with, for example, an embodiment of the variable heavy chain HCDR1 region described in SEQ ID NO: 44, an HCDR2 region having the amino acid sequence of SEQ ID NO: 52, selected from any one of SEQ ID NOs: 45, 50, 51, 52, 53, 54, 55, 56, 57, 9, 59, preferably SEQ ID NO: 52, and the HCDR3 region described in SEQ ID NO: 46.

[0069] Therefore, a very preferred embodiment of the present invention relates to an antibody or antibody fragment comprising a light chain including the LCDR1 region of SEQ ID NO: 3, the LCDR2 region of SEQ ID NO: 4, and the LCDR3 region of SEQ ID NO: 47, combined with the variable heavy chain HCDR1 region of SEQ ID NO: 44, the HCDR2 region of SEQ ID NO: 52, and the HCDR3 region of SEQ ID NO: 46.

[0070] In a preferred embodiment, the antibody or antibody fragment comprises a variable light chain containing the LCDR3 region described in SEQ ID NO: 18 combined with a variable heavy chain containing the HCDR2 region described in SEQ ID NO: 30, or a variable light chain containing the LCDR3 region described in SEQ ID NO: 49 combined with a variable heavy chain containing the HCDR2 region described in SEQ ID NO: 60.

[0071] More preferably, the antibody or antibody fragment includes, for example, a variable light chain containing the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and the LCDR3 region described in SEQ ID NO: 9, as contained in the variable chains of CAN1005016_VL (SEQ ID NO: 39) and CAN1005016_VH (SEQ ID NO: 40), and / or a variable heavy chain containing the HCDR1 region described in SEQ ID NO: 6, the HCDR2 region described in SEQ ID NO: 19, and the HCDR3 region described in SEQ ID NO: 8.

[0072] In alternative preferred embodiments, the antibody or antibody fragment includes, for example, a variable light chain comprising the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and the LCDR3 region described in SEQ ID NO: 47, as contained in the variable chains of CAN1005019_VL (SEQ ID NO: 65) and CAN1005019_VH (SEQ ID NO: 66), and / or a variable heavy chain comprising the HCDR1 region described in SEQ ID NO: 44, the HCDR2 region described in SEQ ID NO: 52, and the HCDR3 region described in SEQ ID NO: 46. [Table 1-1] [Table 1-2] [Table 1-3]

[0073] In consensus sequences 18, 30, 49, and 60, the sequences may contain any of the amino acids at the specific positions in parentheses, separated by " / " as "alternatives". For example, the position (G / S / W) in sequence number 60 could be glycine, serine, or tryptophan.

[0074] In addition to the CDR1, CDR2, and CDR3 regions, the variable light chain domain and heavy chain domain include framework regions 1 to 4 from the N-terminus to the C-terminus. The antibody or antibody fragment of the present invention may include one, several, or all of the light chain variable domain framework sequences having an amino acid sequence selected from any one of SEQ ID NOs: 31, 32, 33, or 34, and / or one, several, or all of the heavy chain variable domain framework sequences having an amino acid sequence selected from any one of SEQ ID NOs: 35, 36, 37, or one, several, or all of the heavy chain variable domain framework sequences having an amino acid sequence selected from any one of SEQ ID NOs: 61, 62, 63, or 64. A heavy chain variable domain comprising HCDR1 and HCDR3 as described in SEQ ID NOs. 6 and 8, and an HCDR2 sequence selected from any one of SEQ ID NOs. 5, 9, 10, 11, 12, 13, 14, 15, 16, and 17, preferably comprises a framework region selected from any one of SEQ ID NOs. 35, 36, 37, and 38, and a heavy chain variable domain comprising HCDR1 and HCDR3 as described in SEQ ID NOs. 44 and 46, and an HCDR2 sequence selected from any one of SEQ ID NOs. 51, 51, 52, 53, 54, 55, 56, 57, 58, and 59, preferably comprising a framework region selected from any one of SEQ ID NOs. 61, 62, 63, and 64.

[0075] In preferred embodiments, any antibody or antibody fragment of the present invention comprises a light chain variable domain described in SEQ ID NO: 1 or 41, more preferably SEQ ID NO: 39 or 65, and / or a heavy chain variable domain described in SEQ ID NO: 2 or 42, more preferably SEQ ID NO: 40 or 66, more preferably a light chain variable domain described in SEQ ID NO: 39 combined with a heavy chain variable domain described in SEQ ID NO: 40, or a light chain variable domain described in SEQ ID NO: 65 combined with a heavy chain variable domain described in SEQ ID NO: 66.

[0076] The present invention further provides antibodies or antibody fragments described herein, comprising at least one LCDR, HCDR, framework region, or variable domain chain having a sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 1 to SEQ ID NOs. 66. Such antibodies or antibody fragments may be provided by introducing at least one, for example, one, two, three, or four mutations in the form of amino acid substitutions or deletions into the amino acid sequences of SEQ ID NOs. Preferably, the mutations are conservative amino acid substitutions.

[0077] In a further embodiment, the present invention relates to an antibody or antibody fragment that cross-competes with the above-mentioned antibody fragment, preferably comprising the light chain variable domain described in SEQ ID NO: 39 and / or the heavy chain variable domain described in SEQ ID NO: 40, or the light chain variable domain described in SEQ ID NO: 65 and / or the heavy chain variable domain described in SEQ ID NO: 66.

[0078] An antibody or antibody fragment is an antibody or antibody fragment of any of the preceding claims that specifically binds to canine PD-L1 with a dissociation constant (Kd) of less than about 20 nM, 18 nM, 16 nM, 14 nM, 12 nM, preferably less than about 10 nM, 8 nM, 6 nM, more preferably less than about 5 nM, 4 nM, 3 nM, and most preferably less than about 2 nM. The dissociation constant for the binding of an IgG antibody to a monovalent PD-L1 antigen is determined. The canine PD-L1 to which the disclosed antibodies and antibody fragments bind is characterized, for example, by the amino acid sequence of NCBI reference sequence: NP_001278901.1: FTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQHSSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQRISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINATANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTHFMILGPFLLLLGVVLAVTFCLKKHGRMMDVEKCCTRDRNSKKRNDIQFEET (SEQ ID NO: 67).

[0079] This antibody has at least about 2×10 4 [M -1 s -1 , preferably at least about 5×10 4 [M -1 s -1 , more preferably at least about 1×10 5 [M -1 s -1 of K on rate, and / or a K -3 [s -1 lower than about 1×10 -4 [s -1 , preferably lower than about 5×10 -4 [s -1 , or lower than about 2.5×10 off [s 5 [M -1 s-1 ] of K on , and approximately 2.5 × 10 -4 [s -1 K lower than ] off The dissociation constant for the binding of the IgG antibody to the monovalent PD-L1 antigen is determined. In another embodiment, the antibody has at least about 7.5 × 10 4 [M -1 s -1 ] of K on , and approximately 2.5 × 10 -4 [s -1 K lower than ] off It has the following properties. The dissociation constant for the binding of IgG antibodies to the monovalent PD-L1 antigen is determined.

[0080] The antibodies or antibody fragments of the present invention can interfere with or block the interaction between canine PD-L1 (cPD-L1) and canine PD-1 (cPD-1), as confirmed by Example 8. For example, the antibodies or antibody fragments of the present invention have an IC50 lower than about 100 nM, preferably lower than about 50 nM, or lower than about 20 nM, more preferably lower than about 10 nM, or lower than about 5 nM, most preferably lower than about 3 nM, or even lower than about 2 nM. 50 The IC of the interaction between cPD-L1 and cPD-1 can be blocked by this value. 50 The value can be determined, for example, by a ligand binding inhibition assay based on biolayer interference (BLI) or an ELISA ligand binding inhibition assay (ELISA IBIA).

[0081] In a further embodiment, the antibody or antibody fragment of the present invention is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, Fab'-SH, or VHH.

[0082] Any antibody or antibody fragment of the present invention may contain an Fc fragment or fragment selected from canine immunoglobulin G isotype A (also known as HC-A, HCA, or caIgG-A), immunoglobulin G isotype B (also known as HC-B, HCB, or caIgG-B), immunoglobulin G isotype C (also known as HC-C, HCC, or caIgG-C), or immunoglobulin G isotype D (also known as HC-D, HCD, or calgG-D). Preferably, the Fc domain is selected from isotype B. The Fc domain contained in the antibody or antibody fragment may have a wild-type sequence or a mutant sequence.

[0083] The selection of antibody isotypes is crucial depending on the therapeutic application, and it is necessary to consider whether the involvement of fluid or cellular components of the immune system is advantageous or whether it would lead to undesirable side effects of the drug. For example, therapeutic antibodies against tumor cell growth or pathogens should have potent effector function. In contrast, targeting soluble mediators or cell surface receptors in healthy cells to prevent receptor-ligand interactions typically requires the absence of any CDC or ADCC activity to prevent targeted cell death or undesirable cytokine secretion. Disease areas where silent antibody formats are needed include, but are not limited to, inflammatory diseases (e.g., rheumatoid arthritis, psoriasis, inflammatory bowel disease), allergies (e.g., asthma), pain (e.g., osteoarthritis pain, cancer pain, back pain), and eye diseases (e.g., age-related macular degeneration). Depending on the target, the absence of CDC or ADCC may also be desirable in antibodies for cancer treatment, such as the PD-L1-targeting antibodies of the present invention.

[0084] The term “Fc fragment” refers to a fragment of immunoglobulin containing at least part or all of the constant heavy chain region 2 (C2 or CH2) and constant heavy chain region 3 (C3 or CH3), or a crystallizable fragment of immunoglobulin obtained by papain digestion. A fragment is understood to be part of a larger polypeptide sequence. Therefore, a “fragment” typically has an amino acid sequence attached to the C-terminus and / or N-terminus. The terms “C2” or “CH2,” and “C3” or “CH3,” may be used interchangeably. Furthermore, the terms “Fc region” and “Fc domain,” unless otherwise specified, may be used interchangeably when referring to immunoglobulin Fc CH2 and CH3 sequences. In the context of the present invention, the boundaries between the CH2 and CH3 regions of canine immunoglobulin isotypes HC-A, HC-B, HC-C, and HC-D are defined according to Tang et al. (Tang L, Sampson C, Dreitz MJ, McCall C (2001) Cloning and characterization of cDNAs encoding four different canine immunoglobulin gamma chains. Vet Immunol Immunopathol. 80(3-4):259-70), which is incorporated herein by reference.

[0085] Wild-type Fc fragments of different isotypes may have the sequences disclosed in Table 2.

[0086] Table 2: Different isotypes of wild-type canine Fc sequences JPEG2026511256000004.jpg203164

[0087] The Fc fragment contained in the antibody or antibody fragment may further include substitutions in any of the wild-type sequences disclosed in Table 2. The possible substitutions are disclosed in WO2021 / 165417A1, which is incorporated herein by reference. Specifically, the Fc fragment includes, compared to the wild-type Fc fragment, at least one substitution of an amino acid selected from at least one of the amino acids at positions 235, 239, 270, and / or 331. Preferably, the mutation is in the Fc fragment from isotype B of canine IgG.

[0088] Preferably, the Fc fragment contains at least two substitutions of amino acids selected from at least two of the amino acids at positions 234, 235, 239, 270, and / or 331. More preferably, these two amino acids are 235 and 239; 235 and 270; 235 and 331; 239 and 270; 239 and 331; 270 and 331; 234 and 235; 234 and 239; 234 and 270; or 234 and 331.

[0089] In another preferred embodiment, the Fc fragment comprises at least three substitutions of amino acids selected from at least three of the amino acids at positions 234, 235, 239, 270, and / or 331. More preferably, these three amino acid positions are 235, 239, and 270; 239, 270, and 331; 235, 270, and 331; or 235, 239, and 331 in the wild-type Fc sequence disclosed in Table 2.

[0090] In another preferred embodiment, the present invention relates to a polypeptide comprising at least a canine or feline Fc fragment, wherein the Fc fragment comprises at least four substitutions of amino acids selected from amino acids L235, S239, D270, and P331, more preferably 235, 239, 270, and 331. Most preferably, the Fc fragment comprises mutant L235A, S239A, D270A, and P331G compared to the wild-type Fc fragment.

[0091] Each substitution in the Fc fragment resulted in a reduction in binding affinity to C1q and / or the Fc receptor compared to the polypeptide containing the corresponding wild-type Fc fragment. Under physiological conditions of an undamaged immune system, the reduction or decrease in binding to C1q and / or FcγRI leads to a reduction or complete elimination of the immune effector function of complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) induction. The reduction or decrease in binding of polypeptides containing at least one substitution in the Fc fragment to C1q and / or FcγRI, and / or the resulting reduction or complete elimination of CDC or ADCC, is commonly referred to as silencing. In the canine isotype B Fc fragment, the Fc fragment maintains its ability to bind to the neonatal Fc receptor (FcRn) and protein A.

[0092] The antibody or antibody fragment may preferably include the above-mentioned Fc fragment in which CDC or ADCC is reduced or completely eliminated for use in inflammatory diseases, allergies, pain, and eye diseases.

[0093] In a preferred embodiment, the antibody comprises a light chain containing a light chain variable domain (VL) as disclosed herein, which is linked to the Fc fragment (containing CH-2 and CH-3 domains) described above by a CH domain, and a heavy chain containing a lambda constant domain (CL) and a heavy chain variable domain (VH). The CH-1 domain may be linked to the CH-2 domain via an amino acid sequence referred to as a "hinge" or alternatively a "hinge region". Preferred canine lambda constant domains (CL) and canine CH-1 domains are known in the art. The two heavy chains are preferably linked to each other by disulfide bonds, and each heavy chain is preferably also linked to one of the light chains by disulfide bonds.

[0094] In certain embodiments of the present invention, an antibody comprising a light chain and a heavy chain may include the light chain described in SEQ ID NO: 72 or 74 and (or the heavy chain described in SEQ ID NO: 73 or 75, preferably the light chain described in SEQ ID NO: 72 combined with the heavy chain described in SEQ ID NO: 73, or the light chain described in SEQ ID NO: 74 combined with the heavy chain described in SEQ ID NO: 75).

[0095] Table 3: Full-length antibody sequences: JPEG2026511256000005.jpg177165

[0096] In preferred embodiments of the present invention, the antibody or antibody fragment is a complete canine antibody or antibody fragment. In preferred embodiments of the present invention, the antibody or antibody fragment is an isolated antibody or antibody fragment. Preferably, the antibody or antibody fragment of the present invention is a monoclonal antibody or antibody fragment.

[0097] In a further embodiment, the antibody or antibody fragment is a recombinant antibody or antibody fragment. A “recombinant” antibody is an antibody produced in cells of a different species than the species from which the antibody's genome originates. Suitable cells for recombinant expression of the antibodies of the present invention include, in particular, mammalian cells such as primate or non-primate animal cells, yeast cells, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 cells, and CHO cells, as well as cell lines derived therefrom, such as 293-6E, DG44, CHO-S, and CHO-K cells, and hybridoma cells. Also in the context of the present invention, synthetic or semi-synthetic antibodies are considered recombinant antibodies.

[0098] In further embodiments, the present invention relates to a pharmaceutical composition comprising, optionally, a pharmaceutically acceptable carrier or excipient, an antibody or antibody fragment as described herein. The "pharmaceutical composition" is a composition comprising the antibody or antibody fragment of the present invention and additional compounds that are toxicologically acceptable and enable the storage and administration of the antibody or antibody fragment of the present invention to a subject to be treated, and that enable the antibody or antibody fragment to exert its intended pharmacological and biological activity.

[0099] Pharmaceutically acceptable carriers or excipients include drugs that are nontoxic to cells or mammals exposed to the carrier or excipient at the dosage and concentration used, such as diluents, stabilizers, adjuvants, or other types of excipients. Examples of pharmaceutically acceptable carriers include alumina; aluminum stearate; lecithin; serum proteins such as human serum albumin, canine or other animal albumin; buffers such as phosphates, citrates, tromethamine, or HEPES buffer; glycine; sorbic acid; 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, or magnesium trisilicate; polyvinylpyrrolidone, cellulosic substances; polyethylene glycol; sucrose; mannitol; or amino acids, including arginine, but not limited to these.

[0100] The present invention further relates to antibodies or antibody fragments or pharmaceutical compositions described herein for therapeutic use, or to methods for treating subjects in need of treatment, preferably dogs. Preferably, the treatment or method of treatment is for treating a disease. A method for treating a disease according to the present invention comprises the step of administering an antibody or antibody fragment or pharmaceutical composition described herein to a patient in need of treatment, preferably a dog. Preferably, the disease is cancer, or an inflammatory disease or autoimmune disease. Preferably, cancer is colorectal cancer, melanoma, oral malignant melanoma, osteosarcoma, mast cell tumor, angiosarcoma, hepatocellular sarcoma, squamous cell carcinoma, nasal adenocarcinoma, transitional cell carcinoma, and anal sac adenocarcinoma, soft tissue cancer, mammary gland cancer, histiocytic sarcoma, diffuse large B cell carcinoma, gastric cancer, e.g., gastric adenocarcinoma, lymphoma, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, and head and neck cancer.

[0101] In a further embodiment, the antibodies or antibody fragments described herein may be used in diagnostic assays, diagnostic methods, etc. The method or assay may include a step of detecting canine PD-L1.

[0102] In a further embodiment, the present invention relates to a polynucleotide or a plurality of polynucleotides encoding the antibody or antibody fragment of the present invention. These polynucleotides or a plurality of polynucleotides may be isolated polynucleotides. These polynucleotides or a plurality of polynucleotides may be contained in a vector such as a plasmid or artificial chromosome. These polynucleotides may be operably ligated to transcriptional and translational control sequences. In this context, the term “operably ligated” means that the transcriptional and translational control sequences function to functionally transcribe and translate the antibody or antibody fragment to express the encoded antibody or antibody fragment.

[0103] The vector and / or polynucleotide or multiple polynucleotides may be contained within a cell. The cell is preferably a host cell suitable for recombinant expression of an antibody or antibody fragment. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells, yeast cells, plant cells, and insect cells. Non-limiting and exemplary mammalian cells include, but are not limited to, NSO cells, 293 cells, and CHO cells, as well as cell lines derived therefrom, such as 293-6E, DG44, CHO-S, and CHO-K cells. [Examples]

[0104] Generation of canine antibodies specific to canine PD-L1 A collection of canine antibodies that specifically bind to canine PD-L1 was prepared. The sequence of each antibody was isolated from the synthetic complete canine antibody library disclosed in WO2018 / 234438A1 using phage display. The obtained antibodies inhibit the binding of canine PD-1 to canine PD-L1. Affinity maturation was performed on individual inhibitory clones to increase binding affinity while maintaining epitope specificity.

[0105] Example 1: Generation and screening of anti-canine PD-L1 antibodies Phage display selection may be performed as described below, or by other methods known to those skilled in the art. Different panning strategies (e.g., solid-phase panning, solution panning, semi-solution panning, Fc capture panning) were applied to increase the possibility of discovering diverse binding antibodies. Selection for cPD-L1 as an Fc fusion protein (cPD-L1_hFc, Sino Biological, catalog: 70110-D02H) was performed using solid-phase panning, as well as solution and semi-solution panning, in three rounds, as described below. Due to the nature of the antigen as an Fc fusion protein, phages were blocked against human IgG to eliminate potential Fc-reactive candidates. Common to all panning strategies used was the reduction of antigen load in each round while increasing stringency of washing. Phage output after three rounds of panning was screened in ELISA format after subcloning into bacterial Fab expression vectors.

[0106] Solid-phase panning For solid-phase panning, cPD-L1_hFc was immobilized on the surface of a microtiter plate (Maxisorp, 96-well flat-bottom) at room temperature for at least 1 hour. For blocking, 5 μg / ml human serum IgG (huIgG, Jackson ImmunoResearch, catalog: 009-000-003) was immobilized on the plate in parallel, and together with cPD-L1, a concentration of 3 μg / ml was used in the first round of panning. For each selection, 250 μl / well of antigen diluted in PBS (phosphate-buffered saline, pH 7.4) was used. After incubation, the wells were washed with PBST (PBS supplemented with 0.05% Tween-20), and then 300 μl / well of blocking buffer (Chemiblocker, Merck-Millipore) was added.

[0107] In parallel, library phages were blocked in blocking buffer at room temperature for 1 hour. Due to the presence of hFc tagging, 10 μg / ml of human serum IgG was added to the blocking buffer.

[0108] For each panning subcode, 20 ml of 2×YT medium was incubated with E. coli ER2738 from M9 minimal agar plates in a phage-free workspace, and the culture was then used for infection with the selected phage. 0.6 OD 600 The culture was shaken at 160 rpm and 37°C until it reached nm. The E. coli culture was kept on ice until infection by eluted phages was required.

[0109] After antigen coating and blocking, the blocked phages were transferred to their respective wells and incubated at room temperature for 1 hour. Several washing steps were performed with PBST and PBS to remove nonspecific or weakly bound phages. Standard washing steps were applied during the first panning (5 rapid washes with PBST, 3 washes with PBST for 5 minutes, and 3 rapid washes with PBS). Stringency was adapted in subsequent rounds by increasing the number and duration of washing steps according to the phage output titer.

[0110] Following antigen-phage incubation and washing, the bound phages were eluted by adding trypsin (250 μl of 10 μg / ml trypsin solution in PBS, 30 minutes at 37°C) to cleave the proteinase-sensitive linker between the antibody fragment and the gIII protein. Each selected phage suspension was transferred to a pre-warmed E. coli ER2738 culture and incubated in a 37°C water bath for 45 minutes without shaking. The bacterial culture was centrifuged, the supernatant was removed, and the pellet was resuspended in 2×YT medium. The bacteria were plated onto LB / Cam agar plates and incubated overnight at 37°C. The following day, the bacteria were scraped from the plates using freezing medium (2×YT medium containing 34 μg / ml chloramphenicol (Cam), 1% glucose, and 15% glycerol), and aliquots were stored at -80°C before preparing phages for subsequent panning rounds.

[0111] Panning of solutions and semi-solutions In solution and semi-solution panning, magnetic beads (GE, Sera-Mag streptavidin-coated magnetic particles, catalog #30152104010150) were used in combination with biotinylated cPD-L1_hFc (SinoBiological, catalog: 70110-D02H-B) to either capture antigen-phage complexes on the bead surface or immobilize antigens, respectively. To reduce phage selection for the magnetic beads used, neutraavidin-coated beads (GE, Sera-Mag neutraavidin-coated magnetic particles, catalog #78152104010150) were used as an alternative to the streptavidin-coated beads described above.

[0112] Before panning, the beads were washed and blocked. For semi-solution panning, the phages were also loaded with antigen. For this purpose, 250 μl of beads for each selection were transferred to a 2 ml low-binding tube, the beads were captured with a magnetic particle separator, and the storage solution was removed. The beads were then washed three times with PBS, the beads were collected using a magnet, and the wash buffer was removed. The beads were then blocked in a blocking solution (100% Chemiblock) at room temperature for 1 hour. In parallel, the phages were blocked. For this purpose, the required amount of phage was mixed with Chemiblock containing 10 μg / ml huIgG for blocking and incubated at room temperature for at least 1 hour. In addition, the blocked phages were pre-absorbed onto empty magnetic beads to remove sticky phages. To remove biotin-specific and hFc-tag-specific phages before panning, the phages were incubated in 96-well plates coated with 5 μg / ml BSA-biotin for a full 45 minutes, and in wells coated with 5 μg / ml huIgG overnight at 4°C, respectively.

[0113] After blocking the beads and blocking / pre-adsorption the phages, the biotinylated antigen was added to the phage solution and incubated at room temperature for 1 hour with rotation. To capture the phage / antigen complex, the blocking buffer was removed from the beads, and the phages were added to bind the biotinylated cPD-L1_hFc at room temperature for 20 minutes. Nonspecific phages were then removed by washing (3 rapid washes with PBST, 3 rapid washes with PBST for 5 minutes, 3 rapid washes with PBS). Wash stringency was adapted between rounds depending on the panning output. In the final wash step, the magnetic beads with the captured antigen-phage complexes were transferred into new low-binding tubes.

[0114] To elute specific phages, 300 μl of trypsin was added while rotating at 37°C for 30 minutes. Each selected phage suspension was then transferred to 20 ml of pre-warmed E. coli ER2738 culture and incubated in a 37°C water bath for exactly 45 minutes without shaking. The bacterial cultures were centrifuged at 4600 rpm for 5 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in 600 μl of 2×YT medium, plated onto a large LB / Cam agar plate, and incubated overnight at 37°C. The following day, using a sterile Drygalski spatula, bacteria were scraped from the plate with 1–3 mL of frozen medium (2×YT medium containing 34 μg / ml Cam, 1% glucose, and 15% glycerol), aliquots were stored at -80°C, and phages were prepared for subsequent panning rounds.

[0115] Similar to solution panning, semi-solution panning was performed with the following modifications: Instead of capturing the phage complex bound to the biotinylated target from solution using streptavidin or neutraavidin magnetic beads, the respective antigens were immobilized on the beads before blocking the beads. Thus, the panning mode reflects selection in the solid phase but allows for better target orientation and washing conditions compared to panning where the antigens are coated on the surface of a microtiter plate. Similar to solid-phase panning, three rounds were performed for each, with reduced cPD-L1_hFc concentration and increased washing stringency.

[0116] Phage preparation In each phage preparation, a phagemid-containing bacterial suspension or glycerol stock is inoculated into the inoculum (2×YT medium containing 34 μg / ml Cam and 1% glucose) to produce an OD of approximately 0.2. 600 This occurred. OD of approximately 0.5-0.6 600The cultures were incubated at 37°C for 60–120 minutes with shaking until the desired result was reached. VCSM13 helper phage was added and incubated at 37°C for 30 minutes without shaking, then at 37°C for 30 minutes with shaking at 250 rpm. The bacteria were then spun down, and the helper phage containing the supernatant was discarded. Phage-infected bacteria were resuspended in induction medium (2×YT medium containing 50 μg / ml carbenicillin, 50 μg / ml kanamycin (Kan), and 0.2 mM IPTG) and incubated in a phage shaker at 22°C for 18-20 hours with shaking at 200 rpm. The following day, the bacteria were spun down and the supernatant containing antibody-presenting phages was transferred to a new tube. For phage precipitation, 1 / 5 volume of ice-cold PEG / NaCl was added to the phage-containing supernatant, mixed, and incubated on ice for at least 30 minutes with gentle shaking. The precipitated phages were spun down at 10000×g at 4°C for at least 30 minutes. The supernatant was quantitatively removed and the phage pellet was resuspended in an appropriate volume of PBS. For short periods, the phages were stored rotating at 4°C, or for longer periods, they were frozen at -80°C.

[0117] Subcloning and Screening Plate Generation After multiple rounds of panning, polyclonal phage output was subcloned into the bacterial Fab expression vector pCaBx. Antibody-coding fragments were excised from the phage display vector using adjacent restriction enzymes, isolated by preparative agarose gel electrophoresis (1.0% agarose), and the fragments were DNA-purified from gel sections using a suitable gel extraction kit. Ligation reactions with inserts and pre-cut pCaBx vectors, as well as subsequent transformation in chemically competent E. coli BL21(DE3), were performed according to standard procedures.

[0118] A round-bottom 96-well plate (e.g., Thermo Fisher, catalog #262162) was filled with 80 μl / well of all-in-one medium (2×YT medium containing 34 μg / ml Cam, 0.1% glucose, and 0.5 mM IPTG). Single colonies from the agar plate used in the subcloning procedure were inoculated into the wells of the expression plate. The plate was incubated at 37°C for 5 hours with shaking at 600 rpm, and then incubated overnight at 22°C with shaking at 600 rpm. To prepare crude bacterial lysates, so-called BEL lysates, for screening purposes, 30 μl / well of lysis buffer (2×BBS containing 2.5 mg / ml lysozyme, 4 mM EDTA, and 13 U / ml benzonase) was added, and the mixture was incubated at 22°C for 1 hour with shaking at 600 rpm. Next, 30 μl / well of blocking buffer (1x PBS containing 5% powdered milk) was added, and the plates were incubated at 22°C for 1 hour with shaking at 600 rpm. The plates were stored at -20°C or used directly for screening.

[0119] Screening ELISA and identification of unique clones For screening, antibody fragments containing lysate (BEL lysate) were tested for binding to antigens immobilized on Maxisorp microtiter plates. Following antigen immobilization on the corresponding surface, the plates were washed three times with PBST and then blocked at room temperature for 1 hour with 5% milk in PBST. BEL lysate, control antibody, and negative control were transferred to plates and incubated at room temperature for 1 hour. The plates were washed three times with PBST, and then the detection antibody was diluted with 0.5% milk in PBST, added to the plates, and incubated at room temperature for 1 hour. For detection, anti-FLAG antibody (Sinobiological, 109143-MM13) was used. The plates were washed five times with PBST, and detection of the bound antibody was performed using QuantaBlu reagent according to the manufacturer's instructions on a Tecan Genius Reader (excitation filter: 320 nm, absorption filter: 430 nm).

[0120] Individual clones identified by screening in ELISA format were Sanger sequenced. Therefore, Miniprep cultures from each positive hit were inoculated from glycerol stocks of individual samples. Plasmids were purified using standard protocols and sent to an external service provider for sequencing using primers that cover all CDR regions, which vary depending on the library design.

[0121] Following screening, approximately 65 different antibody variants were identified. Two sequence-specific candidates with promising binding properties, referred to as CAN1005001, containing the VL and VH domains described in SEQ ID NOs. 41 and 42, and CAN1005010, also containing the VL and VH domains described in SEQ ID NOs. 1 and 2, were further characterized.

[0122] Example 2: Conjugation ELISA using initial clones CAN1005001 and CAN1005010 from panning. An ELISA setup was used to characterize the binding of CAN1005001 and CAN1005010 to target cPD-L1. The cPD-L1 fusion protein was immobilized directly onto plates at a concentration of approximately 57 nM. The coated plates were washed three times with PBS-T, followed by blocking in a Chemiblocker (Merck-Millipore, 2170) at room temperature for 1 hour. After blocking, serial dilutions of the antibody solution were added to the plates, incubated at room temperature for 1 hour, washed, and a suitable detection antibody conjugated to HRP was added. The screening ELISA was performed as described above. The results of this experiment are presented together with Example 3.

[0123] Example 3: Ligand binding inhibition assay using initial clones CAN1005001 and CAN1005010 To confirm the ability of CAN1005001 and CAN1005010 to block the interaction between canine PD-1 and canine PD-L1, a ligand binding inhibition assay was set up using an ELISA format. Immobilized cPD-L1_hFc cells were immobilized and presented on a plate, and accessible binding sites were blocked with CAN1005001 and CAN1005010, respectively. The lack of accessible binding sites for PD-1 was then analyzed by measuring residual PD-1 binding to PD-L1_hFc cells.

[0124] ELISA ligand binding inhibition assay (LBI) Generally, a 57 nM cPD-L1 fusion protein was directly immobilized on the plate overnight at 4°C. For LBI ELISA, a black 384-well Maxisorp plate was used. The coated plate was washed three times with PBS-T, followed by blocking in a Chemiblocker (Merck-Millipore, 2170) at room temperature for 1 hour. The blocked plate was washed three times with PBS-T, followed by the addition of a titration-series of blocking antibodies, and incubated at room temperature for 1 hour. The plate was then washed three more times with PBS-T, and PD-1-biotin (1x biotinylation via Avi-tag, Sinobiological catalog: 70109-D27H-B) was added at a concentration of typically 4 μg / ml (206 nM), and incubated at room temperature for 1 hour. This concentration of canine PD-1-biotin was previously identified as optimal by titrating the binding of PD-1-biotin to immobilized canine PD-L1_hFc. After incubation with ligand PD-1, the plates were washed three times with PBS-T, and a detection reagent suitable for biotinylated PD-1, typically streptavidin-HRP, was added and incubated at room temperature for 1 hour. Subsequently, the plates were washed five times, followed by the addition of Quanta Blue, and measured for screening ELISA as described above.

[0125] The results are shown in Figure 3. CAN1005001 and CAN1005010 show dose-dependent binding to cPD-L1. Consistently, increasing the amount of both antibodies efficiently blocks the binding of cPD-1 to immobilized cPD-L1. CAN1005001 showed an EC50 value of 0.4 nM and an IC50 value of 4.6 nM. 50 The value was calculated.

[0126] Example 4: Affinity maturation and screening in Fab format for CAN1005001 and CAN1005010 LBI experiments revealed a subset of clones, including CAN1005001 and CAN1005010, capable of blocking the interaction between canine PD-1 and canine PD-L1. To further increase the binding strength and efficacy of each candidate, affinity maturation strategies were initiated.

[0127] The maturation approach focused on two sites within these antibodies: within the heavy chain, HCDR2 was modified, while on the light chain, LCDR3 was removed and replaced by the respective maturation modules. The maturation modules were designed in a way that reflects the natural diversity within each CDR, lacking key PTM sites and being highly diversified. For clarity, the "VH mature clone" contains the original HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, but with a modified HCDR2 region, while the "VL mature clone" has a distinct LCDR3 sequence, but the remaining CDRs are identical to the parent clone. Notably, it is also possible to combine the matured chains to generate so-called cross-clones, modifying both VL and VH. There is a common understanding of the correlation between library size and the opportunity for strong affinity improvement. Therefore, the objective was to prepare libraries in the range of over 1.00E+07 variants.

[0128] Prior to affinity maturation, selected candidate Fab code inserts were digested via EcoRI / NcoI and ligated into phage display vectors according to standard procedures. Subsequently, VH or VL stuffer sequences were ligated into these candidates, and these were then replaced by HCRD2 or LCDR3 maturation modules.

[0129] For the preparation of the vector skeleton for introducing the mature module, the standard procedure is followed to excavate the VH packing and then again by restricted digestion using BbsI and KpnI or VL packing with BssHII and MfeI.

[0130] In VL maturation libraries, the mature modules of the LCDR3 region are cloned into the vector skeleton using BbsI and KpnI as described above. In VH maturation, the HCDR2 modules are similarly introduced by cloning BssHII and MfeI.

[0131] For library cloning, highly electrocompetent ER2738 cells were used for transformation, and DNA was desalted by precipitation prior to electrotransformation. For this purpose, the ligation sample was adjusted to 50 μl by adding sterile ddH2O and 1 μl glycogen, as well as 500 μl 2-butanol, and incubated on a rotating plate at room temperature for at least 5 minutes. The precipitated DNA was then spun down on a benchtop centrifuge at maximum speed at 4°C for 30 minutes. The supernatant was discarded, and the DNA pellet was washed with 500 μl of pre-cooled 70% ethanol. The sample was again centrifuged at maximum speed at 4°C for 15 minutes to remove the ethanol, and the DNA pellet was air-dried for approximately 15 minutes. The pellet was resuspended in 5 μL of ddH2O per ligation approach.

[0132] For each library, two transformations were performed on ER2738 cells using a BTX electroporator (settings: 25 μF, 200 Ω, 1.6 kV) according to the manufacturer's instructions. Immediately after pulse, the cells were transferred to 950 μl of pre-warmed recovery medium and incubated at 37°C for 1 hour with gentle shaking at 200 rpm. A small aliquot of the recovered culture was set aside for library size determination. The remaining culture was spun down at 4600 × g for 10 minutes, resuspended in 400 μl of 2 × YT, plated onto two large LB / Glu / Carb agar plates, and incubated overnight at 37°C. The following day, bacteria were scraped from the plates using pre-cooled LB medium containing 20% ​​glycerol, aliquots were prepared, and stored at -80°C until subsequent phage preparation.

[0133] Library size was determined using an Eddy Jet spiral plater, exceeding 2.00E+08 clones per library. QC was performed by colony PCR and sequencing of 10 clones per library to confirm diversification and absence of parental clones, and viable cell counting was performed to assess the quality of the frozen glycerol stocks.

[0134] Essentially, phage pretreatment was performed as described above (Example 1), but it was scaled up to reflect the requirement of encompassing a larger number of individual clones in the library and this greater diversity at the time of phage production.

[0135] Mature panning was carried out essentially as described above (Example 1), primarily using solution panning. In contrast to initial panning, mature panning was carried out under more stringent conditions. This was achieved by reducing the antigen concentration during panning, i.e., using a lower range of target amounts in the first panning round, similar to the third round of initial panning. The washing step was also extended and increased in number. In addition, k offThe procedure included a selection step. Using magnetic streptavidin beads, antigen-phage complexes were captured. After washing the beads to remove unwanted phages, a 10-fold molar excess of non-biotinized antigen was added to the washing buffer and incubated overnight. During this step, low-affinity antibodies dissociate from the captured antigen on the beads but find alternative interaction partners in the solution rather than on the bead surface. Therefore, only high-affinity antibodies will be recovered. off After the selection step, the supernatant containing the low-affinity antibody was removed, the beads were washed again with PBS, and eluted as previously described.

[0136] Maturation panning successfully identified 186 clones that showed superior signals compared to their respective parent clones. This included maturation panning campaigns against other parent clones. For parent clone CAN1005010, clone CAN1005010L1 (containing SEQ ID NO: 9) from the LCDR3 maturation campaign and clone CAN1005010H2 (containing SEQ ID NO: 19) from the HCDR2 maturation campaign were selected for further study. Similarly, for parent clone CAN1005001, clone CAN1005001L1 (containing SEQ ID NO: 47) from the LCDR3 maturation campaign and clone CAN1005001H3 (containing SEQ ID NO: 52) from the HCDR2 maturation campaign were selected for further study.

[0137] Fab Capture ELISA Screening To identify derivatives with higher affinity, we used an alternative ELISA setup for screening to better normalize BEL expression levels and more efficiently distinguish Fabs in terms of their affinity.

[0138] In contrast to the previous screening ELISA setup, Maxisorp plates were coated overnight with a low-density anti-canine Fab antibody at 1 μg / ml. The coated plates were washed three times with PBS-T and then packed with diluted BEL. After BEL incubation and washing with PBS-T, serial dilutions of cPD-L1-biotin were added to the plates. Detection was performed with streptavidin-HRP, followed by five stringent washes with PBS-T and Quanta Blue substrate. Measurements were performed as described previously (Example 1). The resulting data are shown in Figure 4.

[0139] Compared to the parent clone, derivatives from both heavy and light chain maturation exhibited significantly better binding to cPD-L1. This is expressed by EC50 values ​​of 362 pM for CAN1005001L1 and 97 pM for CAN1005001H3, compared to 820 pM for the parent clone CAN1005001. EC50 values ​​of 139 pM and 121 pM were determined for CAN1005010L1 and CAN1005010H2, respectively. The parent clone CAN1005010 was not included in the experiments shown.

[0140] Example 5: Cross-cloning with different mature heavy and light chains derived from CAN1005010 and CAN1005001. To further increase the affinity of CAN1005010 derivatives, CAN1005010L1 and CAN1005010H2 (including SEQ ID NOs. 9 and 19), their respective mature light and heavy chains were combined into a single Fab, sharing only HCDR3 with CAN1005010 and containing both mature LCDR3 and HCDR2. In the same manner, their respective mature light and heavy chains from the CAN1005001 derivative and CAN1005001H3 (including SEQ ID NOs. 47 and 52) were combined into a single Fab.

[0141] The resulting clones were designated CAN1005016 and CAN1005019, respectively. Cloning was performed according to the standard procedure described below, which involved initially digesting both clones, one acting as the vector backbone and the other as the insert. In this case, the LCDR3 mature clones CAN1005010L1 (containing SEQ ID NO: 39) and CAN1005001L1 (containing SEQ ID NO: 65) acted as the vector backbone. The resulting Fabs were tested with Fab Capture ELISA as previously reported in Example 4.

[0142] Consumption of restrictions Both vectors were digested with 5 μg of MfeI and XhoI to excise the heavy chain coding regions. Restriction digestion was performed at 37°C for 1 hour, followed by inactivation at 80°C for 20 minutes. The skeletal sample digests further contained FastAP to inhibit vector migration. For CAN1005010L1, the heavy chain-deficient vector was excised from the agarose gel and purified by standard procedures. The corresponding insert from clone CAN1005010H2 was also applied to an agarose gel, the insert was excised, and purified by standard procedures.

[0143] Ligation After restricted digestion, the insert was ligated to the vector skeleton. A 5:1 insert-to-vector mix was prepared and T4 ligase (NEB, catalog: M0202S) was added according to standard instructions. Ligation was performed overnight at 16°C for 16 hours. The final ligation product was directly transformed into chemically competent E. coli cells. 10 μl of ligation product was mixed with 100 μl of competent cells and incubated on ice for 30 minutes. The cells were then placed in a 42°C water bath for 10–45 seconds, depending on the type of E. coli cells. After rescuing the transformed cells in fresh 2×YT medium for 1 hour, the cells were centrifuged and resuspended in appropriate volumes for plating onto LB agar plates containing the respective selected antibiotics for each vector.

[0144] Fab Capture ELISA Screening Screening was performed as described in Example 4. Fab binding factors of CAN1005016 and CAN1005019 were loaded onto plate-conjugated anti-canine Fab capture antibody, followed by the addition of a serial titration of cPD-L1_hFc-biotin. Detection was performed using streptavidin-HRP. Parental clone CAN1005001 was included as a control. Curve fit and EC50 values ​​were analyzed using GraphPad Prism. The results are shown in Figure 5.

[0145] CAN1005016 was generated through a combination of CAN1005010L1 and CAN1005010H2, while CAN1005019 was generated from a combination of CAN1005001L1 and CAN1005001H3. For CAN1005016, an EC50 of 87 pM was determined, demonstrating improvement compared to its parent clone, as shown in Figure 5. For CAN1005019, an EC50 of 138 pM was determined.

[0146] Example 6: Grating-bound interferometry (GCI) measurement for characterizing anti-PD-L1IgG To further characterize the dynamic characteristics of our candidates CAN1005016 and CAN1005019, we used waveRAPID in the GCI system according to the procedure described below to evaluate the K D This was quantified.

[0147] Binding dynamics studies were conducted using a PAG sensor chip (Creoptix, Malvern Panalytical brand) with the Creoptix® WAVEdelta system. All experiments were performed at a temperature of 25°C and a sampling rate of 10 Hz, using HBS-EP (Cytiva) as the experimental buffer. Data acquisition and evaluation were performed using WAVEcontrol software version 4.5.13.

[0148] Prior to ligand capture, the sensor tip was prepared by injecting 100 mM sodium borate, 1 M NaCl, pH 9 (Xantec) for 180 seconds. The antibody was diluted to 1 μg / ml in HBS-EP and injected at approximately 300 pg / mm³ through flow channel 2. 2 The analyte was captured down to its density. Flow channel 1 was left blank to function as a reference surface. For kinetic data acquisition, PD-L1-his was diluted to 200 nM in HBS-EP and injected into the ligand and reference surface during repeated analyte pulses (waveRAPID) with increasing duration at a flow rate of 60 μl / min. PD-L1-his was injected for a total duration of 180 seconds, followed by measurement of complex dissociation in the experimental buffer for 30 minutes. Blank injections were performed to allow for dual reference of the data, and a 0.5% DMSO pulse injection (experimental buffer containing 0.5% DMSO) was used as the analyte concentration-adjusted calibration curve. The data was corrected (X and Y offsets, DMSO calibration, dual reference), and kinetic parameters were obtained by fitting a simple 1:1 interaction model (global fit). The results are shown in Figure 6.

[0149] Regarding CAN1005016, the K is 0.77nM. D It was decided that for CAN1005019, the K is 1.7nM. D This was determined. For comparison, the parent clone CAN1005010H2 has a metric of 2.8nM. D It possesses [the characteristic]. Therefore, cross-cloning of CAN1005016 resulted in more than a three-fold improvement in affinity.

[0150] Example 7: Generation of binding profiles of CAN1005016 to cPD-L1_hFc using ELISA and FACS. An ELISA setup was used to characterize the binding of CAN1005016 to target cPD-L1. ELISA was performed using cPD-L1_hFc or cPD-L1_MBP directly immobilized on plates, following the standard procedure described below. Additionally, binding to cPD-L1-transfected HEK293c18 cells was examined by flow cytometry (FACS).

[0151] ELISA The cPD-L1 fusion protein was immobilized directly onto the plate at a common concentration of 57 nM. For the conjugation ELISA, a black 384-well Maxisorp plate was used. This was carried out overnight at 4°C. The coated plate was washed three times with PBS-T, followed by blocking for 1 hour in a Chemiblocker (Merck-Millipore, 2170) at room temperature. After blocking, serial dilutions of each antibody were added to the plate, typically covering concentrations ranging from single-digit ng / ml to single-digit μg / ml. The antibodies were incubated at room temperature for 1 hour, followed by washing, and a suitable detection antibody conjugated to HRP was added. For the screening ELISA, the measurements were performed as described above. The results are shown in Figure 7.

[0152] Mature cross-cloned IgGs CAN1005016 and CAN1005019 showed dose-dependent binding to cPD-L1, exhibiting EC50 values ​​of 76 pM and 49 pM, respectively. This represents a significant improvement over the EC50 value of 400 pM for CAN1005001 IgG measured in Example 3.

[0153] Transfection of HEK293c18 cells 1 × 10 in FCS with DMEM + 10% 6HEK293c18 cells were seeded in a 6-well plate at a cell / well density. The transfection mix was prepared the following day. Transfection was performed using jetPRIME transfection reagent (Polyplus, catalog: 101000046). 2 μg of plasmid encoding wild-type canine PD-L1 was diluted in 200 μl of jetPRIME buffer per well. The solutions were mixed, 4 μl of transfection reagent was added, the mixture was then mixed again, and the mixture was centrifuged. The adherent cell medium was then replaced with fresh medium, and the transfection mix was carefully added onto the cells. The plate containing the transfected cells was returned to the incubator and incubated for 24 hours.

[0154] The following day, cells were collected using PBS + 2 mM EDTA (PBS / EDTA) by gentle pipetting, counted, and 2 × 10⁻¹⁶ cells were prepared for FACS staining. 5 The cells / well were transferred to a 96-well V-bottom plate.

[0155] FACS staining The cells were washed once by adding 200 μl of PBS / EDTA per well and centrifuging at 1500 rpm for 5 minutes. The supernatant was carefully decanted by holding the plate upside down over waste and dried on a paper towel. The cells were then stained for dead cells with Zombie violet dye (Biolegend, catalog 423114) diluted 1:500 in PBS / EDTA for 15 minutes in the dark at 4°C. The cells were then washed with FACS buffer (PBS / EDTA + 2% FCS) as described above. Serial titrations of CAN1005016 and CAN1005019, typically in the range of 10 ng / ml to 10 μg / ml, were prepared by diluting with FACS buffer and added to the respective wells, and incubated in the dark at 4°C for 30 minutes. After incubation, the cells were washed three times with FACS buffer, the detection antibody goat-anti-canine-IgG FITC (ThermoFisher, catalog: #A18764) was added, and the cells were incubated at 4°C in the dark for 30 minutes. Finally, the cells were washed three more times, then resuspended in 200 μl of FACS buffer and measured directly. The results are shown in Figure 8.

[0156] Both candidates recognize cell-bound cPD-L1, as demonstrated by FACS against HEK293c18 cells transfected with the target protein. EC50 values ​​of 10.5 nM were determined for CAN1005016 and 11.8 nM for CAN1005019. No staining was observed with an unrelated control antibody.

[0157] Example 8: Ligand Binding Inhibition Assays Using CAN1005016 and CAN1005019 in ELISA and BLI Settings ELISA ligand binding inhibition assay (LBIA) To confirm the ability of CAN1005016 and CAN1005019 to block the interaction between canine PD-1 and canine PD-L1, ligand binding inhibition assays were set up using an ELISA format. cPD-L1_hFc was immobilized on plates, and accessible binding sites were blocked with each candidate. The lack of accessible binding sites for PD-1 was analyzed by measuring residual PD-1 binding to PD-L1_hFc.

[0158] The cPD-L1 fusion protein was immobilized directly onto plates overnight at a common concentration of 57 nM at 4°C. For the lbia ELISA, a black 384-well Maxisorp plate was used. The coated plates were washed three times with PBS-T, followed by blocking in a Chemiblocker (Merck-Millipore, 2170) at room temperature for 1 hour. The blocked plates were washed three times with PBS-T, followed by the addition of a titration-series of blocking antibodies, and incubated at room temperature for 1 hour. The plates were then washed three more times with PBS-T, and PD-1-biotin (1x biotinylation via Avi-tag, Sinobiological catalog: 70109-D27H-B) was added at a concentration of typically 4 μg / ml (206 nM), and incubated at room temperature for 1 hour. This concentration of canine PD-1-biotin was previously identified as optimal by titrating the binding of PD-1-biotin to immobilized canine PD-L1_hFc. After incubation with ligand PD-1, the plate was washed three times with PBS-T, and a detection reagent suitable for biotinylated PD-1, typically streptavidin-HRP, was added and incubated at room temperature for 1 hour. Subsequently, the plate was washed five times, followed by the addition of Quanta Blue, and measured for screening ELISA as described above. The results are shown in Figure 9.

[0159] Both candidates can efficiently block the interaction between cPD-L1 and cPD-1. CAN1005016 uses 0.95 nM, and CAN1005019 uses 1.32 nM. 50 The value has been determined.

[0160] Ligand binding inhibition assay based on biolayer interference (BLI) (CAN1005016 only) In biolayer interferometry, the reverse configuration was tested. Here, PD-1 was immobilized on the sensor, and a mixture of cPD-L1_hFc and the anti-PD-L1 antibody CAN1005016 competed for binding to cPD-L1. All reagents were diluted with Kinetics Buffer (Sartorius, 18-1105).

[0161] Biotinylated canine PD-1 (cPD-1-Bio) was immobilized on a streptavidin sensor (Sartorius, catalog: 18-5019). Therefore, after a baseline phase of 3 cP 0 seconds, a 5 μg / ml D-1-Bio solution was immobilized for 45 seconds until a signal of 1–2 nm was reached. This was followed by a 30-second baseline in Kinetics buffer. Subsequently, pre-mixed solutions of CAN1005016 (10 nM–100 nM) and 100 nM cPD-L1 were associated with the packed sensor for 150 seconds. After association, a 120-second dissociation was recorded. Measurements were performed using a BLItz system (Pall Fortebio).

[0162] Pre-incubation of cPD-L1 with stoichiometric amounts of CAN1005016 results in blocking of the interaction site to cPD-1 and absence of binding of cPD-L1 to immobilized cPD-1, supporting results from ELISA-based ligand binding inhibition assays. Using non-stoichiometric amounts of CAN1005016 (1:10), binding of PD-L1 to immobilized cPD-1 is partially blocked, as demonstrated in the intermediate signal of cPD-L1 binding. Stoichiometric pre-incubation with unrelated canine control IgG has only a minor effect on the binding of cPD-L1 to cPD-1.

[0163] Example 9: CAN1005016 and its binding to canine cell lines expressing PD-L1. To validate the binding of candidate antibodies to endogenous canine PD-L1, PD-L1 was stained on IFN-γ-treated canine cell lines using CAN1005016 and CAN1005019. The canine squamous cell carcinoma cell line SCC1 has been reported to express canine PD-L1 after IFN-γ stimulation (Pantelyushin et al. 2021).

[0164] Treatment of SCC1 cells with IFN-γ SCC1 cells were cultured in DMEM high-glucose medium (Sigma-Aldrich, catalog: D6429) supplemented with 15% FCS (PAN Biotech, catalog: P30-3302), non-essential amino acids, and penicillin-streptomycin (Thermo-Fisher, catalog: 15140122). Cells were isolated with trypsin (Thermo Fisher, catalog: 12604013), washed, and resuspended in fresh medium. Cells were counted, with 1 × 10⁶ cells per well. 6 The cells were seeded in a 6-well plate. The cells were incubated at 37°C in a 5% CO2 incubator for 3 hours, followed by treatment with 10 ng / ml IFN-γ for 2 days. Control cells without IFN-γ treatment were cultured together.

[0165] FACS staining of SCC1 cells treated with CAN1005016 and CAN1005019 After treatment, cells were separated with trypsin (Thermo Fisher, catalog: 12604013), filtered through a 70 μm or 100 μm cell strainer, and washed twice with PBS-EDTA. From this point, the cells were kept cold at 4°C. Cells were stained with Zombie Violet live / dead stain (Biolegend, catalog: 423114), and equal numbers of cells were distributed into 96-well plates for staining. Treated cells and control cells were stained separately for 1 hour with different concentrations of biotinylated CAN1005016, CAN1005019, and control IgG diluted in FACS buffer (PBS-EDTA + 2% FCS). Cells were then washed three times with FACS buffer by centrifugation at 1500 rpm at 4°C. Streptavidin-allophycocyanin (Biolegend, catalog: 405207) was used as the detection antibody and stained for 30 minutes. After incubation, the cells were washed three times again as previously described, resuspended in 200 μl of FACS buffer, and then measured using a Beckman Coulter Cytoflex LX.

[0166] CAN1005016 and CAN1005019 bind to PD-L1-expressing canine cancer cell lines. Treatment with IFN-γ, which induces physiological upregulation of canine PD-L1, results in improved binding of anti-PD-L1 IgG to SCC1 cells. This supports the ability of CAN1005016 and CAN1005019 to recognize cPD-L1 in a cellular context.

[0167] Example 10: Expression, purification, and thermal stability of CAN1005016 and CAN1005019 Plasmids encoding CAN1005016 and CAN1005019 were separately transfected into suspension HEK293-Freestyle (HEK293-F) cells. IgG purification was performed by protein A affinity chromatography followed by size exclusion chromatography (SEC). Details are described below. The structural characteristics of CAN1005016 and CAN1005019 were further characterized by differential scanning fluorescence (nanoDSF).

[0168] HEK293-F cell transfection and IgG expression HEK293-F cells were cultured in Freestyle medium (ThermoFisher, catalog: 12338026) in an incubator at 37°C, 5% CO2, and under constant rotation of 135 rpm. Approximately 18–24 hours before transfection, HEK293-F cells were counted and transfected to an appropriate volume for expression, typically 1.5 × 10⁶ cells in 25–100 ml of Freestyle medium. 6 Cells were seeded at a density of cells / ml. The following day, the transfection mix was prepared according to the cell volume. Generally, 3 μg of plasmid and 9 μg of PEI per ml of culture volume were used, and the scale was adjusted accordingly. The transfection mix was added dropwise to the cells while stirring the flask. After incubating the transfected cells for 5-6 hours, the culture was diluted with fresh, pre-warmed Freestyle medium containing 5 mM valproic acid (VPA) as an additive to a final concentration of 2.5 mM VPA. Expression was typically performed for 5-7 days, and the supernatant was collected by centrifugation and filtration.

[0169] Purification of CAN1005016 and CAN1005019 Both IgGs were purified via protein A affinity chromatography using standard buffers for binding and elution. Eluted IgG was concentrated to less than 5 ml using a centrifugal concentrator (Amicon, 100 kDa, catalog: ACS510024) and applied to a Superdex200 16 / 600 pg size exclusion column operated according to standard procedures. Major peaks were collected, analyzed via SDS-PAGE, and pooled. Purification from transient, unoptimized expression cultures typically yields high-purity IgG of 10–25 mg / L.

[0170] nanoDSF and DLS measurements For parallel DLS and nanoDSF measurements (Nanotemper Technologies GmbH), FORMOscreen® preformulation studies were conducted using the Prometheus PANTA system. All experiments were performed twice using high-sensitivity capillaries (Nanotemper Technologies GmbH), and data analysis was performed using integrated datasets for each test condition. Data analysis and evaluation were performed using PANTA Analysis software version 1.4.4.

[0171] A protein stock (concentration = 29.04 μM) was centrifuged at 4°C and 21,000 g for 1 hour. The supernatant was separated and its concentration was measured again (concentration after centrifugation = 28.72 μM). Under each FORMOscreen® condition, 1.32 μl of protein stock supernatant was mixed with 4 μl of 5× FORMOscreen buffer stock and 14.68 μl of ddH2O to a final volume of 20 μl with a final protein concentration of 1.9 μM per 1× FORMOscreen® buffer condition.

[0172] Samples were prepared in a 384-well AURORA microplate and incubated at 40°C for 7 days before measurement. DLS measurements were then performed with 10 acquisitions per capillary tube and a measurement time of 10 μs per acquisition. NanoDSF measurements were performed with a heating gradient of 1°C / min from 20°C to 95°C.

[0173] Table 4: DLS measurement results JPEG2026511256000006.jpg47164

[0174] Example 11 Manufacturing of CAN1005016 CAN1005016 used in this hospitalized animal study was produced in genetically engineered CHO cells according to standard bioreactor fermentation techniques. Supernatant production was carried out using a high-expression stable cell pool at a suitable supply batch process scale (5 L). Cells were removed from the cell suspension by centrifugation for supernatant collection. mAbs were purified from the supernatant using one-step protein A affinity chromatography, formulated, and filtered sterile. The formulation is designed to stabilize the monoclonal antibody at low temperatures and allow for antibody freeze / thaw. It consists of the antibody formulated in histidine buffer at pH 5.9, with trehalose added as a cryoprotectant and Tween® 80 added to prevent aggregation.

[0175] Example 12 In vivo trial of CAN10005016 To investigate the efficacy of an anti-canine PD-L1 antibody, CAN10005016 was tested in a 14-year-old, mixed-breed female dog (14.3 kg) with stage IV oral melanoma and distant lung metastases. At enrollment, the size of the primary oral tumor lesion was 50 × 49 mm. CAN1005016 was administered intravenously at a dose of 5 mg / kg body weight over 30 minutes for four doses at 2-week intervals, followed by monthly doses.

[0176] Safety was assessed at each treatment session, scheduled using the Veterinary Cooperative Oncology Group (VCOG) criteria (Vet Comp Oncol. 2016;14:417-46). To monitor systemic toxicity, 5 ml of blood was collected at each antibody administration to obtain CBC, serum biochemistry, and thyroid profiles. No adverse events were reported throughout the study period. Antitumor response was assessed on a monthly basis by physical examination, chest X-ray, and abdominal ultrasound, and tumor size was measured and recorded by caliper. Antitumor response was classified according to the RECIST criteria (Nguyen SM et al., Vet Comp Oncol. 2015;13:176-83).

[0177] After five doses, the affected animal achieved complete oral remission. After the seventh dose, the dog remained in complete oral remission. Treatment was extended to a total of 10 doses. 229 days after registration, the animal was alive and clinically in good condition, but recurrent maxillary melanoma and progressive lung metastases were observed.

[0178] Throughout the veterinary literature, dogs with stage IV melanoma have been documented to have a poor prognosis, with a median survival time of 60–80 days when treated with surgery, radiotherapy, and / or chemotherapy (Bergman, PJ Canine oral melanoma. Clin. Tech. Small Anim. Pract. 2007;22:55; Kawabe M, et al., J Am Vet Med Assoc. 2015 247:1146; Tuohy JL, et al., J Am Vet Med Assoc. 2014;245:1266). At the time of preparation of this patent application, the affected animals were still alive, with overall survival exceeding 300 days. These results suggest that anti-PD-L1 therapy can be considered a promising treatment option for treating diseases of high medical need, such as malignant melanoma.

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Claims

1. The LCDR1 region described in Sequence ID No. 3, the LCDR2 region described in Sequence ID No. 4, and the LCDR3 region described in Sequence ID No. 18 or Sequence ID No. 49, and / or Including the HCDR1 region described in SEQ ID NO: 6 or SEQ ID NO: 44, the HCDR2 region described in SEQ ID NO: 30 or SEQ ID NO: 60, and the HCDR3 region described in SEQ ID NO: 8 or SEQ ID NO: 46, Antibody or antibody fragment.

2. An LCDR3 region having an amino acid sequence selected from SEQ ID NOs. 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 43, 47, 48, preferably SEQ ID NO. 9 or 47, and / or The antibody or antibody fragment according to claim 1, comprising an HCDR2 region having an amino acid sequence selected from SEQ ID NOs: 7, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, preferably SEQ ID NO: 19 or 52.

3. It includes a variable light chain containing the LCDR3 region described in SEQ ID NO: 18, combined with a variable heavy chain containing the HCDR2 region described in SEQ ID NO: 30, or it includes a variable light chain containing the LCDR3 region described in SEQ ID NO: 49, combined with a variable heavy chain containing the HCDR2 region described in SEQ ID NO:

60. Preferably, it includes a variable light chain comprising the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and the LCDR3 region described in SEQ ID NO: 5, combined with the variable heavy chain HCDR1 region described in SEQ ID NO: 6, the HCDR2 region described in SEQ ID NO: 19, and the HCDR3 region described in SEQ ID NO: 8, or Preferably, the light chain includes the LCDR1 region described in SEQ ID NO: 3, the LCDR2 region described in SEQ ID NO: 4, and the LCDR3 region described in SEQ ID NO: 47, combined with the variable heavy chain HCDR1 region described in SEQ ID NO: 44, the HCDR2 region described in SEQ ID NO: 52, and the HCDR3 region described in SEQ ID NO:

46. The antibody or antibody fragment according to any one of claims 1 to 2.

4. One, some, or all of the light chain variable domain framework sequences having amino acid sequences selected from SEQ ID NOs: 31, 32, 33, and 34, and / or A heavy chain variable domain framework sequence comprising one, some, or all of the amino acid sequences selected from SEQ ID NOs: 35, 36, 37, and 38, or one, some, or all of the heavy chain variable domain framework sequences comprising the amino acid sequences selected from SEQ ID NOs: 61, 62, 63, and 64. The antibody or antibody fragment according to any one of claims 1 to 3.

5. Light chain variable domains as described in Sequence ID No. 1, 39, 41, or 65, and / or heavy chain variable domains as described in Sequence ID No. 2, 40, 42, or 66, Preferably, the light chain variable domain described in SEQ ID NO: 39 is combined with the heavy chain variable domain described in SEQ ID NO: 40, or the light chain variable domain described in SEQ ID NO: 65 is combined with the heavy chain variable domain described in SEQ ID NO:

66. The antibody or antibody fragment according to any one of claims 1 to 4.

6. The system includes at least one LCDR, HCDR, framework region, or variable domain chain containing a sequence having at least 85% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 66. The antibody or antibody fragment according to any one of claims 1 to 5.

7. A substance that specifically binds to canine PD-L1 and / or optionally cross-competes with an antibody fragment described in any one of claims 1 to 6, and / or optionally interferes with or blocks the interaction between canine PD-L1 and canine PD-1. The antibody or antibody fragment according to any one of claims 1 to 6.

8. Specifically bind to canine PD-L1 with a dissociation constant (Kd) of less than about 20 nM, preferably less than about 10 nM, more preferably less than about 5 nM, and most preferably less than about 2 nM, and / or at least about 2 × 10 off [M -1 s -1 , preferably at least about 5 × 10 4 [M -1 s -1 of K on rate, and / or at a K -3 rate lower than about 1 × 10 -1 [s -4 , preferably lower than about 5 × 10 -1 [s off rate to bind to canine PD-L1, The antibody or antibody fragment according to any one of claims 1 to 7.

9. The antibody or antibody fragment according to any one of claims 1 to 8, which is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, Fab'-SH, and VHH.

10. An antibody or antibody fragment according to any one of claims 1 to 9, comprising an IgG isotype IgG-A, IgG-B, IgG-C, or IgG-D, most preferably an Fc domain or fragment selected from IgG isotype IgG-B.

11. Compared to the wild-type Fc fragment (and the corresponding amino acid position therein), at least one of amino acids at positions 235, 239, 270, and / or 331 is selected. Preferably, the antibody or antibody fragment according to claim 10, comprising at least one amino acid substitution at a position selected from at least one of L235, S239, D270, and / or P331, and more preferably, the Fc fragment comprising mutants L235A, S239A, D270A, and P331G compared to the wild-type Fc fragment (with the corresponding amino acid position).

12. The antibody according to any one of claims 1 to 11, comprising the light chain described in SEQ ID NO: 72 combined with the heavy chain described in SEQ ID NO: 73, or the light chain described in SEQ ID NO: 74 combined with the heavy chain described in SEQ ID NO:

75.

13. The antibody or antibody fragment according to any one of claims 1 to 12, wherein the antibody or antibody fragment is, optionally, a recombinant canine antibody or antibody fragment, or optionally, an isolated antibody or antibody fragment.

14. A pharmaceutical composition comprising an antibody or antibody fragment according to any one of claims 1 to 13 for use in the treatment of a subject requiring treatment, or an antibody or antibody fragment according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier or excipient.

15. An antibody according to any one of claims 1 to 13, or a polynucleotide or a plurality of polynucleotides encoding an antibody, or A vector or a plurality of vectors comprising the polynucleotide or a plurality of polynucleotides, or the vector composition or a cell comprising the polynucleotide or a plurality of polynucleotides.

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