Anti-canine PD-L1 antibodies

Abstract

The present invention relates to an anti-canine PD-L1 antibody. The antibody is preferably a whole canine antibody. The invention further relates to epitopes of canine PD-L1 that bind to and inhibit binding of canine PD-1 to PD-L1 by these antibodies. The invention further relates to the use of the antibodies of the invention in the treatment of dogs, including cancer treatment.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to EP 23164436.0, filed on March 27, 2023, which is incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present invention relates to anti-canine PD-L1 antibodies. The antibodies are preferably fully canine antibodies. The invention further relates to an epitope of canine PD-L1 that is bound by and inhibits the binding of canine PD-1 to PD-L1. The invention further relates to the use of the antibodies of the invention in the treatment of dogs, including the treatment of cancer. BACKGROUND

[0004] In the field of human medicine, the advent of immune checkpoint inhibitors (ICIs) has led to many advances in the immunotherapeutic management of cancer, especially malignant melanoma. The anti-CTLA-4 monoclonal antibody ipilimumab was the first ICI to be approved and showed efficacy in humans with advanced / metastatic melanoma. Subsequently, nivolumab and pembrolizumab, both anti-PD-1 antibodies, were approved for the treatment of advanced melanoma. Since May 2006, the FDA and / or EMA have approved a total of six anti-PD1 / PD-L1 specific ICIs for various cancer indications: pembrolizumab (anti-PD1, IgG4kappa), nivolumab (anti-PD1, IgG4), cemiplimab (anti-PD1, IgG4), atezolizumab (anti-PD-L1, IgG1, silent Fc form), avelumab (anti-PD-L1, IgG1, wild-type Fc form), and durvalumab (anti-PD-L1, IgG1, silent Fc form) (Ai et al. 2020).

[0005] ICIs are a true breakthrough in the field of human melanoma therapy, which has significantly improved the prognosis of responding patients. Therefore, veterinary medicine is currently turning its attention to the use of ICIs as a potentially effective systemic therapy also for dogs with tumors. However, experience is limited in the case of dogs. Studies and reports have been made on 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, (Igase et al. 2020; Maekawa et al. 2017; Mason et al. 2021).

[0006] PD-1 is a 55 kDa type I transmembrane glycoprotein containing an extracellular Ig variable type (V-type) domain that binds to its ligands 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 detrimental. It plays a critical role in reducing ineffective or destructive immune response regulation. Thus, PD-1 maintains immune tolerance by suppressing detrimental responses against self-proteins. On the other hand, PD-1 leads to malignant cell expansion by impairing protective immune responses. Inhibition of the immune response is achieved by the binding of PD-L1, a ligand of PD-1, to PD-1.

[0007] PD-L1 is a 33 kDa type I transmembrane glycoprotein that contains Ig V-like and Ig C-like domains in the extracellular domain, and a short cytoplasmic region, without known signaling motifs (Sanmamed and Chen 2014). PD-L1 is normally expressed by macrophages, some activated T and B cells, DCs, and some epithelial cells, especially under inflammatory conditions (Han et al. 2020). Moreover, PD-L1 is overexpressed in a variety of tumors, where it binds to PD-1, inhibits the proliferation of PD-1 -positive cells, and is involved in the immune evasion of tumors, leading to treatment failure (Ohaegbulam et al. 2015). Such tumor tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, and pancreatic cancer, as well as head and neck tumors.

[0008] 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). The binding of PD-L1 on tumor cells to PD-1 on immune cells can attenuate T cell-mediated immune surveillance, leading to a lack of immune response against tumors, and even to T cell apoptosis (Iwai et al. 2017).

[0009] In summary, aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancer. Importantly, PD-L1 expression has also been demonstrated in multiple canine cancers, particularly oral malignant melanoma (OMM) (36 / 40 patients); (Maekawa et al. 2016). Other PD-L1 positive cancers include osteosarcoma, hemangiosarcoma, mast cell tumor, breast 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). Until recently, canine B-cell lymphoma was demonstrated by flow cytometry to have higher expression of PD-L1 on neoplastic lymphocytes compared to normal B-cells (Hartley et al. 2018). Increased PD-L1 expression was associated with a high risk of progression and lymphoma-related death regardless of treatment (Aresu et al. 2021). This finding is consistent with recent evidence found in humans that upregulation of PD-L1 in tumor cells enables the tumor to evade the host’s immune system and increase chemoresistance. PD-1 expression on tumor infiltrating lymphocytes obtained from oral melanoma was high, suggesting that lymphocytes in this cancer type can have been exhausted (Maekawa et al. 2016). Another study on canine melanoma cell lines and tumor infiltrating macrophages also showed similar results with upregulation of PD-L1 expression after exposure to interferon-gamma, suggesting an important mechanism of tumor-mediated T-cell suppression (Hartley et al. 2017).

[0010] Few attempts have been made to design therapeutic antibody candidates against canine ICI, but clinical experience is still in its infancy. Chimeric rat-dog anti-PD-L1 (Maekawa et al. 2017) and “caninized” anti-CTLA-4 (Mason et al. 2021) and anti-PD-1 (Igase et al. 2020) monoclonal antibodies (mAbs) have been developed.

[0011] Maekawa and colleagues demonstrated that a canine chimeric PD-L1 monoclonal antibody, termed c4G12, enhanced cytokine production and proliferation of canine peripheral blood mononuclear cells (Maekawa et al. 2017). More importantly, in a pilot clinical study using c4G12, one of seven dogs with oral malignant melanoma and one of two dogs with undifferentiated sarcoma were observed to have an anti-tumor response (Maekawa et al. 2017). In a follow-up study of 29 dogs with a diagnosis of primary OMM and confirmed lung metastasis, an anti-tumor response and increased overall survival time after c4G12 treatment were also reported (Maekawa et al. 2021).

[0012] Notably, this canine chimeric antibody has a variable light chain (VL) and a variable heavy chain (VH) from rat origin (representing an important source of development of anti-drug antibodies (ADAs)). The authors do not comment on whether any of the treated dogs in the study had ADAs. However, after repeated administration, ADAs can reduce the drug efficacy of the compound. In addition, there is an increased risk of anaphylactic reactions after administration of chimeric antibodies, common side effects include fever, chills, headache, nausea, vomiting, diarrhea, skin rash or weakness.

[0013] The generation of species-specific mAbs with better safety profile is expected to be technically challenging and the experience of developing antibodies for companion animals is only in gradual development. There are only a few technical approaches available for generating therapeutic antibodies for companion animals such as dogs or cats, i.e. modification of existing compounds and use of transgenic animals. Methods to "caninize" or "felinize" antibodies have been published (Gearing et al. 2016; Gearing et al. 2013). However, even minor changes in the protein sequence of an antibody can lead to significant loss of efficacy and changes in biophysical properties, making such approaches time-consuming and prone to failure. More advanced techniques involve transgenic rodents expressing canine immunoglobulins (Wabl 5 / 23 / 2017). The drawback is the need to sacrifice animals in the initial antibody discovery process and the immunization process is almost uncontrollable. In this regard, in vitro selection methods such as phage display offer a great advantage as these methods allow a customized antibody selection process. Until recently, synthetic phage display libraries containing fully canine antibody fragments have been published (Tiller et al. 6 / 21 / 2018).

[0014] The present invention relates to fully canine antibodies against canine PD-L1 derived from a species-specific canine phage display library with high target specificity and good biophysical properties useful for various canine malignoma therapies.

[0015] The citation of any reference in this disclosure should not be construed as an admission that such reference is available as "prior art" to the application. SUMMARY

[0016] The present invention relates to an antibody or antibody fragment binding to canine Programmed Death Ligand 1 (canine PD-L1). The antibody or antibody fragment is characterized by its complementarity determining regions (CDRs) or its light and / or heavy variable domains.

[0017] In a first aspect, the inventors identified antibodies or antibody fragments that can comprise a light chain CDR1 (LCDR1) region according to SEQ ID No.: 3, a light chain CDR2 (LCDR2) region according to SEQ ID No.: 4, and / or a light chain CDR3 (LCDR3) region according to SEQ ID No.: 18 or SEQ ID No.: 49. In addition, the antibodies or antibody fragments can comprise a heavy chain CDR 1 (HCDR1) region according to SEQ ID No.: 6 or SEQ ID No.: 44, a heavy chain CDR 2 (HCDR2) region according to SEQ ID No.: 30 or SEQ ID No.: 60, and / or a heavy chain CDR 3 (HCDR3) region according to SEQ ID No.: 8 or SEQ ID No.: 45.

[0018] By maturation of antibodies having a light chain variable domain according to SEQ ID No.: 1 (comprising a LCDR3 according to SEQ ID No.: 5) in combination with a heavy chain variable domain according to SEQ ID No.: 2 (comprising a HCDR2 according to SEQ ID No.: 7), several high affinity antibodies comprising a LCDR3 region having an amino acid sequence selected from any one of SEQ ID No.: 9, 10, 11, 12, 13, 14, 15, 16, and 17 and a HCDR2 region having an amino acid sequence selected from any one of SEQ ID No.: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 were obtained. Based on these LCDR3 regions, the inventors identified a first anti-PD-L1 high affinity LCDR3 consensus sequence according to SEQ ID No.: 18 and a first anti-PD-L1 high affinity HCDR2 consensus sequence according to SEQ ID No.: 30.

[0019] In an alternative aspect of the application, several high affinity antibodies comprising a LCDR3 region having an amino acid sequence selected from SEQ ID No.: 47 or 48 and a HCDR2 region having an amino acid sequence selected from any one of SEQ ID No.: 50, 51, 52, 53, 54, 55, 56, 57, 58 and 59, preferably SEQ ID No.: 19 or 52, were obtained by maturation of an antibody having a light chain variable domain according to SEQ ID No.: 41 (comprising a LCDR3 according to SEQ ID No.: 43) in combination with a heavy chain variable domain according to SEQ ID No.: 42 (comprising a HCDR2 according to SEQ ID No.: 45). Based on these LCDR3 regions, the inventors identified a second anti-PD-L1 high affinity LCDR3 consensus sequence according to SEQ ID No.: 49, and a second anti-PD-L1 high affinity HCDR2 consensus sequence according to 60.

[0020] Thus, the antibody or antibody fragment according to the application can comprise a high affinity LCDR3 consensus sequence according to SEQ ID No.: 18 or SEQ ID No.: 49 and / or a high affinity HCDR2 consensus sequence according to SEQ ID No.: 30 or 60.

[0021] Preferably the antibody or antibody fragment comprises a light chain comprising a combination of the various LCDR1, LCDR2 and LCDR 3 disclosed herein, and / or a heavy chain comprising a combination of the various HCDR1, HCDR2 and HCDR 3 disclosed herein. Most preferably the antibody or antibody fragment comprises a light chain comprising a combination of the LCDR1, LCDR2 and LCDR 3 disclosed herein, and a heavy chain comprising a combination of the HCDR1, HCDR2 and HCDR 3 disclosed herein.

[0022] In a preferred embodiment, the antibody or antibody fragment comprises a variable light chain comprising a LCDR3 region according to SEQ ID No.: 18 in combination with a variable heavy chain comprising a HCDR2 region according to SEQ ID No.: 30, or a variable light chain comprising a LCDR3 region according to SEQ ID No.: 49 in combination with a variable heavy chain comprising a HCDR2 region according to SEQ ID No.: 60.

[0023] In a particularly preferred embodiment, the present application provides an anti-PD-L1 antibody having surprisingly high affinity, said anti-PD-L1 antibody comprising a light chain variable domain according to SEQ ID No.: 39 in combination with a heavy chain variable domain according to SEQ ID No.: 40, or comprising a light chain variable domain according to SEQ ID No.: 65 in combination with a heavy chain variable domain according to SEQ ID No.: 66.

[0024] The antibody or antibody fragment is preferably a fully canine antibody, optionally a recombinant canine antibody or antibody fragment. The antibody or antibody fragment can be used to treat a disease in a subject in need thereof, preferably a canine subject. The disease can for example be a cancer or an inflammatory or autoimmune disease.

[0025] Notably, the fully canine antibody according to the present application can overcome the immunogenicity deficiencies associated with chimeric antibodies derived from non-canine species such as rodent sources. Importantly, the chimeric antibodies described in the literature only display weak affinities which can severely limit their use as therapeutic antibodies.

[0026] Furthermore, the present application relates to a pharmaceutical composition comprising an antibody or antibody fragment, a polynucleotide encoding said antibody or antibody fragment, a vector comprising one or more polynucleotides disclosed herein. In a preferred embodiment, the pharmaceutical composition of the present application comprises a therapeutically effective amount of an antibody of the present application, further comprising a pharmaceutically acceptable carrier.

[0027] In one or more embodiments, the antibody or antibody fragment of the present application provides a method of treating a PD-L1 related disorder. In one or more embodiments, the PD-L1 related disorder is a cancer. In one or more embodiments, the type of cancer is selected from, but not limited to, melanoma, lung cancer, bladder cancer, renal cell carcinoma, head and neck cancer, breast cancer, esophageal cancer, and lymphoma. In a preferred embodiment, the PD-L1 disorder is melanoma.

[0028] In one or more aspects, the present application provides a method of treating a PD-L1 related disorder in a subject, said method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition of the present application.

[0029] In one or more embodiments, the present application provides a host cell producing any one or more of the antigen binding proteins of the present application.

[0030] In one or more embodiments, the present application provides a vector comprising any one or more of the polynucleotides encoding the antibody or antibody fragment of the present application.

[0031] In one or more embodiments, the present application provides a host cell comprising any one or more of the polynucleotides of the present application.

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

[0033] In one or more embodiments, the present application provides a host cell comprising any one or more of the nucleic acids of the present application.

[0034] In one or more aspects, the present application provides a method of producing an antigen binding protein of the present application by culturing a host cell of the present application under conditions such that the antigen binding protein is produced, and subsequently isolating the antigen binding protein from the host cell or the culture medium of the host cell.

[0035] In one or more aspects, the present application provides a kit comprising an antibody or antibody fragment of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 : Mature consensus sequences for LCDR3 (Figure 1A) and HCDR2 regions (Figure IB) based on CAN1005010.

[0037] Figure 2: Consensus sequences for mature LCDR3 (Figure 2A) and HCDR2 regions (Figure 2B) based on CAN1005001.

[0038] Figure 3: Neutralization of cPD-L1 binding and cPD-L1 / cPD-1 interaction by CAN1005001 and CAN1005010

[0039] Figure 4: Concentration dependent binding of CAN1005010 L1 and CAN1005010 H2 (Figure 4.A), CAN1005001 L1, CAN1005001 H3 and CAN1005001 (Figure 4.B) in a cPD-L1-biotin ELISA assay, and derived IC 50 values (Figure 4.C).

[0040] Figure 5: Concentration dependent binding of CAN1005016 (Figure 5.A) and CAN1005019 (Figure 5.B) in a cPD-L1-biotin ELISA assay.

[0041] Figure 6: Affinity measurements of CAN1005016 (Figure 6.A) and CAN1005019 (Figure 6.B) by GCI through 2BIND.

[0042] Figure 7: cPD-L1 binding of IgG candidates derived from cross-clonal maturation. Titration series of biotinylated CAN1005016 (Figure 7.A) and CAN1005019 (Figure 7.B) were added to plate-bound cPD-L1_hFc.

[0043] Figure 8: FACS binding of HEK293c18 cells transfected with target proteins CAN1005016 (Figure 8A), CAN1005019 (Figure 8B) and control IgG (Figure 8C). Figure 8C

[0044] Figure 9: ELISA-based ligand binding inhibition assay of CAN1005016 (Figure 9A), CAN1005019 (Figure 9B).

[0045] Figure 10 Figure 10: BLI-based PD-1 binding inhibition assay of CAN1005016 antibody candidate.

[0046] Definitions

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0048] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0049] In the context of quantities, "about" refers to an average deviation of + / - 20%, preferably + / - 10%, most preferably + / - 5% from the indicated value. For example, a quantity of about 20 mg / ml means 20 mg / ml + / - 6 mg / ml, preferably 20 mg / ml + / - 4 mg / ml, most preferably 20 mg / ml + / - 2 mg / ml. This also includes the value itself without any deviation. + + This also includes the value itself without any deviation.

[0050] All ranges recited in the specification and claims of this application include all numerical values and values from the range surrounding the recited range. The ranges of the present application are explicitly named and recited as including all integer, decimal and fractional values within the range. The term "about" can be used to describe a range.

[0051] ​​​As used herein, the term "antibody" or "polypeptide binder" includes intact antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chains thereof. A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region of an IgG, IgA, or IgD antibody comprises three domains, CH1, CH2, and CH3, while the heavy chains of an IgM and an IgE antibody comprise four domains, CH1, CH2, CH3, CH4. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FW). Each VHand VLcomprises three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. A polypeptide comprising the FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4 of the variable regions of the heavy and light chains can be referred to as a "VH or VL polypeptide". The variable regions of the heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) through Fc receptors and to the first component (C1q) of the classical complement system.

[0052] The ranges of the framework regions and CDRs of human antibodies have been precisely defined (see Kabat, 1991, J. Immunol. 147: 915-920.; Chothia and 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 framework regions of an antibody (i.e., the combined framework regions of the constituent light and heavy chains) are used to position and align the CDRs, which are primarily responsible for binding to an antigen. However, while canine antibodies can be partially aligned with human antibodies, the numbering schemes mentioned above are not ideally suited to describe the amino acid positions in an antibody heavy or light chain sequence. In the present invention, the following numbering scheme is used:

[0053] The antibody heavy chain is defined as VH FW1-HCDR1-FW2-HCDR2-FW3-HCDR3-FW4. Framework 1 (FW1) comprises 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 5 amino acids in length and is defined by positions X31 to X35 (X31, X32, X33, X34, X35). Framework 2 (FW2) is 14 amino acids in length and is defined by positions X36 to X49 (X36, X37, X38, X39, X40, X41, X42, X43, X44, X45, X46, X47, X48, X49). HCDR2 is defined by positions X50 to 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 to X94 and is 32 amino acids in length. HCDR3 is defined by positions X95 to X102 (X98, X99, X100, X100a, X100b, X100c, X100d, X100e, X100f, X100g, X101, X102, ). The length of this CDR is variable, represented by positions X100a to X100g which can or can not carry an amino acid. For the sake of clarity, if one of these positions is vacant, then the positions following up to X101 are also vacant. Framework 4 (FW4) is defined by positions X103 to X113 (X103, X104, X105, X106, X107, X108, X109, X111, X112, X113). The general concept of the numbering scheme is also depicted in Figure 9.

[0054] The antibody light chain is defined as VL FW1-LCDR1-FW2-LCDR2-FW3-LCDR3-FW4. Framework 1 (FW1) is defined by positions Y1 to Y23, with one length variation at position 10, for clarity, position Y10 can be vacant or have 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 11 amino acids in length and is defined by positions 24 to 34 (Y24, Y25, Y26, Y27, Y28, Y29, Y30, Y31, Y32, Y33, Y34). Framework 2 (FW2) is defined by positions 35 to 49 (Y35, Y36, Y37, Y38, Y39, Y40, Y41, Y42, Y43, Y44, Y45, Y46, Y47, Y48, Y49). LCDR2 is 7 amino acids in length and is defined by positions 50 to 56 (Y50, Y51, Y52, Y53, Y44, Y55, Y56). Framework 3 (FW3) is defined by 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 by positions Y89 to Y97 (Y89, Y90, Y91, Y92, Y93, Y94, Y95, Y95a, Y95b, Y95c, Y96, Y97). This CDR has a length variation, indicated by positions Y95a to Y95c which can or can not carry an amino acid. For clarity, if one of these positions is vacant, then the positions following up to Y91 are also vacant. Framework 4 (FW4) is defined by positions Y98 to Y107 (Y98, Y99, Y100, Y101, Y102, Y103, Y104, Y105, Y106, Y107).

[0055] The terms "antigen-binding portion" of an antibody or "fragment" are used interchangeably herein. These terms refer to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. It has been shown that 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 portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment, (Ward et al., 1989, Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). The preferred antigen-binding portion or fragment of an antibody is a Fab fragment. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988), Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies include one or more "antigen binding portions" of an antibody. These antibody fragments are obtained using conventional techniques known to those with ordinary skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen binding portions can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). One type of single domain antibody is a heavy chain variable domain (heavy chain antibody's heavy chain variable domain), referred to herein as "VHH", which is derived from an 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 binder derived from cartilaginous fish with heavy chain antibodies (IgNAR, "immunoglobulin new antigen receptor").Antigen-binding portions of antibodies can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies). Antigen-binding portions can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) called a single chain Fv (scFv; see, e.g., Bird et al., 1988 Science 242: 423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. USA 85: 5879-5883) that form a

[0056] The term“isolated” means that a compound, which can be, for example, an antibody or antibody fragment, is substantially free of other antibodies or antibody fragments of different antigenic specificities. In addition, an isolated antibody or antibody fragment can be substantially free of other cellular material and / or chemicals. Thus, in certain aspects, the provided antibodies are isolated antibodies that are separated from antibodies of different specificities. An isolated antibody can be a monoclonal antibody. An isolated antibody can be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to an epitope, isotype, or variant of a target can have cross-reactivity with other related antigens, e.g., antigens from other species (e.g., species homologs).

[0057] The term“fully canine antibody,” as used herein, refers to an antibody having variable regions in which both the framework and CDR regions are derived from canine sequences. For example, both the framework and CDR regions can be derived from canine sequences. In addition, if the antibody contains a constant region, that constant region is also derived from such canine sequences, e.g., canine germline sequences or mutated versions of canine germline sequences. The canine antibodies of the application can include amino acid residues that are not encoded by canine sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation).

[0058] The term“monoclonal antibody” or“monoclonal antibody composition,” as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0059] The term“germline” refers to a fully germline sequence and except for germline sequences that have been modified or engineered with minor mutations in the amino acid sequence, such as for the purposes of removing undesirable post-translational modification (PTM) sites, removing undesirable cysteines, optimizing the antibody (e.g., affinity, half-life), or introducing a desired restriction site or modifications resulting from errors in synthesis, amplification, or cloning.

[0060] As used herein, "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with the target at multiple sites through weak noncovalent forces, the more interactions, the stronger the affinity.

[0061] As used herein, the term "K D " refers to the dissociation constant, obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as molar concentration (M). KD values for antigen binding molecules, e.g., monoclonal antibodies, can be determined using methods established in the art. Methods for determining K D of antigen binding molecules, e.g., monoclonal antibodies, are SET (soluble equilibrium titration) or surface plasmon resonance using a biosensor system such as a Biacore® system.

[0062] The light chain variable domain and the heavy chain variable domain described herein are "combined" in reference to the light chain and the heavy chain pairing with each other. The pairing can be between different domains of one polypeptide chain comprising a VL polypeptide sequence and a VH polypeptide sequence, e.g., in a scFv, or between two polypeptide chains comprising a VL polypeptide sequence and a VH polypeptide sequence, e.g., in a full-length antibody or a Fab fragment.

[0063] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) incorporating the recombinant polynucleotide.

[0064] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids, such as hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon bonded to a hydrogen, a carboxyl, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise function in a manner similar to the naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a different structure from an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids are identified herein according to the one-letter or three-letter codes well known in the art.

[0065] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which 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 specified, a particular polypeptide sequence explicitly includes conservatively modified variants thereof.

[0066] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity," mean that two or more sequences are the same with respect to the identical or percent identical amino acid residues or nucleotides over a specified comparison window or designated region. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same, e.g., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity, over a specified comparison window or designated region, when compared and aligned for maximum correspondence, e.g., as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity is over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. Thus, a "substantially identical sequence" according to the present application can include any of the disclosed sequences with one or two or three or four or five amino acid exchanges, preferably one to three, more preferably one or two amino acid exchanges. Preferably, the exchanges can be conservative amino acid exchanges.

[0067] For sequence comparison, typically one sequence acts as the reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the program parameters. Determining percent sequence identity in the present application can be performed in a variety of ways known to one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALINETM (DNASTAR) software. One of skill in the art could routinely determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared. Unless otherwise indicated, alignment will be performed at the default settings of the alignment tool.

[0068] 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 terms are intended to also include progeny of the original cell which do not contain the identical sequence, but which have been modified in some way. For example, a mutation in a sequence contained in the recombinant host cell can be introduced deliberately, for example, by means of PCR mutagenesis, or by the mere fact that a sequence encoding a polypeptide can be modified by the cellular machinery.

[0069] The term "vector" refers to a polynucleotide molecule capable of trans-porting another polynucleotide to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA technologies are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector for recombinant DNA

[0070] By "cross-competition" is meant the ability of an antibody, antibody fragment, or other antigen binding moiety to interfere with the binding of other antibodies, antibody fragments, or antigen binding moieties 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 interferes with the binding of another antibody, antibody fragment, or antigen binding moiety to a particular antigen, and thus whether it can be said to cross-compete in accordance with the present application, can be determined using a standard competitive binding assay. One suitable assay involves the use of Biacore technology (e.g., by using a BlAcore 3000 instrument (Biacore, Uppsala, Sweden)) which can measure the extent of interaction using surface plasmon resonance technology. DETAILED DESCRIPTION

[0071] In a general aspect, the present application relates to an antibody or antibody fragment comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4 and / or a LCDR3 region according to SEQ ID No.: 18 or SEQ ID No.: 49. The antibody or antibody fragment can further comprise a HCDR1 region according to SEQ ID No.: 6 or SEQ ID No.: 44, a HCDR2 region according to SEQ ID No.: 30 or SEQ ID No.: 60 and / or a HCDR3 region according to SEQ ID No.: 8 or SEQ ID No.: 46.

[0072] The antibodies and antibody fragments described herein specifically bind to canine Programmed Cell Death 1 Ligand 1 (PD-L1), which can be characterized, for example, by the amino acid sequence according to NCBI Reference Sequence: NP_001278901.1 or according to SEQ ID NO: 67.

[0073] By maturation of an antibody having a light chain CAN1005010_VL variable domain according to SEQ ID No.: 1 (with a LCDR3 (CAN1005010_VL_LCDR3) according to SEQ ID No.: 5) in combination with a heavy chain variable domain according to SEQ ID No.: 2 (CAN1005010_VH) comprising a HCDR2 (CAN1005010_VH_HCDR2) according to SEQ ID No.: 7, several high affinity antibodies comprising a 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 a 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 were obtained. Based on these LCDR3 regions, the inventors could identify a first anti-PD-L1 high affinity LCDR3 consensus sequence according to SEQ ID No.: 18 and a first anti-PD-L1 high affinity HCDR2 consensus sequence according to SEQ ID No.: 30. The alignment leading to the LCDR3 consensus sequence according to SEQ ID No.: 18 and the HCDR2 consensus sequence according to SEQ ID No.: 30 is shown in Figure 1.

[0074] In an alternative aspect of the application, several high affinity antibodies comprising a LCDR3 region having an amino acid sequence selected from any one of SEQ ID No.: 47 and 48 and a HCDR2 region having an amino acid sequence selected from any one of SEQ ID No.: 50, 51, 52, 53, 54, 55, 56, 57, 9 and 59, preferably SEQ ID No.: 19 or 52, were obtained by maturation of an antibody having a light chain variable domain according to SEQ ID No.: 41 (CAN1005001_VL) comprising a LCDR3 according to SEQ ID No.: 43 (CAN1005001_VL_LCDR3) in combination with a heavy chain variable domain according to SEQ ID No.: 42 (CAN1005001_VH) comprising a HCDR2 according to SEQ ID No.: 45 (CAN1005001_VH_HCDR2). Based on these LCDR3 and HCDR3 regions, the inventors identified a second anti-PD-L1 high affinity LCDR3 consensus sequence according to SEQ ID No.: 49 and a second anti-PD-L1 high affinity HCDR2 consensus sequence according to SEQ ID No.: 60. The alignment leading to the LCDR3 consensus sequence according to SEQ ID No.: 49 and the HCDR2 consensus sequence according to SEQ ID No.: 60 is shown in Figure 2.

[0075] According to one aspect, the antibody or antibody fragment according to the application can comprise a LCDR3 region having an amino acid sequence selected from any one of SEQ ID No.: 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 43, 47 and 48. Preferably, the antibody comprises a LCDR3 according to SEQ ID No.: 9 or SEQ ID No.: 47.

[0076] Furthermore, the antibody or antibody fragment according to the application can comprise a HCDR2 region having an amino acid sequence selected from any one of SEQ ID No.: 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 comprises a HCDR2 according to SEQ ID No.: 19 or 52.

[0077] In one embodiment, the variable light chain can comprise a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4, and a LCDR3 region selected from any one of SEQ ID No.: 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, preferably SEQ ID No.: 5. This light chain can for example be combined with another independent embodiment of a variable heavy chain HCDR1 region according to SEQ ID No.: 6, a HCDR2 region having an amino acid sequence selected from any one of SEQ ID No.: 7, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, preferably SEQ ID No.: 19, and a HCDR3 region according to SEQ ID No.: 8.

[0078] Thus, a highly preferred embodiment of the application relates to an antibody or antibody fragment comprising a variable light chain comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4, and a LCDR3 region according to SEQ ID No.: 5, in combination with a variable heavy chain HCDR1 region according to SEQ ID No.: 6, a HCDR2 region according to SEQ ID No.: 19, and a HCDR3 region according to SEQ ID No.: 8.

[0079] In one embodiment, the variable light chain can comprise a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4, and a LCDR3 region according to SEQ ID No.: 43, 47, 48, preferably SEQ ID No.: 47. This light chain can for example be combined with an embodiment of a variable heavy chain HCDR1 region according to SEQ ID No.: 44, a HCDR2 region having an amino acid sequence selected from any one of SEQ ID No.: 45, 50, 51, 52, 53, 54, 55, 56, 57, 9, 59, preferably SEQ ID No.: 52, and a HCDR3 region according to SEQ ID No.: 46.

[0080] Thus, a highly preferred embodiment of the application relates to an antibody or antibody fragment comprising a light chain comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4 and a LCDR3 region according to SEQ ID No.: 47, in combination with a variable heavy chain HCDR1 region according to SEQ ID No.: 44, a HCDR2 region according to SEQ ID No.: 52 and a HCDR3 region according to SEQ ID No.: 46.

[0081] In a preferred embodiment, the antibody or antibody fragment comprises a variable light chain comprising a LCDR3 region according to SEQ ID No.: 18, in combination with a variable heavy chain comprising a HCDR2 region according to SEQ ID No.: 30, or a variable light chain comprising a LCDR3 region according to SEQ ID No.: 49, in combination with a variable heavy chain comprising a HCDR2 region according to SEQ ID No.: 60.

[0082] More preferably, the antibody or antibody fragment comprises a variable light chain comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4 and a LCDR3 region according to SEQ ID No.: 9, and / or a variable heavy chain comprising a HCDR1 region according to SEQ ID No.: 6, a HCDR2 region according to SEQ ID No.: 19 and a HCDR3 region according to SEQ ID No.: 8; the variable light chain and / or the variable heavy chain are for example comprised in the CAN1005016_VL (SEQ ID No.: 39) and CAN1005016_VH (SEQ ID No.: 40) variable chains.

[0083] In an alternative preferred embodiment, the antibody or antibody fragment comprises a variable light chain comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4 and a LCDR3 region according to SEQ ID No.: 47, and / or a variable heavy chain comprising a HCDR1 region according to SEQ ID No.: 44, a HCDR2 region according to SEQ ID No.: 52 and a HCDR3 region according to SEQ ID No.: 46; the variable light chain and / or the variable heavy chain are for example comprised in the CAN1005019_VL (SEQ ID No.: 65) and CAN1005019_VH (SEQ ID No.: 66) variable chains.

[0084] Table 1 : Sequences of antibodies and antibody fragments

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] In the consensus sequences 18, 30, 49 and 60, the sequence can comprise any amino acid at the specific position in parentheses, and / is separated "as an alternative." For example, position (G / S / W) in SEQ ID No 60 can be glycine, serine or tryptophan.

[0091] In addition to the CDR1 region, CDR2 region and CDR3 region, the variable light domain and the variable heavy domain comprise framework regions 1 to 4 in the direction from the N-terminus to the C-terminus. The antibody or antibody fragment according to the application can comprise one, several or all of the light chain variable domain framework sequences having an amino acid sequence selected from any one of SEQ ID No.: 31, 32, 33, 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 No.: 35, 36, 37, 38 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 No.: 61, 62, 63, 64. A heavy chain variable domain comprising HCDR1 and HCDR3 according to SEQ ID No 6 and 8 and HCDR2 sequence selected from any one of SEQ ID No.: 5, 9, 10, 11, 12, 13, 14, 15, 16, 17 preferably comprises a framework region selected from any one of SEQ ID No.: 35, 36, 37, 38, and a heavy chain variable domain comprising HCDR1 and HCDR3 according to SEQ ID No 44 and 46 and HCDR2 sequence selected from any one of SEQ ID No.: 51, 51, 52, 53, 54, 55, 56, 57, 58, 59 preferably comprises a framework region selected from any one of SEQ ID No.: 61, 62, 63 and 64.

[0092] In a preferred embodiment, the antibody or antibody fragment according to any one of the present application comprises a light chain variable domain according to SEQ ID No.: 1 or 41, more preferably SEQ ID No.: 39 or 65, and / or a heavy chain variable domain according to SEQ ID No.: 2 or 42, preferably SEQ ID No.: 40 or 66, more preferably a light chain variable domain according to SEQ ID No.: 39 in combination with a heavy chain variable domain according to SEQ ID No.: 40, or a light chain variable domain according to SEQ ID No.: 65 in combination with a heavy chain variable domain according to SEQ ID No.: 66.

[0093] The present application further provides an antibody or antibody fragment as described herein, wherein the antibody or antibody fragment comprises at least one LCDR, HCDR, framework region or variable domain chain having a sequence with at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID No.: 1 to SEQ ID No.: 66. Such an antibody or antibody fragment can be provided by introducing at least one, e.g. one, two, three, four mutations in the form of amino acid substitutions or amino acid deletions in the amino acid sequence of SEQ ID No.: 1 to SEQ ID No.: 66. Preferably, the mutations are conservative amino acid substitutions.

[0094] In a further aspect, the present application relates to an antibody or antibody fragment cross- competing with the above described antibody fragments, preferably with an antibody or antibody fragment comprising a light chain variable domain according to SEQ ID No.: 39 and / or a heavy chain variable domain according to SEQ ID No.: 40; or comprising a light chain variable domain according to SEQ ID No.: 65, and / or a heavy chain variable domain according to SEQ ID No.: 66.

[0095] The antibody or antibody fragment according to any one of the preceding claims, wherein the antibody or antibody fragment 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, 5 nM, and most preferably less than about 2 nM. The dissociation constant for the binding of the IgG antibody to the monovalent PD-L1 antigen is determined. The disclosed antibody and antibody fragment binding to canine PD-L1 is characterized, for example, by the amino acid sequence of FITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQHSSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQRISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINATANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTHFMILGPFLLLLGVVLAVTFCLKKHGRMMDVEKCCTRDRNSKKRNDIQFEET (SEQ ID No.: 67) according to the NCBI reference sequence: NP_001278901.1.

[0096] Antibodies can be at least approximately 2 x 10⁻⁶. 4 [M -1 s -1 Preferably at least about 5 x 10 4 [M -1 s -1 More preferably about 1x10 5 [M -1 s -1 K on Rate, and / or at less than about 1 x 10 -3 [s -1 Preferably below about 5 x 10 -4 [s -1 or less than approximately 2.5 x 10 -4 [s -1 K off The rate of binding to canine PD-L1. In one embodiment, the antibody's K... on For at least approximately 1 x 10 5 [M -1 s -1 ], and K off Below approximately 2.5 x 10 -4 [s-1 ]. The dissociation constant of the binding of the IgG antibody to the monovalent PD-L1 antigen is determined. In another embodiment, the K on of at least about 7.5 x 10 4 [M -1 s -1 ] and a K off of lower than about 2.5 x 10 -4 [s -1 ]. The dissociation constant of the binding of the IgG antibody to the monovalent PD-L1 antigen is determined.

[0097] The antibody or antibody fragment according to the present application can interfere with or block the interaction between canine PD-L1 (cPD-L1) and canine PD-1 (cPD-1), as demonstrated in Example 8. For example, the antibody or antibody fragment can block the interaction between cPD-L1 and cPD-1 with an IC 50 value of lower than about 100 nM, preferably lower than about 50 nM, or lower than 20 nM, more preferably lower than about 10 or lower than about 5 nM, most preferably lower than about 3 nM or even lower than about 2 nM. The IC 50 value of the interaction of cPD-L1 with cPD-1 can be determined, for example, by a biolayer interferometry (BLI)-based ligand binding inhibition assay or an ELISA ligand binding inhibition assay (ELISA lbia).

[0098] According to a further aspect, the antibody or antibody fragment according to the present application is an antibody fragment selected from Fv, scFv, Fab, Fab', F(ab')2, Fab'-SH or VHH.

[0099] The antibody or antibody fragment according to any one of the present application can comprise a Fc fragment or a fragment thereof selected from the canine isotype A of immunoglobulin G (also referred to as HC-A, HCA, calgG-A), the isotype B of immunoglobulin G (also referred to as HC-B, HCB, calgG-B), the isotype C of immunoglobulin G (also referred to as HC-C, HCC, calgG-C) or the isotype D of immunoglobulin G (also referred to as HC-D, HCD, calgG-D). Preferably, the Fc domain is selected from the isotype B. The Fc domain comprised in the antibody or antibody fragment can have a wild-type sequence or a mutated sequence.

[0100] According to the therapeutic application, the selection of the respective antibody isotype is crucial and needs to be considered whether binding to the humoral or cellular component of the immune system is beneficial or even can lead to unwanted side effects of the drug. For example, a therapeutic antibody against tumor cell growth or a pathogen should have a strong effector function. In contrast, targeting soluble mediators or cell surface receptors of healthy cells to prevent receptor-ligand interactions usually requires the absence of any CDC or ADCC activity to prevent target cell death or unwanted cytokine secretion. Fields of diseases requiring silent antibody formats contain, but are not limited to, inflammatory diseases (e.g. rheumatoid arthritis, psoriasis, inflammatory bowel disease), allergies (e.g. asthma), pain (e.g. osteoarthritic pain, cancer pain, lower back pain) and ocular diseases (e.g. age-related macular degeneration). Depending on the target, the absence of CDC or ADCC can be desirable in antibodies for the treatment of cancer (e.g. antibodies targeting PD-L1 according to the present application).

[0101] The term "Fc fragment" relates to a fragment of an immunoglobulin comprising at least part or all of the constant heavy chain region 2 (C2 or CH2) and the constant heavy chain region 3 (C3 or CH3), or the fragment of an immunoglobulin obtained by papain digestion which is a crystallizable fragment of an immunoglobulin. The fragment is understood to be part of a larger polypeptide sequence. Thus, the "fragment" will typically have amino acid sequences bound to the C-terminus and / or N-terminus. The terms "C2" or "CH2" and the terms "C3" or "CH3" can be used interchangeably. Furthermore, the terms "Fc region" and "Fc domain" can be used interchangeably when referring to the immunoglobulin Fc CH2 and CH3 sequences, unless explicitly stated otherwise. In the context of the present application, the boundaries of the CH2 and CH3 regions of the 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.

[0102] Wild-type Fc fragments of different isotypes can have sequences as disclosed in Table 2.

[0103] Table 2: Wild-type canine Fc sequences of different isotypes

[0104]

[0105]

[0106] The Fc fragment comprised in the antibody or antibody fragment can further comprise a substitution in any of the wild-type sequences disclosed in Table 2. Substitutions that can be comprised are disclosed in WO 2021 / 165417 Al, which is incorporated herein by reference. In particular, the Fc fragment comprises at least one substitution of an amino acid selected from at least one of amino acid positions 235, 239, 270, and / or 331 relative to a wild-type Fc fragment. Preferably, the mutation is in an Fc fragment of the isotype B from canine IgG.

[0107] Preferably, the Fc fragment comprises at least two substitutions of an amino acid selected from at least two of the amino acids at positions 234, 235, 239, 270, and / or 331. More preferably, the 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.

[0108] In another preferred embodiment, the Fc fragment, wherein the Fc fragment comprises at least three substitutions of an amino acid selected from at least three of amino acid positions 234, 235, 239, 270, and / or 331. More preferably, the three amino acid positions are 235, 239, and 270; 239, 270, and 331; 235, 270, and 331; or 235, 239, and 331 in a wild-type Fc sequence disclosed in Table 2.

[0109] In another preferred embodiment, the present application relates to a polypeptide comprising at least one canine or feline Fc fragment, wherein the Fc fragment comprises at least four amino acid substitutions selected from amino acid positions 234, 235, 239, 270, and 331, more preferably 235, 239, 270, and 331, and most preferably the amino acids L235, S239, D270, and P331. Most preferably, the Fc fragment comprises the mutations L235A, S239A, D270A, and P331G relative to a wild-type Fc fragment.

[0110] The corresponding substitution in the Fc fragment results in reduced binding affinity to Clq and / or Fc receptor relative to a polypeptide comprising the corresponding wild-type Fc fragment. Under physiological conditions, where the immune system is not compromised, reduced or diminished binding to Clq and / or FcyRI results in reduced or complete ablation of the immune effector function of complement-dependent cytotoxicity (CDC) and induction of antibody-dependent cellular cytotoxicity (ADCC). Reduced or diminished binding of a polypeptide comprising at least one substitution in the Fc fragment to Clq and / or FcyRI and / or the resulting reduction or complete ablation of CDC or ADCC is often also referred to as silencing. In the Fc fragment of canine isotype B, the Fc fragment retains its ability to bind to the neonatal Fc receptor (FcRn) and to Protein A.

[0111] The antibody or antibody fragment can comprise an Fc fragment with reduced or complete ablation of CDC or ADCC as described above, preferably for use in inflammatory diseases, allergies, pain and ocular diseases.

[0112] In a preferred embodiment, the antibody comprises a light chain comprising a light chain variable domain (VL) and a lambda constant domain (CL) as disclosed herein and a heavy chain comprising a heavy chain variable domain (VH) linked via a CH domain to an Fc fragment comprising a CH-2 and a CH-3 domain as described above. The CH-1 domain can be linked to the CH-2 domain via an amino acid sequence which is referred to as "hinge" or alternatively as "hinge region". Suitable 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 via disulfide bonds and each heavy chain is preferably linked to one of the light chains via a disulfide bond.

[0113] In a particular embodiment of the application, the antibody comprising a light chain and a heavy chain can comprise a light chain according to SEQ ID No: 72 or 74 and / or a heavy chain according to SEQ ID No: 73 or 75, preferably a light chain according to SEQ ID No: 72 in combination with a heavy chain according to SEQ ID No: 73 or a light chain according to SEQ ID No: 74 in combination with a heavy chain according to SEQ ID No: 75.

[0114] Table 3: Full length antibody sequences:

[0115]

[0116]

[0117] In a preferred aspect of the application, the antibody or antibody fragment is a fully canine antibody or antibody fragment. In a preferred aspect of the application, the antibody or antibody fragment is an isolated antibody or antibody fragment. Preferably, the antibody or antibody fragment according to the application is a monoclonal antibody or antibody fragment.

[0118] In a further aspect, the antibody or antibody fragment is a recombinant antibody or antibody fragment. A "recombinant" antibody is an antibody produced in a cell of a different species than the species from which the genome of the antibody is derived. Suitable cells for recombinant expression of an antibody according to the application include, inter alia, 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. In the context of the present application, synthetic or semi-synthetic derived antibodies are also considered recombinant antibodies.

[0119] In a further aspect, the present application relates to a pharmaceutical composition comprising an antibody or antibody fragment as described herein, optionally together with a pharmaceutically acceptable carrier or excipient. A "pharmaceutical composition" is a composition comprising an antibody or antibody fragment according to the application and a further compound which is toxicologically acceptable and which enables the storage of the antibody or antibody fragment according to the application and its administration to a subject in need of treatment and allows the antibody or antibody fragment to exert its intended pharmacological and biological activity.

[0120] A pharmaceutically acceptable carrier or excipient can include an agent that is non-toxic to the cells or mammals exposed thereto at the doses and concentrations employed, such as a diluent, stabilizer, adjuvant, or other type of excipient. Examples of pharmaceutically acceptable carriers include aluminum oxide; aluminum stearate; lecithin; serum proteins, such as human serum albumin, canine or other animal albumin; buffers, such as phosphate, citrate, tromethamine, or HEPES buffers; 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, cellulose-based substances; polyethylene glycol; sucrose; mannitol; or amino acids, including but not limited to arginine.

[0121] The present application further relates to an antibody or antibody fragment or a pharmaceutical composition as described herein for use in the treatment of a subject, preferably a canine subject, in need thereof or a method of treatment thereof. Preferably, the treatment or method of treatment is for the treatment of a disease. According to the present application, the method of treatment of a disease encompasses the step of administering an antibody or antibody fragment or a pharmaceutical composition as described herein to a patient in need of treatment, preferably to a canine subject. Preferably, the disease is a cancer or an inflammatory disease or an autoimmune disease. Preferably, the cancer is colorectal cancer, melanoma, oral malignant melanoma, osteosarcoma, mastocytoma, angiosarcoma, hepatocellular sarcoma, squamous carcinoma, adenocarcinoma of the nasal gland, transitional cell carcinoma, anal cystadenocarcinoma, soft tissue carcinoma, breast carcinoma, histiocytic sarcoma, diffuse large B-cell carcinoma, gastric carcinoma (e.g., gastric adenocarcinoma), lymphoma, non-small cell lung carcinoma, small cell lung carcinoma, renal cell carcinoma, head and neck carcinoma.

[0122] According to a further aspect, the antibody or antibody fragment described herein can be used in a diagnostic assay, a diagnostic method, etc. The method or assay can comprise the step of detecting canine PD-L1.

[0123] In a further aspect, the present application relates to a polynucleotide or polynucleotides encoding an antibody or antibody fragment according to the present application. The polynucleotide or polynucleotides can be an isolated polynucleotide. The polynucleotide or polynucleotides can be comprised in a vector, such as a plasmid or an artificial chromosome. The polynucleotide can be operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the transcriptional and translational control sequences functionally act to transcribe and translate the antibody or antibody fragment to express the encoded antibody or antibody fragment.

[0124] The vector and / or the polynucleotide or polynucleotides can be comprised in a cell. Preferably, the cell is a host cell suitable for recombinant expression of the 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 exemplary mammalian cells include, but are not limited to, NSO cells, 293 cells, and CHO cells, and cell lines derived therefrom, e.g., 293-6E, DG44, CHO-S, and CHO-K cells.

[0125] Examples

[0126] Generation of canine antibodies specific for canine PD-L1

[0127] A series of canine antibodies was generated that specifically bind to canine PD-L1. Sequences of the respective antibodies were isolated using phage display from a synthetic fully canine antibody library disclosed in WO 2018 / 234438 Al. The obtained antibodies inhibit the binding of canine PD-1 to canine PD-L1. Affinity maturation was performed on individual inhibitory clones to increase the binding affinity while maintaining epitope specificity.

[0128] Example 1:

[0129] Generation and screening of anti-canine PD-L1 antibodies

[0130] Phage display selection can be done as described below or by another method known to the person skilled in the art. To increase the likelihood of finding different binding antibodies, different panning strategies were applied (e.g. solid phase panning, solution panning, semi-solution panning, Fc-capture panning). Selection against cPD-L1 as Fc fusion protein (cPD-L1 hFc; Sino Biological; Cat. 70110-D02H) was performed in 3 rounds of solid phase panning as well as solution and semi-solution panning as described below. Due to the nature of the antigen as Fc-fusion protein, phage against human IgG was blocked to eliminate potential Fc-reactive candidates. A common point of all panning strategies applied was a decreasing amount of antigen per round while increasing washing stringency. Phage output after 3 rounds of panning was subcloned into a bacterial Fab expression vector before screening in ELISA format.

[0131] Solid phase panning

[0132] When performing solid phase panning, cPD-L1 hFc was immobilized on the surface of a microtiter plate (Maxisorp, 96-well flat bottom) for at least 1 hour at room temperature. In the first round of panning, a concentration of 3 pg / ml of cPD-L1 was used, together with 5 pg / ml of human serum IgG (huIgG; Jackson ImmunoResearch; Cat.: 009-000-003) for blocking, which was also immobilized on the plate. For each selection, 250 mI / 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) before adding 300 mI / well of blocking buffer (Chemiblocker, Merck-Millipore).

[0133] At the same time, the phage library was blocked with blocking buffer for 1 h at room temperature. Due to the hFc-tag, 10 pg / ml human serum IgG was added to the blocking buffer.

[0134] For each panning subcode, 20 ml 2xYT medium was incubated with E. coli ER2738 from M9 minimal agar plates in phage working space and the culture was then used to infect the selected phage. The culture was shaken at 160 rpm and 37°C until an OD of 0.6 was reached 600 nm. The E. coli culture was kept on ice until the eluted phage needed to be infected.

[0135] After antigen coating and blocking, the blocked phage was transferred to the respective well and incubated for 1 h at room temperature. To remove non-specific or weakly bound phage, several washing steps with PBST and PBS were performed. In the first panning, a standard washing procedure (5x PBST fast, 3x PBST for 5 min, 3x PBS fast) was applied. Depending on the phage output titer, the stringency was adjusted in the next round by increasing the number and time of washing steps.

[0136] After antigen-phage incubation and washing, the bound phage was eluted by adding trypsin (250 mI of 10 pg / ml trypsin solution in PBS for 30 min at 37°C) to cleave the protease-sensitive linker between the antibody fragment and the gIII protein. Each selected phage suspension was transferred separately into a pre-warmed E. coli ER2738 culture and incubated for 45 min at 37°C in a water bath without shaking. The bacterial culture was centrifuged, the supernatant was removed and the pellet was resuspended in 2xYT medium. The bacteria were plated on LB / Cam agar plates and incubated overnight at 37°C. The next day, the bacteria were scraped from the plates with frozen medium (2xYT medium containing 34 pg / ml chloramphenicol (Cam), 1% glucose and 15% glycerol) and aliquots were stored at -80°C before phage were prepared for the subsequent panning round.

[0137] Solution and semi-solution panning

[0138] For solution and semi-solution panning, magnetic beads (GE Healthcare, Sera-Mag Streptavidin-coated magnetic particles, Cat. No. 30152104010150) were used in combination with biotinylated cPD-L1_hFc (Yikai, Cat. No. 70110-D02H-B) to capture antigen-phage complexes or to immobilize antigens on the bead surface, respectively. To reduce phage selection against the used magnetic beads, the streptavidin-coated beads mentioned before were replaced by neutravidin-coated beads (GE Healthcare, Sera-Mag Neutravidin-coated magnetic particles, Cat. No. 78152104010150).

[0139] Before panning, beads were washed and blocked. For semi-solution panning, phage were also loaded with antigen. To this end, 250 μΐ beads per selection were transferred to 2 ml low-binding tubes, beads were captured with a magnetic particle separator, and storage solution was removed. Then, beads were washed 3 times with PBS, and beads were collected using a magnet to remove the wash buffer. Subsequently, beads were blocked in blocking solution (100% Chemiblock) for 1 hour at room temperature. In parallel, phage were blocked. To this end, the required volume of phage was mixed with Chemiblock containing 10 μg / ml huIgG for blocking and incubated for at least 1 hour at room temperature. In addition, blocked phage were pre-absorbed on empty magnetic beads to remove sticky phage. To remove biotin-specific and hFc-tag-specific phage before panning, phage were also incubated in 96-well plate wells coated with 5 μg / ml BSA-biotin for 45 minutes and in wells coated with 5 μg / ml huIgG overnight at 4°C, respectively.

[0140] After blocking of beads and blocking / pre-absorption of phage, biotinylated antigen was added to the phage solution and incubated for 1 hour at room temperature with rotation. To capture phage / antigen complexes, blocking buffer was removed from the beads, and phage were added to allow binding of biotinylated cPD-L1_hFc for 20 minutes at room temperature. Subsequently, non-specific phage were removed by washing (3x PBST fast, 3x PBST for 5 minutes, 3x PBS fast). Washing stringency was adjusted from round to round according to panning output. In the last washing step, magnetic beads with captured antigen-phage complexes were transferred to fresh low-binding tubes.

[0141] For elution of specific phage, 300 μΐ of trypsin was added for 30 minutes at 37°C. Subsequently, each selected phage suspension was transferred into 20 ml of pre-warmed E. coli ER2738 culture, respectively, and incubated exactly for 45 minutes at 37°C in a water bath without shaking. The bacterial culture was centrifuged for 5 minutes at 4600 rpm at 4°C and the supernatant was discarded. The pellet was resuspended in 600 μΐ of 2xYT medium and plated on large LB / Cam agar plates and incubated overnight at 37°C. The next day, bacteria were scraped from the plates with 1-3 mL of frozen medium (2xYT medium containing 34 μg / ml Cam, 1% glucose and 15% glycerol) using a sterile Drygalski spatula and aliquots were stored at -80°C before phage were prepared for the subsequent panning round.

[0142] Semi-solution panning was performed in a similar way as solution panning with the following modification: the respective antigen was already fixed on the beads before blocking the beads, instead of using streptavidin or neutravidin magnetic beads to capture phage complexes bound to biotinylated target from solution. Thus, the panning mode reflects a selection to a solid phase compared to panning where the antigen is coated on the surface of a microtiter plate, but allows better target orientation and washing conditions.

[0143] Similar to solid phase panning, 3 rounds were performed, each with decreasing cPD-L1_hFc concentration and increasing washing stringency.

[0144] Phage preparation

[0145] For each phage preparation, the inoculation medium (2xYT medium containing 34 μg / ml Cam and 1% glucose) was inoculated with the phagemid containing the bacterial suspension or the glycerol stock, resulting in an OD 600 of about 0.2. The culture was incubated for 60-120 minutes at 37°C with shaking until an OD 600VCSM13 helper phage was added and incubated at 37°C for 30 minutes without shaking and then at 37°C for 30 minutes with shaking at 250 rpm. Subsequently, the bacteria were spun down and the supernatant containing the helper phage was discarded. The phage infected bacteria were resuspended in induction media (2xYT media containing 50 pg / ml Carbenicillin, 50 pg / ml Kanamycin (Kan) and 0.2 mM IPTG) and incubated at 22°C for 18-20 hours in a phage shaker at 200 rpm. The next day, the bacteria were spun down and the supernatant containing phage presenting the antibody 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 with gentle shaking for at least 30 minutes. The precipitated phage was spun down at 10000 x g for at least 30 minutes at 4°C. The supernatant was quantitatively removed and the phage pellet was resuspended in a sufficient volume of PBS. The phage was stored at 4°C for short periods or at -80°C for longer periods.

[0146] Subcloning and generation of screening plates

[0147] After multiple rounds of panning, the polyclonal phage output was subcloned into the bacterial Fab expression vector pCaBx. The antibody encoding fragments were removed from the phage display vector using flanking restriction enzymes, separated using preparative agarose gel electrophoresis (1.0% agarose) and the fragments from the gel slices were DNA purified using appropriate gel extraction kits. According to standard procedures, ligation reactions were performed with the inserts and pre-cut pCaBx vector and subsequently transformed in chemically competent E. coli BL21 (DE3).

[0148] Expression plates (well of round bottom 96 well plates (e.g. Thermo Fisher, Cat. No. 262162) filled with 80 μl / well of all-in-one medium (2x YT medium containing 34 μg / ml Cam, 0.1 % glucose and 0.5 mM IPTG) and inoculated with single colonies from agar plates of the subcloning procedure. The plates were incubated at 37 °C with 600 rpm shaking for 5 h and afterwards overnight at 22 °C with 600 rpm shaking. For the preparation of crude bacterial lysates for screening purposes, so-called BEL lysates, 30 μl / well of lysis buffer (2x BBS containing 2.5 mg / ml lysozyme, 4 mM EDTA and 13 U / ml nuclease (Benzonase)) were added and incubated at 22 °C with 600 rpm shaking for 1 h. Then, 30 μl / well of blocking buffer (1x PBS containing 5 % milk powder) were added and incubated at 22 °C with 600 rpm shaking for 1 h. The plates were stored at -20 °C or directly used for screening.

[0149] Screening ELISA and identification of unique clones

[0150] For screening, the binding of lysates containing antibody fragments (BEL lysates) to antigens immobilized on Maxisorp microtiter plates was tested. After immobilization of the antigens on the respective surface, the plates were washed three times with PBST and subsequently blocked with 5 % milk powder in PBST for 1 h at room temperature. The BEL lysates, control antibodies and negative controls were transferred to the plates and incubated for 1 h at room temperature. The plates were washed 3 times with PBST and subsequently detection antibodies were diluted in PBST 0.5 % milk powder and added to the plates and incubated for 1 h at room temperature. For detection, an anti-FLAG antibody (Sigma Aldrich, 109143-MM13) was used. The plates were washed 5 times with PBST and detection of bound antibodies was performed using QuantaBlu reagent on a Tecan Genious reader (excitation filter: 320 nm, emission filter: 430 nm) according to the manufacturer’s instructions.

[0151] Individual clones identified by screening in ELISA format were subjected to Sanger sequencing. Therefore, micro-preparation cultures from the respective positive hits were inoculated from the glycerol stocks of the individual samples. The plasmids were purified with standard protocols and sent to an external service provider for sequencing using primers covering all CDR regions of the library design variable.

[0152] Approximately 65 different antibody variants were identified after screening. Two sequence unique candidates with promising binding properties were characterized in more detail, called CAN1005001 (which comprises VL and VH domains according to SEQ ID Nos: 41 and 42) and CAN1005010 (which comprises VL and VH domains according to SEQ ID Nos: 1 and 2).

[0153] Example 2:

[0154] Binding ELISA with initial clones from panning CAN1005001 and CAN1005010

[0155] To characterize the binding of CAN1005001 and CAN1005010 to their target cPD-L1, an ELISA setup was used. The cPD-L1 fusion protein was directly immobilized on the plate, usually at a concentration of 57 nM. The coated plates were washed 3 times with PBS-T, followed by blocking with Chemiblocker (Merck Millipore; 2170) for 1 hour at room temperature. After blocking, a dilution series of antibody solution was added to the plates, incubated for 1 hour at room temperature, followed by washing and addition of the appropriate detection antibody coupled to HRP. Measurements were taken as described above for the screening ELISA. The results of this experiment are shown together with Example 3.

[0156] Example 3:

[0157] Ligand binding inhibition assay with initial clones CAN1005001 and CAN1005010

[0158] To demonstrate 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 was present on the plate and the accessible binding site was blocked with CAN1005001 and CAN1005010, respectively. The lack of accessible binding site for PD-1 was then analyzed to measure the residual binding of PD-1 to PD-L1_hFc.

[0159] ELISA ligand binding inhibition assay (LBI)

[0160] cPD-L1 fusion protein was directly immobilized on plates at 4°C overnight, usually at a concentration of 57 nM. For the LBII ELISA, black 384 well Maxisorp plates were used. Coated plates were washed 3 times with PBS-T, followed by blocking with Chemiblocker (Merck Millipore; 2170) for 1 hour at room temperature. Blocked plates were washed 3 times with PBS-T, followed by addition of a titration series of blocking antibodies and incubation for 1 hour at room temperature. Subsequently, plates were washed again 3 times with PBS-T and PD-1-biotin (1x biotinylated through Avi-tag; Yiergen, Cat: 70109-D27H-B) was added and incubated for 1 hour at room temperature, usually at a concentration of 4 pg / ml (206 nM). This concentration of canine PD-1-biotin was identified as optimal concentration by titration of PD-1-biotin binding to immobilized canine PD-L1_hFc. After incubation with the ligand PD-1, plates were washed 3 times with PBS-T, the appropriate detection reagent for biotinylated PD-1 was added, usually streptavidin-HRP, and incubated for 1 hour at room temperature. Subsequently, plates were washed 5 times, followed by addition of Quanta Blue and measurement as described above for the screening ELISA.

[0161] The results are depicted in Figure 3. CAN1005001 and CAN1005010 show dose-dependent binding to cPD-L1. In line with this, increasing the amount of both antibodies efficiently blocked the binding of cPD-1 to immobilized cPD-L1. For CAN1005001, an EC50 value of 0.4 nM was calculated, and an IC 50 value of 4.6 nM.

[0162] Example 4:

[0163] Affinity maturation of CAN1005001 and CAN1005010 and screening in Fab format

[0164] The LBII experiment showed that a subset of some clones, including CAN1005001 and CAN1005010, can block the interaction between canine PD-1 and canine PD-L1. An affinity maturation strategy was initiated to further increase the binding strength and potency of the respective candidates.

[0165] The maturation approach focuses on two sites within these antibodies: in the heavy chain, HCDR2 is modified, while on the light chain, LCDR3 is removed and replaced by a corresponding maturation module. The maturation modules are designed in a way that reflects the natural diversity within the corresponding CDR, without key PTM sites, and are highly diverse. For the sake of clarity, a "VH-matured clone" contains the original HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3, but the HCDR2 region is changed, while a "VL-matured clone" has a unique LCDR3 sequence, while the remaining CDRs are identical to the parental clone. It is worth noting that the matured chains can also be combined to generate so-called crossover clones with modifications in both VL and VH. It is generally accepted that there is a correlation between library size and the chance of strong affinity improvement. Therefore, the goal is to make libraries with a range of more than 1.00E+07 variants.

[0166] Prior to affinity maturation, the Fab-encoding inserts of the selected candidates were digested by EcoRI / Ncol and ligated into the phage display vector according to standard procedures. Afterwards, VH or VL stuffer sequences were ligated into the candidates before they were replaced by HCRD2 or LCDR3 maturation modules.

[0167] For the preparation of the vector backbone for the introduction of the maturation modules, the stuffer sequences were removed again by restriction digestion using BssHII and MfeI for the VH stuffer sequence and BbsI and Kpnl for the VL stuffer sequence according to standard procedures.

[0168] For the VL maturation library, the maturation module of the LCDR3 region was cloned into the vector backbone using BbsI and Kpnl as described above. For VH maturation, the HCDR2 module was introduced by BssHII and MfeI cloning analogously.

[0169] For library cloning, high-competence ER2738 cells were used for transformation and DNA was desalted by precipitation prior to electroporation. For this, the ligation sample was adjusted to 50 μΐ with sterile ddH20 and 1 μΐ of glycogen and 500 μΐ of 2-butanole were added and incubated for at least 5 min at room temperature on a rotator. Then, the precipitated DNA was spun down in a table centrifuge at maximum speed for 30 min at 4°C. The supernatant was discarded, the DNA pellet was washed with 500 μΐ of pre-cooled 70% ethanol. The sample was again centrifuged for 15 min at maximum speed at 4°C, the ethanol was removed and the DNA pellet was air-dried for about 15 min. The pellet was resuspended in 5 μΐ of ddH20 according to the ligation protocol.

[0170] For each library, 2 transformations in ER2738 cells were performed using a BTX electroporator (settings: 25 μΐ, 200 Ω, 1.6 kV) according to the manufacturer's instructions. Immediately after pulsing, the cells were transferred into 950 μΐ pre-warmed recovery medium and incubated at 37°C with 200 rpm gentle shaking for 1 hour. A small aliquot of the recovered culture was saved for library size determination. The remaining culture was spun down at 4600 x g for 10 minutes, resuspended in 400 μΐ 2xYT, plated on 2 large LB / Glu / Carb agar plates and incubated at 37°C overnight. The next day, the bacteria were scraped off the plates using pre-cooled LB medium containing 20% glycerol, aliquots were prepared and stored at -80°C until subsequent phage preparation.

[0171] Library sizes were determined using an Eddy Jet spiral plater and each library exceeded 2.00E+08 clones. QC was performed by colony PCR and 10 clones per library were sequenced to confirm diversity and absence of parental clones and a live cell count was performed to assess the quality of the frozen glycerol stock.

[0172] Phage preparation was performed essentially as described above (Example 1) but on a larger scale to reflect the higher number of individual clones within the library and the requirement to also cover this greater diversity in phage production.

[0173] Mature panning was performed essentially as described above (Example 1) but mainly using solution panning. In contrast to the initial panning, the mature panning was performed under more stringent conditions. This was achieved by lowering the antigen concentration during panning, i.e. for the first round of panning, a lower range of target amounts was used as in the third round of initial panning. In addition, the washing steps were prolonged and the number was also increased. Furthermore, a k off Selection step. After capturing the antigen-phage complex using magnetic streptavidin beads and washing the beads to remove unwanted phage, a 10-fold molar excess of non-biotinylated antigen was added to the washing buffer and incubated overnight. During this step, low affinity antibodies will dissociate from the antigen captured on the beads and search for other interaction partners in solution rather than on the bead surface. Thus, only high affinity antibodies are recovered. k off After the selection step, the supernatant containing the lower affinity antibodies was removed, the beads were washed again with PBS and elution was performed as described before.

[0174] Mature panning successfully identified 186 clones that showed better signal than the corresponding parental clones. This included the mature panning campaign of other parental clones. For parental clone CAN1005010, clones CAN1005010L1 (comprising SEQ ID NO: 9) from the LCDR3 mature campaign and CAN1005010H2 (comprising SEQ ID NO: 19) from the HCDR2 mature campaign were selected for further study. Respectively, for parental clone CAN1005001, clones CAN1005001L1 (comprising SEQ ID NO: 47) from the LCDR3 mature campaign and CAN1005001H3 (comprising SEQ ID NO: 52) from the HCDR2 mature campaign were selected for further study.

[0175] Fab-capture ELISA screening

[0176] To identify derivatives with higher affinity, a screening was performed using an alternative ELISA setup to better normalize BEL expression levels and more effectively discriminate Fabs according to their affinity.

[0177] In contrast to the previous screening ELISA setup, Maxisorp plates were coated with low density anti-canine Fab antibody at 1 pg / ml overnight. Coated plates were washed 3 times with PBS-T before loading with diluted BEL. After BEL incubation and washing with PBS-T, a dilution series of cPD-L1-biotin was added to the plates. Detection was performed with streptavidin-HRP followed by 5 more stringent washes with PBS-T and addition of Quanta Blue substrate. Measurement was performed as previously described (Example 1). The resulting data is shown in Figure 4.

[0178] Derivatives from both heavy and light chain maturation were identified that bound cPD-L1 significantly better than the parental clones. This is manifested in EC50 values of 362 pM for CAN1005001L1 and 97 pM for CAN1005001H3 compared to 820 pM for parental clone CAN1005001. For CAN1005010L1 and CAN1005010H2, EC50 values of 139 pM and 121 pM were determined, respectively. The experiment shown does not include parental clone CAN1005010.

[0179] Example 5:

[0180] Cross-cloning of different mature heavy and light chains from CAN1005010 and CAN1005001

[0181] To further increase the affinity of the CAN1005010 derivative CAN1005010L1 and CAN1005010H2 (comprising SEQ ID NO: 9 and 19), the respective mature light and heavy chains were combined in a single Fab, which shares only the HCDR3 with CAN1005010 and contains both the mature LCDR3 and HCDR2. In the same way, the respective mature light and heavy chains from the CAN1005001 derivative and CAN1005001H3 (comprising SEQ ID NO: 47 and 52) were combined in a single Fab.

[0182] The resulting clones were named CAN1005016 and CAN1005019, respectively. Cloning was performed according to standard procedures as described below. This involved the primary digestion of two clones, one of which served as the vector backbone and the other as the insert. In this case, the LCDR3 matured clones CAN1005010L1 (comprising SEQ ID NO: 39) and CAN1005001L1 (comprising SEQ ID NO: 65) served as the vector backbone. The resulting Fabs were tested in a Fab-capture ELISA as previously reported in Example 4.

[0183] Restriction digestion

[0184] 5 μg of both vectors were digested with Mfel and Xhol to remove the heavy chain coding region. The restriction digest was performed at 37°C for 1 hour, followed by inactivation at 80°C for 20 minutes. The backbone sample digest further contained FastAP to inhibit vector degradation. For CAN1005010L1, the vector lacking the heavy chain was excised from an agarose gel and purified by standard procedures. The corresponding insert derived from clone CAN1005010H2 was also applied to an agarose gel and excised and purified by standard procedures.

[0185] Ligation

[0186] After restriction digestion, the insert was ligated into the vector backbone. A 5:1 mixture of insert to vector was prepared according to standard instructions and T4 ligase (NEB; Cat: M0202S) was added. Ligation was performed at 16°C, overnight, 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 water bath at 42°C for 10-45 seconds, depending on the type of E. coli cells. After rescuing the transformed cells in fresh 2xYT media for 1 hour, the cells were centrifuged and resuspended in a volume for plating on LB agar plates containing the respective selection antibiotic for the vector.

[0187] Fab-capture ELISA screening

[0188] The screening was performed as described in Example 4. The Fab conjugates of CAN1005016 and CAN1005019 were loaded onto plate-bound anti-canine Fab capture antibody, followed by addition of a titration series of cPD-L1_hFc-biotin. Detection was performed with streptavidin-HRP. The parental clone CAN1005001 was included as a control. Curve fitting and EC50 values were analyzed with GraphPad Prism. The results are shown in Figure 5.

[0189] CAN1005016 was generated by combining CAN1005010L1 and CAN1005010H2, while CAN1005019 was generated by combining CAN1005001L1 and CAN1005001H3. For CAN1005016, an EC50 of 87 pM was determined, demonstrating an improvement compared to its parental clone, as depicted in Figure 5. For CAN1005019, an EC50 of 138 pM was determined.

[0190] Example 6:

[0191] Grating-coupled interferometry (GCI) for characterization of anti-PD-L1 IgG

[0192] To further characterize the kinetic properties of the candidates CAN1005016 and CAN1005019, the Kd of the candidates was determined in a GCI system using waveRAPID according to the following procedure. D Quantification.

[0193] Binding kinetics studies were performed using PAG sensor chips (Creoptix, Malvern Panalytical brand) on a Creoptix® WAVEdelta system. All experiments were performed using HBS-EP (Cytiva) as running buffer at a temperature of 25 °C and a sampling rate of 10 Hz. Data acquisition and evaluation were performed using WAVEcontrol software (version 4.5.13).

[0194] Prior to ligand capture, the sensor chip was conditioned by a 180 seconds injection of 100 mM sodium borate, 1 M NaCl, pH 9 (Xantec). Antibodies were diluted to 1 pg / ml in HBS-EP and captured to a density of approximately 300 pg / mm 2The flow channel 1 is left blank and serves as a reference surface. For the collection of kinetic data, PD-L1-his was diluted to 200 nM in HBS-EP and injected at a flow rate of 60 μΐ / min in waveRAPID with increasing duration of analyte pulses onto the ligand and reference surface. PD-L1-his was injected with a total duration of 180 seconds, followed by a measurement of complex dissociation in running buffer for 30 minutes. Blank injections were performed to enable double referencing of the data and a 0.5% DMSO pulse injection (running buffer containing 0.5% DMSO) was used as analyte concentration adjustment calibration curve. The data were corrected (X and Y offset, DMSO calibration, double referencing) and fitted with a simple 1 : 1 interaction model (global fit) to obtain the kinetic parameters. The results are shown in Figure 6.

[0195] For CAN1005016, Kd was determined to be 0.77 nM and for CAN1005019, Kd was determined to be 1.7 nM. In comparison, Kd for the parental clone CAN1005010 H2 was 2.8 nM. Thus, the cross-cloning improved the affinity of CAN1005016 more than 3-fold. D For CAN1005016, Kd was determined to be 0.77 nM and for CAN1005019, Kd was determined to be 1.7 nM. In comparison, Kd for the parental clone CAN1005010 H2 was 2.8 nM. Thus, the cross-cloning improved the affinity of CAN1005016 more than 3-fold. D For CAN1005016, Kd was determined to be 0.77 nM and for CAN1005019, Kd was determined to be 1.7 nM. In comparison, Kd for the parental clone CAN1005010 H2 was 2.8 nM. Thus, the cross-cloning improved the affinity of CAN1005016 more than 3-fold. D For CAN1005016, Kd was determined to be 0.77 nM and for CAN1005019, Kd was determined to be 1.7 nM. In comparison, Kd for the parental clone CAN1005010 H2 was 2.8 nM. Thus, the cross-cloning improved the affinity of CAN1005016 more than 3-fold.

[0196] Example 7:

[0197] Generation of binding profile of CAN1005016 to cPD-L1_hFc with ELISA and FACS

[0198] To characterize the binding of CAN1005016 to its target cPD-L1, an ELISA setup was used. ELISAs were performed with cPD-L1_hFc or cPD-L1_MBP directly immobilized on the plate according to standard procedures as described below. In addition, binding to HEK293c18 cells transfected with cPD-L1 was tested in flow cytometry (FACS).

[0199] ELISA

[0200] cPD-L1 fusion protein was directly immobilized on the plate, typically at a concentration of 57 nM. For the binding ELISA, black 384-well Maxisorp plates were used. This was performed at 4°C, overnight. Coated plates were washed 3 times with PBS-T, followed by a 1-hour blocking with Chemiblocker (Merck Millipore; 2170) at room temperature. After blocking, a dilution series of the respective antibody was added to the plates, typically covering a concentration range of single-digit ng / ml to single-digit pg / ml. Antibodies were also incubated for 1 hour at room temperature, followed by washing and addition of a suitable detection antibody coupled to HRP. Measurements were performed as described above for the screening ELISA. Results are shown in Figure 7.

[0201] Mature and cross-cloned IgG CAN1005016 and CAN1005019 showed dose-dependent binding to cPD-L1 with EC50 values of 76 pM and 49 pM, respectively. This is a significant improvement over the 400 pM EC50 value measured for the CAN1005001 IgG in Example 3.

[0202] Transfection of HEK293c18 cells

[0203] HEK293c18 cells were seeded at a density of 1 x 10 6 Cells were seeded at a density of 2 x 105cells / well in 6-well plates in DMEM + 10% FCS. The next day, the transfection mix was prepared. Transfection was performed with jetPRIME transfection reagent (Polyplus, Cat: 101000046). Per well, 2 pg of plasmid encoding wild-type canine PD-L1 was diluted in 200 pl of jetPRIME buffer. The solution was mixed and 4 pl of transfection reagent was added, followed by mixing and centrifugation of the mixture. Then, the medium of the adherent cells was replaced with fresh medium and the transfection mix was carefully added to the cells. The plates containing the transfected cells were put back into the incubator and incubated for 24 hours.

[0204] The next day, cells were harvested by gentle pipetting with PBS + 2 mM EDTA (PBS / EDTA), counted, and 2 x 105cells / well were transferred to 96-well V-bottom plates for FACS staining. 5

[0205] FACS staining

[0206] ​Cells were washed once by adding 200 mΐ PBS / EDTA per well and centrifuged at 1500 rpm for 5 min. The supernatant was carefully decanted by inverting the plate over the waste and dried on paper towels. Then, cells were stained with the dye Zombie violet (Biolegend; Cat: 423114) diluted 1 :500 in PBS / EDTA for 15 min at 4°C in the dark to find dead cells. Subsequently, cells were washed with FACS buffer (PBS / EDTA + 2% FCS) as described above. A titration series of CAN1005016 and CAN1005019, usually in the range of 10 ng / ml to 10 pg / ml, was prepared, diluted in FACS buffer and added to the respective wells to incubate for 30 min at 4°C in the dark. After incubation, cells were washed 3 times with FACS buffer and the detection antibody goat anti-canine IgG FITC (Thermo Fisher; Cat. No: A18764) was added and incubated for 30 min at 4°C in the dark. Finally, cells were washed again 3 times and then resuspended in 200 mΐ FACS buffer and measured directly. Results are shown in Figure 8.

[0207] Both candidates recognized cell-bound cPD-L1 as confirmed by FACS of HEK293 c18 cells transfected with the target protein. For CAN1005016 an EC50 value of 10.5 nM was determined and for CAN1005019 an EC50 value of 11.8 nM. No staining was observed for the unrelated control antibody.

[0208] Example 8:

[0209] Ligand binding inhibition assay with CAN1005016 and CAN1005019 in ELISA and BLI settings

[0210] ELISA ligand binding inhibition assay (LIBIA)

[0211] To confirm the ability of CAN1005016 and CAN1005019 to block the interaction between canine PD-1 and canine PD-L1, a ligand binding inhibition assay was set up using an ELISA format. cPD-L1 hFc was immobilized on the plate and the accessible binding site was blocked with the respective candidate. The lack of accessible binding site for PD-1 was analyzed to measure the residual binding of PD-1 to PD-L1 hFc.

[0212] cPD-L1 fusion protein was directly immobilized on the plate at 4°C overnight, typically at a concentration of 57 nM. For the lbia ELISA, black 384-well Maxisorp plates were used. Coated plates were washed 3 times with PBS-T, followed by blocking with Chemiblocker (Merck Millipore; 2170) for 1 hour at room temperature. Blocked plates were washed 3 times with PBS-T, followed by addition of a titration series of blocking antibodies and incubation for 1 hour at room temperature. Subsequently, plates were washed again 3 times with PBS-T and PD-1-biotin (1x biotinylated through Avi-tag; Yikai; Cat: 70109-D27H-B) was added and incubated for 1 hour at room temperature, typically at a concentration of 4 pg / ml (206 nM). This concentration of canine PD-1-biotin was identified as optimal concentration by titration of PD-1-biotin binding to immobilized canine PD-L1_hFc. After incubation with the ligand PD-1, plates were washed 3 times with PBS-T, the appropriate detection reagent for biotinylated PD-1 was added, typically streptavidin-HRP, and incubated for 1 hour at room temperature. Subsequently, plates were washed 5 times, followed by addition of Quanta Blue and measurement as described above for the screening ELISA. Results are shown in Figure 9.

[0213] Both candidates blocked the interaction between cPD-L1 and cPD-1 efficiently. For CAN1005016, an IC 50 value of 0.95 nM was determined and for CAN1005019, an IC 50 value of 1.32 nM.

[0214] Ligand binding inhibition assay based on BioLayer Interferometry (BLI) (CAN1005016 only)

[0215] The reverse setup was tested in BioLayer Interferometry. Here, PD-1 was immobilized on the sensor and a mixture of cPD-L1_hFc and anti-PD-L1 antibody CAN1005016 competed for binding to cPD-L1. All reagents were diluted in Kinexa Kinetics Buffer (Sartorius; 18-1105).

[0216] Biotinylated canine PD-1 (cPD-1-Bio) was immobilized on a streptavidin sensor (Sartorius; Cat: 18-5019). Thus, after a baseline phase of 30 seconds, a 5 pg / ml cPD-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, a premixed solution of 100 nM cPD-L1 with different concentrations of CAN1005016 (10 nM - 100 nM) was associated with the loaded sensor for 150 seconds. After association, a dissociation of 120 seconds was recorded. Measurements were performed on a BLItz system (Pall Fortebio)

[0217] Pre-incubation of cPD-L1 with stoichiometric amounts of CAN1005016 resulted in a blockade of the interaction site with cPD-1 and a lack of binding of cPD-L1 to immobilized cPD-1, confirming the results of the ELISA-based ligand binding inhibition assay. When using non-stoichiometric amounts of CAN1005016 (1 :10), the binding of PD-L1 to immobilized cPD-1 was partially blocked, as evidenced in the intermediate signal of cPD-L1 binding. Stoichiometric pre-incubation with an unrelated canine control IgG had only a slight influence on the binding of cPD-L1 to cPD-1.

[0218] Example 9:

[0219] Binding of CAN1005016 and CAN1005016 to canine cell lines expressing PD-L1

[0220] To verify the binding of the antibody candidates to endogenous canine PD-L1, PD-L1 staining of IFN-g treated canine cell lines was performed using CAN1005016 and CAN1005019. It was reported that the canine squamous cell carcinoma cell line SCC1 expresses canine PD-L1 upon stimulation with IFN-g (Pantelyushin et al. 2021).

[0221] Treatment of SCC1 cells with IFN-g

[0222] SCC1 cells were cultivated in DMEM high glucose medium (Sigma-Aldrich, Cat: D6429) supplemented with 15% FCS (PAN Biotech, Cat: P30-3302), non-essential amino acids and penicillin-streptomycin (Thermo Fisher Scientific, Cat: 15140122). Cells were detached with trypsin (Thermo Fisher Scientific, Cat: 12604013), washed and resuspended in fresh medium. Cells were counted and plated at 1 x 105per well in a 96-well format.6 Cells were seeded in 6-well plates. Cells were incubated in an incubator for 3 hours at 37°C, 5% CO2, followed by treatment with 10 ng / ml IFN-g for 2 days. Control cells without IFN-g treatment were incubated at the same time.

[0223] FACS staining of SCC1 cells treated with CAN1005016 and CAN1005019

[0224] After treatment, cells were detached with trypsin (Thermo Scientific, Cat: 12604013), filtered through a 70 pm or 100 pm cell strainer and washed twice with PBS-EDTA. From this point on, cells were kept cold at 4°C. Cells were stained with Zombie violet live / dead stain (Biolegend, Cat: 423114) and the same number of cells were distributed in a 96-well plate for staining. Treated cells and control cells were stained with biotinylated CAN1005016, CAN1005019 and control IgG at different concentrations for 1 hour, diluted in FACS buffer (PBS-EDTA + 2% FCS), respectively. Cells were then washed 3 times with FACS buffer by centrifugation at 1500 rpm at 4°C. As detection antibody, streptavidin- allophycocyanin was used and stained for 30 minutes (Biolegend, Cat: 405207). After incubation, cells were washed again 3 times as described before and resuspended in 200 mΐ FACS buffer before measurement with a Beckman Coulter Cytoflex LX.

[0225] CAN1005016 and CAN1005019 bind to canine cancer cell lines expressing PD-L1. Treatment with IFN-g triggers a physiological upregulation of canine PD-L1, leading to improved binding of anti-PD-L1 IgG to SCC1 cells. This confirms the ability of CAN1005016 and CAN1005019 to recognize cPD-L1 in a cellular context.

[0226] Example 10:

[0227] Expression, purification and thermal stability of CAN1005016 and CAN1005019

[0228] Plasmids encoding CAN1005016 and CAN1005019 were transfected into suspension HEK293-Freestyle (HEK293-F) cells, respectively. Purification of IgG was performed by protein A affinity chromatography followed by size exclusion chromatography (SEC). Details are described below. Structural properties of CAN1005016 and CAN1005019 were further characterized by differential scanning fluorimetry (nanoDSF).

[0229] Transfection of HEK293-F cells and IgG expression

[0230] HEK293-F cells were cultured in Freestyle medium (Thermo Fisher Scientific; catalog: 12338026) at 37°C and 5% CO2 in an incubator with constant rotation at 135 rpm. Approximately 18-24 hours before transfection, HEK293-F cells were counted at a concentration of 1.5 x 10⁻⁶ cells / mL. 6 Cells are seeded at a density of 100 cells / ml in an appropriate volume for expression, typically in 25 ml to 100 ml of Freestyle medium. The next day, a transfection mixture is prepared based on the cell volume. Generally, 3 µg plasmid and 9 µg PEI are used per ml of culture volume, scaled accordingly. The transfection mixture is added dropwise to the cells while stirring the flask. The transfected cells are incubated for 5–6 hours, then diluted with fresh, preheated Freestyle medium containing 5 mM valproic acid (VPA) (as an additive), resulting in a final concentration of 2.5 mM VPA. Expression typically takes 5–7 days, and the supernatant is harvested by centrifugation and filtration.

[0231] Purification of CAN1005016 and CAN1005019

[0232] Both IgGs were purified by protein-A affinity chromatography, using standard buffers for binding and elution. The eluted IgG was concentrated to a volume < 5 ml using a centrifuge (Amicon, 100 kDa, catalog: ACS510024) and applied to a Superdex 200 16 / 600 pg size size exclusion column operated according to standard procedures. The main peak was collected, analyzed by SDS-PAGE, and pooled. Purification from transient, non-optimized expression cultures typically yields high-purity IgG of 10–25 mg / L.

[0233] nanoDSF and DLS measurements

[0234] FORMOscreen® pre-mixed studies were conducted on a Prometheus PANTA system for parallel DLS and nanoDSF measurements (Nanotemper Technologies GmbH). All experiments were performed in duplicate using a high-sensitivity capillary (Nanotemper Technologies GmbH), and data analysis was performed using a merged dataset for each test condition. Data analysis and evaluation were performed using PANTA Analysis software (version 1.4.4).

[0235] The protein stock solution (concentration = 29.04 µM) was centrifuged at 21.000 g for 1 hour at 4°C. The supernatant was separated and the concentration was measured again (concentration after centrifugation = 28.72 µM). For each FORMOscreen® condition, 1.32 µl of the protein stock solution supernatant was mixed with 4 µl of the 5X FORMOscreen buffer stock solution and 14.68 µl of ddH20 to reach a final volume of 20 µl with a final protein concentration of 1.9 µM per 1X FORMOscreen® buffer condition.

[0236] The samples were prepared in 384-well AURORA microplates and incubated at 40°C for 7 days before measurement. Next, DLS measurements were performed, 10 acquisitions per capillary with a measurement time of 10 µs per acquisition. nanoDSF measurements were performed from 20°C to 95°C with a heating rate of 1°C / min.

[0237] Table 3: DLS measurement results

[0238] Example 11

[0239] Preparation of CAN1005016

[0240] CAN1005016 for this inpatient study was produced in genetically engineered CHO cells according to standard bioreactor fermentation techniques. The supernatant was produced using a high expressing stable cell pool in a suitable fed-batch process scale (5L). To harvest the supernatant, the cells were removed from the cell suspension by centrifugation. The mAb was purified from the supernatant using a one-step protein A affinity chromatography, formulated and sterile filtered. The formulation has been developed for the stabilization of the monoclonal antibody for storage at low temperature and allows freeze / thaw of the antibody. The formulation consists of the antibody formulated in a histidine buffer at pH 5.9, with trehalose added as a cryoprotectant, Tween® 80 to prevent aggregation.

[0241] Example 12

[0242] In vivo testing of CAN10005016

[0243] To investigate the efficacy of the anti-canine PD-L1 antibody, CAN10005016 was tested in a female dog (14 years, mixed breed, 14.3 kg) with a stage IV oral melanoma and distant metastasis to the lungs. At the time of inclusion, the primary tumor lesion in the oral cavity was 50 x 49 mm in size. CAN1005016 was administered intravenously at a dose of 5 mg / kg body weight within 30 minutes, four times every two weeks, and then once a month. At each scheduled treatment session, safety was assessed using the Veterinary Co-operative Oncology Group (VCOG) criteria (Veterinary Co-operative Oncology Group (Vet Comp Oncol). 2016; 14: 417-46.) To monitor systemic toxicity, 5 ml of blood were taken at each antibody administration to obtain CBC, serum biochemistry, and thyroid profile. No adverse events were reported throughout the study period. For the anti-tumor response, the dog was evaluated monthly by physical examination, chest radiography, and abdominal ultrasound, tumor size was measured with a caliper and recorded. Anti-tumor responses were classified according to RECIST criteria (Nguyen SM, et al. Vet Comp Oncol. 2015; 13: 176-83.)

[0244] After five administrations, the patient was in complete oral remission. After the seventh administration, the oral cavity of this dog was still in complete remission. The treatment was prolonged and a total of 10 administrations were reached. 229 days after inclusion, the patient was alive and in good clinical condition, but recurrent maxillary melanoma and progressive lung metastases were observed.

[0245] In the veterinary literature, dogs with stage IV melanoma have been described as having a poor prognosis, with a median survival time of 60 to 80 days when treated with surgery, radiotherapy, and / or chemotherapy (Bergman, P. J. 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 filing this patent application, the patient was still alive, with a total survival time > 300 days. These results suggest that anti-PD-Ll therapy can be considered a promising treatment option for treating diseases with high medical need, such as malignant melanoma.

[0246] References

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[0249] Aresu, Luca; Marconato, Laura; Martini, Valeria; Fanelli, Antonella; Licenziato, Luca; Foiani, Greta et al. (2021): “Prognostic Value of PD-L1, PD-1 and CD8A in Canine Diffuse Large B-Cell Lymphoma Detected by RNAscope”. In: Veterinary sciences 8 (7). DOI: 10.3390 / vetsci8070120.

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[0251] Bellucci, Roberto; Martin, Allison; Bommarito, Davide; Wang, Kathy; Hansen, Steen H.; Freeman, Gordon J.; Ritz, Jerome (2015): “Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression”. In: Oncoimmunology 4 (6), e1008824. DOI: 10.1080 / 2162402X.2015.1008824.

[0252] Cascio, Matthew J.; Whitley, Elizabeth M.; Sahay, Bikash; Cortes-Hinojosa, Galaxia; Chang, Lung-Ji; Cowart, Jonathan et al. (2021): “Canine osteosarcoma checkpoint expression correlates with metastasis and T-cell infiltrate”. In: Veterinary Immunology and Immunopathology 232, p. 110169. DOI: 10.1016 / j.vetimm.2020.110169.

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[0254] Gearing, David P.; Virtue, Elena R.; Gearing, Robert P.; Drew, Alexander C. (2013): “A fully caninized anti-NGF monoclonal antibody for pain relief in dogs”. In: BMC veterinary research 9, p. 226. DOI: 10.1186 / 1746-6148-9-226.

[0255] Han, Yanyan; Liu, Dandan; Li, Lianhong (2020): “PD-1 / PD-L1 pathway: current researches in cancer”. In: American Journal of Cancer Research 10 (3), pp. 727-742.

[0256] Hartley, G.; Elmslie, R.; Dow, S.; Guth, A. (2018): “Checkpoint molecule expression by B and T cell lymphomas in dogs”. In: Veterinary and comparative oncology 16 (3), pp. 352-360. DOI: 10.1111 / vco.12386.

[0257] Hartley, G.; Faulhaber, E.; Caldwell, A.; Coy, J.; Kurihara, J.; Guth, A. et al. (2017): “Immune regulation of canine tumour and macrophage PD-L1 expression”. In: Veterinary and comparative oncology 15 (2), pp. 534-549. DOI: 10.1111 / vco.12197.

[0258] Igase, Masaya; Nemoto, Yuki; Itamoto, Kazuhito; Tani, Kenji; Nakaichi, Munekazu; Sakurai, Masashi et al. (2020): “A pilot clinical study of the therapeutic antibody against canine PD-1 for advanced spontaneous cancers in dogs”. In: Scientific reports 10 (1), p. 18311. DOI: 10.1038 / s41598-020-75533-4.

[0259] Iwai, Yoshiko; Hamanishi, Junzo; Chamoto, Kenji; Honjo, Tasuku (2017): “Cancer immunotherapies targeting the PD-1 signalling pathway”. In: Journal of biomedical science 24 (1), p. 26. DOI: 10.1186 / s12929-017-0329-9.

[0260] Maekawa, Naoya; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Adachi, Mami; Takagi, Satoshi et al. (2014): “Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade”. In: PLoS ONE 9 (6), e98415. DOI: 10.1371 / journal.pone.0098415.

[0261] Maekawa, Naoya; Konnai, Satoru; Nishimura, Maki; Kagawa, Yumiko; Takagi, Satoshi; Hosoya, Kenji et al. (2021): “PD-L1 immunohistochemistry for canine cancers and clinical benefit of anti-PD-L1 antibody in dogs with pulmonary metastatic oral malignant melanoma”. In: NPJ precision oncology 5 (1), p. 10. DOI: 10.1038 / s41698-021-00147-6.

[0262] Maekawa, Naoya; Konnai, Satoru; Okagawa, Tomohiro; Nishimori, Asami; Ikebuchi, Ryoyo; Izumi, Yusuke et al. (2016): “Immunohistochemical Analysis of PD-L1 Expression in Canine Malignant Cancers and PD-1 Expression on Lymphocytes in Canine Oral Melanoma”. In: PLoS ONE 11 (6), e0157176. DOI: 10.1371 / journal.pone.0157176.

[0263] Maekawa, Naoya; Konnai, Satoru; Takagi, Satoshi; Kagawa, Yumiko; Okagawa, Tomohiro; Nishimori, Asami et al. (2017): “A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma”. In: Scientific Reports 7 (1), p. 8951. DOI: 10.1038 / s41598-017-09444-2.

[0264] Mason, Nicola J.; Chester, Nicholas; Xiong, Ailian; Rotolo, Antonia; Wu, Ying; Yoshimoto, Sho et al. (2021): “Development of a fully canine anti-canine CTLA4 monoclonal antibody for comparative translational research in dogs with spontaneous tumors”. In: mAbs 13 (1), p. 2004638. DOI: 10.1080 / 19420862.2021.2004638.

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[0266] Pantelyushin, Stanislav; Ranninger, Elisabeth; Guerrera, Diego; Hutter, Gregor; Maake, Caroline; Markkanen, Enni et al. (2021): “Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs”. In: Cancers 13(4). DOI: 10.3390 / cancers13040785.

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Claims

1. An antibody or antibody fragment comprising: a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4, and a LCDR3 region according to SEQ ID No.: 18 or SEQ ID No.: 49; and / or a HCDR1 region according to SEQ ID No.: 6 or SEQ ID No.: 44, a HCDR2 region according to SEQ ID No.: 30 or SEQ ID No.: 60, and a HCDR3 region according to SEQ ID No.: 8 or SEQ ID No.:

46.

2. The antibody or antibody fragment according to claim 1, comprising a LCDR3 region having an amino acid sequence selected from the group consisting of SEQ ID No.: 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 43, 47, 48; preferably SEQ ID No.: 9 or 47; and / or a HCDR2 region having an amino acid sequence selected from the group consisting of SEQ ID No.: 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. The antibody or antibody fragment according to any one of the preceding claims, comprising a variable light chain comprising a LCDR3 region according to SEQ ID No.: 18 in combination with a variable heavy chain comprising a HCDR2 region according to SEQ ID No.: 30, or a variable light chain comprising a LCDR3 region according to SEQ ID No.: 49 in combination with a variable heavy chain comprising a HCDR2 region according to SEQ ID No.: 60; preferably a variable light chain comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4, and a LCDR3 region according to SEQ ID No.: 5 in combination with a variable heavy chain HCDR1 region according to SEQ ID No.: 6, a HCDR2 region according to SEQ ID No.: 19, and a HCDR3 region according to SEQ ID No.: 8; or preferably a light chain comprising a LCDR1 region according to SEQ ID No.: 3, a LCDR2 region according to SEQ ID No.: 4, and a LCDR3 region according to SEQ ID No.: 47 in combination with a variable heavy chain HCDR1 region according to SEQ ID No.: 44, a HCDR2 region according to SEQ ID No.: 52, and a HCDR3 region according to SEQ ID No.:

46.

4. The antibody or antibody fragment according to any one of the preceding claims, comprising one, several or all of the heavy chain variable domain framework sequences selected from the group consisting of the amino acid sequences of SEQ ID No.: 35, 36, 37, 38 or one, several or all of the heavy chain variable domain framework sequences selected from the group consisting of the amino acid sequences of SEQ ID No.: 61, 62, 63, 64.

5. The antibody or antibody fragment according to any one of the preceding claims, comprising a light chain variable domain according to SEQ ID No.: 1, 39, 41 or 65 and / or a heavy chain variable domain according to SEQ ID No.: 2, 40, 42 or 66, preferably a light chain variable domain according to SEQ ID No.: 39 in combination with a heavy chain variable domain according to SEQ ID No.: 40 or a light chain variable domain according to SEQ ID No.: 65 in combination with a heavy chain variable domain according to SEQ ID No.:

66.

6. The antibody or antibody fragment according to any one of the preceding claims, wherein the antibody or antibody fragment comprises at least one LCDR, HCDR, framework region or variable domain chain comprising a sequence having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID No.: 1 to SEQ ID No.:

66.

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

8. The antibody or antibody fragment according to any one of the preceding claims, wherein 9. The antibody or antibody fragment according to any one of the preceding claims, The antibody or antibody fragment binds specifically 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 wherein the antibody or antibody fragment binds with a dissociation constant (Kd) of at least about 2 x 10^6 nM. 4 [M -1 s -1 Preferably at least about 5 x 10 4 [M -1 s -1 K on Rate, and / or at less than about 1 x 10 -3 [s -1 Preferably below about 5 x 10 -4 [s -1 K off Rate of binding to canine PD-L1. wherein the antibody is an antibody fragment selected from the group consisting of Fv, scFv, Fab, Fab', F(ab')2, Fab'-SH, VHH.

10. The antibody or antibody fragment according to any one of the preceding claims, comprising an Fc domain or fragment thereof selected from the group consisting of IgG isotypes IgG-A, IgG-B, IgG-C or IgG-D, most preferably selected from the group consisting of IgG isotypes IgG-B.

11. The antibody or antibody fragment according to claim 10, wherein the Fc fragment is ​ comprises at least one substitution of an amino acid at a position selected from the group consisting of amino acid positions 235, 239, 270 and / or 331 relative to the corresponding amino acid position in a wild-type Fc fragment, preferably the amino acid is selected from at least one of L235, S239, D270 and / or P331, more preferably wherein the Fc fragment comprises the mutations L235A, S239A, D270A and P331G relative to the corresponding amino acid position in the wild-type Fc fragment.

12. The antibody according to any one of claims 1 to 11, comprising a light chain according to SEQ ID No: 72 in combination with a heavy chain according to SEQ ID No: 73, or a light chain according to SEQ ID No: 74 in combination with a heavy chain according to SEQ ID No:

75.

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

14. The antibody or antibody fragment according to one of the preceding claims, for use in treating a subject in need thereof, or a pharmaceutical composition comprising the antibody or antibody fragment according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier or excipient.

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

Images