BR112017006203B1 - ISOLATED ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF AND PHARMACEUTICAL COMPOSITIONS - Google Patents

Abstract

This specification describes antibodies including caninized antibodies against canine PD-L1 with specific properties. The document refers to the epitopes of canine PD-L1 that bind to these antibodies, as well as to anti-canine PD-L1 antibodies that bind to these epitopes, and to the use of the caninized anti-canine PD-L1 antibodies in the treatment of cancer in dogs.

Description

CROSS-REFERENCE TO RELATED ORDERS

[001] This application claims the benefit of U.S. Provisional Application Serial No. 62/057,541, filed on September 30, 2014, and U.S. Provisional Application Serial No. 62/172,511, filed on June 8, 2015, the contents of which are hereby incorporated by reference in their entireties.

FIELD OF INVENTION

[002] The present invention relates to anti-canine PD-L1 antibodies with specific properties. The present invention also relates to caninized antibodies against canine PD-L1 that have specific sequences and a high binding affinity for canine PD-L1. The present invention further relates to epitopes of canine PD-L1 that bind to these antibodies, as well as to anti-canine PD-L1 antibodies that bind to these epitopes. The invention further relates to the use of the antibodies of the present invention in the treatment of dogs, including in the treatment of cancer.

FUNDAMENTALS OF THE INVENTION

[003] An immunoinhibitory receptor that is primarily expressed on activated T and B cells, Programmed cell death receptor 1, also known as programmed death receptor 1 (PD-1), is a member of the CD28-related immunoglobulin and T-lymphocyte-associated protein 4 (CTLA-4) superfamily. PD-1 and similar family members are type I transmembrane glycoproteins that contain an extracellular Ig-type V variable domain that binds its ligands and a cytoplasmic tail that binds signaling molecules. The cytoplasmic tail of PD-1 contains two tyrosine-based signaling moieties, an ITIM (immunoreceptor tyrosine-based inhibition moiety) and an ITSM (immunoreceptor tyrosine-based switching moiety).

[004] PD-1 attenuates T cell responses when bound to programmed cell death ligand 1, also known as programmed death ligand 1 (PD-L1), and/or programmed cell death ligand 2, also known as programmed death ligand 2 (PD-L2). Binding of either of these ligands to PD-1 downregulates antigen receptor signaling. Blocking PD-L1 binding to PD-1 enhances tumor-specific CD8+ T cell immunity, also helping to eliminate tumor cells by the immune system. The three-dimensional structure of murine PD-1 as well as the cocrystal structure of mouse PD-1 with human PD-L1 is reported [Zhang et al., Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 3011-3016 (2008)].

[005] PD-L1 and PD-L2 are type 1 transmembrane ligands that contain both IgV- and IgC-like domains in the extracellular region, along with short cytoplasmic regions with unknown signaling portions. Both PD-L1 and PD-L2 are constitutively expressed, or can be induced, on a variety of cell types, including non-hematopoietic tissues as well as several tumor types. PD-L1 is not only expressed on B, T, myeloid, and dendritic cells (DCs), but also on peripheral cells such as microvascular endothelial cells and in non-lymphoid organs, e.g., the heart or lung. In contrast, PD-L2 is found only on macrophages and DCs. The expression pattern of PD-1 ligands suggests that PD-1 plays a role in maintaining peripheral tolerance and may additionally serve to regulate autoreactive T and B cell responses in the periphery.

[006] In any case, it is now abundantly clear that PD-1 and PD-L1 play critical roles in at least some human cancers, presumably by mediating immune evasion. Accordingly, PD-L1 has been shown to be expressed on a variety of human and mouse tumors, and to be inducible by IFN-Y in most PD-L1-negative tumor cell lines [Iwai et al., Proc. Natl. Acad. Sci. U.S.A. 99:12293-12297 (2002); Strome et al., Cancer Res., 63:6501-6505 (2003)]. Furthermore, expression of PD-1 on tumor-infiltrating lymphocytes and/or PD-L1 on tumor cells has been identified in several human primary tumor biopsies. Such tumor tissues include lung, liver, ovarian, cervical, skin, colon, glioma, bladder, breast, kidney, esophageal, gastric, oral squamous cell, urothelial cell, and pancreatic cancers, as well as head and neck tumors [Brown et al., J. Immunol. 170:1257–1266 (2003); Dong et al., Nat. Med. 8:793–800 (2002); Wintterle et al., Cancer Res. 63:7462–7467 (2003); Strome et al., Cancer Res., 63:6501–6505 (2003); Thompson et al., Cancer Res. 66:3381–5 (2006); Thompson et al., Clin. Cancer Res. 13: 1757–1761 (2007); Nomi et al., Clin.Cancer Res. 13: 2151–2157. (2007)]. More compellingly, PD ligand expression on tumor cells has been correlated with poor prognosis of human cancer patients across multiple tumor types [reviewed in Okazaki and Honjo, Int. Immunol. 19: 813–824 (2007)].

[007] Furthermore, Nomi et al. [Clin. Cancer Res. 13: 2151-2157 (2007)] demonstrated the therapeutic efficacy of blocking PD-L1 binding to PD-1 in a murine model of aggressive pancreatic cancer by administering antibody targeting either PD-1 or PD-L1. These antibodies effectively promoted the infiltration of tumor-reactive CD8+ T cells into the tumor, resulting in the upregulation of antitumor effectors including IFN-Y, granzyme B, and perforin. Similarly, the use of antibodies to block PD-L1 and PD-1 binding significantly inhibited tumor growth in a mouse model of squamous cell carcinoma [Tsushima et al, Oral Oncol. 42: 268-274 (2006)].

[008] In other studies, transfection of a murine mast cell line with PD-L1 led to decreased tumor cell lysis when cocultured with a tumor-specific CTL clone. Lysis was restored when anti-PD-L1 monoclonal antibody was added [Iwai et al, Proc. Natl. Acad. Sci. U.S.A. 99: 12293-12297 (2002)]. In vivo, blocking the PD1/PD-L1 interaction has been shown to increase the efficacy of adoptive T cell transfer therapy in a mouse tumor model [Strome et al, Cancer Res. 63: 6501-6505 (2003)]. Additional evidence for a role for PD-1 and PD-L1 in cancer treatment comes from experiments with PD-1 knockout mice, in which myeloma cells expressing PD-L1 grew only in wild-type animals (resulting in tumor growth and associated animal death) but not in PD-1-deficient mice [Iwai Y. et al., Proc. Natl. Acad. Sci. U.S.A. 99:12293–12297 (2002)]. More recently, humanized murine monoclonal antibodies against human PD-1 have demonstrated initial success in human cancer therapy [see, for example, documents 8,354,509 B2, 8,008,449 B2, and 7,595,048 B2].

[009] Antibodies to PD-L1 may also be useful in chronic viral infections. Memory CD8+ T cells generated after acute viral infection are highly functional and constitute an important component of protective immunity. In contrast, chronic infections are often characterized by varying degrees of functional impairment (exhaustion) of virus-specific T cell responses, and this defect is a major reason for the inability of the host to eliminate the persistent pathogen. Although functional effector T cells are initially generated during the early stages of infection, they gradually lose function over the course of a chronic infection. Barber et al. [Nature 439: 682–687 (2006)] demonstrated that mice infected with a laboratory strain of chronic LCMV produced high levels of virus in the blood and other tissues. These mice initially developed a robust T cell response but eventually succumbed to the infection after T cell exhaustion. Barber et al. found that the decline in the number and function of effector T cells in chronically infected mice could be reversed by injecting an antibody that blocked the interaction between PD-1 and PD-L1.

[010] Canine antibodies (also referred to as immunoglobulin G or IgG) are large tetrameric proteins of about 150 kd. Each IgG protein comprises two identical light chains of about 25 kd each and two identical heavy chains of about 50 kd each. There are four known IgG heavy chain subclasses of canine IgG, and they are referred to as IgGA, IgGB, IgGC, and IgGD. There are two types of light chains; the kappa and lambda chains. Each of the kappa or lambda light chains is composed of a variable domain (VL) and a constant domain (CL). Each of the two heavy chains consists of a variable domain (VH) and three constant domains, called CH-1, CH-2, and CH-3. The CH-1 domain is linked to the CH-2 domain through an amino acid sequence referred to as the “hinge” or alternatively as the “hinge region.” In humans, IgG exists in one of four subclasses referred to as IgG1, IgG2, IgG3, and IgG4. The subclass of IgG is determined largely by the sequence of the hinge region, which differs among the four IgG subclasses. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to one of the light chains also through a disulfide bond.

[011] Digestion of IgG antibodies with the enzyme papain breaks the antibody molecule at the hinge region and results in the formation of three fragments. Two of these fragments are identical, and each consists of the light chain held together with the VH and CH1 domains of the heavy chain. These fragments are called “Fab” fragments and contain the antigen-binding sites of the antibody. The third fragment that results from digestion with papain is called “Fc” and contains the remainder of the two heavy chains held together by disulfide bonds. The Fc thus contains a dimer consisting of the CH2 and CH3 domains of each of the two heavy chains. Although the Fab allows the antibody to bind to its cognate epitope, the Fc allows the antibody to mediate immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[012] It is well known in the art that IgG antibodies mediate effector functions, such as ADCC and ADCP, through binding of their Fc portion to a family of proteins known as Fcv receptors, while CDC is mediated through binding of the Fc to the first complement component, C1q. It is also well known in the art that different IgG subclasses differ in their abilities to mediate these effector functions. For example, human IgG1 exhibits strong ADCC and CDC, while IgG4 exhibits no or weak ADCC and CDC. Furthermore, methods for identifying which IgG subclasses exhibit or do not have effector functions are well known in the art.

[013] Approaches that rely on the use of monoclonal antibodies for therapeutic purposes require the design of suitable antibodies or antibody fragments to achieve the desired therapeutic response. For example, some therapeutic approaches for cancer require that therapeutic antibodies have increased effector functions, while others require that effector functions be significantly reduced or completely eliminated. The increase or elimination of effector functions can be achieved by introducing one or more amino acid mutations (substitutions) into the Fc portion of the antibody in order to increase or reduce binding to Fcv receptors and the first complement component. There are numerous reports in the prior art describing amino acid substitutions that can be introduced into an antibody molecule to modulate its effector functions. For example, Shields et al., [J. of Biol. Chem., 276 (9): 6591–6604 (2001)] reported that an asparagine-to-alanine substitution (N297A), which results in a nonglycosylated antibody, significantly reduced antibody binding to several Fcv receptors. Furthermore, Shields et al reported that an aspartic acid-to-alanine substitution (D265A) also significantly reduced antibody binding to Fcv receptors. Each of the N297A and D265A substitutions was also shown to significantly impair CDC. There are other similar reports identifying possible substitutions to reduce or eliminate effector function in antibodies [e.g., Sazinsky et al., Proc.Nat.Acad.Sci.,105:20167- 20172 (2008), Alegre et al., Transplantation, 57:1537-1543 (1994), Hutchins et al., Proc.Nat.Acad.Sci. 92:11980-11984 (1994), McEarchem et al., Blood,109:1185-1192 (2007)].

[014] Citation of any reference in this document should not be construed as an admission that such reference is available as “prior art” to the present application.

SUMMARY OF THE INVENTION

[015] The present invention provides antibodies and antigen-binding fragments thereof (including isolated antibodies and isolated antigen-binding fragments thereof) that bind to canine programmed death ligand 1 (canine PD-L1) with specificity. In particular embodiments, the antibodies and fragments thereof are mammalian antibodies. In more particular embodiments, the mammalian antibodies are murine (i.e., mouse) antibodies. In a related aspect of the present invention, the isolated antibodies are caninized antibodies. In specific embodiments, the caninized antibodies are caninized (e.g., mouse) mammalian anti-canine PD-L1 antibodies. The antibodies and antigen-binding fragments thereof of the present invention bind to canine PD-L1 and can block the binding of canine PD-L1 to canine programmed death receptor 1 (PD-1). The present invention further provides the use of such antibodies or antigen-binding fragments thereof in the treatment of disease, e.g., in the treatment of cancer in canines.

[016] In particular embodiments, the antibodies or antigen-binding fragments thereof that bind to canine PD-L1 with specificity comprise three light chain complementarity determining regions (CDRs): light chain CDR 1 (CDRL1), light chain CDR 2 (CDRL2), and light chain CDR 3 (CDRL3); and three heavy chain CDRs: heavy chain CDR 1 (CDRH1), heavy chain CDR 2 (CDRH2), and heavy chain CDR 3 (CDRH3).

[017] In certain embodiments, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 13. In other embodiments, the CDRH1 comprises a conservatively modified variant of SEQ ID NO: 13. In still other embodiments, the CDRH1 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 13 that comprises canonical structure class 1. In still other embodiments, the CDRH1 comprises an amino acid sequence of SEQ ID NO: 19. In still other embodiments, the CDRH1 comprises a conservatively modified variant of SEQ ID NO: 19. In still other embodiments, the CDRH1 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 19 that comprises canonical structure class 1.

[018] In certain embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 14. In other embodiments, the CDRH2 comprises a conservatively modified variant of SEQ ID NO: 14. In still other embodiments, the CDRH2 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 14 that comprises the canonical structure class 3B. In still other embodiments, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 20. In still other embodiments, the CDRH2 comprises a conservatively modified variant of SEQ ID NO: 20. In still other embodiments, the CDRH2 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 20 that comprises the canonical structure class 3B.

[019] In certain embodiments, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 15. In other embodiments, the CDRH3 comprises a conservatively modified variant of SEQ ID NO: 15. In still other embodiments, the CDRH3 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 15 that comprises the canonical structure class of 10. In still other embodiments, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 21. In still other embodiments, the CDRH3 comprises a conservatively modified variant of SEQ ID NO: 21. In still other embodiments, the CDRH3 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 21 that comprises the canonical structure class of 8.

[020] In certain embodiments, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 16. In other embodiments, the CDRL1 comprises a conservatively modified variant of SEQ ID NO: 16. In still other embodiments, the CDRL1 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 16 that comprises the canonical structure class of 2. In still other embodiments, the amino acid sequence of the CDRL1 comprises SEQ ID NO: 22. In still other embodiments, the CDRL1 comprises a conservatively modified variant of SEQ ID NO: 22. In still other embodiments, the CDRL1 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 22 that comprises the canonical structure class of 3.

[021] In certain embodiments, the CDRL2 comprises the amino acid sequence of SEQ ID NO: 17. In other embodiments, the CDRL2 comprises a conservatively modified variant of SEQ ID NO: 17. In still other embodiments, the CDRL2 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 17, which comprises the canonical structure class of 1. In still other embodiments, the CDRL2 comprises SEQ ID NO: 23. In still other embodiments, the CDRL2 comprises a conservatively modified variant of SEQ ID NO: 23. In still other embodiments, the CDRL2 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 23, which comprises the canonical structure class of 1.

[022] In certain embodiments, the CDRL3 comprises the amino acid sequence of SEQ ID NO: 18. In other embodiments, the CDRL3 comprises a conservatively modified variant of SEQ ID NO: 18. In still other embodiments, the CDRL3 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 18 that comprises the canonical structure class of 1. In still other embodiments, the CDRL3 comprises the amino acid sequence of SEQ ID NO: 24. In still other embodiments, the CDRL3 comprises a conservatively modified variant of SEQ ID NO: 24. In still other embodiments, the CDRL3 comprises a variant (e.g., a functionally conservative variant) of SEQ ID NO: 24 that comprises the canonical structure class of 1.

[023] The present invention further provides combinations of two or more of the CDRs of the present invention (or variants thereof) in a given antibody, for example, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 15 and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18.

[024] In specific embodiments, the antibodies and antigen-binding fragments thereof that bind to canine PD-L1 comprise a CDRH1 comprising an amino acid sequence of SEQ ID NO: 13, a conservatively modified variant of SEQ ID NO: 13, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 13 that comprises the canonical structure class of 1; a CDRH2 comprising the amino acid sequence of SEQ ID NO: 14, a conservatively modified variant of SEQ ID NO: 14, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 14 that comprises the canonical structure class of 3B; a CDRH3 comprising SEQ ID NO: 15, a conservatively modified variant of SEQ ID NO: 15, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 15 that comprises canonical structure class 10; a CDRL1 comprising an amino acid sequence of SEQ ID NO: 16, a conservatively modified variant of SEQ ID NO: 16, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 16 that comprises canonical structure class 2; a CDRL2 comprising the amino acid sequence of SEQ ID NO: 17, a conservatively modified variant of SEQ ID NO: 17, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 17 that comprises canonical structure class 1; and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18, a conservatively modified variant of SEQ ID NO: 18, or a variant (e.g., a function-conservative variant) of SEQ ID NO: 18 that comprises the canonical structure class 1.

[025] In related embodiments, the antibodies and antigen-binding fragments thereof that bind to canine PD-L1 comprise a CDRH1 comprising an amino acid sequence of SEQ ID NO: 19, a conservatively modified variant of SEQ ID NO: 19, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 19 that comprises the canonical structure class of 1; a CDRH2 comprising the amino acid sequence of SEQ ID NO: 20, a conservatively modified variant of SEQ ID NO: 20, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 20 that comprises the canonical structure class of 3B; a CDRH3 comprising SEQ ID NO: 21, a conservatively modified variant of SEQ ID NO: 21, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 21 that comprises the canonical structure class of 8; a CDRL1 comprising an amino acid sequence of SEQ ID NO: 22, a conservatively modified variant of SEQ ID NO: 22, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 22 that comprises the canonical structure class of 3; a CDRL2 comprising the amino acid sequence of SEQ ID NO: 23, a conservatively modified variant of SEQ ID NO: 23, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 23 that comprises the canonical structure class of 1; and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 24, a conservatively modified variant of SEQ ID NO: 24, or a variant (e.g., a functionally conservative variant) of SEQ ID NO: 24 that comprises the canonical structure class of 1.

[026] Accordingly, in a particular aspect of the present invention, the invention further provides caninized anti-canine PD-L1 antibodies. In certain embodiments, the caninized anti-canine PD-L1 antibodies are caninized mammalian (e.g., murine) anti-canine PD-L1 antibodies. In specific embodiments, the caninized anti-canine PD-L1 antibodies (e.g., caninized mammalian anti-canine PD-L1 antibodies, such as caninized murine anti-canine PD-L1 antibodies) comprise a cFc that has been genetically modified to enhance, decrease, or eliminate one or more effector functions. In particular such embodiments, the genetically modified cFc decreases or eliminates one or more effector functions. In other particular embodiments, the genetically modified cFc enhances one or more effector functions.

[027] In certain embodiments, the genetically modified cFc region is a genetically modified canine IgGB Fc region. In another such embodiment, the genetically modified cFc region is a genetically modified canine IgGC Fc region. In a particular embodiment, the effector function is antibody dependent cytotoxicity (ADCC) that is increased, decreased, or eliminated. In another embodiment, the effector function is complement dependent cytotoxicity (CDC) that is increased, decreased, or eliminated. In yet another embodiment, the cFc region has been genetically modified to increase, decrease, or eliminate ADCC and CDC.

[028] The present invention further provides canine frameworks and/or complete heavy chains comprising the genetically modified cFc regions. Accordingly, the present invention provides complete antibody heavy chains wherein the complete heavy chains comprise the genetically modified cFc regions of the present invention and the CDRs of the present invention. Such complete heavy chains may also be combined with the corresponding canine light chains (kappa or lambda) to form a complete antibody. In particular embodiments of this type, the resulting antibody binds to canine PD-L1.

[029] In certain embodiments, the genetically modified cFc region comprises the amino acid sequence of SEQ ID NO: 66 (or SEQ ID NO: 68) wherein one to seven of the following amino acid residues are substituted with another amino acid residue at the indicated positions: P4, D31, N63, G64, T65, A93, or P95. The amino acid replacing P4, D31, N63, G64, T65, A93, and/or P95 is individually selected from one of the other 19 naturally occurring standard amino acids as listed in Table 1 below. The present invention further provides variants of the genetically modified cFc regions that comprise an amino acid sequence that is 90%, 95%, 98% or 99% identical to the amino acid sequence of such genetically modified cFc regions and that retain at least 50%, 75%, 90%, 95% or more of the increase, decrease or elimination of ADCC and/or CDC, such as the genetically modified cFc regions that comprise the amino acid sequence of SEQ ID NO: 66 (or SEQ ID NO: 68) in which one or more of the following amino acid residues have been substituted: i.e., at P4, D31, N63, G64, T65, A93 or P95.

[030] In other embodiments, two to five of the following amino acid residues are substituted with another amino acid residue at the indicated positions: P4, D31, N63, G64, T65, A93, or P95. In particular embodiments of this type, the genetically modified cFc region comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, with the following substitutions: P4A, D31A, N63A, A93G, and P95A. In related embodiments, the genetically modified cFc region comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, with the following substitutions: P4A, D31A, N63A, and P95A. In other embodiments, the genetically modified cFc region comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, with substitutions at D31A and at N63A. In particular embodiments of this type, the aspartic acid residue at position 31 is replaced with a glutamic acid residue, an asparagine residue or an alanine residue, while the asparagine residue at position 63 is replaced with a glutamine residue, a histidine residue or an alanine residue. In a more particular embodiment of this type, the genetically modified cFc region comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, with the following substitutions: D31A and N63A. In particular embodiments, the genetically modified cFc region is encoded by the nucleotide sequence of SEQ ID NO: 65 or SEQ ID NO: 67, comprising nucleotide changes that correspond to the amino acid sequences they encode.

[031] In other embodiments, the genetically modified cFc region comprises the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68, with the substitution at A93. In a particular such embodiment, the substitution is A93G. In a related embodiment, the substitution is A93S. As described in U.S. Provisional Application No. 62/030,812, filed July 30, 2014, incorporated herein in its entirety, the A93G substitution leads to improved C1q complement binding, which is indicative of increased CDC activity.

[032] In related embodiments, the genetically modified cFc region comprises a hinge region comprising the amino acid sequence of SEQ ID NO: 45. In other embodiments, the genetically modified Fc region comprises a hinge region comprising the amino acid sequence of SEQ ID NO: 46. In still other embodiments, the genetically modified Fc region further comprises a hinge region comprising the amino acid sequence of SEQ ID NO: 47. In still other embodiments, the genetically modified Fc region further comprises a genetically modified hinge region comprising the amino acid sequence of SEQ ID NO: 48.

[033] In alternative embodiments, the present invention provides a canine IgGD Fc region with a genetically modified hinge region of a canine IgGD antibody, a hinge region of a canine IgGA antibody, a hinge region of a canine IgGB antibody, or a hinge region of a canine IgGC antibody. Furthermore, the present invention provides full-length antibody heavy chains, wherein the full-length heavy chains comprise the canine IgGD Fc region of the present invention with a genetically modified hinge region of a canine IgGD antibody, a hinge region of a canine IgGA antibody, a hinge region of a canine IgGB antibody, or a hinge region of a canine IgGC antibody. Such full-length heavy chains can also be combined with the corresponding canine light chains (kappa or lambda) to form a full-length antibody.

[034] Accordingly, the present invention provides a canine IgGD Fc region that further comprises a genetically modified hinge region of a canine IgGD antibody. In particular embodiments of this type, the canine IgGD Fc region and the genetically modified hinge region comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that is 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6, comprising a proline residue at position 10 (P10). In a more particular embodiment, the canine IgGD Fc region and the genetically modified hinge region is encoded by the nucleotide sequence of SEQ ID NO: 5. In other embodiments, the canine IgGD Fc region further comprises a hinge region of a canine IgGA antibody. In particular embodiments of this type, the canine IgGD Fc region and hinge region comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In a more particular embodiment, the canine IgGD Fc region and hinge region are encoded by the nucleotide sequence of SEQ ID NO: 7. In still other embodiments, the canine IgGD Fc region further comprises a hinge region of a canine IgGB antibody. In particular embodiments of this type, the genetically engineered canine IgGD Fc region and hinge region comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that is 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6, comprising a proline residue at position 10 (P10). In a more particular embodiment, the canine IgGD Fc region and hinge region are encoded by the nucleotide sequence of SEQ ID NO: 9. In still other embodiments, the canine IgGD Fc region further comprises a hinge region of a canine IgGC antibody. In particular embodiments of this type, the canine IgGD cFc region comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In a more particular embodiment, the canine IgGD cFc region and hinge region are encoded by the nucleotide sequence of SEQ ID NO: 11. The present invention further provides caninized antibodies comprising these caninized IgGD Fc regions and hinge regions. In a particular embodiment, the caninized antibody or antigen-binding fragment thereof binds to canine programmed death receptor 1 (canine PD-1) with specificity.

[035] The present invention therefore provides caninized anti-canine PD-L1 antibodies having specificity and/or having a high binding affinity for canine PD-L1. In particular embodiments, the caninized anti-canine PD-L1 antibodies also have the ability to block the binding of canine PD-L1 to canine PD-1. Such caninized antibodies or antigen-binding fragments thereof that specifically bind to canine PD-L1 may comprise a canine IgG heavy chain of the present invention and a canine kappa or lambda light chain. In particular embodiments, the caninized anti-canine PD-L1 antibodies are caninized murine anti-canine PD-L1 antibodies. The present invention also relates to the use of such caninized antibodies in the treatment of diseases, such as cancer and/or due to infections.

[036] In particular embodiments, the caninized anti-canine PD-L1 antibody comprises a genetically modified cFc region of the present invention. In alternative embodiments, the caninized anti-canine PD-L1 antibody comprises the Fc region of canine IgGD with a genetically modified hinge region of a canine IgGD antibody, a hinge region of a canine IgGA antibody, a hinge region of a canine IgGB antibody, or a hinge region of a canine IgGC antibody. The present invention further provides such caninized anti-canine PD-L1 antibodies comprising the canine frameworks of the present invention in combination with CDRs obtained from mouse anti-canine PD-L1 antibodies, namely three light chains of the CDRs: light chain CDR 1 (CDRL1), light chain CDR 2 (CDRL2), and light chain CDR 3 (CDRL3); and three heavy chain CDRs: heavy chain CDR 1 (CDRH1), heavy chain CDR 2 (CDRH2), and heavy chain CDR 3 (CDRH3).

[037] In particular embodiments, the caninized murine anti-canine PD-L1 antibodies comprise the genetically modified cFc region of the IgGB or IgGC of the present invention or, alternatively, the Fc region of canine IgGD, together with a genetically modified hinge region of a canine IgGD antibody, a hinge region of a canine IgGA antibody, a hinge region of a canine IgGB antibody, or a hinge region of a canine IgGC antibody in combination with CDRs obtained from mouse anti-canine PD-L1 antibodies. Furthermore, the present invention provides not only caninized mouse anti-canine PD-L1 antibodies with specific CDRs as detailed herein, but further provides caninized mouse anti-canine PD-L1 antibodies comprising conservatively modified variants of those CDRs, as well as variants that comprise (e.g., share) the same canonical structure.

[038] Accordingly, in particular embodiments, the caninized anti-canine PD-L1 antibody further comprises complementarity determining regions (CDRs) wherein the CDRs have canonical structures of: H1-1, H2-3B, and H3-10, respectively for the heavy chain CDR1, CDR2, and CDR3. In even more particular embodiments, the CDRs for the corresponding light chains have canonical structures of: L1-2, L2-1, and L3-1, respectively for the light chain CDR1, CDR2, and CDR3. In other embodiments, the caninized anti-canine PD-L1 antibody further comprises complementarity determining regions (CDRs) wherein the CDRs have canonical structures of: H1-1, H2-3B, and H3-8, respectively for the heavy chain CDR1, CDR2, and CDR3. In even more particular embodiments of this type, the CDRs for the corresponding light chains have canonical structures of: L1-3, L2-1, and L3-1, respectively, for the light chain CDR1, CDR2, and CDR3.

[039] In more particular embodiments, the caninized antibody of the present invention, or antigen-binding fragment thereof, comprises one or more of a heavy chain complementarity determining region 1 (VH CDR1) having an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19. In other embodiments, the heavy chain complementarity determining region 2 (VH CDR2) comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 20. In yet another embodiment, the heavy chain complementarity determining region 3 (VH CDR3) comprises an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 21. In a particular embodiment of this type, the caninized antibody or antigen-binding fragment comprises both a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19 and a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 20. ID NO: 14 or SEQ ID NO: 20.

[040] In another such embodiment, the caninized antibody or antigen-binding fragment comprises both a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19 and a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 21. In still another such embodiment, the caninized antibody or antigen-binding fragment comprises both a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 20 and a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 21. In still another such embodiment, the caninized antibody or antigen-binding fragment comprises a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19, a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 20 and a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 21.

[041] In particular embodiments, the caninized antibody or antigen-binding fragment further comprises a complementarity determining region of light chain 1 (CDR1 VL) comprising an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 22. In related embodiments, the complementarity determining region of light chain 2 (CDR2 VL) comprises an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23. In yet another embodiment, the complementarity determining region of light chain 3 (CDR3 VL) comprises an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 24. In a particular such embodiment, the caninized antibody or antigen-binding fragment comprises both a CDR1 VL comprising an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 22 and a CDR2 VL comprising an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23. SEQ ID NO: 23.

[042] In another such embodiment, the caninized antibody or antigen-binding fragment comprises both a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 22 and a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 24. In still another such embodiment, the caninized antibody or antigen-binding fragment comprises both a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23 and a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 24. In still another such embodiment, the caninized antibody or antigen-binding fragment comprises a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 22, a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23 and a CDR3 VL comprising an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 24.

[043] The present invention further provides caninized antibodies comprising the amino acid sequence of SEQ ID NO: 26 or that is 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 26 and SEQ ID NO: 28 or that is 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, or antigen-binding fragments of such caninized antibodies. The present invention also provides caninized antibodies comprising the amino acid sequence of SEQ ID NO: 30 or that is 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30 and SEQ ID NO: 32 or that is 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32, or antigen-binding fragments of such caninized antibodies.

[044] In particular embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) P, A, G, or S at position 242, (ii) A, G, or S at position 269, (iii) A, G, or S at position 301, (iv) G, P, or A at position 302, (v) T, A, G, or S at position 303, (vi) A, G, or S at position 331, and (vii) P, A, G, or S at position 333. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) P, A, G, or S at position 240, (ii) A, G, or S at position 267, (iii) A, G, or S at position 299, (iv) G, P, or A at position 300, (v) T, A, G, or S at position 301, (vi) A, G, or S at position 329, and (vii) P, A, G, or S at position 331. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) P, A, G, or S at position 238, (ii) A, G, or S at position 265, (iii) A, G, or S at position 297, (iv) G, P, or A at position 298, (v) T, A, G, or S in heading 299, (vi) A, G or S in heading 327 and (vii) P, A, G, or S in heading 329.

[045] In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) P, A, G, or S at position 242, (ii) A at position 269, (iii) A at position 301, (iv) G, P, or A at position 302, (v) T, A, G, or S at position 303, (vi) A, G, or S at position 331, and (vii) P, A, G, or S at position 333. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) P, A, G, or S at position 240, (ii) A at position 267, (iii) A at position 299, (iv) G, P, or A at position 300, (v) T, A, G, or S at position 301, (vi) A, G, or S at position 329, and (vii) P, A, G, or S at position 331. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) P, A, G, or S at position 238, (ii) A at position 265, (iii) A at position 297, (iv) G, P, or A at position 298, (v) T, A, G, or S at position 299, (vi) A, G, or S at position 327 and (vii) P, A, G, or S in heading 329.

[046] In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) A at position 242, (ii) A at position 269, (iii) A at position 301, (iv) P at position 302, (v) A at position 303, (vi) G at position 331, and (vii) A at position 333. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) A at position 240, (ii) A at position 267, (iii) A at position 299, (iv) P at position 300, (v) A at position 301, (vi) G at position 329, and (vii) A at position 331. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) A at position 238, (ii) A at position 265, (iii) A at position 297, (iv) P at position 298, (v) A at position 299, (vi) G at position 327, and (vii) A at position 329.

[047] In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) P at position 242, (ii) A, G, or S at position 269, (iii) A, G, or S at position 301, (iv) G at position 302, (v) T at position 303, (vi) A at position 331, and (vii) P at position 333. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) P at position 240, (ii) A, G, or S at position 267, (iii) A, G, or S at position 299, (iv) G at position 300, (v) T at position 301, (vi) A at position 329, and (vii) P at position 331. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) P at position 238, (ii) A, G, or S at position 265, (iii) A, G or S in heading 297, (iv) G in heading 298, (v) T in heading 299, (vi) A in heading 327 and (vii) P in heading 329.

[048] In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) P at position 242, (ii) A at position 269, (iii) A at position 301, (iv) G at position 302, (v) T at position 303, (vi) A at position 331, and (vii) P at position 333. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) P at position 240, (ii) A at position 267, (iii) A at position 299, (iv) G at position 300, (v) T at position 301, (vi) A at position 329, and (vii) P at position 331. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) P at position 238, (ii) A at position 265, (iii) A at position 297, (iv) G at position 298, (v) T at position 299, (vi) A at position 327, and (vii) P at position 329.

[049] In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) P, A, G, or S at position 242, (ii) A, G, or S at position 269, (iii) A, G, or S at position 301, (iv) G at position 302, (v) T at position 303, (vi) A, G, or S at position 331, and (vii) P, A, G, or S at position 333. In other such embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) P, A, G, or S at position 240, (ii) A, G, or S at position 267, (iii) A, G, or S at position 299, (iv) G at position 300, (v) T at position 301, (vi) A, G, or S at position 329, and (vii) P, A, G, or S at position 331. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) P, A, G, or S at position 238, (ii) A, G, or S at position 265, (iii) A, G, or S at position 297, (iv) G at position 298, (v) T at position 299, (vi) A, G, or S at position 327, and (vii) P, A, G, or S in heading 329.

[050] In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) P, A, G, or S at position 242, (ii) A at position 269, (iii) A at position 301, (iv) G at position 302, (v) T at position 303, (vi) A, G, or S at position 331, and (vii) P, A, G, or S at position 333. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30 ... position 240, (ii) A at position 267, (iii) A at position 299, (iv) G at position 300, (v) T at position 301, (vi) A, G, or S at position 329, and (vii) P, A, G, or S at position 331. In other such embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) P, A, G, or S at position 238, (ii) A at position 265, (iii) A at position 297, (iv) G at position 298, (v) T at position 299, (vi) A, G, or S at position 327, and (vii) P, A, G, or S at position 329.

[051] In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26) comprising (i) A at position 242, (ii) A at position 269, (iii) A at position 301, (iv) G at position 302, (v) T at position 303, (vi) G at position 331, and (vii) A at position 333. In still other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 28 or 30) comprising (i) A at position 240, (ii) A at position 267, (iii) A at position 299, (iv) G at position 300, (v) T at position 301, (vi) G at position 329, and (vii) A at position 331. In other such embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 32 (or 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32) comprising (i) A at position 238, (ii) A at position 265, (iii) A at position 297, (iv) G at position 298, (v) T at position 299, (vi) G at position 327, and (vii) A at position 329. Furthermore, the present invention provides a caninized antibody, or an antigen-binding fragment thereof, further comprising a canine light chain comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 44.

[052] Accordingly, the present invention further provides a caninized antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. In another embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 40 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[053] In yet another embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In yet another embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 40 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[054] The present invention further provides a caninized antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. In another embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[055] The present invention further provides a caninized antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 28 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In another embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 30 and a light chain comprising the amino acid sequence of SEQ ID NO: 38. In a related embodiment, the caninized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[056] The present invention further provides nucleic acids that encode any of the amino acid sequences of the present invention, including the CDRs, cFc regions, the cFc regions with hinge regions, and the heavy chains and light chains of the caninized antibodies of the present invention. The present invention further provides expression vectors comprising one or more of the nucleic acids of the present invention. The present invention further provides host cells comprising one or more expression vectors of the present invention and methods for expressing the CDRs, cFc regions and/or the cFc regions with hinge regions, and/or the heavy chains and/or the light chains of the caninized antibodies of the present invention using such host cells. The present invention also provides host cells that have been genetically modified to express the CDRs, cFc regions and/or cFc regions with hinge regions, and/or the heavy chains and/or the light chains of the caninized antibodies of the present invention in the absence of such vectors. In particular embodiments, these nucleic acids, expression vectors, polypeptides, or host cells of the invention are useful in methods of making an antibody.

[057] In particular embodiments, the antibody is a recombinant antibody or an antigen-binding fragment thereof. In related embodiments, the heavy chain variable domain and the light chain variable domain are joined by a flexible linker to form a single chain antibody.

[058] In certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof (e.g., an isolated antibody or an antigen-binding fragment thereof) that binds to canine PD-L1 with specificity, and that when bound to canine PD-L1, the antibody or antigen-binding fragment thereof binds to at least one amino acid residue in the amino acid sequence of SEQ ID NO: 82. In related embodiments, the antibody or antigen-binding fragment thereof that binds to PD-L1 with specificity also binds to at least one amino acid residue in the amino acid sequence of SEQ ID NO: 83. In still other embodiments, the antibody or antigen-binding fragment thereof that binds to canine PD-L1 with specificity also binds to at least one amino acid residue in the amino acid sequence of SEQ ID NO: 82 and to at least one amino acid residue in the amino acid sequence of SEQ ID NO: 83. In related ... In particular embodiments, such antibodies and/or antigen-binding fragments thereof block the binding of canine PD-L1 to canine PD-1. In related embodiments, the antibody is a monoclonal antibody. In particular such embodiments, the antibody is a murine anti-canine PD-L1 monoclonal antibody. In further particular embodiments, the monoclonal antibody is a caninized antibody. In still more particular embodiments, the monoclonal antibody is a caninized murine anti-canine PD-L1 antibody.

[059] In certain embodiments, the antibody, or an antigen-binding fragment thereof, binds to 2 to 5 amino acid residues of SEQ ID NO: 82. In other embodiments, the antibody or antigen-binding fragment thereof binds to 6 to 12 amino acid residues of SEQ ID NO: 82. In still other embodiments, the isolated antibody or antigen-binding fragment thereof binds to 13 to 20 amino acid residues of SEQ ID NO: 82. In related embodiments, the antibody, or an antigen-binding fragment thereof, binds to 2 to 5 amino acid residues of SEQ ID NO: 83. In other embodiments, the antibody or antigen-binding fragment thereof binds to 6 to 11 amino acid residues of SEQ ID NO: 83.

[060] In still other embodiments, monoclonal antibodies or antigen-binding fragments thereof are provided which cross-compete for binding to canine PD-L1 with one or more of the anti-canine PD-L1 antibodies of the present invention. In particular embodiments, the cross-competing antibodies and antigen-binding fragments thereof bind to canine PD-L1 and block the binding of canine PD-L1 to canine PD-1. In more particular embodiments, the monoclonal antibodies or antigen-binding fragments thereof cross-compete with 4F9 (or an antibody having the 6 CDRs of 4F9) for binding to canine PD-L1. In other more particular embodiments, the monoclonal antibodies or antigen-binding fragments cross-compete with 5F12 (or an antibody having the 6 CDRs of 5F12) for binding to canine PD-L1. In still other embodiments, monoclonal antibodies or antigen-binding fragments thereof cross-compete with 4F9 and 5F12 for binding to canine PD-L1.

[061] In particular embodiments, a monoclonal antibody of the invention is a murine antibody. In other embodiments, the monoclonal antibody is a caninized antibody. In more particular embodiments, a monoclonal antibody of the invention is a caninized murine antibody.

[062] Furthermore, the present invention provides antibodies (e.g., caninized antibodies) to canine PD-L1 that comprise the CDRs of the present invention or variants of the CDRs that have the corresponding canonical structures set forth herein, and/or that bind to the amino acid sequence of SEQ ID NP: 82 and/or 83 of PD-L1. In particular embodiments of this type, the dissociation constant (Kd) for caninized antibody-canine PD-L1 binding is 1 X 10-5 to 1 X 10-12 M. In more particular embodiments, the caninized antibodies to canine PD-L1 comprise variants of the CDRs of the present invention that have the corresponding canonical structures set forth herein, and bind to the amino acid sequence of SEQ ID NP: 82 and/or 83 of PD-L1. The present invention therefore includes caninized antibodies and antigen-binding fragments thereof that bind to canine PD-L1 with specificity, and when they bind to canine PD-L1, the antibody binds to at least one amino acid residue of the amino acid sequence of SEQ ID NO: 82 and/or 83 of PD-L1. In particular embodiments, the antibodies and antigen-binding fragments thereof bind to canine PD-L1 and block the binding of canine PD-L1 to canine PD-1. Accordingly, in particular embodiments, when binding to canine PD-L1, the caninized antibody (including antibodies having one or more variant CDRs, e.g., a variant such as, but not limited to, a conservatively modified variant and/or a variant comprising a defined canonical structure class) binds to at least one amino acid residue of an epitope of PD-L1, e.g., the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83.

[063] The present invention further provides caninized antibodies or antigen-binding fragments thereof that bind to canine PD-L1 with a dissociation constant (Kd) that is less than 1 X 10-12 M (e.g., 1 X 10-13 M, or even less). In other embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with a dissociation constant of 1 X 10 M to 1 X 10 M. In more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with a dissociation constant of 1 X 10 M to 1 X 10 M. In still more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with a dissociation constant of 1 X 10 M to 1 X 10 M. In still more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with a dissociation constant of 1 X 10 M to 1 X 10 M.

[064] The present invention also provides caninized antibodies or antigen-binding fragments thereof that bind to canine PD-L1 with an association ratio (kon) that is greater than 1 X 107 M-1s-1. In other embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with an association ratio of 1 X 102 M-1s-1 to 1 X 107 M-1s-1. In more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with an association ratio of 1 X 103 M-1s-1 to 1 X 106 M-1s-1. In even more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with an association ratio of 1 X 10 M-1s-1 to 1 X 10 M-1s-1. In even more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with an association ratio of 1 X 10 M-1s-1 to 1 X 10 M-1s-1.

[065] The present invention also provides caninized antibodies or antigen-binding fragments thereof that bind to canine PD-L1 with a dissociation rate (koff) that is less than 1 X 10-7 s-1. In particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with a dissociation rate of 1 X 10-3 s-1 to 1 X 10-8 s-1. In more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with a dissociation rate of 1 X 10-4 s-1 to 1 X 10-7 s-1. In even more particular embodiments, the caninized antibodies or antigen-binding fragments thereof bind to canine PD-L1 with an off-rate of 1 X 10-5 s-1 to 1 X 10-7 s-1.

[066] In related embodiments, the antibodies (e.g., caninized antibodies) or antigen-binding fragments thereof stimulate antigen-specific memory responses to a tumor or pathogen. In particular embodiments, the antibodies (e.g., caninized antibodies) or antigen-binding fragments thereof stimulate an antibody response in vivo. In other particular embodiments, the antibodies (e.g., caninized antibodies) or antigen-binding fragments thereof stimulate an immune response in an animal subject. In more specific embodiments, the animal subject is a canine. In a related embodiment, the animal subject is a feline.

[067] Accordingly, any of the antibodies (e.g., caninized antibodies) of the present invention may exhibit one, two, three, four, five, or all of these properties, i.e., the aforementioned dissociation constants with canine PD-L1, the aforementioned binding rates for binding to canine PD-L1, the aforementioned dissociation rates for separation of the caninized antibody-canine PD-L1 binding complex, stimulation of antigen-specific memory responses to a tumor or pathogen, stimulation of an antibody response in vivo, and/or stimulation of an immune response in an animal subject.

[068] In more particular embodiments, the antibodies and/or antigen-binding fragments thereof of the present invention bind to canine PD-L1 and also block the binding of canine PD-L1 to PD-1. In even more particular embodiments, the antibodies and/or antigen-binding fragments thereof of the present invention bind to canine PD-L1 and block the binding of canine PD-L1 to PD-1.

[069] As indicated above, the antibodies (and antigen-binding fragments thereof) of the present invention, including certain of the aforementioned antibodies (and antigen-binding fragments thereof), can be monoclonal antibodies (and antigen-binding fragments thereof), mammalian antibodies (and antigen-binding fragments thereof), e.g., murine (mouse) antibodies (and antigen-binding fragments thereof), caninized antibodies (and antigen-binding fragments thereof), including caninized murine antibodies (and antigen-binding fragments thereof). In certain embodiments, the antibodies (and antigen-binding fragments thereof) are isolated.

[070] In particular embodiments, the antibody is a recombinant antibody or an antigen-binding fragment thereof. In related embodiments, the heavy chain variable domain and the light chain variable domain are joined by a flexible linker to form a single chain antibody.

[071] In particular embodiments, the antibody or antigen-binding fragment is a Fab fragment.

[072] In other embodiments, the antibody or antigen-binding fragment is a Fab' fragment. In other embodiments, the antibody or antigen-binding fragment is a (Fab')2 fragment. In still other embodiments, the antibody or antigen-binding fragment is a diabody. In particular embodiments, the antibody or antigen-binding fragment is a domain antibody. In particular embodiments, the antibody or antigen-binding fragment is a camelized single domain antibody.

[073] In particular embodiments, a caninized murine anti-canine PD-L1 antibody, or antigen-binding fragment, enhances the immune response of the animal subject (e.g., canine or feline) being treated.

[074] The present invention further provides nucleic acids (including isolated nucleic acids) encoding any of the antibodies and portions thereof (including CDRs) of the present invention. In certain embodiments, the present invention provides nucleic acids (including isolated nucleic acids) encoding any of the light chains of the caninized antibodies or portions thereof of the present invention. Similarly, the present invention provides nucleic acids (including isolated nucleic acids) encoding any of the heavy chains of the caninized antibodies or portions thereof of the present invention. The present invention further provides expression vectors comprising one or more of the nucleic acids (including isolated nucleic acids) of the present invention. Accordingly, the present invention provides nucleic acids encoding the caninized murine anti-canine PD-L1 antibodies or portions thereof of the present invention. In related embodiments, such antibodies or antigen-binding fragments can be used for the preparation of a medicament for treating cancer in a canine and/or feline subject. Alternatively, or in conjunction, the present invention provides the use of any of the antibodies or antibody fragments of the present invention for diagnostic use. In still additional embodiments, a kit is provided that comprises any of the caninized antibodies or antigen-binding fragments described herein. In specific embodiments, an expression vector is provided that comprises an isolated nucleic acid encoding any of the caninized murine anti-canine PD-L1 antibodies or antigen-binding fragments of the invention. The present invention further provides host cells that comprise one or more expression vectors of the present invention. In particular embodiments, such nucleic acids, expression vectors, or polypeptides of the invention are useful in methods of making an antibody.

[075] The present invention further provides antigenic peptides (including isolated antigenic peptides) consisting of 80 or fewer amino acid residues comprising the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83. In related embodiments, the antigenic peptides (including isolated peptides) consisting of 60 or fewer amino acid residues comprising the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83. In related embodiments, the antigenic peptides (including isolated peptides) consist of 11 to 45 amino acid residues comprising the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83. In still other embodiments, the antigenic peptides consist of 5 to 20 amino acid residues of the amino acid sequence of SEQ ID NO: 83. In still other embodiments, the antigenic peptides consist of 10 to 15 amino acid residues comprising the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83. at 5 to 11 amino acid residues of the amino acid sequence of SEQ ID NO: 83.

[076] The present invention further provides antigenic peptides (including isolated peptides) consisting of 80 or fewer amino acid residues that comprise an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83, and binds to an isolated mammalian antibody, or an antigen-binding fragment thereof of the present invention. In related embodiments, antigenic peptides (including isolated antigenic peptides) consisting of 60 or fewer amino acid residues that comprise an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83, and binds to an isolated mammalian antibody, or an antigen-binding fragment thereof of the present invention. In other embodiments, the peptides consist of 5 to 20 amino acid residues of an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 82 and binds to an isolated mammalian antibody, or an antigen-binding fragment thereof. In other embodiments, the peptides consist of 5 to 11 amino acid residues of an amino acid sequence that is 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 83 and binds to an isolated mammalian antibody, or an antigen-binding fragment thereof. In particular embodiments, the mammalian antibody is 4E9. In other embodiments, the mammalian antibody is 5F12.

[077] The present invention further provides fusion proteins comprising any of the above antigenic peptides. In a particular embodiment, the fusion protein comprises such an antigenic peptide and an Fc region of a non-canine mammalian IgG antibody. In a more particular embodiment, the fusion protein comprises an Fc region of a non-canine mammalian IgG antibody. In certain embodiments, the non-canine mammalian IgG antibody is a murine IgG. In alternative embodiments, the non-canine mammalian IgG antibody is a human IgG. In other embodiments, the non-canine mammalian IgG antibody is an equine IgG. In still other embodiments, the non-canine mammalian IgG antibody is a murine IgG. In still other embodiments, the non-canine mammalian IgG antibody is a bovine IgG.

[078] In particular embodiments, the non-canine mammalian IgG antibody is an IgG1. In other embodiments, the non-canine mammalian IgG antibody is an IgG2a. In still other embodiments, the non-canine mammalian IgG antibody is an IgG3. In still other embodiments, the non-canine mammalian IgG antibody is an IgG4.

[079] In other embodiments, the fusion protein comprises any of the above antigenic peptides and maltose binding protein. In still other embodiments, the fusion protein comprises any of the above antigenic peptides and beta-galactosidase. In still other embodiments, the fusion protein comprises any of the above antigenic peptides and glutathione S-transferase. In still other embodiments, the fusion protein comprises any of the above antigenic peptides and thioredoxin. In still other embodiments, the fusion protein comprises any of the above antigenic peptides and Gro EL. In still other embodiments, the fusion protein comprises any of the above antigenic peptides and NusA.

[080] The present invention further provides nucleic acids (including isolated nucleic acids) encoding the antigenic peptides and corresponding fusion proteins of the present invention. The present invention also provides expression vectors comprising such nucleic acids and host cells comprising one or more expression vectors of the present invention.

[081] Furthermore, the present invention includes pharmaceutical compositions comprising anti-canine PD-L1 antibodies or antigen-binding fragments thereof of the present invention, antigenic peptides (including isolated antigenic peptides) of canine PD-L1, fusion proteins comprising the antigenic peptides of canine PD-L1 of the present invention, nucleic acids (including isolated nucleic acids) encoding the antigenic fragments and/or fusion proteins of the present invention, expression vectors comprising such nucleic acids or any combination thereof, and a pharmaceutically acceptable carrier or diluent.

[082] In particular embodiments, such pharmaceutical compositions comprise a canine anti-PD-1 antibody or an antigen-binding fragment thereof. In more particular embodiments, the canine anti-PD-1 antibody is a caninized murine canine anti-PD-1 antibody or an antigen-binding fragment of a caninized murine canine anti-PD-1 antibody. In related embodiments, such pharmaceutical compositions further comprise a canine anti-CTLA-4 antibody or an antigen-binding fragment thereof. In particular embodiments, the canine anti-CTLA-4 antibody is a caninized murine canine anti-CTLA-4 antibody, or an antigen-binding fragment of a caninized murine canine anti-CTLA-4 antibody.

[083] Accordingly, the present invention provides pharmaceutical compositions comprising one, two, three or more of the following: anti-canine PD-L1 antibodies, an anti-canine PD-1 antibody, an anti-canine CTLA-4 antibody, an antigen-binding fragment of an anti-canine PD-L1 antibody, an antigen-binding fragment of an anti-canine PD-1 antibody, or an antigen-binding fragment of an anti-canine CTLA-4 antibody. In particular embodiments, such anti-canine protein antibodies (i.e., anti-canine PD-L1, PD-1 or CTLA-4) or antigen-binding fragments thereof are murine anti-canine protein antibodies. In other such anti-canine protein antibodies or antigen-binding fragments thereof are caninized anti-canine protein antibodies. In more particular embodiments, the anti-canine protein antibodies or antigen-binding fragments thereof are caninized murine anti-canine protein antibodies.

[084] Furthermore, the present invention provides methods of increasing the activity of an immune cell, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In certain embodiments, the method is used for the treatment of cancer. In other embodiments, the method is used in the treatment of an infection or an infectious disease. In still other embodiments, a caninized antibody of the present invention or antigen-binding fragment thereof is used as a vaccine adjuvant. In particular embodiments, a pharmaceutical composition comprising a caninized murine anti-canine PD-L1 antibody, or antigen-binding fragment thereof, can be administered prior to, following, or simultaneously with a caninized murine anti-canine PD-1 antibody, or antigen-binding fragment thereof, and/or a caninized murine anti-canine CTLA-4 antibody, or antigen-binding fragment thereof.

[085] These and other aspects of the present invention will be better appreciated by reference to the following Brief Description of the Drawings and the Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[086] Figure 1 shows the ELISA results for the reactivity of two mouse anti-canine PD-L1 mAbs against canine PD-L1 as a function of OD 650/490 versus mAb log (nM). Both mAbs, designated 4F9 and 5F12, demonstrate strong and dose-dependent binding to canine PD-L1.

[087] Figure 2 shows ligand blocking with mouse and canine anti-PD-L1 mAbs. Two mAbs, designated 4F9 and 5F12, were tested for their ability to inhibit PD-L1 binding to PD-1 expressed on CHO cells. Both mAbs block PD-L1 binding to PD-1, although mAb 4F9 is a stronger inhibitor than 5F12. DETAILED DESCRIPTION Abbreviations Throughout the detailed description and examples of the invention, the following abbreviations will be used: ADCC Antibody-dependent cellular cytotoxicity CDC Complement-dependent cytotoxicity CDR Complementarity-determining region in immunoglobulin variable regions defined for human antibodies using the Kabat numbering system CHO Chinese hamster ovary EC50 concentration resulting in 50% efficacy or binding ELISA Enzyme-linked immunosorbent assay FR Antibody framework region: the immunoglobulin variable regions, excluding the CDR regions. HRP Horseradish peroxidase IFN interferon IC50 concentration resulting in 50% inhibition IgG Immunoglobulin G Kabat An immunoglobulin alignment and numbering system for human antibodies pioneered by Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] mAb Monoclonal antibody (also Mab or MAb) MES 2-(N-morpholino)ethanesulfonic acid (MOA) Mechanism of action NHS Normal human serum PCR Polymerase chain reaction PK Pharmacokinetics SEB Staphylococcal enterotoxin B TT Tetanus toxoid V region The segment of human IgG chains that is variable in sequence among different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain. VH Variable region of immunoglobulin heavy chain VK Variable region of immunoglobulin kappa light chain

DEFINITIONS

[088] In order that the invention may be more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined elsewhere, all other technical and scientific terms used in this document have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs.

[089] As used herein, including in the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly specifies otherwise.

[090] “Activation”, when applied to cells or receptors, refers to the activation or treatment of a cell or receptor with a ligand, unless otherwise indicated by the context or explicitly. “Ligand” encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogues, muteins and antibody-derived binding compounds. “Ligand” also encompasses small molecules, e.g., cytokine peptide mimetics and antibody peptide mimetics. “Activation” may refer to cellular activation, as regulated by internal mechanisms as well as by external or environmental factors.

[091] The “activity” of a molecule may describe or refer to the molecule’s binding to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, modulation of the activities of other molecules, and the like. The “activity” of a molecule may also refer to activity in modulating or maintaining interactions between cells, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. “Activity” may also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity]/[mg protein], concentration in a biological compartment, or the like. “Activity” may refer to modulation of components of the innate or adaptive immune systems.

[092] “Administration” and “treatment,” as they apply to an animal, e.g., a canine experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition to the animal, e.g., a canine experimental subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses the contact of a reagent with the cell, as well as the contact of a reagent with a fluid, where the fluid is in contact with the cell. “Treating” or “treatment” means administering a therapeutic agent, such as a composition containing an antibody or antigen-binding fragment of the present invention, internally or externally to a veterinary subject (e.g., canine or feline) having one or more symptoms of disease, or suspected of having a disease for which the agent has therapeutic activity. In this sense, “administration” and “treatment” also include in vitro and ex vivo treatments, for example, of a cell, by a reagent, diagnostic, binding compound or by another cell.

[093] Typically, the agent is administered in an amount effective to alleviate and/or ameliorate one or more disease symptoms in the individual or population treated, either by inducing regression or inhibiting progression of such symptom(s) to any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to as the “therapeutically effective amount”) may vary depending on factors such as the disease state, the age and weight of the individual (e.g., canine), and the ability of the pharmaceutical composition to elicit a desired response in the individual. Whether a disease symptom has been attenuated or ameliorated may be assessed by any clinical measure commonly used by veterinarians or other health care providers skilled in assessing the severity or stage of progression of that symptom. Although an embodiment of the present invention (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptom(s) of the target disease in every subject, it should alleviate the symptom(s) of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student's t-test, the chi2 test, the Mann and Whitney U test, the Kruskal-Wallis test (H-test), the Jonckheere-Terpstra test, and the Wilcoxon test.

[094] The term “immune response” refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or by the liver (including antibodies, cytokines, and complement) that results in the selective damage, destruction, or elimination from the mammalian body (e.g., canine body) of cancer cells, cells or tissues infected with pathogens, or invading pathogens.

[095] The term “individual” includes any organism, preferably an animal, and more preferably a mammal (e.g., canine, feline or human), and most preferably a canine.

[096] As used herein, the term “canine” includes all domestic dogs, Canis lupus familiaris or Canis familiaris, unless otherwise indicated.

[097] As used herein, the term “feline” refers to any member of the family Felidae. Members of this family include wild, zoo, and domestic members, as well as any member of the subfamilies Felinae, e.g., cats, lions, tigers, cougars, jaguars, leopards, snow leopards, panthers, mountain lions, cheetahs, bobcats, bobcats, caracals, or any crossbreeds thereof. Cats also include domestic cats, purebred and/or mixed-breed companion cats, show cats, laboratory cats, cloned cats, and wild or feral cats.

[098] Canine PD-1 has been found to comprise the amino acid sequence of SEQ ID NO: 50 [U.S. Provisional Application No. 61/918,946, filed December 20, 2013, the contents of which are incorporated herein in their entirety]. In a specific embodiment, canine PD-1 is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 49.

[099] Canine PD-1 has been found to comprise the amino acid sequence of SEQ ID NO: 56 [U.S. Provisional Application No. 61/918,946, filed December 20, 2013, supra]. In a specific embodiment, canine PD-L1 is encoded by a nucleotide sequence comprising SEQ ID NO: 55.

[0100] As used herein, a “substitution of an amino acid residue” with another amino acid residue in an amino acid sequence of, for example, an antibody, is equivalent to “substituting an amino acid residue” with another amino acid residue and denotes that a specific amino acid residue at a specific position in the amino acid sequence has been replaced by (or substituted for) a different amino acid residue. For example, such a substitution is denoted as P4A of an Fc region of an IgGB or IgGC amino acid sequence, and in that case, the proline residue at amino acid position 4 of the amino acid sequence of the Fc region of an IgGB or the Fc region of an IgGC has been replaced by an alanine residue.

[0101] Accordingly, such substitutions may be particularly engineered, i.e., by purposefully replacing an alanine with a serine at a specific position in the amino acid sequence, e.g., by recombinant DNA technology. Alternatively, a particular amino acid residue or set of amino acid residues of an antibody may be replaced by one or more amino acid residues through more natural selection processes, e.g., based on the ability of the antibody produced by a cell to bind to a given region on that antigen, e.g., one containing an epitope or portion thereof, and/or for the antibody to comprise a particular CDR that maintains the same canonical structure as the CDR it is replacing. Such substitutions may produce “variant” CDRs and/or variant antibodies.

[0102] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are ideally aligned. As used herein, an amino acid sequence is 100% “identical” to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% “identical” to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. Sequence comparison is performed on a contiguous block of amino acid residues comprised of a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that might otherwise alter the correspondence between the two amino acid sequences are taken into account.

[0103] Sequence similarity includes both identical residues and non-identical and biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable are discussed below.

[0104] The following references refer to BLAST algorithms commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al., J. Mol. Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. Meth.3:266-272 (1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141(1996); Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang, J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al., Comput. Chem. 17:149-163 (1993); Hancock, J.M. et al., Comput. Appl. Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, (1978); Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, vol. 5, suppl. 3.” (1978), M.O. Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F., J. Mol. Biol. 219:555-565 (1991); States, D.J., et al., Methods 3:66-70 (1991); Henikoff, S., et al., Proc. Natl. Academic. Sci. USA 89:10915-10919 (1992); Altschul, S. F., et al., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S., et al., Proc. Natl. Academic. Sci USA 87:2264-2268 (1990); Karlin, S., et al., Proc. Natl. Academic. Sci. USA 90:58735877 (1993); Dembo, A., et al., Ann. Prob. 22:2022-2039(1994); and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), pp. 1-14, Plenum, New York (1997).

Caninized anti-canine antigen antibodies

[0105] As used herein, an antibody is considered to specifically bind to a polypeptide comprising a given antigen sequence (in this case, a portion of the amino acid sequence of a canine antigen, e.g., canine PD-L1) if it binds to polypeptides comprising that portion of the amino acid sequence of the canine antigen, e.g., canine PD-L1, but does not bind to other canine proteins lacking that portion of the canine antigen sequence, e.g., canine PD-L1. For example, an antibody that specifically binds to a polypeptide comprising canine PD-L1 may bind to a FLAG®-tagged form of canine PD-L1, but will not bind to other FLAG®-tagged canine proteins with specificity. An antibody, or binding compound derived from the antigen-binding site of an antibody, binds to its canine antigen, or a variant or mutein thereof, “with specificity” when it has an affinity for that canine antigen, or a variant or mutein thereof, that is at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than its affinity for any other canine antigen tested.

[0106] As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in its broadest sense and encompasses, specifically, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), caninized antibodies, fully canine antibodies, chimeric antibodies, and camelized single domain antibodies. “Parent antibodies” are antibodies obtained by exposing an immune system to an antigen prior to modifying the antibodies for an intended use, such as caninizing an antibody for use as a canine therapeutic antibody.

[0107] As used herein, “antibody fragment” or “antigen-binding fragment” refers to antigen-binding fragments of antibodies, i.e., antibody fragments that retain the ability to specifically bind to the antigen bound by the full-length antibody, e.g., fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies, linear antibodies, single-chain antibody molecules, e.g., scFc; nanobodies, and multispecific antibodies formed from antibody fragments.

[0108] A “Fab fragment” is composed of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” may be the cleavage product of papain from an antibody.

[0109] A “fragment crystallizable” (“Fc”) region contains two heavy chain fragments (i.e., two identical polypeptides) comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are joined by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. In the present invention, the amino acid sequence for each of the four Fc fragments of IgG is based on the identified boundary of the CH1 and CH2 domains as determined by Tang et al. [Vet. Immunol. Immunopathol. 80:259-270 (2001)].

[0110] A “Fab’ fragment” contains a light chain and a portion of a heavy chain that contains the VH domain and the CH1 domain, and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab’ fragments to form an F(ab’)2 molecule.

[0111] An “F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab')2 fragment” may be the pepsin cleavage product of an antibody.

[0112] The “Fv region” comprises the variable regions of both the heavy and light chains, but lacks the constant regions.

[0113] The term “single-chain Fv” or “scFv” refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. [See, Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113 Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); WO 88/01649; and U.S. Pat. Nos. 4,946,778 and 5,260,203.]

[0114] As used herein, the term “canonical structure” refers to the local conformation that can be adopted by each of the hypervariable regions of the light and heavy chains of an antibody within the framework in which they reside. For each hypervariable region, there are a small number of canonical structures (usually denoted by single integers such as 1 or 2, etc.) that can be predicted with great accuracy from the amino acid sequences of the corresponding hypervariable region (particularly within the context of its framework amino acid sequence, as provided below for the corresponding murine anti-canine PD-L1 variable domains). These canonical structures can be determinative of whether a modification of the amino acid sequence of a given CDR will result in retention or loss of the ability to bind its antigen-binding partner [see Chothia and Lesk, Canonical Structures for the hypervariable regions of immunoglobulins, J. Mol. Biol. 196:901-917(1987); Chothia et al., Conformation of immunoglobulin hypervariable regions, Nature, 34:877-883(1989); and Al-Lazikani et al., Standard Conformations for the canonical structures of immunoglobulins, J. Mol. Biol. 273:927-948 (1997)].

[0115] A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more VH regions are covalently joined with a linker peptide to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0116] A “bivalent antibody” comprises two antigen-binding sites. In some cases, both binding sites have the same antigen specificities. However, bivalent antibodies can be bispecific (see below).

[0117] In certain embodiments, the monoclonal antibodies herein also include camelized single domain antibodies. [See, e.g., Muyldermans et al., Trends Biochem. Sci. 26:230 (2001); Reichmann et al., J. Immunol. Methods 231:25 (1999); WO 94/04678; WO 94/25591; U.S. 6,005,079]. In one embodiment, the present invention provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed.

[0118] As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains from another chain and create two antigen-binding sites. [See EP 0 404 097 B1; WO 93/11161; and Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). For a review of engineered antibody variants, see generally [Holliger and Hudson Nat. Biotechnol. 23:1126-1136 (2005)].

[0119] Typically, an antibody or antigen-binding fragment of the invention retains at least 10% of its binding activity to canine PD-L1 (compared to the parent antibody) when such activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the canine antigen, (e.g., PD L1) in terms of binding affinity to the parent antibody. It is also intended that a caninized antibody or antigen-binding fragment of the invention may include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “functionally conserved variants” of the antibody) that do not substantially alter its biological activity.

[0120] “Isolated antibody” refers to the purified state and, in such context, means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell fragments and growth medium. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compound as described herein.

[0121] As used herein, a “chimeric antibody” is an antibody that has the variable domain of a first antibody and the constant domain of a second antibody, where the first and second antibodies are from different species. [U.S. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)]. Typically, the variable domains are obtained from an antibody from an experimental animal (the “parent antibody”), such as a rodent, and the constant domain sequences are obtained from antibodies from the animal subject, e.g., canine, such that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a canine subject than the parent (e.g., rodent) antibody.

[0122] As used herein, the term “caninized antibody” refers to forms of the antibodies that contain sequences from both canine and non-canine (e.g., murine) antibodies. In general, the caninized antibody will include substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-canine immunoglobulin (e.g., comprising 6 murine anti-canine PD-L1 CDRs, as exemplified below) and all or substantially all of the canine frames.

[0123] The term “wholly canine antibody” refers to an antibody that comprises only canine immunoglobulin protein sequences. A wholly canine antibody may contain murine carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody that comprises only mouse immunoglobulin sequences. Alternatively, a wholly canine antibody may contain rat carbohydrate chains if produced in a mouse, a rat cell, or a hybridoma derived from a rat cell. Similarly, “rat antibody” refers to an antibody that comprises only rat immunoglobulin sequences.

[0124] The variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are generally the same.

[0125] Typically, the variable domains of the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), situated within relatively conserved framework (FR) regions. The CDRs are usually flanked by framework regions, allowing binding to a specific epitope. In general, from the N-terminus to the C-terminus, both the variable domains of the light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain for human antibodies generally follows the definitions in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat, et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol. Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].

[0126] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues of a “complementarity determining region” or “CDR” (i.e., CDRL1, CDRL2, and CDRL3 in the light chain variable domain and CDRH1, CDRH2, and CDRH3 in the heavy chain variable domain). [See Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), defining the CDR regions of a human antibody by sequence; also see Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), defining the CDR regions of an antibody by structure]. As used herein, the term “framework” or “FR” residues refers to those variable domain residues that are not the hypervariable region residues defined herein as CDR residues.

[0127] As used herein, the term “canine framework” refers to the amino acid sequence of the heavy chain and light chain of a canine antibody other than the hypervariable region residues defined herein as CDR residues. In both chains, the amino acid sequences of the native canine CDRs are replaced with the corresponding foreign CDRs (e.g., those of a mouse antibody). Optionally, the heavy and/or light chains of the canine antibody may contain some foreign non-CDR residues, e.g., in order to preserve the conformation of the foreign CDRs in the canine antibody and/or to modify the Fc function, as exemplified below.

[0128] As used herein, an “anti-canine PD-L1 antibody” refers to an antibody that has been raised against PD-L1 (e.g., in a mammal such as a mouse or rabbit) and that specifically binds to canine PD-L1. An antibody that “specifically binds to canine PD-L1,” or an antibody that “specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 56,” is an antibody that exhibits preferential binding to canine PD-L1 compared to other antigens, e.g., that binds to canine PD-L1 “with specificity.” Binding does not require absolute binding specificity. An anti-canine PD-L1 antibody is considered “specific” for canine PD-L1 if its binding is determinant of the presence of canine PD-L1 in a sample, or if it is capable of altering the activity of canine PD-L1 without unduly interfering with the activity of other molecules in a canine sample, e.g., without producing undesirable results such as false positives in a diagnostic context or side effects in a therapeutic context. The degree of specificity required for an anti-canine PD-L1 antibody may depend on the intended use of the antibody, and to any degree is determined by its suitability for use for an intended purpose. The antibody, or the binding compound derived from the antigen-binding site of an antibody, of the contemplated method binds to its antigen, or a variant or mutein thereof, with an affinity that is at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for any other antigen.

[0129] Accordingly, the present invention provides canine anti-PD-L1 antibodies, or antigen-binding fragments thereof (including in isolated form) that bind to canine PD-L1 (e.g., with specificity), and uses of such antibodies or fragments thereof. In specific embodiments, murine anti-canine PD-L1 CDRs of murine anti-canine PD-L1 antibodies are provided, which have been demonstrated to both bind to canine PD-L1 and block the binding of canine PD-L1 to its receptor, e.g., canine PD-1. These CDRs can be inserted into a modified canine framework of the present invention to make a caninized murine anti-canine PD-L1 antibody, as exemplified herein.

[0130] More specifically, a “caninized murine anti-canine PD-L1 antibody” of the present invention refers to an antibody comprising the three heavy chain CDRs and the three light chain CDRs of a murine anti-canine PD-L1 antibody, together with a canine framework or a modified canine framework. A modified canine framework comprises one or more amino acid changes, as exemplified in the present invention, that further optimize the efficacy of the caninized antibody, e.g., to increase, reduce, or eliminate the effector functions of the antibody, to increase its binding to the canine antigen, e.g., to canine PD-L1, and/or to increase its ability to block the binding of the canine antigen, e.g., canine PD-L1, to its natural binding partner (e.g., to canine PD-1, in the case where the antigen is canine PD-L1).

[0131] “Homology” refers to the similarity between two polynucleotide sequences or between two polypeptide sequences when they are perfectly aligned. When a position in the two sequences being compared is occupied by the same base or monomeric subunit of the amino acid, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology is the number of homologous positions shared by the two sequences divided by the total number of positions compared x 100. For example, if 6 of the 10 positions in the two sequences are coincident or homologous when the sequences are ideally aligned, then the two sequences are 60% homologous. Comparison is generally made when two sequences are aligned to provide maximum percentage homology.

[0132] “Isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, that is not associated with all or a portion of a polynucleotide to which the isolated polynucleotide is found in nature, or that is linked to a polynucleotide to which it is not linked in nature. For purposes of this description, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” the specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

[0133] The phrase “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0134] A nucleic acid is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, the DNA for a presequence or secretory leader is operably linked to the DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0135] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary cell of the individual and cultures derived therefrom without regard to the number of transfers. It should be understood that not all progeny will have precisely identical DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same biological function or activity as tested for the originally transformed cell are included. Where distinct designations are intended, this will be clear from the context.

[0136] As used herein, “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from the JOINSOLVER® germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al. [Nucleic Acids Res. 33: D256-D261 (2005)].

Properties of caninized antibodies

[0137] In canine animals, there are four IgG heavy chains referred to as A, B, C, and D. These heavy chains represent four different subclasses of IgG, which are referred to as IgGA, IgGB, IgGC, and IgGD. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270 (2001)]. The DNA and amino acid sequences for these heavy chains are also available from the GenBank databases. For example, the amino acid sequence for the heavy chain of IgGA has accession number AAL35301.1, IgGB has accession number AAL35302.1, IgGC has accession number AAL35303.1, and IgGD has accession number (AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda. The DNA and amino acid sequences of these chains can be obtained from the GenBank databases. For example, the amino acid sequence of the kappa light chain has accession number ABY 57289.1 and the lambda light chain has accession number ABY 55569.1. In the present invention, the amino acid sequence for each of the four Fc fragments of canine IgG is based on the identified boundary of the CH1 and CH2 domains as determined by Tang et al, above.

[0138] The development of a therapeutic monoclonal antibody is a complex process that involves the coordination of a complex set of activities to generate the desired antibody. These include optimizing the antibody's specificity, affinity, functional activity, expression level in engineered cell lines, long-term stability, elimination or enhancement of effector functions, and development of commercially viable manufacturing and purification methods. Considering the objectives of the present invention and in addition to the ability to activate immune cells, a caninized or canine monoclonal antibody against canine PD-L1 ideally possesses three additional attributes: 1. lack of effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), 2. relatively long half-life in vivo; and 3. be readily purified on a large scale using industry standard technologies, such as those based on protein A chromatography.

[0139] None of the naturally occurring canine IgG subclasses satisfy all of these criteria. For example, IgGB can be purified using protein A, but has a high level of ADCC activity. IgGC also has considerable ADCC activity. On the other hand, IgGA binds weakly to protein A, but exhibits undesirable ADCC activity. Furthermore, neither IgGC nor IgGD can be purified on protein A columns, although IgGD exhibits no ADCC activity. Furthermore, IgGC has a short serum half-life, since it does not bind to the canine FcRn receptor. The present invention overcomes this difficulty by providing modified canine IgG antibodies specific for canine antigens, e.g., canine PD-L1; such antibodies lack the effector functions such as ADCC and CDC, exhibit relatively long half-lives, and can be readily purified using industry standard protein A chromatography.

[0140] Until now, genetically modified canine IgGs that lack the ADCC and CDC effector functions and that, in addition, could be purified by protein A chromatography, have not been previously described. As described in the present invention, a single substitution at a position in canine IgG that is analogous to that in human and mouse IgG, such as N297A or D265A, does not completely eliminate the ADCC and CDC effector functions in the corresponding canine antibody. For example, although each of the N297 and D265 substitutions in human or murine antibodies results in abrogation of binding to Fcv and C1q receptors, no single substitution completely abrogated the binding of canine antibodies to C1q. Instead, as further described below, in order to eliminate both ADCC and CDC in canine antibodies of the IgGB or IgGC subclasses, it was found necessary to make a double substitution in the canine antibody Fc, combining both an asparagine-to-alanine substitution and an aspartic acid-to-alanine substitution. Furthermore, quite unexpectedly, a substitution that had been shown to reduce effector functions in human antibodies actually resulted in an increase in binding of the corresponding canine IgG to FcvR and C1q.

[0141] In order to generate canine IgGB and IgGC variants that lack effector functions, modified canine IgGB or modified canine IgGc heavy chains can be generated. A total of seven amino acid residues that are present in both of these canine fragment crystallizable regions (cFcs) have been identified for such possible substitution. These seven amino acid residues are: P4, D31, N63, G64, T65, A93, and P95 for both the amino acid sequence of SEQ ID NO: 66 for the canine IgGB Fc; and the amino acid sequence of SEQ ID NO: 68 for the canine IgGC Fc. In this sense, the amino acid sequence of SEQ ID NO: 2 differs from SEQ ID NO: 66 in having the amino acid residues at positions: 4, 31, 63, 64, 65, 93 and 95, which are proline (P), aspartic acid (D), asparagine (N), glycine (G), threonine (T), alanine (A) and proline (P), respectively, in the amino acid sequence of SEQ ID NO: 66 as “X” (or “Xaa”, with the three-letter code) for all seven positions, meaning that these seven amino acid positions can be any of the twenty natural amino acids (see the list in column 1 of Table 1 below). Similarly, the amino acid sequence of SEQ ID NO: 4 differs from SEQ ID NO: 68 in having the amino acid residues at positions 4, 31, 63, 64, 65, 93, and 95 listed as “X” (or “Xaa,” with the three-letter code) for all seven positions, meaning that these seven amino acid positions could be any of the twenty naturally occurring amino acids. The amino acid sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 1, while the amino acid sequence of SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0142] In one embodiment, the cFc comprises the amino acid sequence of SEQ ID NO: 66, with the following substitutions P4 (A, G, or S), D31(A, G, or S), N63 (A, G, or S), G64 (A or P), T65(A, G, or S), A93 (G, or S), and P95(A, G, or S); wherein P4 (A, G, or S) means that the proline residue at position 4 is replaced by an alanine, glycine, or serine residue, and similarly, G64 (P or A) means that the glycine residue at position 64 is replaced by a proline or an alanine residue, etc.). In a particular embodiment, the cFc comprises the amino acid sequence of SEQ ID NO: 66 with the following substitutions: P4A, D31A, N63A, G64P, T65A, A93G, and P95A.

[0143] In a related embodiment, the cFc comprises the amino acid sequence of SEQ ID NO: 4, which contains 7 amino acids designated as Xaa, with the following amino acid residues: A4, A31, A63, G64, T65, G93, and A95, i.e., the amino acid sequence of SEQ ID NO: 68, with the following five (5) amino acid residue changes: P4A, D31A, N63A, A93G, and P95A, and the remaining two amino acid residues of the seven, G64 and T65, being retained from the amino acid sequence of SEQ ID NO: 68.

[0144] All of the amino acid sequences of SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32 contain “X” (or “Xaa” with the three-letter code) at seven amino acid positions, meaning that these seven amino acid positions can be any of the twenty naturally occurring amino acids listed in column 1 of Table 1 below. Notably, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32 comprise the amino acid sequence of either SEQ ID NO: 2 or SEQ ID NO: 4 within their respective sequences. Specific examples of the amino acid residues at one or more of these seven positions of the amino acid sequences are outlined above and below and are therefore included within the genus of the individual amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32, as well as in the caninized antibodies comprising these sequences.

[0145] Table 10 provided below specifically correlates the seven amino acid positions that may be substituted, as described herein, of the cIgGB Fc (SEQ ID NO: 66 and SEQ ID NO: 2) and the cIgGC Fc (SEQ ID NO: 68 and SEQ ID NO: 4) with those of the complete canine heavy chains comprising these cFc amino acid sequences, namely SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32. Accordingly, the actual position in the complete IgGB or IgGC sequence may be readily coordinated with that of the cFc it comprises through the use of Table 10 below.

[0146] In particular embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 26 comprising (i) P, A, G, or S at position 242, (ii) D, A, G, or S at position 269, (iii) N, A, G, or S at position 301, (iv) G, P, or A at position 302, (v) T, A, G, or S at position 303, (vi) A, G, or S at position 331, and (vii) P, A, G, or S at position 333. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 28 or 30 comprising (i) P, A, G, or S at position 240, (ii) D, A, G, or S at position 267, (iii) N, A, G, or S at position 299, (iv) G, P, or A at position 300, (v) T, A, G, or S at position 301, (vi) A, G, or S at position 329, and (vii) P, A, G, or S at position 331. In other embodiments, the heavy chain of an antibody comprises the amino acid sequence of SEQ ID NO: 31 comprising (i) P, A, G, or S at position 238, (ii) D, A, G, or S at position 265, (iii) N, A, G, or S at position 297, (iv) G, P, or A at position 298, (v) T, A, G, or S at position 299, (vi) A, G, or S at position 327, and (vii) P, A, G, or S at position 329.

[0147] The present invention also provides modified canine IgGDs comprising a hinge region of IgGA, IgGB or IgGC in place of their native IgGD hinge region. Alternatively, the hinge region of the IgGD can be genetically modified by replacing a serine residue with a proline residue, as shown in Table 5. Such modifications can lead to a canine IgGD without the fab arm exchange. Modified canine IgGDs can be constructed using standard methods of recombinant DNA technology [e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)]. To construct such variants, the nucleic acids encoding the amino acid sequence of the canine IgGD can be modified so as to encode the modified IgGDs. The modified nucleic acid sequences are then cloned into expression plasmids for protein expression. Nucleic acids encoding IgGD Fcs with the substituted hinge region are exemplified by the nucleotide sequences of SEQ ID NOs: 7, 9, and 11, which encode the amino acid sequences of SEQ ID NOs: 8, 10, and 12. A nucleic acid encoding a canine IgGD Fc with a modified IgGD hinge region comprises the nucleotide sequence of SEQ ID NO: 5, which encodes the amino acid sequence of SEQ ID NO: 6.

[0148] The present invention further provides full-length canine heavy chains that can correspond with corresponding light chains to form a caninized antibody. Accordingly, the present invention further provides caninized murine anti-canine canine antigen antibodies (including isolated caninized murine anti-canine PD-L1 antibodies) and methods of using the antibodies or antigen-binding fragments thereof in the treatment of disease, e.g., in the treatment of cancer in canines.

[0149] Furthermore, the present invention provides caninized murine anti-canine PD-L1 antibodies or antigen-binding fragments that bind to canine PD-L1 and block the binding of canine PD-1 to canine PD-L1. In certain embodiments, the caninized murine anti-canine PD-L1 antibodies comprise a modified canine IgGB Fc, a modified canine IgGC Fc, or a modified canine IgGD without the fab arm exchange, as described herein.

[0150] The antibody, or antigen-binding fragment thereof that binds to canine antigen, e.g., canine PD-L1, can comprise one, two, three, four, five, or six of the complementarity determining regions (CDRs) of a murine anti-canine antibody as described herein. The one, two, three, four, five, or six CDRs can be independently selected from the CDR sequences provided below. In a further embodiment, the antibody or antigen-binding fragment thereof that binds to canine PD-L1 comprises a canine antibody kappa light chain comprising a murine light chain CDR-1, CDR-2, and/or CDR-3 and a canine antibody heavy chain IgG comprising a murine heavy chain CDR-1, CDR-2, and/or CDR-3. Accordingly, the present invention further provides the complete canine heavy chains which can then be combined, for example, with the corresponding light chains to form a caninized antibody [see Table 2 below, in which the sequences of two sets of murine anti-canine PD-L1 CDRs are provided, for example, 4F9 and 5F12 are provided].

[0151] In other embodiments, the invention provides antibodies or antigen-binding fragments thereof that bind to PD-L1, with specificity, and have canine antibody kappa light chains comprising from one to six different CDRs comprising at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 16, 17, 18, 22, 23, and/or 24, and canine antibody heavy chain IgG comprising from one to six different CDRs comprising at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the amino acid sequences of SEQ ID NOs: 13, 14, 15, 19, 20, or 21, further exhibiting the binding and functional properties desired. In another embodiment, the antibody or antigen-binding fragment of the present invention comprises a canine framework comprising one of the IgG heavy chain sequence with a kappa light chain, having one or more of the aforementioned CDR amino acid sequences with 0, 1, 2, 3, 4, or 5 conservative or non-conservative amino acid substitutions, while exhibiting the desired binding and functional properties.

[0152] “Conservatively modified variants” or “conservative substitution” refers to replacements of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that changes can often be made without altering the biological activity of the protein. Those skilled in the art recognize that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity [see, e.g., Watson et al., Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. Furthermore, structurally or functionally similar amino acid substitutions are less likely to disrupt biological activity. Exemplary conservative substitutions are defined in Table I immediately below.TABLE 1EXEMPLARY CONSERVATIVE AMINO ACID SUBSTITUTIONS

[0153] Functionally conserved variants of the antibodies of the invention are also contemplated by the present invention. “Functionally conserved variants,” as used herein, refers to antibodies or fragments in which one or more amino acid residues have been altered without altering a desired property, such as antigen affinity and/or specificity. Such variants include, but are not limited to, the replacement of one amino acid with another that has similar properties, such as the conservative amino acid substitutions in Table I above.

Nucleic acids

[0154] The present invention further comprises nucleic acids encoding the immunoglobulin chains of caninized murine canine anti-PD-L1 antibodies and antigen-binding fragments thereof described herein (see Examples below).

[0155] Also included in the present invention are nucleic acids encoding immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% identical, at least about 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of the CDRs and/or cFcs and/or canine antibodies provided herein, when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to provide the greatest match between the respective sequences over the full length of the respective reference sequences. The present invention further provides nucleic acids encoding immunoglobulin polypeptides comprising amino acid sequences that are at least about 70% similar, at least about 80% similar, more preferably at least about 90% similar, and most preferably at least about 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of the reference amino acid sequences when the comparison is performed with a BLAST algorithm, wherein the parameters of the algorithm are selected to provide the greatest match between the respective sequences over the entire length of the respective reference sequences.

[0156] This present invention also provides expression vectors comprising the nucleic acids (including isolated nucleic acids) of the invention, wherein the nucleic acid is operably linked to control sequences that are recognized by a host cell when the host cell is transfected with the vector. Also provided are host cells comprising an expression vector of the present invention and methods for producing the antibody, or antigen-binding fragment thereof described therein, comprising culturing a host cell with an expression vector encoding the antibody or antigen-binding fragment in culture medium, and isolating the antigen or antigen-binding fragment thereof from the host cell or culture medium.

[0157] A caninized murine anti-canine PD-L1 antibody can, for example, be produced recombinantly by methods that are known in the art. Mammalian cell lines available as hosts for expression of the antibodies, or fragments described herein, are well known in the art and include many immortalized cell lines available from the American Collection of Cell Cultures (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and various other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding portion, or fragment thereof, the light chain and/or antigen-binding fragment thereof are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

[0158] Antibodies can be recovered from the culture medium using standard protein purification methods. Furthermore, expression of the antibodies of the invention (or other portions thereof) from production cell lines can be enhanced with various known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach to enhancing expression under certain conditions. The GS system is discussed in whole or in part together with European Patent Nos. 0 216 846, 0 256 055 and 0 323 997, and with European Patent Application No. 89303964.4.

[0159] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of the glycoproteins produced in the transgenic cell line or animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules of the present invention, or comprising the amino acid sequences provided herein, comprise the present invention, regardless of the glycosylation pattern that the antibodies may have. Likewise, in particular embodiments, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because such antibodies have typically been shown to exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo [see, e.g., Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); and U.S. Pat. Nos. 6,946,292 and 7,214,775].

[0160] The present invention further includes antibody fragments of the caninized murine canine anti-PD-L1 antibodies described herein. The antibody fragments include F(ab)2 fragments, which can be produced by enzymatic cleavage of an IgG, e.g., with pepsin. Fab fragments can be produced, e.g., by reduction of F(ab)2 with dithiothreitol or mercaptoethylamine. A Fab fragment is a VL-CL chain attached to a VH-CH1 chain by a disulfide bond. An F(ab)2 fragment corresponds to two Fab fragments which, in turn, are linked by two disulfide bonds. The Fab portion of an F(ab)2 molecule includes a portion of the Fc region between which are disulfide bonds. An FV fragment is a VL or VH region.

[0161] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g., a canine constant region, such as the canine heavy chain constant region of IgGA, IgGB, IgGC, and IgGD0, or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g., a canine light chain constant region, such as a canine kappa or lambda light chain region, or a variant thereof. By way of example and not limitation, the canine heavy chain constant region may be from IgGB, and the canine light chain constant region may be from kappa.

Antibody Manipulation

[0162] The caninized murine canine anti-PD-L1 antibodies of the present invention can be engineered to include modifications to the canine framework of a parent (i.e., canine) monoclonal antibody, for example, to improve the properties of the antibody, as detailed below.

Cross-Blocking Antibodies and Epitope Binding

[0163] Cross-blocking antibodies and antigen-binding fragments thereof that cross-compete with 4F9 and/or 5F12, including caninized cross-blocking antibodies and antigen-binding fragments thereof, are part of the present invention. In addition, antibodies and antigen-binding fragments thereof that bind to the same epitope as any of the anti-canine PD-L1 antibodies or fragments thereof of the present invention are also part of the present invention. Cross-blocking antibodies and antigen-binding fragments can be identified based on their ability to cross-compete with 4F9 and/or 5F12 in standard binding assays (e.g., BIACore®, ELISA, or flow cytometry). For example, standard ELISA assays can be used, in which a recombinant canine PD-L1 protein is immobilized on the plate, one of the antibodies is fluorescently labeled, and the ability of the unlabeled antibodies to compete with the labeled antibody is assessed. Additionally or alternatively, the BIAcore® assay can be used to assess the ability of antibodies to cross-compete. The ability of a test antibody to inhibit the binding of, for example, 4F9 and/or 5F12 to canine PD-L1 demonstrates that the test antibody can compete with 4F9 and/or 5F12 for binding to canine PD-L1 and thus can, in some cases, bind to the same epitope on canine PD-L1 as 4F9 and/or 5F12. In more particular embodiments, cross-blocking antibodies and antigen-binding fragments thereof that cross-compete with 4F9 and/or 5F12 and that also bind to the same epitope on canine PD-L1 as 4F9 and/or 5F12 are also part of the present invention.

Fusion Protein and Peptide Vaccines

[0164] Peptides comprising epitopes (or portions thereof) recognized by canine anti-PD-L1 mAbs and fusion proteins comprising such peptides can be used as vaccines to elicit antibodies that block the binding of PD-L1 to PD-1 and result in activation of T cells and enhancement of the immune response. Such vaccines may be useful as therapeutic vaccines for diseases such as cancer or to act as enhancers of the immune response to other vaccines. To use such peptides as vaccines, one or more of these peptides can be coupled chemically or through recombinant DNA technology techniques to a carrier protein to increase the immunogenicity of such peptides and elicit peptide-specific antibodies. Techniques for coupling the peptides to the carrier proteins are known to those skilled in the art. Peptide (and corresponding fusion protein) vaccines can be used to vaccinate animals, for example, by the intramuscular (IM), subcutaneous (S/C), oral, spray, or in ovo routes (see also below). Such vaccines can be used as subunit proteins expressed from bacterial, viral, yeast, or baculovirus virus systems. Alternatively, such peptide (or fusion protein) vaccines can be administered following administration of a variety of viral or bacterial vectors expressing such peptides or fusion proteins, as can be practiced by methods known to those skilled in the art. Peptide or fusion protein vaccines can be administered in doses of 1 to 1000 μg and can optionally contain an adjuvant and a pharmaceutically acceptable carrier.

Pharmaceutical Compositions and Administration

[0165] To prepare pharmaceutical or sterile compositions of a caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof, they may be admixed with a pharmaceutically acceptable carrier or excipient. [See, e.g., Remington's Pharmaceutical Sciences and the U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0166] Formulations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, suspensions, or aqueous solutions [see, for example, Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY]. In one embodiment, the anti-PD-L1 antibodies of the present invention are diluted to an appropriate concentration in a sodium acetate solution at pH 5 to 6, and NaCl or sucrose is added for tonicity. Additional agents, such as polysorbate 20 or polysorbate 80, may be added to increase stability.

[0167] The toxicity and therapeutic efficacy of antibody compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or in experimental animals, for example, to determine the LD50 (lethal dose for 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index (LD50/ED50). In particular aspects, antibodies that exhibit high therapeutic indices are desirable. Data obtained from such cell culture assays and animal studies can be used in formulating a dosage range for use in canines. The dosage of such compounds is preferably in a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range, depending on the dosage form employed and the route of administration.

[0168] The mode of administration may vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, by insufflation, topical, cutaneous, transdermal or intraarterial.

[0169] In embodiments the caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof can be administered by an invasive route, such as by injection. In other embodiments of the invention, a murine anti-canine PD-L1 antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol administration. Administration by non-invasive routes (e.g., orally, e.g., in a pill, capsule, or tablet) is also within the scope of the present invention.

[0170] The pharmaceutical compositions described in the present invention may also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include: U.S. Patent No. 4,487,603, which describes an implantable microinfusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,447,233, which describes a medication infusion pump for administering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which describes a variable-flow implantable infusion apparatus for continuous drug delivery; and U.S. Patent No. 4,439,196, which describes an osmotic drug delivery system having multi-chambered compartments. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0171] Alternatively, a caninized murine anti-canine PD-L1 antibody can be administered to a site via a systemic route, e.g., by injecting the antibody directly into a pathogen-induced lesion or arthritic joint characterized by immunopathology, often in a depot or sustained-release formulation. Additionally, the antibody can be administered in a targeted drug delivery system, e.g., in a liposome coated with a tissue-specific antibody, targeting, for example, the arthritic joint or pathogen-induced lesion characterized by immunopathology. The liposomes will be targeted to and selectively taken up by the afflicted tissue.

[0172] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of target cells in the biological matrix. Preferably, the administration regimen provides sufficient therapeutic antibody to effect improvement in the target disease state while minimizing undesirable side effects. Accordingly, the amount of biological material administered depends, in part, on the specific therapeutic antibody and the severity of the condition being treated. Guidance on selecting appropriate doses of therapeutic antibodies is available [see, for example, Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY (1991); Bach (ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY (1993); Baert, et al. New Engl. J Med 348:601-608 (2003); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J Med 344:783-792 (2001); Beniaminovitz et al. New Engl. J Med 342:613-619 (2000); Ghosh et al. New Engl. J Med 348:24-32 (2003); Lipsky et al. New Engl. J Med 343:1594-1602 (2000)].

[0173] Determination of the appropriate dose is made by the veterinarian, for example, using parameters or factors known or suspected in the art to affect treatment. Generally, the dose starts with an amount somewhat lower than the optimal dose and is increased by small increments thereafter until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measures include some of the symptoms, for example, the inflammation or level of inflammatory cytokines produced.

[0174] The antibodies or antigen-binding fragments thereof described herein may be provided by continuous infusion, or by doses administered, for example, daily, 1 to 7 times a week, weekly, every other week, monthly, bimonthly, quarterly, semi-annually, annually, etc. The doses may be provided, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. A total weekly dose is usually at least 0.05 μg/kg of body weight, more usually at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/mL, 10 mg/kg, 25 mg/kg, 50 mg/kg, or more [see, for example, Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. New Engl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg. Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother. 52:133-144 (2003)]. Doses may also be given to achieve a predetermined target concentration of a caninized murine anti-canine PD-L1 antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/mL or greater. In other embodiments, a caninized murine anti-canine PD-L1 antibody of the present invention is administered subcutaneously or intravenously on a weekly, every other week, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg/subject.

[0175] As used herein, “inhibit,” “treat,” or “treatment” includes a delay in the development of symptoms associated with a disorder and/or a reduction in the severity of symptoms of such a disorder. The terms further include the amelioration of existing uncontrolled or unwanted symptoms, the prevention of additional symptoms, and the amelioration or prevention of the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred in a vertebrate subject having a disorder, disease, or symptom, or having the potential to develop such a disorder, disease, or symptom.

[0176] As used herein, the terms “therapeutically effective amount,” “therapeutically effective dose,” and “effective amount” refer to an amount of a caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective in causing a measurable improvement in one or more symptoms of a disease or condition or the progression of such a disease or condition. A therapeutically effective dose further refers to the amount of the binding compound sufficient to result in at least a partial improvement in symptoms, e.g., in the treatment, cure, prevention, or amelioration of the relevant clinical condition, or in an increase in the rate of treatment, cure, prevention, or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic measure or parameter of at least 10%; generally of at least 20%; preferably of at least about 30%; more preferably of at least 40%, and most preferably of at least 50%. An effective amount may also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.

Other combination therapies

[0177] As described above, a caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof can be co-administered with one or more other therapeutic agents (such as a chemotherapeutic agent). The antibody can be related to the agent (such as an immune complex) or can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or it can be co-administered with other known therapies.

[0178] In particular combination therapies, a caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof can be co-administered with a caninized murine anti-canine PD-1 antibody or antigen-binding fragment thereof [e.g., as described in U.S. Provisional Application Serial No. 61/918,946, filed December 20, 2013, and U.S. Provisional Application Serial No. 62/030,812, filed July 30, 2014, Provisional Application Serial No. 62/092,496, filed December 16, 2014, PCT/EP2014/078655 (WO2015091911), and PCTEP2014/078653 (WO2015/091910), the contents of which are all incorporated herein by reference in two entireties] and/or a caninized murine anti-canine cytotoxic T-lymphocyte-associated protein 4 antibody or antigen-binding fragment thereof. Accordingly, any combination of these three caninized murine anti-canine protein antibodies (i.e., anti-canine PD-L1, PD-1 or CTLA-4) or antigen-binding fragments thereof may be co-administered. For example, the caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof may be administered prior to, subsequent to, or concurrently with a caninized murine anti-canine PD-1 antibody or antigen-binding fragment thereof, and/or with a caninized murine CTLA-4 antibody or antigen-binding fragment thereof. Furthermore, the combination of the caninized murine anti-canine PD-L1 antibody or antigen-binding fragment thereof with the caninized murine anti-canine PD-1 antibody or antigen-binding fragment thereof, and/or the caninized murine CTLA-4 antibody or antigen-binding fragment thereof may also be co-administered with other known therapies, i.e., before, after, or at the same time.

Kits

[0179] Further provided are kits comprising one or more components including, but not limited to, an antibody or antigen-binding fragment as discussed herein that specifically binds to PD-L1 (e.g., a caninized murine anti-canine PD-1 antibody or antigen-binding fragment thereof) in association with one or more additional components including, but not limited to, a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof, and/or a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof, and/or a caninized murine anti-canine CTLA-4 antibody or antigen-binding fragment thereof, a pharmaceutically acceptable carrier, and/or a chemotherapeutic agent as discussed herein. In alternative embodiments, the kit may comprise one or more peptides comprising an epitope or portion thereof (or a fusion protein comprising the epitope or portion thereof) recognized by canine anti-PD-L1 mAbs, as discussed above. The binding composition and/or the chemotherapeutic agent or the peptide comprising an epitope or portion thereof (or a fusion protein comprising the epitope or portion thereof) may be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

[0180] In one embodiment, the kit includes a binding composition of the present invention, e.g., a caninized murine anti-canine PD-L1 (or an antigen-binding fragment thereof) or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and/or a pharmaceutical composition thereof and/or a chemotherapeutic agent in another container (e.g., in a sterile glass or plastic vial) and/or a caninized murine anti-canine PD-1 (or an antigen-binding fragment thereof) and/or a caninized murine anti-canine CTLA-4 (or an antigen-binding fragment thereof) or a pharmaceutical composition thereof in one or more other containers (e.g., in a sterile glass or plastic container).

[0181] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit may also include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices, as discussed above. The kit may also include a package insert including information about the pharmaceutical compositions and dosage forms in the kit. Generally, this information assists pet owners and veterinarians in using the included pharmaceutical compositions effectively and safely. For example, the following information about a combination of the invention may be provided in the package insert: pharmacokinetic studies, pharmacodynamics, clinical studies, efficacy parameters, indications and use, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, form of presentation, appropriate storage conditions, references, manufacturer/distributor information, and patent information.

[0182] For convenience, a specific antibody or binding agent described in the present invention may be provided in a kit, that is, a packaged combination of reagents in predetermined quantities, with instructions for performing the diagnostic or detection assay. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents may vary widely to encompass concentrations in the reagent solution that significantly optimize the sensitivity of the assay. In particular, the reagents may be provided as dry, usually lyophilized, powders including excipients that, on dissolution, will provide a reagent solution of the appropriate concentration.

EXAMPLES EXAMPLE 1 PD-1 AND PD-L1 CANINES Canine PD-1 and PD-L1 sequences:

[0183] U.S. Provisional Application Nos. 61/918,946, filed December 20, 2013, and 62/030,812, filed July 30, 2014, incorporated herein by reference in their entireties, provide: the complete nucleotide sequence of PD-1 (cPD 1) of SEQ ID NO: 49 [SEQ ID NO: 69 includes the signal sequence]; the corresponding translated amino acid sequence of SEQ ID NO: 50 [SEQ ID NO: 70 includes the signal sequence]; the nucleotide sequence encoding the extracellular domain (ECD) of canine PD-1, SEQ ID NO: 51; the amino acid sequence of the ECD of canine PD-1, SEQ ID NO: 52; the nucleotide sequence of the canine PD-1 ECD plus a GT linker and the Fc portion of the human IgG1 Fc gene, SEQ ID NO: 53; and the amino acid sequence of the canine PD-1 ECD plus a GT linker and the Fc portion of the human IgG1 Fc gene, SEQ ID NO: 54 [SEQ ID NO: 81 includes the signal sequence].

[0184] U.S. Provisional Application Nos. 61/918,946, filed December 20, 2013, and 62/030,812, filed July 30, 2014, incorporated herein by reference in their entireties, provide: the complete nucleotide sequence of canine PD-1 (cPD-L1) of SEQ ID NO: 55 [SEQ ID NO: 71 includes the signal sequence]; the corresponding translated amino acid sequence of SEQ ID NO: 56 [SEQ ID NO: 72 includes the signal sequence]; the nucleotide sequence encoding the extracellular domain (ECD) of canine PD-L1, SEQ ID NO: 57; the amino acid sequence of the ECD of canine PD-L1, SEQ ID NO: 58; the nucleotide sequence of the canine PD-1 ECD plus a GT linker and the Fc part of the human IgG1 Fc gene, SEQ ID NO: 59; and the amino acid sequence of the PD-1 ECD plus a GT linker and the Fc part of the human IgG1 Fc gene, SEQ ID NO: 60.

Identification and cloning of canine PD-1:

[0185] A nucleic acid encoding a full-length canine PD-1 (cPD-1) was identified through a search of the NCBI Gene Bank databases (accession number XM_543338.4). The translated amino acid sequence (accession number XP_543338.3) corresponded to a putative canine PD-1 protein that was further identified by searching the NCBI Gene Bank protein databases and aligning the identified amino acid sequence with murine, feline, and human PD-1 amino acid sequences. The DNA sequence corresponding to the full-length canine PD-1 gene that was codon optimized for CHO cells was synthesized and cloned into a plasmid designated p96793. Comparison of the predicted canine PD-1 DNA and protein sequences with known PD-1 DNA and known sequences led to the identification of the DNA sequences encoding the canine PD-1 extracellular domain (ECD) and the canine PD-1 ECD amino acid sequence.

[0186] A DNA sequence encoding the canine PD-1 ECD, plus a GT linker and 8 histidine residues, was synthesized and cloned into a plasmid designated LPD2726. A nucleic acid sequence corresponding to the canine PD-1 ECD, plus a GT linker and the Fc portion of the IgG1 Fc gene was chemically synthesized and cloned into a plasmid designated LPD2727. The canine PD-1 ECD and the Fc portion of the human IgG1 Fc comprise the amino acid sequences of SEQ ID NO: 81 (including the signal sequence).

Identification and cloning of canine PD-L1:

[0187] A nucleic acid encoding a full-length canine PD-L1 was identified through a search of the NCBI Gene Bank databases (accession number XM_541302.4). The translated amino acid sequence (accession number XP-541302.4) corresponding to the putative canine PD-L1 protein was identified by searching the NCBI Gene Bank protein databases and aligning the identified sequence with known mouse and human PD-L1 sequences.

[0188] Comparison of the DNA encoding canine PD-L1 with known PD-L1 sequences identified the DNA sequence corresponding to the canine PD-L1 ECD domain (which was codon optimized for CHO cells). The predicted amino acid sequence of the canine PD-L1 ECD is SEQ ID NO: 58. DNA encoding the PD-L1 ECD plus the GT linker and 8 histidine residues was synthesized and cloned into a plasmid designated LPD2695.

[0189] A DNA sequence encoding the amino acid sequence of the canine PD-L1 ECD plus the GT linker and the Fc portion of the human IgG1 Fc was chemically synthesized and cloned into a plasmid designated LPD2697. The canine PD-1 ECD plus the GT linker and the Fc portion of the human IgG1 comprises the amino acid sequences of SEQ ID NO: 60.

Expression of PD-1 and PD-L1 proteins:

[0190] Expression plasmids encoding the PD-1ECD-HIS, PD-1ECD-Fc, PD-L1 ECD-HIS, and PD-L1ECD-Fc proteins were transfected into HEK 293 cells and the proteins were purified from the supernatant of the transfected cells using protein A for the Fc-fusion proteins or nickel (Ni2+) column chromatography for the HIS-tagged proteins. The purified proteins were used for: ELISA or binding assays as detailed below. The expressed proteins were analyzed by SDS-PAGE gels.

[0191] Full-length canine PD-1 DNA sequence: The signal sequence is underlined and in bold. The nucleotide sequence SEQ ID NO:49 is missing the signal sequence. atggggagccggcggggggccctggccgctcgtctggggccgtgctgcagctggggctggtggccaggatggctcct agactcccctgacaggccctggagccgctcaccttctccccggcgcagctcacggtgcaggagggagagaac gccacgttcacctgcagcctggccgacatccccgacagcttcgtgctcaactggtaccgcctgagccccccgcaac cagacggacaagctggccgccttccaggaggaccgcatcgagccggggccgggacaggcgcttccgcgtcatgc ggctgcccaacgggcgggacttccacatgagcatcgtcgctgcgcgcctcaacgacagcggcatctacctgtgc ggggccatctacctgccccccaacacacagatcaacgagagtccccgcgcagagctctccgtgacggagagaa ccctggagccccccacacagagccccagccccccacccagactcagcggccagttgcaggggctggtcatcggc gtcacgagcgtgctggtgggtgtcctgctactgctgctgctgacctgggtcctggccgctgtcttccccagggcca cccgaggtgcctgtgtgtgcgggagcgaggacgagcctctgaaggagggccccgatgcagcgcccgtcttcacc ctggactacggggagctggacttccagtggcgagagaagacgccggagccccccggcgccctgtgccccggagc agaccgagtatgccaccatcgtcttcccgggcaggccggcgtccccgggccgcagggcctcggccagcagcctg cagggagcccagcctccgagccccgaggacggacccggcctgtggcccctctgaAmino acid sequence of full-length canine PD-1: signal sequence underlined and in bold. The amino acid sequence of SEQ ID NO: 50 is missing the signal sequence.MGSRRGPWPLVWAVLQLGWWPGWL LDSPDR PWSPLTFSPAQLTVQEGENATFTCSL ADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVMRLPNGRDFHMSIVAARL NDSGIYLCGAIYLPPNTQINESPRAELSVTERTLEPPTQSPSPPPRLSGQLQGLVIGVTSVLV GVLLLLLLTWVLAAVFPRATRGACVCGSEDEPLKEPLKEPVFTLDYGELDFQWREKTPE PPAPCAPEQTEYATIVFPGRPASPGR RASASSLQGAQPPSPEDGPGLWPLCanine PD-1 extracellular domain DNA sequence: SEQ ID NO: 51 (codon optimized for expression in CHO cells) ctggattcccccgacagaccctggagccctctcaccttctcccctgcccagctgaccgtccaggaaggcgagaat gccaccttcacctgcagcctcgccgacatccccgacagcttcgtgctgaactggtacagactgagccccaggaac cagaccgacaagctggccgctttccaggaggacaggatcgaacccggcagggacaggaggtttagggtcatga ggctgcccaacggcagggacttccacatgtccatcgtggccgccagactgaacgactccggcatctacctgtgcg gcgctatctacctgccccccaacacccagatcaacgagagccccagggccgaactgagcgtgacagagagaac cctggaacctcccacccagagcccttcccctcctcctagactgagcggacagctgcagggcctggtg Canine PD-1 extracellular domain: SEQ ID NO: 52LDSPDRPWSPLTFSPAQLTVQEGENATFTCSLADIPDSFVLNWYRLSPRNQTDKLAAFQE DRIEPGRDRRFRVMRLPNGRDFHMSIVAARLNDSGIYLCGAIYLPPNTQINESPRAELSVT ERTLEPPTQSP SPPPRLSGQLQGLVCanine PD-1 extracellular domain-Fc of human IgG1: SEQ ID NO: 53 (codon optimized for expression in HEK-293 cells) ctggattcccccgacagaccctggagccctctcaccttctcccctgcccagctgaccgtccaggaaggcgagaat gccaccttcacctgcagcctcgccgacatccccgacagcttcgtgctgaactggtacagactgagccccaggaac cagaccgacaagctggccgctttccaggaggacaggatcgaacccggcagggacaggaggtttagggtcatga ggctgcccaacggcagggacttccacatgtccatcgtggccgccagactgaacgactccggcatctacctgtgcg gcgctatctacctgccccccaacacccagatcaacgagagccccagggccgaactgagcgtgacagagagaac cctggaacctcccacccagagcccttcccctcctcctagactgagcggacagctgcagggcctggtgggtaccga caaaactcacacatgcccaccgtgcccagcacctgaactcctgggggaccgtcagtcttcctcttccccccaaa acccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacc ctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagca gtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtaca agtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccg agaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctgg tcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatggggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggca gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccct gtctccgggtaaatgaExtracellular domain of canine PD-1 - human IgG1 Fc fusion protein: signal sequence underlined and in bold: the amino acid in SEQ ID NO: 54 is missing the signal sequence.MNFLLSWVHWSLALLLYLHHAKWSQALDSPDRPWSPLTFSPAQLTVQEGENATFTCSL ADIPDSFVLNWYRLSPRNQTDKLAAFQEDRIEPGRDRRFRVMRLPNGRDFHMSIVAARL NDSGIYLCGAIYLPPNTQINESPRAELSVTERTLEPPTQSPSPPPRLSGQLQGLVGTDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKComplete canine PD-L1 DNA sequence: signal sequence underlined and in bold. The nucleotide sequence SEQ ID NO: 55 is without the signal sequence. ttgcactaatcgtctactgggaaatggaggataaaaaaattatacaatttgtgaatggaaaggaagacctgaaa gttcagcacagcagctacagccagagggctcagctattgaaggaccagctcttcttggggaaggctgcgcttca gatcacagatgtgagattgcaggatgcaggggtttactgctgcttgatcggctatggcggtgctgactacaagcg gattactttgaaagttcatgccccgtaccgcaacatcagccaaagaatttctgtggatcctgtcacctctgaacat gaactaatgtgtcaggctgagggttaccctgaggctgaagtcatctggacaagcagtgaccaccgagtcctgag tggcaaaaccaccatcactaattccaatagggaagagaagcttttcaatgtgaccagcacgctgaacatcaatg caacagctaatgagattttctactgcacttttcaaagatcaggtcctgaggaaacaatactgccgagttggtcat cccagaacgactgcccgttccagcaagtgagaggactcatttcatgattctgggacctttcctgttgcttcttggtg tagtcctggcagtcactttctgtctaaaaaaacatgggagaatgatggatgtggaaaaatgttgcacccgagata ggaactcaaagaaacgaaatgatatacaatttgaagagacataaPD-L1 complete canine: signal sequence underlined and in bold. The nucleotide sequence SEQ ID NO: 56 is missing the signal sequence. MRMFSVFTFMAYCHLLKAFTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEM EDKKIIQFVNGKEDLKVQHSSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGG ADYKRITLKVHAPYRNISQRISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTIT NSNREEKLFNVTSTLNINATANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTHFMILG PFLLLLGVVLAVTFCLKKHGRMMDVEKCCTRDRNSKKRNDIQFEETCanine PD-L1 extracellular domain DNA sequence: SEQ ID NO: 57 (codon optimized for expression in CHO cells) tttaccatcaccgtgtccaaggacctgtacgtggtcgagtacggcggcaatgtgaccatggagtgcaagttcccc gtggagaagcagctgaacctgttcgccctcatcgtgtactgggagatggaggacaagaagatcatccagttcgt gaacggcaaggaggacctgaaggtgcagcactccagctactcccagagagcccagctgctgaaggaccagct gttcctgggcaaggccgccctgcagatcaccgacgtgagactgcaggacgccggcgtgtattgctgcctgatcg gctacggaggcgccgactacaagaggatcaccctgaaggtgcatgcaccctacaggaacatcagccagaggat cagcgtcgatcccgtgaccagcgagcacgagctgatgtgccaagccgagggctatcccgaggccgaagtgatct ggaccagcagcgaccacagggtcctgagcggcaagaccaccatcaccaacagcaacagggaggagaagctgt tcaacgtgaccagcaccctcaacatcaacgccaccgccaacgagatcttctactgcaccttccagaggagcggc cccgaagagaacaacaccgccgagctggtgatccccgagagactgcctgtgcctgccagcgagaggacccac Canine PD-L1 extracellular domain protein: SEQ ID NO: 58 FTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQH SSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQ RISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINAT ANEIFYCTFQRSGPE ENNTAELVIPERLPVPASERTH Canine PD-L1 extracellular domain - human IgG1 Fc DNA sequence: SEQ ID NO: 59 (codon optimized for expression in HEK-293 cells)tttaccatcaccgtgtccaaggacctgtacgtggtcgagtacggcggcaatgtgaccatggagtgcaagttcccc gtggagaagcagctgaacctgttcgccctcatcgtgtactgggagatggaggacaagaagatcatccagttcgt gaacggcaaggaggacctgaaggtgcagcactccagctactcccagagagcccagctgctgaaggaccagct gttcctgggcaaggccgccctgcagatcaccgacgtgagactgcaggacgccggcgtgtattgctgcctgatcg gctacggaggcgccgactacaagaggatcaccctgaaggtgcatgcaccctacaggaacatcagccagaggat cagcgtcgatcccgtgaccagcgagcacgagctgatgtgccaagccgagggctatcccgaggccgaagtgatct ggaccagcagcgaccacagggtcctgagcggcaagaccaccatcaccaacagcaacagggaggagaagctgt tcaacgtgaccagcaccctcaacatcaacgccaccgccaacgagatcttctactgcaccttccagaggagcggc cccgaagagaacaacaccgccgagctggtgatccccgagagactgcctgtgcctgccagcgagaggacccacg gtaccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcc ccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccac gaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggg aggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaag gagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggc agccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgac ctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaac tacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctgtctccgggtaaatgaExtracellular domain of canine PD-L1-human IgG1 Fc fusion protein: SEQ ID NO: 60FTITVSKDLYVVEYGGNVTMECKFPVEKQLNLFALIVYWEMEDKKIIQFVNGKEDLKVQH SSYSQRAQLLKDQLFLGKAALQITDVRLQDAGVYCCLIGYGGADYKRITLKVHAPYRNISQ RISVDPVTSEHELMCQAEGYPEAEVIWTSSDHRVLSGKTTITNSNREEKLFNVTSTLNINAT ANEIFYCTFQRSGPEENNTAELVIPERLPVPASERTHGTDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

EXAMPLE 2 MURINE CANINE ANTI-PD-L1 ANTIBODIES Generation of canine anti-PD-L1 monoclonal antibodies:

[0192] A total of three Balb/c mice were immunized multiple times (with 10 μg each time) over a 17-day period. The immunizing antigen was the canine ECD-Fc PD-L1 fusion protein. Following immunization, serum was collected from each mouse and tested for reactivity with the canine ECD-HIS-tagged PD-L1 protein. Spleen cells from the mouse with the highest titer of anti-PD-L1 ECD-HIS in serum were fused with the P3X63Ag8.653 myeloma cell line. Approximately 2 weeks after fusion, supernatant from the putative hybridoma cells were tested by ELISA for their reactivity with the ECD-HIS-tagged PD-L1 protein. Hybridomas producing strong positive signals in the ELISA were subcloned by limiting dilution and retested for reactivity to the canine ECD-HIS-tagged protein PD-L1.

Confirmation of monoclonal antibody reactivity against canine PD-L1:

[0193] The reactivity of hybridoma-secreted antibodies to the canine PD-L1 ECD was confirmed by ELISA. Hybridoma cells were cultured using CELLine bioreactors (Integra-biosciences) for 10 to 30 days. Cells were initially maintained in DMEM supplemented with 4 mM L-glutamine and 10% Gibco ultra-low IgG fetal bovine serum (FBS). Hybridoma cells were seeded into CELLine bioreactor cell chambers at a cell density of approximately 2 x 106 cells/mL in 15 mL of the same medium with the FBS concentration increased to 20%. The outer chamber was filled with 1 L of nutrient medium (DMEM with 4 mM L-glutamine and 2% standard FBS). Hybridoma cells in the cell chamber were expanded to approximately 2.5 × 107 cells/mL for 3 to 7 days. Then, 10 mL of the cell suspension was collected from the cell chamber and replaced with fresh medium to allow cell re-expansion and subsequent collections. This procedure was repeated as needed to obtain the appropriate amount of mAb from each hybridoma clone. The collected cell suspensions were centrifuged, and the supernatants were filtered through 0.2-μm filter membranes. For antibody purification, each clone supernatant was purified using a 5 mL protein G Sepharose 4 fast-flow column (GE Healthcare) by gravity flow. After washing with Tris-EDTA pH 8.0 (TE) buffer 8.0, bound antibodies were eluted using 0.1 M glycine buffer, pH 2.7, followed by pH neutralization using 1 M Tris, pH 8.0. Antibodies were concentrated and buffer exchanged to phosphate-buffered saline (PBS) using Centriprep YM-10,10 kDa NMWL spin filter units (Millipore). Antibody concentrations were quantified using spectrophotometry.

[0194] Canine anti-PD-L1 mAbs were tested for reactivity with canine PD-L1-hFc fusion protein by ELISA as follows: canine PD-L1-hFc fusion protein is diluted to 10 μg/mL in coating buffer (carbonate/bicarbonate pH 9.0) and dispensed at 100 μL/well into 96-well flat-bottom ELISA plates (NUNC). Plates are incubated at 4 °C overnight. Plates are then washed three times with phosphate-buffered saline containing 0.05% Tween-20 (PBST). Next, 200 μL of blocking buffer (5% nonfat milk in PBST) is added to each well and plates are incubated at 37 °C for 60 minutes. The plates are then washed three times with PBST. Then, 100 μL of test mAbs diluted in blocking buffer are added to the first wells of the appropriate columns. The test mAbs are then diluted two-fold to the appropriate plate position. After incubating the plates at 37 °C for 60 minutes, the plates are washed three times with PBST. Then, 100 μL per well of a 1:2,000 dilution of a goat anti-mouse IgG conjugated to horseradish peroxidase (KPL) is added to the plates, which are then incubated at 37 °C for 60 minutes. The plates are then washed three times with PBST and 100 μL/well of 3,3′,5,5′-tetramethylbenzidine substrate (TMB) (from KPL) is added to the plates. The color reaction is allowed to develop for 5 to 20 minutes at 37 °C before measuring absorbance at 650 nm.

CHO cells expressing canine PD-1 protein:

[0195] The full-length canine PD-1 gene was cloned into plasmid p96793. In this plasmid, PD-1 protein expression is induced by an hCMV promoter. DXB11 CHO (dhfr-) cells were maintained in MEM-alpha (Gibco) supplemented with 10% fetal bovine serum. Transfection of CHO cells with plasmid p96793 was performed in 75 cm2 flasks containing approximately 6 x 106 cells by liposome-mediated gene delivery using lipofectamine (Invitrogen). After 48 hours, cells were passaged into nucleoside-free MEM-alpha medium supplemented with 10% FBS and 400 μg/mL hygromycin B (selective medium). Limiting dilution cloning was performed on the pool of hygromycin-resistant dhfr+ cells. Clones were evaluated for canine PD-1 expression by immunofluorescence assay. Briefly, cell monolayers were fixed in 96-well plates with 80% acetone. The fixed and dried cell monolayers were then incubated for 1 hour with polyclonal goat anti-human PD-1 antibody (R&D Systems). Plates were washed with PBS and then incubated for 1 hour with a fluorescein-labeled rabbit anti-goat IgG antibody (KPL). Plates were washed with PBS. Clones showing fluorescence were expanded and cell stocks were established.

Ligand blockade by mouse anti-PD-L1 mAbs:

[0196] A cell-based ELISA (CELISA) assay based on the CHO cell line expressing canine PD-1 was used to demonstrate the ability of mouse anti-canine PD-L1 antibodies to block the interaction between (e.g., block binding of) canine PD-L1 and its receptor canine PD-1. Ligand blocking was confirmed using this assay together with canine PD-L1/Fc protein h as follows: 1. Seed CHO cPD-1 cells in 96-well plates and grow the cells to 95 to 100% confluence. General guidelines for plating CHO cells, on day -3: 1 x 103 c/well (1 x 105 c/mL) -2: 2x104 c/well (2x105 c/mL) -1: 4x104 c/well (4x105 c/mL) 2. English: Dilute 3-fold anti-cPDL1 mAbs in CHO medium, starting at 30 μg/mL, at 100 μL/well. Add cPD-L1-hFc at 4 μg/mL in CHO medium, at 100 μL/well, co-incubate in a dilution plate at 37°C, in 5% CO2 with shaking for 60 min. 3. Aspirate cell culture media from cell-coated plates, wash plates with 3x PBS + 0.05% Tween20 and 1x CHO medium. 4. Add co-incubated cPD-L1/PD-L1 Fc mAbs from the dilution plate to the cell-coated plate. 100 uL/well. Incubate at 370°C, 5% CO2 with shaking for 60 min. 5. Wash plates 6x with PBS + 0.05% Tween 20 (using manual wash protocol). 6. Add anti-human Fc-HRP (Calbiochem) (1:2500) in CHO medium at 100 uL/well and incubate for 30 to 60 min at 370C/5% CO2. 7. Wash plates 5x with PBS + 0.05% Tween20 (using manual wash protocol). 8. Add 100 μL/well TMB microwell substrate. Incubate at room temperature for 10 min. Use a Pierce one-step substrate. 9. Stop with 1.5 M phosphoric acid at 100 μL/well. 10. Measure A450 - A620 in an ELISA reader.

Cloning and identification of DNA sequences corresponding to the variable regions of mouse canine anti-PD-L1 mAbs:

[0197] The DNA sequence of the mouse VH and VL chains and the DNA sequences encoding their CDRs are identified after isolation of mRNA from each hybridoma using standard molecular biology methods. The sequences of the variable regions of the heavy and light chains of the two antibodies exemplified in the present invention are provided in Table 12 below. The SEQ ID Nos.: of the predicted amino acid sequences of the CDRs of these hybridomas are listed in Table 2 below: TABLE 2 AMINO ACID SEQUENCES OF THE CDRS

EXAMPLE 3 CANINIZATION AND CHARACTERIZATION OF CANINIZED ANTIBODIES

[0198] In order to produce caninized antibodies, it was necessary to identify the DNA sequence encoding the heavy and light chains of canine IgG. The nucleotide and amino acid sequences of the canine heavy chain can be obtained from the NCBI gene and protein databases. There are four known IgG subclasses of canine IgG: IgGA, IgGB, IgGC, and IgGD, and two types of light chains: kappa and lambda. Table 7 lists the amino acids and nucleotides of the SEQ ID Nos. of the unmodified canine Fc fragments.

[0199] Without wishing to be bound to any specific approach, the process for producing anti-PD-L1 monoclonal antibody variants with various canine and mouse sequence contents involved the following general scheme:i) Determine the nucleotide sequence of the VH and VL chains of mouse mAbs;ii) Identify the H and L chain CDRs of mouse mAbs;iii) Identify a suitable H and L chain from canine IgG;iv) Determine the nucleotide sequence of the H and L chains of IgG;v) Replace the nucleotide sequence encoding the CDRs of the endogenous canine H and L chain with the nucleotide sequences encoding the respective mouse CDRs. Likewise, optionally replace some residues from the canine framework with selected residues from the mouse framework regions;vi) Synthesize the nucleotide from step (v) and insert it into a suitable expression plasmid; transfect plasmids into appropriate cells, e.g. HEK 293 cells;vii) Purify the expressed antibody from HEK 293 supernatant; andviii) Test the antibody for binding to canine PD-L1.

EXAMPLE 4 GENETICALLY MODIFIED CANINE IgGs

[0200] In order to generate canine IgG variants that lack effector functions, several mutant canine IgGB heavy chains have been generated [see U.S. Provisional Application No. 62/030,812, filed July 30, 2014, and incorporated herein in its entirety by reference]. Such variants may include one of the following single or combined substitutions in the Fc portion of the heavy chain amino acid sequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. Variant heavy chains (i.e., containing such amino acid substitutions) were cloned into expression plasmids and transfected into HEK 293 cells along with a plasmid containing the gene encoding a light chain. Intact antibodies expressed and purified from HEK 293 cells were evaluated for binding to FcvRI and C1q to assess their potential for mediating immune effector functions. Table 3 lists examples of the plasmids encoding the genetically modified caninized heavy chains, the caninized heavy chains; and the genetic modifications in these heavy chains. The variant heavy chains were used for evaluation of effector function in the genetically modified mAbs. All heavy chains comprised the CDRs of murine anti-canine PD-1 antibodies. [See U.S. Provisional Application No. 62/030,812, supra.] TABLE 3 TABLE 4cFc MODIFIED OR NATIVE cFc WITH HINGE SEQUENCES * The substitutions are at P4, D31, N63, G64, T65, A93, and P95 of the amino acid sequences of SEQ ID NOs: 2 and 4; or in the nucleotides that encode the amino acids for the nucleotide sequences of SEQ ID NOs: 1 and 3. # Single amino acid substitution, as shown in Table 5 below, in the hinge region of IgGD.TABLE 5HINGE REGION SEQUENCES # Single amino acid substitution of a serine to a proline as in bold and underlined.TABLE 6Canine PD-1/PD-L1 sequences TABLE 7 NATIVE cFc SEQUENCES TABLE 8CDR AMINO ACID SEQUENCES TABLE 9 INDIVIDUAL REPLACED CANINIZED HEAVY CHAINS Potential specific substitutions are at P4, D31, N63, G64, T65, A93, and P95TABLE 10CORRELATION OF AMINO ACID RESIDUE POSITIONS # # Correlation of the amino acid residue positions of the native and substituted cFcs with those of the corresponding substituted canine heavy chains. The first column lists the SEQ ID Nos.; the remaining columns list the corresponding amino acid positions. For the two native amino acid sequences (SEQ ID Nos. 66 and 68), the one-letter codes for the natural amino acid residues are also provided. TABLE 11 INDIVIDUAL UNSUBSTITUTED CANIN HEAVY AND LIGHT CHAINS TABLE 12 VARIABLE REGIONS OF HEAVY AND LIGHT CHAINS Modified FcFc cIgGB [SEQ ID NO: 2]LGGXSVFIFPPKPKDTLLIARTPEVTCVVVXLDPEDPEVQISWFVDGKQMQTAKTQPREEQFXXXYRVVSVLPIGHQDWLKGKQFTCKVNNKXLXSPI ERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHE ALHNHYTQESLSHSPGKFc cIgGB [SEQ ID NO: 1]ctgggcggcnnnagcgtgtttatttttccgccgaaaccgaaagataccctgctgattgcgcgcaccccgg aagtgacctgcgtggtggtgnnnctggatccggaagatccggaagtgcagattagctggtttgtggatggcaaa cagatgcagaccgcgaaaacccagccgcgcgaagaacagtttnnnnnnnnntatcgcgtggtgagcgtgctg ccgattggccatcaggattggctgaaaggcaaacagtttacctgcaaagtgaacaacaaannnctgnnnagc ccgattgaacgcaccattagcaaagcgcgcggccaggcgcatcagccgagcgtgtatgtgctgccgccgagcc gcgaagaactgagcaaaaacaccgtgagcctgacctgcctgattaaagatttttttccgccggatattgatgtgg aatggcagagcaacggccagcaggaaccggaaagcaaatatcgcaccaccccgccgcagctggatgaagatg gcagctattttctgtatagcaaactgagcgtggataaaagccgctggcagcgcggcgatacctttatttgcgcgg tgatgcatgaagcgctgcataaccattatacccaggaaagcctgagccatagcccgggcaaaFc cIgGC [SEQ ID NO: 4]LGGXSVFIFPPKPKDILVTARTPTVTCVVVXLDPENPEVQISWFVDSKQVQTANTQP REEQSXXXYRVVSVLPIGHQDWLSGKQFKCKVNNKXLXSPIEEIISKTPGQAHQPNVYVLP PSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKL SVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGKFc cIgGC [SEQ ID NO: 3]ctgggcggcnnnagcgtgtttatttttccgccgaaaccgaaagatattctggtgaccgcgcgcaccccga ccgtgacctgcgtggtggtgnnnctggatccggaaaacccggaagtgcagattagctggtttgtggatagcaa caggtgcagaccgcgaacacccagccgcgcgaagaacagagcnnnnnnnnntatcgcgtggtgagcgtgct gccgattggccatcaggattggctgagcggcaaacagtttaaatgcaaagtgaacaacaaannnctgnnnag cccgattgaagaaattattagcaaaaccccgggccaggcgcatcagccgaacgtgtatgtgctgccgccgagcc gcgatgaaatgagcaaaaacaccgtgaccctgacctgcctggtgaaagatttttttccgccggaaattgatgtgg aatggcagagcaacggccagcaggaaccggaaagcaaatatcgcatgaccccgccgcagctggatgaagatg gcagctattttctgtatagcaaactgagcgtggataaaagccgctggcagcgcggcgatacctttatttgcgcgg tgatgcatgaagcgctgcataaccattatacccagattagcctgagccatagcccgggcaaaFab Fc cIgGD arm swap (S—>P at hinge D)[SEQ ID NO: 6]PKESTCKCIPPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISW FVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISK ARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAP QLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGKFab Fc cIgGD arm swap (S—>P at hinge D)[SEQ ID NO: 5]ccgaaagaaagcacctgcaaatgcattccgccgtgcccggtgccggaaagcctgggcggcccgagcgt gtttatttttccgccgaaaccgaaagatattctgcgcattacccgcaccccggaaattacctgcgtggtgctggat ctgggccgcgaagatccggaagtgcagattagctggtttgtggatggcaaagaagtgcataccgcgaaaaccc agccgcgcgaacagcagtttaacagcacctatcgcgtggtgagcgtgctgccgattgaacatcaggattggctg accggcaaagaatttaaatgccgcgtgaaccatattggcctgccgagcccgattgaacgcaccattagcaaagc gcgcggccaggcgcatcagccgagcgtgtatgtgctgccgccgagcccgaaagaactgagcagcagcgatacc gtgaccctgacctgcctgattaaagatttttttccgccggaaattgatgtggaatggcagagcaacggccagccg gaaccggaaagcaaatatcataccaccgcgccgcagctggatgaagatggcagctattttctgtatagcaaact gagcgtggataaaagccgctggcagcagggcgatacctttacctgcgcggtgatgcatgaagcgctgcagaac cattataccgatctgagcctgagccatagcccgggcaaaArm swap fab Fc cIgGD (hinge A) [SEQ ID NO: 8]FNECRCTDTPPCPVPEPLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQIS WFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTI SKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTT APQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK Fab Fc cIgGD arm swap (hinge A) [SEQ ID NO: 7]tttaacgaatgccgctgcaccgataccccgccgtgcccggtgccggaaccgctggggcggcccgagcgtgt ttatttttccgccgaaaccgaaagatattctgcgcattacccgcaccccggaaattacctgcgtggtgctggatct gggccgcgaagatccggaagtgcagattagctggtttgtggatggcaaagaagtgcataccgcgaaaacccag ccgcgcgaacagcagtttaacagcacctatcgcgtggtgagcgtgctgccgattgaacatcaggattggctgac cggcaaagaatttaaatgccgcgtgaaccatattggcctgccgagcccgattgaacgcaccattagcaaagcgc gcggccaggcgcatcagccgagcgtgtatgtgctgccgccgagcccgaaagaactgagcagcagcgataccgt gaccctgacctgcctgattaaagatttttttccgccggaaattgatgtggaatggcagagcaacggccagccgga accggaaagcaaatatcataccaccgcgccgcagctggatgaagatggcagctattttctgtatagcaaactga gcgtggataaaagccgctggcagcagggcgatacctttacctgcgcggtgatgcatgaagcgctgcagaaccat tataccgatctgagcctgagccatagcccgggcaaaFab arm swap Fc cIgGD (hinge B) [SEQ ID NO: 10]PKRENGRVPRPPDCPKCPAPEMLGPSVFIFPPKPKDILRITRTPEITCVVLDLGRED PEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLP SPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGKFab Arm Swap Fc cIgGD (Hinge B) [SEQ ID NO: 9]ccgaaacgcgaaaacggccgcgtgccgcgcccgccggattgcccgaaatgcccggcgccggaaatgct gggcggcccgagcgtgtttatttttccgccgaaaccgaaagatattctgcgcattacccgcaccccggaaattac ctgcgtggtgctggatctgggccgcgaagatccggaagtgcagattagctggtttgtggatggcaaagaagtgc ataccgcgaaaacccagccgcgcgaacagcagtttaacagcacctatcgcgtggtgagcgtgctgccgattgaa catcaggattggctgaccggcaaagaatttaaatgccgcgtgaaccatattggcctgccgagcccgattgaacg caccattagcaaagcgcgcggccaggcgcatcagccgagcgtgtatgtgctgccgccgagcccgaaagaactg agcagcagcgataccgtgaccctgacctgcctgattaaagatttttttccgccggaaattgatgtggaatggcag agcaacggccagccggaaccggaaagcaaatatcataccaccgcgccgcagctggatgaagatggcagctatt ttctgtatagcaaactgagcgtggataaaagccgctggcagcagggcgatacctttacctgcgcggtgatgcatg aagcgctgcagaaccattataccgatctgagcctgagccatagcccgggcaaaFab arm swap Fc cIgGD (C hinge) [SEQ ID NO: 12]AKECECKCNCNNCPCPGCGLLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEV QISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIE RTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYH TTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGKFab arm swap Fc cIgGD (C hinge) [SEQ ID NO: 11]gccaaggagtgcgagtgcaagtgcaactgcaacaactgcccctgccccggctgcggcctgctgggcggc cccagcgtgttcatcttcccccccaagcccaaggacatcctgagaatcaccagaacccccgagatcacctgcgtg gtgctggacctgggcagagaggaccccgaggtgcagatcagctggttcgtggacggcaaggaggtgcacaccg ccaagacccagcccagagagcagcagttcaacagcacctacagagtggtgagcgtgctgcccatcgagcacca ggactggctgaccggcaaggagttcaagtgcagagtgaaccacatcggcctgcccagccccatcgagagaacc atcagcaaggccagaggccaggcccaccagcccagcgtgtacgtgctgccccccagccccaaggagctgagca gcagcgacaccgtgaccctgacctgcctgatcaaggacttcttcccccccgagatcgacgtggagtggcagagc aacggccagcccgagcccgagagcaagtaccacaccaccgccccccagctggacgaggacggcagctacttcc tgtacagcaagctgagcgtggacaagagcagatggcagcagggcgacaccttcacctgcgccgtgatgcacga ggccctgcagaaccactacaccgacctgagcctgagccacagccccggcaagFc nativeFc cIgGA [SEQ ID NO: 62]LGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSR EQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPP SPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLS VDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGKFc cIgGA [SEQ ID NO: 61]ctgggcggccccagcgtgctgatcttcccccccaagcccaaggacatcctgagaatcaccagaaccccc gaggtgacctgcgtggtgctggacctgggcagagaggaccccgaggtgcagatcagctggttcgtggacggca aggaggtgcacaccgccaagacccagagcagagagcagcagttcaacggcacctacagagtggtgagcgtgc tgcccatcgagcaccaggactggctgaccggcaaggagttcaagtgcagagtgaaccacatcgacctgcccag ccccatcgagagaaccatcagcaaggccagaggcagagcccacaagcccagcgtgtacgtgctgccccccagc cccaaggagctgagcagcagcgacaccgtgagcatcacctgcctgatcaaggacttctacccccccgacatcga cgtggagtggcagagcaacggccagcaggagcccgagagaaagcacagaatgacccccccccagctggacg aggacggcagctacttcctgtacagcaagctgagcgtggacaagagcagatggcagcagggcgaccccttcac ctgcgccgtgatgcacgagaccctgcagaaccactacaccgacctgagcctgagccacagccccggcaagFc cIgGD [SEQ ID NO: 64]LGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPRE QQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPPIERTISKARGQAHQPSVYVLPPS PKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVD KSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK Fc cIgGD [SEQ ID NO: 63]ctgggcggccccagcgtgttcatcttcccccccaagcccaaggacatcctgagaatcaccagaacccccg agatcacctgcgtggtgctggacctgggcagagaggaccccgaggtgcagatcagctggttcgtggacggcaa ggaggtgcacaccgccaagacccagcccagagagcagcagttcaacagcacctacagagtggtgagcgtgct gcccatcgagcaccaggactggctgaccggcaaggagttcaagtgcagagtgaaccacatcggcctgcccagc cccatcgagagaaccatcagcaaggccagaggccaggcccaccagcccagcgtgtacgtgctgccccccagcc ccaaggagctgagcagcagcgacaccgtgaccctgacctgcctgatcaaggacttcttcccccccgagatcgac gtggagtggcagagcaacggccagcccgagcccgagagcaagtaccacaccaccgccccccagctggacgag gacggcagctacttcctgtacagcaagctgagcgtggacaagagcagatggcagcagggcgacaccttcacct gcgccgtgatgcacgaggccctgcagaaccactacaccgacctgagcctgagccacagccccggcaagFc cIgGB [SEQ ID NO: 66]LGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQ PREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPPIERTISKARGQAHQPSVYV LPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKL SVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGKFc cIgGB [SEQ ID NO: 65]ctgggcggccccagcgtgttcatcttcccccccaagcccaaggacaccctgctgatcgccagaacccccg aggtgacctgcgtggtggtggacctggaccccgaggaccccgaggtgcagatcagctggttcgtggacggcaa gcagatgcagaccgccaagacccagcccagagaggagcagttcaacggcacctacagagtggtgagcgtgct gcccatcggccaccaggactggctgaagggcaagcagttcacctgcaaggtgaacaacaaggccctgcccagc cccatcgagagaaccatcagcaaggccagaggccaggcccaccagcccagcgtgtacgtgctgccccccagca gagaggagctgagcaagaacaccgtgagcctgacctgcctgatcaaggacttcttcccccccgacatcgacgtg gagtggcagagcaacggccagcaggagcccgagagcaagtacagaaccacccccccccagctggacgagga cggcagctacttcctgtacagcaagctgagcgtggacaagagcagatggcagagaggcgacaccttcatctgc gccgtgatgcacgaggccctgcacaaccactacacccaggagagcctgagccacagccccggcaagFc cIgGC [SEQ ID NO: 68]LGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPEVQISWFVDSKQVQTANTQP REEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVL PPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSK LSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGKFc cIgGC [SEQ ID NO: 67]ctgggcggccccagcgtgttcatcttcccccccaagcccaaggacatcctggtgaccgccagaaccccca ccgtgacctgcgtggtggtggacctggaccccgagaaccccgaggtgcagatcagctggttcgtggacagcaag caggtgcagaccgccaacacccagcccagagaggagcagagcaacggcacctacagagtggtgagcgtgctg cccatcggccaccaggactggctgagcggcaagcagttcaagtgcaaggtgaacaacaaggccctgcccagcc ccatcgaggagatcatcagcaagacccccggccaggcccaccagcccaacgtgtacgtgctgccccccagcag agacgagatgagcaagaacaccgtgaccctgacctgcctggtgaaggacttcttcccccccgagatcgacgtgg agtggcagagcaacggccagcaggagcccgagagcaagtacagaatgacccccccccagctggacgaggac ggcagctacttcctgtacagcaagctgagcgtggacaagagcagatggcagagaggcgacaccttcatctgcg ccgtgatgcacgaggccctgcacaaccactacacccagatcagcctgagccacagccccggcaagIndividual substituted heavy chainscIgGB 4F9-VH3 [SEQ ID NO: 26]EVQLVQSGGDLVKPGGSVRLSCVASGFTFSYAMSWVRQAPGKGLQWMGTISDG GSYTHYPDNLMGRFTFSLDTAKNTAYLQLNSLRAEDTAVYYCARESYDGYYVANWGQG TLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPS VLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKC PAPEMLGGXSVFIFPPKPKDTLLIARTPEVTCVVVXLDPEDPEVQISWFVDGKQMQTAKT QPREEQFXXXYRVVSVLPIGHQDWLKGKQFTCKVNNKXLXSPIERTISKARGQAHQPSVY VLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSK LSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGKDNA of cIgGB 4F9-VH3 [SEQ ID NO: 25]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcggcttcaccttcagctacgccatgagctgggtgagacaggcccccggcaagggcctgcagtggatgg gcaccatcagcgacggcggcagctacacccactaccccgacaacctgatgggcagattcaccttcagcctggac accgccaagaacaccgcctacctgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcgcca gagagagctacgacggctactacgtggccaactggggccagggcaccctggtgaccgtgagcagcgccagcac caccgcccccagcgtgttccccctggcccccagctgcggcagcaccagcggcagcaccgtggccctggcctgcc tggtgagcggctacttccccgagcccgtgaccgtgagctggaacagcggcagcctgaccagcggcgtgcacacc ttccccagcgtgctgcagagcagcggcctgtacagcctgagcagcatggtgaccgtgcccagcagcagatggcc cagcgagaccttcacctgcaacgtggcccaccccgccagcaagaccaaggtggacaagcccgtgcccaagag agagaacggcagagtgcccagaccccccgactgccccaagtgccccgcccccgagatgctgggcggcnnnag cgtgttcatcttcccccccaagcccaaggacaccctgctgatcgccagaacccccgaggtgacctgcgtggtggt gnnnctggaccccgaggaccccgaggtgcagatcagctggttcgtggacggcaagcagatgcagaccgccaa gacccagcccagagaggagcagttcnnnnnnnnntacagagtggtgagcgtgctgcccatcggccaccagg actggctgaagggcaagcagttcacctgcaaggtgaacaacaagnnnctgnnnagccccatcgagagaacc atcagcaaggccagaggccaggcccaccagcccagcgtgtacgtgctgccccccagcagagaggagctgagc aagaacaccgtgagcctgacctgcctgatcaaggacttcttcccccccgacatcgacgtggagtggcagagcaa cggccagcaggagcccgagagcaagtacagaaccacccccccccagctggacgaggacggcagctacttcctg tacagcaagctgagcgtggacaagagcagatggcagagaggcgacaccttcatctgcgccgtgatgcacgagg ccctgcacaaccactacacccaggagagcctgagccacagccccggcaagcIgGC 4F9-VH3 [SEQ ID NO: 28]EVQLVQSGGDLVKPGGSVRLSCVASGFTFSYAMSWVRQAPGKGLQWMGTISDG GSYTHYPDNLMGRFTFSLDTAKNTAYLQLNSLRAEDTAVYYCARESYDGYYVANWGQG TLVTVSSASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSVSLTSGVHTFPS VLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDKPVAKECECKCNCNNCPCP GCGLLGGXSVFIFPPKPKDILVTARTPTVTCVVVXLDPENPEVQISWFVDSKQVQTANTQ PREEQSXXXYRVVSVLPIGHQDWLSGKQFKCKVNNKXLXSPIEEIISKTPGQAHQPNVYVL PPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSK LSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGKcIgGCDNA 4F9-VH3 [SEQ ID NO: 27]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcggcttcaccttcagctacgccatgagctgggtgagacaggcccccggcaagggcctgcagtggatgg gcaccatcagcgacggcggcagctacacccactaccccgacaacctgatgggcagattcaccttcagcctggac accgccaagaacaccgcctacctgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcgcca gagagagctacgacggctactacgtggccaactggggccagggcaccctggtgaccgtgagcagcgccagcac caccgcccccagcgtgttccccctggcccccagctgcggcagccagagcggcagcaccgtggccctggcctgcc tggtgagcggctacatccccgagcccgtgaccgtgagctggaacagcgtgagcctgaccagcggcgtgcacac cttccccagcgtgctgcagagcagcggcctgtacagcctgagcagcatggtgaccgtgcccagcagcagatggc ccagcgagaccttcacctgcaacgtggcccaccccgccaccaacaccaaggtggacaagcccgtggccaagga gtgcgagtgcaagtgcaactgcaacaactgcccctgccccggctgcggcctgctgggcggcnnnagcgtgttca tcttcccccccaagcccaaggacatcctggtgaccgccagaacccccaccgtgacctgcgtggtggtgnnnctg gaccccgagaaccccgaggtgcagatcagctggttcgtggacagcaagcaggtgcagaccgccaacacccag cccagagaggagcagagcnnnnnnnnntacagagtggtgagcgtgctgcccatcggccaccaggactggct gagcggcaagcagttcaagtgcaaggtgaacaacaagnnnctgnnnagccccatcgaggagatcatcagca agacccccggccaggcccaccagcccaacgtgtacgtgctgccccccagcagagacgagatgagcaagaaca ccgtgaccctgacctgcctggtgaaggacttcttcccccccgagatcgacgtggagtggcagagcaacggccag caggagcccgagagcaagtacagaatgacccccccccagctggacgaggacggcagctacttcctgtacagca agctgagcgtggacaagagcagatggcagagaggcgacaccttcatctgcgccgtgatgcacgaggccctgca caaccactacacccagatcagcctgagccacagccccggcaagCigGB 5F12-VH3 [SEQ ID NO: 30]EVQLVQSGGDLVKPGGSVRLSCVASFTFDYYMNWVRQAPGKGLQWIGRWIFPGS GATYYNERFMGKATISADTAKNTAYMQLNSLRAEDTAVYYCLRSDWDVGDFWGQGTL VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVL QSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPA PEMLGGXSVFIFPPKPKDTLLIARTPEVTCVVVXLDPEDPEVQISWFVDGKQMQTAKTQP REEQFXXXYRVVSVLPIGHQDWLKGKQFTCKVNNKXLXSPIERTISKARGQAHQPSVYVL PPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLS VDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK CIgGB DNA 5F12-VH3 [SEQ ID NO: 29]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcttcaccttcgactactacatgaactgggtgagacaggcccccggcaagggcctgcagtggatcggca gatggatcttccccggcagcggcgccacctactacaacgagagattcatgggcaaggccaccatcagcgccga caccgccaagaacaccgcctacatgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcctg agaagcgactgggacgtgggcgacttctggggccagggcaccctggtgaccgtgagcagcgccagcaccaccg cccccagcgtgttccccctggcccccagctgcggcagcaccagcggcagcaccgtggccctggcctgcctggtg agcggctacttccccgagcccgtgaccgtgagctggaacagcggcagcctgaccagcggcgtgcacaccttccc cagcgtgctgcagagcagcggcctgtacagcctgagcagcatggtgaccgtgcccagcagcagatggcccagc gagaccttcacctgcaacgtggcccaccccgccagcaagaccaaggtggacaagcccgtgcccaagagagag aacggcagagtgcccagaccccccgactgccccaagtgccccgcccccgagatgctgggcggcnnnagcgtgt tcatcttcccccccaagcccaaggacaccctgctgatcgccagaacccccgaggtgacctgcgtggtggtgnnn ctggaccccgaggaccccgaggtgcagatcagctggttcgtggacggcaagcagatgcagaccgccaagaccc agcccagagaggagcagttcnnnnnnnnntacagagtggtgagcgtgctgcccatcggccaccaggactgg ctgaagggcaagcagttcacctgcaaggtgaacaacaagnnnctgnnnagccccatcgagagaaccatcag caaggccagaggccaggcccaccagcccagcgtgtacgtgctgccccccagcagagaggagctgagcaagaa caccgtgagcctgacctgcctgatcaaggacttcttcccccccgacatcgacgtggagtggcagagcaacggcc agcaggagcccgagagcaagtacagaaccacccccccccagctggacgaggacggcagctacttcctgtacag caagctgagcgtggacaagagcagatggcagagaggcgacaccttcatctgcgccgtgatgcacgaggccctg cacaaccactacacccaggagagcctgagccacagccccggcaagFc 5F12-VH3-cIgGC [SEQ ID NO: 32]EVQLVQSGGDLVKPGGSVRLSCVASFTFDYYMNWVRQAPGKGLQWIGRWIFPGS GATYYNERFMGKATISADTAKNTAYMQLNSLRAEDTAVYYCLRSDWDVGDFWGQGTL VTVSSASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSVSLTSGVHTFPSVL QSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDKPVAKECECKCNCNNCPCPGC GLLGGXSVFIFPPKPKDILVTARTPTVTCVVVXLDPENPEVQISWFVDSKQVQTANTQPRE EQSXXXYRVVSVLPIGHQDWLSGKQFKCKVNNKXLXSPIEEIISKTPGQAHQPNVYVLPPS RDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSV DKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGKFcDNA 5F12-VH3-cIgGC [SEQ ID NO: 31]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcttcaccttcgactactacatgaactgggtgagacaggcccccggcaagggcctgcagtggatcggca gatggatcttccccggcagcggcgccacctactacaacgagagattcatgggcaaggccaccatcagcgccga caccgccaagaacaccgcctacatgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcctg agaagcgactgggacgtgggcgacttctggggccagggcaccctggtgaccgtgagcagcgccagcaccaccg cccccagcgtgttccccctggcccccagctgcggcagccagagcggcagcaccgtggccctggcctgcctggtg agcggctacatccccgagcccgtgaccgtgagctggaacagcgtgagcctgaccagcggcgtgcacaccttccc cagcgtgctgcagagcagcggcctgtacagcctgagcagcatggtgaccgtgcccagcagcagatggcccagc gagaccttcacctgcaacgtggcccaccccgccaccaacaccaaggtggacaagcccgtggccaaggagtgcg agtgcaagtgcaactgcaacaactgcccctgccccggctgcggcctgctgggcggcnnnagcgtgttcatcttc ccccccaagcccaaggacatcctggtgaccgccagaacccccaccgtgacctgcgtggtggtgnnnctggacc ccgagaaccccgaggtgcagatcagctggttcgtggacagcaagcaggtgcagaccgccaacacccagccca gagaggagcagagcnnnnnnnnntacagagtggtgagcgtgctgcccatcggccaccaggactggctgagc ggcaagcagttcaagtgcaaggtgaacaacaagnnnctgnnnagccccatcgaggagatcatcagcaagac ccccggccaggcccaccagcccaacgtgtacgtgctgccccccagcagagacgagatgagcaagaacaccgtg accctgacctgcctggtgaaggacttcttcccccccgagatcgacgtggagtggcagagcaacggccagcagga no substituted individualFc 4F9-VH3-cIgGA [SEQ ID NO: 34]EVQLVQSGGDLVKPGGSVRLSCVASGFTFSYAMSWVRQAPGKGLQWMGTISDG GSYTHYPDNLMGRFTFSLDTAKNTAYLQLNSLRAEDTAVYYCARESYDGYYVANWGQG TLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPS VLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPPCPVPEP LGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQF NGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKEL SSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSR WQQGDPFTCAVMHETLQNHYTDLSLSHSPGKFc 4F9-VH3-cIgGA [SEQ ID NO: 33]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcggcttcaccttcagctacgccatgagctgggtgagacaggcccccggcaagggcctgcagtggatgg gcaccatcagcgacggcggcagctacacccactaccccgacaacctgatgggcagattcaccttcagcctggac accgccaagaacaccgcctacctgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcgcca gagagagctacgacggctactacgtggccaactggggccagggcaccctggtgaccgtgagcagcgccagcac caccgcccccagcgtgttccccctggcccccagctgcggcagcaccagcggcagcaccgtggccctggcctgcc tggtgagcggctacttccccgagcccgtgaccgtgagctggaacagcggcagcctgaccagcggcgtgcacacc ttccccagcgtgctgcagagcagcggcctgcacagcctgagcagcatggtgaccgtgcccagcagcagatggcc cagcgagaccttcacctgcaacgtggtgcaccccgccagcaacaccaaggtggacaagcccgtgttcaacgag tgcagatgcaccgacacccccccctgccccgtgcccgagcccctgggcggccccagcgtgctgatcttccccccc aagcccaaggacatcctgagaatcaccagaacccccgaggtgacctgcgtggtgctggacctgggcagagagg accccgaggtgcagatcagctggttcgtggacggcaaggaggtgcacaccgccaagacccagagcagagagc agcagttcaacggcacctacagagtggtgagcgtgctgcccatcgagcaccaggactggctgaccggcaagga gttcaagtgcagagtgaaccacatcgacctgcccagccccatcgagagaaccatcagcaaggccagaggcaga gcccacaagcccagcgtgtacgtgctgccccccagccccaaggagctgagcagcagcgacaccgtgagcatca cctgcctgatcaaggacttctacccccccgacatcgacgtggagtggcagagcaacggccagcaggagcccga gagaaagcacagaatgacccccccccagctggacgaggacggcagctacttcctgtacagcaagctgagcgtg gacaagagcagatggcagcagggcgaccccttcacctgcgccgtgatgcacgagaccctgcagaaccactaca ccgacctgagcctgagccacagccccggcaagFc 4F9-VH3 cIgGD [SEQ ID NO: 36]EVQLVQSGGDLVKPGGSVRLSCVASGFTFSYAMSWVRQAPGKGLQWMGTISDG GSYTHYPDNLMGRFTFSLDTAKNTAYLQLNSLRAEDTAVYYCARESYDGYYVANWGQG TLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPS VLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLG GPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNS TYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSS SDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQ QGDTFTCAVMHEALQNHYTDLSLSHSPGKFc 4F9-VH3 cIgGD [SEQ ID NO: 35]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcggcttcaccttcagctacgccatgagctgggtgagacaggcccccggcaagggcctgcagtggatgg gcaccatcagcgacggcggcagctacacccactaccccgacaacctgatgggcagattcaccttcagcctggac accgccaagaacaccgcctacctgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcgcca gagagagctacgacggctactacgtggccaactggggccagggcaccctggtgaccgtgagcagcgccagcac caccgcccccagcgtgttccccctggcccccagctgcggcagcaccagcggcagcaccgtggccctggcctgcc tggtgagcggctacttccccgagcccgtgaccgtgagctggaacagcggcagcctgaccagcggcgtgcacacc ttccccagcgtgctgcagagcagcggcctgtacagcctgagcagcaccgtgaccgtgcccagcagcagatggcc cagcgagaccttcacctgcaacgtggtgcaccccgccagcaacaccaaggtggacaagcccgtgcccaaggag agcacctgcaagtgcatcagcccctgccccgtgcccgagagcctgggcggccccagcgtgttcatcttccccccc aagcccaaggacatcctgagaatcaccagaacccccgagatcacctgcgtggtgctggacctgggcagagagg accccgaggtgcagatcagctggttcgtggacggcaaggaggtgcacaccgccaagacccagcccagagagc agcagttcaacagcacctacagagtggtgagcgtgctgcccatcgagcaccaggactggctgaccggcaagga gttcaagtgcagagtgaaccacatcggcctgcccagccccatcgagagaaccatcagcaaggccagaggccag gcccaccagcccagcgtgtacgtgctgccccccagccccaaggagctgagcagcagcgacaccgtgaccctga cctgcctgatcaaggacttcttcccccccgagatcgacgtggagtggcagagcaacggccagcccgagcccgag agcaagtaccacaccaccgccccccagctggacgaggacggcagctacttcctgtacagcaagctgagcgtgg acaagagcagatggcagcagggcgacaccttcacctgcgccgtgatgcacgaggccctgcagaaccactacac cgacctgagcctgagccacagccccggcaag 4F9-VL3-cL-Kappa [SEQ ID NO: 38]DIVMTQTPLSLSVSPGEPASMSCRASQSISNNLHWYRQKPGQSPQVLVKYASQSIS GVPDRFIGSGSGTDFTLRISRVEADDLGVYYCQQSNSWPQTFGQGTKLELKRNDAQPAV YLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLS STLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD4F9-VL3-cL-Kappa [SEQ ID NO: 37]gacatcgtgatgacccagacccccctgagcctgagcgtgagccccggcgagcccgccagcatgagctgc agagccagccagagcatcagcaacaacctgcactggtacagacagaagcccggccagagcccccaggtgctg gtgaagtacgccagccagagcatcagcggcgtgcccgacagattcatcggcagcggcagcggcaccgacttca ccctgagaatcagcagagtggaggccgacgacctgggcgtgtactactgccagcagagcaacagctggcccca gaccttcggccagggcaccaagctggagctgaagagaaacgacgcccagcccgccgtgtacctgttccagccc agccccgaccagctgcacaccggcagcgccagcgtggtgtgcctgctgaacagcttctaccccaaggacatcaa cgtgaagtggaaggtggacggcgtgatccaggacaccggcatccaggagagcgtgaccgagcaggacagcaa ggacagcacctacagcctgagcagcaccctgaccatgagcagcaccgagtacctgagccacgagctgtacagc tgcgagatcacccacaagagcctgcccagcaccctgatcaagagcttccagagaagcgagtgccagagagtgg acFc 5F12-VH3-cIgGA [SEQ ID NO: 40]EVQLVQSGGDLVKPGGSVRLSCVASFTFDYYMNWVRQAPGKGLQWIGRWIFPGS GATYYNERFMGKATISADTAKNTAYMQLNSLRAEDTAVYYCLRSDWDVGDFWGQGTL VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVL QSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPPCPVPEPLG GPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFN [SEQ ID NO: 39]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcttcaccttcgactactacatgaactgggtgagacaggcccccggcaagggcctgcagtggatcggca gatggatcttccccggcagcggcgccacctactacaacgagagattcatgggcaaggccaccatcagcgccga caccgccaagaacaccgcctacatgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcctg agaagcgactgggacgtgggcgacttctggggccagggcaccctggtgaccgtgagcagcgccagcaccaccg cccccagcgtgttccccctggcccccagctgcggcagcaccagcggcagcaccgtggccctggcctgcctggtg agcggctacttccccgagcccgtgaccgtgagctggaacagcggcagcctgaccagcggcgtgcacaccttccc cagcgtgctgcagagcagcggcctgcacagcctgagcagcatggtgaccgtgcccagcagcagatggcccagc gagaccttcacctgcaacgtggtgcaccccgccagcaacaccaaggtggacaagcccgtgttcaacgagtgca gatgcaccgacacccccccctgccccgtgcccgagcccctgggcggccccagcgtgctgatcttcccccccaagc ccaaggacatcctgagaatcaccagaacccccgaggtgacctgcgtggtgctggacctgggcagagaggaccc cgaggtgcagatcagctggttcgtggacggcaaggaggtgcacaccgccaagacccagagcagagagcagca gttcaacggcacctacagagtggtgagcgtgctgcccatcgagcaccaggactggctgaccggcaaggagttca agtgcagagtgaaccacatcgacctgcccagccccatcgagagaaccatcagcaaggccagaggcagagccc acaagcccagcgtgtacgtgctgccccccagccccaaggagctgagcagcagcgacaccgtgagcatcacctg cctgatcaaggacttctacccccccgacatcgacgtggagtggcagagcaacggccagcaggagcccgagaga aagcacagaatgacccccccccagctggacgaggacggcagctacttcctgtacagcaagctgagcgtggaca agagcagatggcagcagggcgaccccttcacctgcgccgtgatgcacgagaccctgcagaaccactacaccga cctgagcctgagccacagccccggcaagFc 5F12-VH3-cIgGD [SEQ ID NO: 42]EVQLVQSGGDLVKPGGSVRLSCVASFTFDYYMNWVRQAPGKGLQWIGRWIFPGS GATYYNERFMGKATISADTAKNTAYMQLNSLRAEDTAVYYCLRSDWDVGDFWGQGTL VTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVL QSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLGGP SVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTY RVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSD TVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQ GDTFTCAVMHEALQNHYTDLSLSHSPGK Fc 5F12-VH3-cIgGD [SEQ ID NO: 41]gaggtgcagctggtgcagagcggcggcgacctggtgaagcccggcggcagcgtgagactgagctgcgt ggccagcttcaccttcgactactacatgaactgggtgagacaggcccccggcaagggcctgcagtggatcggca gatggatcttccccggcagcggcgccacctactacaacgagagattcatgggcaaggccaccatcagcgccga caccgccaagaacaccgcctacatgcagctgaacagcctgagagccgaggacaccgccgtgtactactgcctg agaagcgactgggacgtgggcgacttctggggccagggcaccctggtgaccgtgagcagcgccagcaccaccg cccccagcgtgttccccctggcccccagctgcggcagcaccagcggcagcaccgtggccctggcctgcctggtg agcggctacttccccgagcccgtgaccgtgagctggaacagcggcagcctgaccagcggcgtgcacaccttccc cagcgtgctgcagagcagcggcctgtacagcctgagcagcaccgtgaccgtgcccagcagcagatggcccagc gagaccttcacctgcaacgtggtgcaccccgccagcaacaccaaggtggacaagcccgtgcccaaggagagca cctgcaagtgcatcagcccctgccccgtgcccgagagcctgggcggccccagcgtgttcatcttcccccccaagc ccaaggacatcctgagaatcaccagaacccccgagatcacctgcgtggtgctggacctgggcagagaggaccc cgaggtgcagatcagctggttcgtggacggcaaggaggtgcacaccgccaagacccagcccagagagcagca gttcaacagcacctacagagtggtgagcgtgctgcccatcgagcaccaggactggctgaccggcaaggagttca agtgcagagtgaaccacatcggcctgcccagccccatcgagagaaccatcagcaaggccagaggccaggccc accagcccagcgtgtacgtgctgccccccagccccaaggagctgagcagcagcgacaccgtgaccctgacctg cctgatcaaggacttcttcccccccgagatcgacgtggagtggcagagcaacggccagcccgagcccgagagc aagtaccacaccaccgccccccagctggacgaggacggcagctacttcctgtacagcaagctgagcgtggaca agagcagatggcagcagggcgacaccttcacctgcgccgtgatgcacgaggccctgcagaaccactacaccga cctgagcctgagccacagccccggcaag5F12-VL3-cLKappa [SEQ ID NO: 44]DIVMTQTPLSLSVSLGEPASISCRSSRSLLHTNGITYLSWYRQKPGQIPQLLIYQMSN LASGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCAQTLGLPRTFGQGTKVEIKRNDAQP AVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTY SLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD5F12-VL3-cLKappa [SEQ ID NO: 43]gacatcgtgatgacccagacccccctgagcctgagcgtgagcctgggcgagcccgccagcatcagctgc agaagcagcagaagcctgctgcacaccaacggcatcacctacctgagctggtacagacagaagcccggccag atcccccagctgctgatctaccagatgagcaacctggccagcggcgtgcccgacagattcagcggcagcggcag cggcaccgacttcaccctgagaatcagcagagtggaggccgacgacgccggcgtgtactactgcgcccagacc ctgggcctgcccagaaccttcggccagggcaccaaggtggagatcaagagaaacgacgcccagcccgccgtgt acctgttccagcccagccccgaccagctgcacaccggcagcgccagcgtggtgtgcctgctgaacagcttctacc ccaaggacatcaacgtgaagtggaaggtggacggcgtgatccaggacaccggcatccaggagagcgtgaccg agcaggacagcaaggacagcacctacagcctgagcagcaccctgaccatgagcagcaccgagtacctgagcc acgagctgtacagctgcgagatcacccacaagagcctgcccagcaccctgatcaagagcttccagagaagcga gtgccagagagtggacMouse anti-canine PDL-1:4F9 variable regionsHeavy chain: DNA sequence [SEQ ID NO: 73]gaagtgcagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgca gcctctggattcactttcagtagctatgccatgtcttgggttcgccagactccggacaagagactggagtgggtcg caaccattagtgatggtggaagttacacccactaccccgacaatttaatgggccgattcaccatctccagagaca atgccaagaacaacctgtacctgcaaatgagccatctgaagtctgacgacacagccatgtattactgtgcacga gagagctatgatggttactacgtggctaactggggccaagggactctggtcactgtctcagcaHeavy chain: amino acid sequence [SEQ ID NO: 74]EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPDKRLEWVATISDGG SYTHYPDNLMGRFTISRDNAKNNLYLQMSHLKSDDTAMYYCARESYDGYYVANWGQG TLVTVSALight chain: DNA sequence [SEQ ID NO: 75]gatattgtgctaactcagtctccagccaccctgtctgtgaatccaggagatagcgtcagtctttcctgcagg gccagccaaagtattagcaacaacctacactggtatcaacaaaaatcacatgagtctccaaggcttctcatcaa gtatgcttcccagtccatctctgggatcccctccaggttcagtggcagtggatcagggacagatttcactctcagt atcaacagtgtggagactgaagattttggaatgtatttctgtcaacagagtaacagctggcctcagacgttcggt ggaggcaccaagctggaaatcaaaLight chain: amino acid sequence [SEQ ID NO: 76] DIVLTQSPATLSVNPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIP SRFSGSG SGTDFTLSINSVETEDFGMYFCQQSNSWPQTFGGGTKLEIKPDL-1 variable regions: Anti-PDL-1 5F12 mouse anticanineHeavy chain: DNA sequence [SEQ ID NO: 77]caggtccagctacagcagtctggacctgagctggtgaagcctggggcttcagtgaagatatcctgcaag gcttctggctacaccttcactgactactatatgaattgggtgaaacagaggcctggacagggacttgagtggatt ggatggatttttcccggaagtggtgctacttactacaatgagaggttcatgggcaaggccacacttactgtggat aaatcttccaacacagcctacatgttgttcagtagcctgacctctgaggactctgcggtctatttctgtttaagatc tgactgggacgtcggggacttctggggccaaggcaccactctcacagtctcctcaHeavy chain: amino acid sequence [SEQ ID NO: 78]QVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQRPGQGLEWIGWIFPG SGATYYNERFMGKATLTVDKSSNTAYMLFSSLTSEDSAVYFCLRSDWDVGDFWGQGTTL TVSSClight chain: DNA sequence [SEQ ID NO: 79]gatattgtgatgacgcaggctgcattctccaatccagtcactcttggaacatcagcttccatctcctgcagg tctagtaggagtctcctacatactaatggcatcacttatttgtcttggtttctgcagaagccaggccagtctcctca gctcctgatttatcagatgtccaaccttgcctcaggagtcccagacaggttcagtagcagtgggtcaggaactga tttcacactgagaatcagtagagtggaggctgaggatgtgggtatttattactgtgctcaaactctaggacttcct cggacgttcggtggaggcaccaagctggaaatcaaaLight chain: amino acid sequence [SEQ ID NO: 80]DIVMTQAAFSNPVTLGTSASISCRSSRSLLHTNGITYLSWFLQKPGQSPQLLIYQMS NLASGVPDR FSSSGSGTDFTLRISRVEAEDVGIYYCAQTLGLPRTFGGGTKLEIK

EXAMPLE 5 EPITOPE MAPPING OF PD-L1 ANTI-CANNIN ANTIBODIES

[0201] The interaction of antibodies with their cognate protein antigens is mediated through the binding of antibody-specific amino acids (paratopes) to specific amino acids (epitopes) of the target antigens. An epitope is an antigenic determinant that elicits a specific reaction by an immunoglobulin. An epitope consists of a group of amino acids on the surface of the antigen. A protein of interest may contain several epitopes that are recognized by different antibodies. The epitopes recognized by antibodies are classified as conformational or linear epitopes. Linear epitopes are formed by a stretch of a continuous amino acid sequence in a protein, whereas conformational epitopes are composed of amino acids that are discontinuous (e.g., very distant) in the primary amino acid sequence but are brought together after three-dimensional folding of the protein.

[0202] Epitope mapping refers to the process of identifying the amino acid sequences (i.e., epitopes) that are recognized by antibodies on their target antigens. Identifying the epitopes recognized by monoclonal antibodies (mAbs) on target antigens has important applications. For example, it can aid in the development of new therapies, diagnostics, and vaccines. Epitope mapping can also aid in the selection of optimized therapeutic mAbs and help elucidate their mechanisms of action. Epitope information on PD-L1 can also elucidate unique cancer epitopes and define the pathogenic or protective effects of vaccines. Epitope identification can also lead to the development of subunit vaccines, based on the chemical or genetic coupling of the identified peptide epitope to a carrier protein or other immunostimulatory agents.

[0203] Epitope mapping can be performed using either polyclonal or monoclonal antibodies and various methods are used for epitope identification depending on the suspected nature of the epitope (i.e. linear or conformational). Mapping of linear epitopes is simpler and relatively easier to perform. For this purpose, commercial services for linear epitope mapping often employ peptide scanning. In this case, a set of overlapping short peptide sequences of the target protein are chemically synthesized and tested for their ability to bind to the antibodies of interest. The strategy is rapid, highly productive and relatively inexpensive to perform. On the other hand, mapping of a discontinuous epitope is more technically challenging and requires more specialized techniques such as x-ray co-crystallography of a monoclonal antibody together with its target protein, hydrogen-deuterium (H/D) exchange, mass spectrometry coupled with enzymatic digestion as well as various other methods known to those skilled in the art.

Mapping of PD-L1 epitopes using mass spectrometry:

[0204] A method based on chemical crosslinking and mass spectroscopy detection was employed to identify the epitopes recognized by the anti-canine PD-L1 mAbs [CovalX® Instrument Incorporated]. The application of this technology for canine PD-L1 epitope mapping resulted in the identification of the epitopes recognized by the indicated mAbs that are listed in Table 13. The results of canine PD-L1 epitope mapping with mAb 4F9 show that this mAb recognizes an epitope located in the extracellular domain of canine PD-L1 comprised of the amino acid sequence represented by SEQ ID NO: 82 and the amino acid sequence represented by SEQ ID NO: 83 [see Table 13]. Furthermore, the results of the epitope mapping experiment with mAb 5F12 show that the mAb recognizes an epitope located in the extracellular domain of canine PD-L1 comprised of the amino acid sequence represented by SEQ ID NO: 82. Notably, the location of the epitopes on canine PD-L1 identified here is reasonably consistent with those recently reported for the corresponding human PD-L1 [Hao et al., J. Mol. Recognit. 28:269-276 (2015)] which also provides further confidence for the above determination. Table 13 PD-L1 EPITOPES RECOGNIZED BY ANTI-CANNINE PD-L1 MABS

[0205] All references cited herein are incorporated by reference to the same extent as if each publication, database record (e.g., Genbank sequences or GeneID records), patent application, or patent were specifically and individually indicated to be incorporated by reference. This incorporation by reference statement is used by applicants pursuant to 37 C.F.R. §1.57(b)(1) to refer to each individual publication, database record (e.g., Genbank sequences or GeneID records), patent application, or patent, each of which is clearly identified in accordance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately contiguous with a dedicated incorporation by reference statement. The inclusion of dedicated incorporation by reference statements, if any, within the specification does not in any way weaken this general incorporation by reference statement. Citation of references in this document is not an acknowledgement that the reference is relevant prior art, nor does it constitute any acknowledgement as to the content or date of such publications or documents.

[0206] The present invention is not to be limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to be within the scope of the appended claims.

[0207] The above-written specification is considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and will be within the scope of the appended claims.

Claims (15)

1. An isolated antibody or antigen-binding fragment thereof that binds to canine programmed cell death ligand 1 (canine PD-L1) comprising: (a) a complementarity determining region (CDR) 1 of the light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 16; (b) a VL CDR2 consisting of the amino acid sequence of SEQ ID NO: 17; (c) a VL CDR3 consisting of the amino acid sequence of SEQ ID NO: 18; (d) a complementarity determining region (CDR) 1 of the heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 13; (e) a VH CDR2 consisting of the amino acid sequence of SEQ ID NO: 14; and(f) a VH CDR3 consisting of the amino acid sequence of SEQ ID NO: 15; wherein the antibody or antigen-binding fragment thereof binds to canine PD-L1 and blocks the binding of canine PD-L1 to canine programmed cell death protein 1 (PD-1). 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a caninized antibody. 3. The caninized antibody or antigen-binding fragment thereof of claim 2, comprising a variant of the canine crystallizable fragment region (cFc region) comprising the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 68; wherein the variant comprises from one to seven amino acid substitutions at the indicated positions of the cFc region selected from the group consisting of P4, D31, N63, G64, T65, A93, and P95, each independently for one of the other 19 naturally occurring standard amino acids. 4. The caninized antibody or antigen-binding fragment thereof according to claim 3, characterized in that the cFc region comprises two substitutions, D31A and N63A. 5. The caninized antibody or antigen-binding fragment thereof of claim 4, further comprising a canine light chain comprising the amino acid sequence of SEQ ID NO: 38. 6. Pharmaceutical composition, characterized by the fact that it comprises the antibody or antigen-binding fragment thereof defined in claim 5 and a pharmaceutically acceptable vehicle or diluent. 7. The caninized antibody or antigen-binding fragment thereof of claim 4, comprising the amino acid sequence of SEQ ID NO: 26, wherein there is a proline residue at amino acid position 242, an alanine residue at amino acid position 269, an alanine residue at amino acid position 301, a glycine residue at amino acid position 302, a threonine residue at amino acid position 303, an alanine residue at amino acid position 331, and a proline residue at amino acid position 333. 8. The caninized antibody or antigen-binding fragment thereof of claim 7, further comprising a canine light chain comprising the amino acid sequence of SEQ ID NO: 38. 9. Pharmaceutical composition, characterized by the fact that it comprises the antibody defined in claim 8 and a pharmaceutically acceptable vehicle or diluent. 10. The caninized antibody or antigen-binding fragment thereof of claim 4, comprising the amino acid sequence of SEQ ID NO: 28, wherein there is a proline residue at amino acid position 240, an alanine residue at amino acid position 267, an alanine residue at amino acid position 299, a glycine residue at amino acid position 300, a threonine residue at amino acid position 301, an alanine residue at amino acid position 329, and a proline residue at amino acid position 331. 11. The caninized antibody or antigen-binding fragment thereof of claim 10, further comprising a canine light chain comprising the amino acid sequence of SEQ ID NO: 38. 12. Pharmaceutical composition, characterized by the fact that it comprises the antibody defined in claim 8 and a pharmaceutically acceptable vehicle or diluent. 13. The caninized antibody or antigen-binding fragment thereof of claim 2, comprising a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34 or SEQ ID NO:36. 14. The caninized antibody or antigen-binding fragment thereof of claim 13, further comprising a canine light chain comprising the amino acid sequence of SEQ ID NO: 38. 15. Pharmaceutical composition, characterized by the fact that it comprises the antibody defined in claim 14 and a pharmaceutically acceptable vehicle or diluent.