Canine antibodies

Abstract

To provide caninized antibodies with specific properties.SOLUTION: The present invention provides a canine fragment crystallizable region (cFc region) of an antibody in which the cFc has been genetically modified to augment, decrease, or eliminate one or more effector functions. The present invention also discloses caninized murine antibodies against canine PD-1 that have a high binding affinity for canine PD-1. The invention further discloses the use of the caninized antibodies of the present invention in the treatment of cancer in dogs.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application is U.S. Provisional Patent Application No. 62 / 030,812, filed on July 30, 2014. U.S. Patent Provisional Application No. 61 / 918,847, filed December 20, 2013 and 2014 This claim asserts priority from U.S. Provisional Patent Application No. 61 / 918,946, filed on December 20. Yes, and all of this content is incorporated herein by reference as a whole.

[0002] The present invention relates to a canine antibody having specific properties. The present invention also relates to an antibody having a specific sequence. and canine antibodies against canine PD-1 that have a high binding affinity to canine PD-1. The present invention further relates to the use of the antibodies of the present invention in the treatment of dogs, including the treatment of cancer. ru. [Background technology]

[0003] Canine antibodies (also called immunoglobulin G or IgG) are large four-cell antibodies of approximately 150 kD. It is a mass protein. Each IgG protein consists of two identical proteins, each approximately 25 kD long. It consists of a light chain and two identical heavy chains, each approximately 50 kD. Four known canine IgGs There are IgG heavy chain subclasses, which are IgGA, IgGB, IgGC, and IgGD. It is called a κ chain. There are two types of light chains: κ chains and λ chains. Each of the κ and λ light chains is It consists of one variable domain (VL) and one constant domain (CL). Each is referred to as a single variable domain (VH), as well as CH-1, CH-2, and CH-3. It consists of three constant domains. The CH-1 domain is either a "hinge" or selectively a "hinge". It is linked to the CH-2 domain by an amino acid sequence called the "sequence region". In humans IgG is divided into four subclasses called IgG1, IgG2, IgG3, and IgG4. It exists in one of the cells. Subclasses of IgG are primarily determined by the sequence of the hinge region. The hinge region sequence differs among the four subclasses of IgG. The two heavy chains are disulfide. They are linked to each other by disulfide bonds, and each heavy chain is also connected to the light chain via disulfide bonds. They are connected to each other.

[0004] The digestion of IgG antibodies by the enzyme papain cleaves the antibody molecule at the hinge region, resulting in three flags. This leads to the formation of a ment. Two of these fragments are identical, and each is a heavy chain. It consists of a light chain bound to the VH and CH1 domains. These fragments are "Fa It is called a "b" fragment and contains the antigen-binding site of the antibody. It is produced from digestion with papain. The third fragment is called "Fc" and is linked by a disulfide bond. It contains the remaining parts of the two heavy chains. Therefore, Fc is each of the CH2 in the two heavy chains It contains a dimer consisting of a CH3 domain. Fab is formed when an antibody binds to its homoepitope. Fc enables the antibody to perform an immunoeffector function, such as antibody-dependent processes. Cellular injury (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cell injury (CDC) ) enables mediation.

[0005] IgG antibodies have an Fc portion. γ A family of proteins known as receptors By combining, mediating effector functions such as ADCC and ADCP As is well known in this field, CDC is mediated through the binding of Fc to C1q, the first component of complement. Different IgG subclasses have different abilities to mediate these effects. Both are well known in this field. For example, human IgG1 shows strong ADCC and CDC, IgG4 may show weak ADCC and CDC or no ADCC and CDC at all. In addition, which IgG subclass exhibits or lacks effector functionality? Methods for identifying them are well known in this field.

[0006] Approaches based on the use of monoclonal antibodies for therapeutic purposes achieve the desired therapeutic response. This requires the design of antibodies or antibody fragments that are suitable for the purpose of achieving cancer. Some therapeutic approaches for this involve therapeutic antibodies having enhanced effector function. And another approach is to have significantly lower effector functionality or effector -Requires that the function be completely removed. Enhancement or removal of effect functions is required. , Fc γ The Fc portion of the antibody enhances or reduces binding to receptors and the first component of complement. This can be achieved by introducing one or more amino acid mutations (substitutions) per minute. This section describes amino acid substitutions that can be introduced into antibody molecules to modify the effector function of antibodies. Furthermore, there are numerous reports of prior art. For example, Shields et al., [J.of Biol. Chem., 276(9):6591-6604 (2001) is from Aspa. This discloses the substitution of lagin with alanine (N297A), which is a nonglycosylated antibody. Bringing about some Fc γ It significantly reduced antibody binding to the receptor. In addition, Shie lds et al. also found that the substitution of aspartic acid with alanine (D265A) is also Fc γ We disclosed that the binding of antibodies to receptors was significantly reduced. (N297A and D265A) Each substitution was shown to significantly reduce CDC. Effector function in antibodies. Other similar reports exist that identify potential substitutions that reduce or eliminate [e.g., Saz] insky et al.,Proc.Nat.Acad.Sci.,105:2016 7-20172(2008), Alegre et al.,Transplantat ion,57:1537-1543(1994), Hutchins et al.,P roc.Nat.Acad.Sci.92:11980-11984(1994), Mc Earchem et al.,Blood,109:1185-1192(2007) ].

[0007] It is an immunosuppressive receptor primarily expressed on activated T and B cells, programmed death Programmed cell death receptor 1, also known as receptor 1 (PD-1), is associated with CD28 and CTLs. It is a member of the immunoglobulin superfamily associated with A-4, as well as PD-1 and similar groups. The family members have an extracellular Ig variable (V) domain that binds to its ligand and PD is a type I transmembrane glycoprotein containing a cytoplasmic tail that binds to signaling molecules. The cytoplasmic tail of -1 consists of two tyrosine-based signaling motifs, ITIM (immunoreceptors). (Tyrosine-based inhibitory motifs) and ITSM (Immune receptor tyrosine-based switch motifs) Contains (Chief).

[0008] PD-1 is a programmed cell death ligand, also known as programmed cell death ligand 1 (PD-L1). Programmed death ligand 1 and / or programmed death ligand 2 (PD-L2) When bound to cell death ligand 2, it attenuates the T cell response. The binding of PD-1 to PD-L1 negatively regulates antigen receptor signaling. Blocking binding to tumor cells helps the immune system clear tumor cells, while also helping tumors specific CD8 + Enhances T-cell immunity. Three-dimensional structure of mouse PD-1 and human PD- The cocrystal structure of L1 and mouse PD-1 has been reported [Zhang et al., Im munity 20:337-347(2004);Lin et al.,Proc. Natl.Acad.Sci.USA 105:3011-3016(2008)].

[0009] PD-L1 and PD-L2 are short cytoplasmic regions that do not possess known signaling motifs. Type I transmembrane containing both IgV-like and IgC-like domains within the extracellular region. They are common ligands. Both PD-L1 and PD-L2 are constitutively expressed or PD-L can be induced in various cell types, including non-hematopoietic tissues and various tumor types. 1 is expressed not only on B cells, T cells, myeloid cells and dendritic cells (DCs), but also on terminal cells. It is also expressed in peripheral cells, such as microvascular endothelial cells, and in non-lymphatic organs, such as the heart or lungs. In contrast, PD-L2 is only found on macrophages and DCs. The expression pattern of ligand 1 plays a role in PD-1's role in maintaining peripheral immune tolerance. Furthermore, it may be useful in regulating autoreactive T cell and B cell responses in the periphery. This suggests that...

[0010] In either case, PD-1, presumably by mediating immune evasion, at least in particular It is now very clear that it plays a very important role in certain human cancers. Therefore Furthermore, PD-L1 has been shown to be expressed in many mouse and human tumors, and PD- Most L1-negative tumor cell lines can be induced by IFN-γ [Iwai et al.] l., Proc.Natl.Acad.Sci.USA99:12293-12 297(2002);Strome et al., Cancer Res.,63:6 501-6505 (2003). Furthermore, PD-1 expression in tumors infiltrating lymphocytes. and / or PD-L1 expression in tumor cells is the same in numerous primary human tumor biopsies. It has been determined that such tumors can occur in the lungs, liver, ovaries, cervix, skin, colon, and nerves. Cancers of glioma, bladder, breast, kidney, esophagus, stomach, oral squamous epithelial cells, urothelial cells, and pancreas This also includes tumors of the head and neck [Brown et al., J.Immunol.1] 70:1257-1266(2003);Dong et al., Nat.Med.8 :793-800(2002);Wintterle et al., Cancer R es.63:7462-7467(2003);Strome et al., Canc. er Res.,63:6501-6505(2003);Thompson et a. l.,Cancer Res.66:3381-5(2006);Thompson e t al., Clin. Cancer Res. 13:1757-1761 (2007) ;Nomi et al.,Clin.Cancer Res.13:2151-215 7. (2007)]. More notably, PD ligand expression in tumor cells is observed in multiple tumor types. It has been correlated with poor prognosis in human cancer patients [Okazaki and Honjo, This topic was reviewed in Int.Immunol. 19:813-824 (2007). .

[0011] Furthermore, Nomi et al. [Clin. Cancer Res. 13:2151] -2157.(2007)] involves administering antibodies against PD-1 or PD-L1. This blocks the binding of PD-L1 to PD-1 in a mouse model of invasive pancreatic cancer. The therapeutic effect of these actions was revealed. These antibodies are tumor-reactive CD8 + T-cell tumors It effectively promotes invasion into the tumor and contains antitumor agents including IFN-γ, granzyme B, and perforin. This resulted in upward adjustment of the effect pedal. Similarly, the coupling of PD-L1 and PD-1 was blocked. The use of antibodies that target tumors significantly inhibited tumor growth in a mouse squamous cell carcinoma model. Tsushima et al.,Oral Oncol.42:268-274(20 06).

[0012] In other studies, transfection of mouse mast cell tumor lines with PD-L1 has shown that tumor When co-cultured with tumor-specific CTL clones, it resulted in reduced lysis of tumor cells. Anti-PD -When an L1 monoclonal antibody was added, solubility was restored [Iwai et al.]. Proc.Natl.Acad.Sci.USA99:12293-12297( (2002). In vivo, blocking the PD-1 / PD-L1 interaction is It has been shown to enhance the efficacy of adoptive T cell transfer therapy in a Uss tumor model [Str ome et al.,Cancer Res.63:6501-6505(2003) Further evidence regarding the role of PD-1 in cancer treatment is available in PD-1 knockout ma This was brought about by an experiment conducted in a Ussian, in which PD-L1 expressing myeloma was observed. The cells proliferate only in wild-type animals (leading to tumor growth and associated animal death), Proliferation did not occur in PD-1 deficient mice [Iwai Y. et al., Proc. Na tl.Acad.Sci.USA99:12293-12297(2002)]. Very recently, antibodies against PD-1 (humanized mouse monoclonal antibodies against human PD-1) (Including the human body) has shown at least initial success in cancer therapy in humans [e.g., U.S. patents]. U.S. Patent No. 8,354,509, U.S. Patent No. 8,008,449, and U.S. Patent No. 7,595 , No. 048].

[0013] Anti-PD-1 antibodies may also be useful in chronic viral infections. Memory CD8 generated after dyeing + T cells are highly functional and are an important component of the protective immune system. In contrast, chronic infection often involves varying degrees of function of the virus-specific T cell response. Characterized by impairment (exhaustion), this defect is the main reason why the host is unable to eliminate the remaining pathogens. This is the reason. Functional effector T cells are initially produced in the early stages of infection, but chronically... During the infection process, they gradually lose their function. Barber et al. [Natur e 439:682-687(2006)] describes mice infected with a laboratory strain of LCMV. However, this can lead to chronic infections that produce high levels of the virus in the blood and other tissues. These mice initially expressed a robust T-cell response, but after T-cell exhaustion, they eventually... They succumbed to the infection. Barber et al. showed that in chronically infected mice the effect Blocking the interaction between PD-1 and PD-L1 leads to a decrease in the number and function of ter T cells. They discovered that the effect could be reversed by injecting antibodies.

[0014] Any reference cited herein does not constitute "prior art" of this application. This should not be interpreted as an approval that it can be used as such. [Prior art documents] [Patent Documents]

[0015] [Patent Document 1] U.S. Patent No. 8,354,509 [Patent Document 2] U.S. Patent No. 8,008,449 [Patent Document 3] U.S. Patent No. 7,595,048 [Non-patent literature]

[0016] [Non-Patent Document 1] Shields et al.,[J.of Biol.Chem.,276(9):6591-6604(2001) [Non-Patent Document 2] Sazinsky et al.,Proc.Nat.Acad.Sci.,105:20167-20172(2008) [Non-Patent Document 3] Alegre et al.,Transplantation,57:1537-1543(1994) [Non-Patent Document 4] Hutchins et al.,Proc.Nat.Acad.Sci.92:11980-11984(1994) [Non-Patent Document 5] McEarchem et al.,Blood,109:1185-1192(2007) [Non-Patent Document 6] Zhang et al.,Immunity 20:337-347(2004)

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[0017] This invention relates to an antibody in which the canine crystalline fragment region (cFc region) is 1 or more. Genetically modified to increase, decrease, or eliminate effector functions. , providing the cFc region of the antibody. In one aspect of the present invention, the genetically modified cFc is To reduce or eliminate one or more effect functions. In another aspect of the present invention Genetically modified cFc increases the effect function by one or more levels.

[0018] In one embodiment, the genetically modified cFc region is genetically modified canine IgG This is the B Fc region. In another such embodiment, the genetically modified cFc region is This is a genetically modified canine IgGC Fc region. In certain embodiments, it is an effector. The function is antibody-dependent cell-mediated cytotoxicity (ADCC), which is increased, decreased, or eliminated. In another embodiment, the effector function is complement-dependent cell injury (CDC), which is It is increased, decreased, or removed. In yet another embodiment, the cFc region is ADCC Genetically modified to increase, decrease, or eliminate both CDC and ru.

[0019] The present invention further relates to canine flames and / or containing genetically modified cFc regions. This provides a full-length heavy chain. Therefore, the present invention provides a full-length heavy chain that is genetically modified according to the present invention. It provides a full-length heavy chain of antibody containing a cFc region. Such a full-length heavy chain also It can also bind to the corresponding canine light chain (κ or λ chain) to form a complete antibody. In certain embodiments of the species, the resulting antibodies specifically bind to specific canine antigens. In one embodiment, the canine antigen is canine PD-1. In yet another embodiment, the canine antigen The source is canine PD-L1. In yet another embodiment, the canine antigen is the α chain of the IL-4 receptor. In another embodiment, the canine antigen is the canine thymic interstitial lymphocyte neogenesis factor protein. cTSLP [for example, the entire content of which is incorporated herein by reference] [See U.S. Patent No. 7,718,772].

[0020] In one embodiment, the genetically modified cFc region consists of the following amino acid residues: P4, D 31, N63, G64, T65, A93, or P95 are indicated as one to seven numbers. SEQ ID NO: 130 (or SEQ ID NO: 130) is replaced by another amino acid residue in the position. It contains the amino acid sequence 132). P4, D31, N63, G64, T65, A93 The amino acids that substitute for and / or P95 are the other 19 marks listed in Table 1 below. It is individually selected from one of the quasi-naturally occurring amino acids. The present invention further relates to such The amino acid sequence of the genetically modified cFc region and 90%, 95%, 98%, or 99% It contains the same amino acid sequence, as well as the following amino acid residues, namely P4, D31 , a sequence in which one or more of N63, G64, T65, A93 or P95 are substituted. Genetically modified amino acid sequence containing number 130 (or sequence number 132) cFc regions: at least 50%, 75%, and 90% of ADCC and / or CDC. Genetically modified cFc regions that retain 95% or more of the increase, decrease, or removal. Provides variants within the region.

[0021] In other embodiments, the following amino acid residues are used: P4, D31, N63, G64, T65, A Two to five amino acid residues at the indicated positions of 93 or P95 are placed by other amino acid residues. It is replaced. In this particular embodiment, the genetically modified cFc region is as follows: Sequence ID: 13 with substitutions: P4A, D31A, N63A, A93G and P95A It contains the amino acid sequence 0 or SEQ ID NO: 132. In related embodiments, it is genetically modified. The modified cFc region has the following substitutions: P4A, D31A, N63A and P95A. It contains the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 132. In other embodiments, it contains the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 132. The genetically modified cFc region has substitutions at D31 and N63 in SEQ ID NO: It contains the amino acid sequence of :130 or SEQ ID NO:132. In this particular embodiment, The aspartic acid residue at position 31 is either a glutamic acid residue, an asparagine residue, or an alanine residue. Substituted with a residue, the asparagine residue at position 63 is replaced with a glutamine residue, a histidine residue, or an a Substituted with a ranin residue. In a more specific embodiment of this type, genetically modified c The Fc region is sequence number 130 or sequence number with the following substitutions: D31A and N63A Contains amino acid sequence number 132. In certain embodiments, genetically modified cF The c region is coded by the nucleotide sequence of SEQ ID NO: 129 or SEQ ID NO: 131. The nucleotide sequence is then determined to correspond to the amino acid sequence it encodes. It contains a change in its properties.

[0022] In another embodiment, the genetically modified cFc region has a substitution at A93. It contains the amino acid sequence of :130 or SEQ ID NO:132. In this particular embodiment, In this example, the substitution is A93G. In the related embodiment, the substitution is A93S. The following is Example 4. As shown, the substitution of A93G increases complement C1q binding activity. This will lead to an increase in [something].

[0023] In related embodiments, the genetically modified cFc region is the amino acid sequence of SEQ ID NO: 109 It further contains a hinge region including a column. In other embodiments, a genetically modified Fc region It further contains a hinge region containing the amino acid sequence of SEQ ID NO: 110. In the application morphology, the genetically modified Fc region contains the amino acid sequence of SEQ ID NO: 111. It further contains a range region. In yet another embodiment, the genetically modified Fc region is Further contains a genetically modified hinge region containing the amino acid sequence of SEQ ID NO: 112. .

[0024] In a selective embodiment, the present invention relates to a genetically modified hinge derived from a canine IgGD antibody. Region, hinge region derived from canine IgGA antibody, hinge region derived from canine IgGB antibody, or This provides a canine IgGD Fc region having a hinge region derived from a canine IgGC antibody. The present invention relates to the full-length heavy chain of an antibody, which has a genetically modified hinge region derived from a canine IgGD antibody. region, hinge region derived from canine IgGA antibody, hinge region derived from canine IgGB antibody, or The present invention contains a canine IgGD Fc region having a hinge region derived from a nu IgGC antibody, It provides the full-length heavy chain of the antibody. Such a full-length heavy chain also provides the corresponding canine light chain (κ It can also bind to the λ chain to form a complete antibody.

[0025] Therefore, the present invention further enhances the genetically modified hinge region derived from canine IgGD antibodies. Provides a canine IgGD Fc region contained in. In a particular embodiment of this type, canine Ig The GD Fc region and the genetically modified hinge region have a proline residue (P10) at position 10. ) containing the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 and 90%, 9 It contains amino acid sequences that are 5%, 98%, or 99% identical. In more specific embodiments... The canine IgGD Fc region and the genetically modified hinge region are the same as those in Sequence ID: 5. Encoded by a creotide sequence. In other embodiments, the canine IgGD Fc region is It further contains a hinge region derived from canine IgGA antibody. In this particular embodiment, The IgGD Fc region and hinge region are based on the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: : Amino acid sequences that are 90%, 95%, 98%, or 99% identical to the amino acid sequence of 8 Contains. In more specific embodiments, the canine IgGD Fc region and hinge region are array Number: Encoded by the nucleotide sequence of 7. In yet another embodiment, canine Ig The GD Fc region further contains a hinge region derived from canine IgGB antibodies. Identification of this type In this embodiment, the canine IgGD Fc region and the hinge region are the amino acid of SEQ ID NO: 10. The amino acid sequence of the acid or SEQ ID NO: 10 is 90%, 95%, 98%, or 99% identical. It contains a certain amino acid sequence. In a more specific embodiment, the canine IgGD Fc region and The hinge region is encoded by the nucleotide sequence of sequence number 9. Further implementations Morphologically, the canine IgGD Fc region further contains a hinge region derived from the canine IgGC antibody. In this particular embodiment, the canine IgGD Fc region and hinge region are arranged in the sequence. Number: 12 amino acid sequence or SEQ ID NO: 12 amino acid sequence and 90%, 95%, 98 It contains amino acid sequences that are % or 99% identical. In a more specific embodiment, canine I The gGD Fc region and hinge region are coded by the nucleotide sequence of SEQ ID NO: 11. The present invention further includes these canine IgGD Fc regions and hinge regions. This provides a canine antibody. In a specific embodiment, the canine antibody or its antigen-binding fragment The reagent specifically binds to canine programmed death receptor 1 (canine PD-1).

[0026] Therefore, the present invention is specific to canine PD-1 and / or highly specific to canine PD-1. The present invention provides a canine anti-canine PD-1 antibody having high binding affinity. In a specific embodiment, canine Anti-canine PD-1 antibodies also have the ability to block the binding of canine PD-1 to canine PD-L1. It also possesses strength. In a specific embodiment, the canine anti-canine PD-1 antibody has high efficacy against canine PD-1. It possesses binding affinity and also has the ability to block the binding of canine PD-1 to canine PD-L2. It comprises a canine antibody or its antigen-binding fragment that specifically binds to canine PD-1. This may contain the canine IgG heavy chain and canine κ or λ light chain of the present invention. In certain embodiments, The canine-modified anti-canine PD-1 antibody is a canine-modified mouse anti-canine PD-1 antibody. Furthermore, such canine antimicrobial agents in the treatment of diseases such as cancer and / or diseases caused by infection. Regarding the use of the body.

[0027] In certain embodiments, the canine anti-canine PD-1 antibody is a genetically modified cF of the present invention. It contains the c region. In a selective embodiment, the canine anti-canine PD-1 antibody is canine IgGD Genetically modified hinge regions derived from antibodies, hinge regions derived from canine IgGA antibodies, canine I Canine I having a hinge region derived from a gGB antibody or a hinge region derived from a canine IgGC antibody The present invention further includes the gGD Fc region obtained from mouse anti-canine PD-1 antibody. R, that is, three light chains CDR: CDR light chain 1 (CDRL1), CDR light chain 2 (CDRL1) 2) and CDR light chain 3 (CDRL3) and three heavy chains CDR: CDR heavy chain 1 (CD Combined with RH1), CDR heavy chain 2 (CDRH2), and CDR heavy chain 3 (CDRH3). The present invention provides a canine anti-canine PD-1 antibody containing the canine flame of the present invention.

[0028] In certain embodiments, the canine mouse anti-canine PD-1 antibody is the IgGB or I of the present invention. The genetically modified cFc region of gGC or the canine IgGD Fc region is used in mouse antimicrobial Genetically modified canine IgGD antibody combined with CDR obtained from canine PD-1 antibody hinge region, hinge region derived from canine IgGA antibody, hinge region derived from canine IgGB antibody or contained together with a hinge region derived from a canine IgGC antibody. Furthermore, the present invention relates to the present specification This document provides not only a canine mouse anti-canine PD-1 antibody with a specific CDR as detailed in the book, but also Furthermore, conservatively modified variants of those CDRs, as well as those having the same canonical structure ( We also provide canine mouse anti-canine PD-1 antibodies containing, for example, shared (mutant) variants.

[0029] Therefore, in certain embodiments, the canine-modified anti-canine PD-1 antibody has a complementarity-determining region (CD). R) has H1-1 and H2-1 respectively for CDR1, CDR2, and CDR3 of the heavy chain. and has the canonical structure of H3-6, i.e., the heavy chain CDR1 has canonical structure class 1 The heavy chain CDR2 has canonical structure class 1, and the heavy chain CDR3 has canonical structure class 6 It further contains a CDR having the corresponding light chain. In a more specific embodiment, it further contains a CDR having the corresponding light chain. The CDRs are L1-3 and L for the light chains CDR1, CDR2, and CDR3, respectively. It has canonical structures 2-1 and L3-1. In other embodiments, it is canine anti-canine PD-1 anti The body has complementarity-determining regions (CDRs) with respect to CDR1, CDR2, and CDR3 of the heavy chain. It further contains CDRs having canonical structures of H1-1, H2-1, and H3-11, respectively. In a more specific embodiment of this type, the CDR for the corresponding light chain is the light chain Regarding CDR1, CDR2, and CDR3, L1-2A, L2-1, and L3- respectively It has the canonical structure of 1. In yet another embodiment, the canine anti-canine PD-1 antibody is complementary The decision region (CDR) is H1 for CDR1, CDR2, and CDR3 of the heavy chain, respectively. It further contains CDR having canonical structures of -1, H2-2A and H3-11. In a more specific embodiment, the CDR for the corresponding light chain is the CDR1 of the light chain. Regarding CDR2 and CDR3, the canonical structures L1-2A, L2-1, and L3-1 respectively It has a structure. In another embodiment, the canine anti-canine PD-1 antibody has a complementarity determining region ( CDR) has H1-1 and H2 in relation to the heavy chains CDR1, CDR2, and CDR3, respectively. It further contains CDRs having canonical structures of -2A and H3-13. In a particular embodiment, the CDR for the corresponding light chain is CDR1, CDR2 of the light chain. The canonical structures for L1-4, L2-1, and L3-1 are for CDR3, respectively.

[0030] In more specific embodiments, the canine antibody or its antigen-binding fragment of the present invention is distributed The amino acid sequence of column number: 27, sequence number: 28, sequence number: 29, or sequence number: 30 It contains one or more heavy chain complementarity determination regions 1 (VH CDR1). Another implementation Morphologically, the heavy chain complementarity determination region 2 (VH CDR2) is represented by sequence number: 31, sequence number: 3 2. Contains the amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35. In another embodiment, the heavy chain complementarity determination region 3 (VH CDR3) is sequence number: 36, Contains the amino acid sequence of sequence number: 37, SEQ ID NO: 38 or SEQ ID NO: 146. In certain embodiments, the canine antibody or antigen-binding fragment is sequence number 27, sequence number VH CDR1 containing amino acid sequence number: 28, SEQ ID NO: 29, or SEQ ID NO: 30 and sequence numbers: 31, 32, 33, 34 or sequence number Contains both VH CDR2 with amino acid sequence number 35. Another such implementation In this state, the canine antibody or antigen-binding fragment is sequence number: 27, sequence number: 28, VH CDR1 containing the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30 and sequence number Contains the amino acid sequence of number: 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 146 It contains both VH and CDR3. In yet another such embodiment, it contains canine antibody The antigen-binding fragments are sequence numbers 31, 32, and 33. VH CDR2 containing amino acid sequence number: 34 or SEQ ID NO: 35 and SEQ ID NO: V containing the amino acid sequence of 36, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 146 It contains both H CDR3. In yet another such embodiment, canine antibody or The antigen-binding fragment is sequence number 27, sequence number 28, sequence number 29 or sequence number Number: 30 contains the amino acid sequence VH CDR1, SEQ ID NO: 31, SEQ ID NO: 32, VH CDR containing the amino acid sequence of column number: 33, SEQ ID NO: 34 or SEQ ID NO: 35 2 and sequence numbers: 36, 37, 38 or 146 It contains VH CDR3 with a minoic acid sequence.

[0031] In certain embodiments, the canine antibody or antigen-binding fragment is SEQ ID NO: 13, Light chain complementarity determination region 1 (VL) containing the amino acid sequence of column number: 14 or sequence number: 15 It also contains CDR1). In related embodiments, the light chain complementarity determining region 2 (VL CDR2) is , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 Or it includes the amino acid sequence of SEQ ID NO: 21. In yet another embodiment, light chain complementarity determination Region 3 (VL CDR3) contains sequence numbers 22, 23, 24, and sequence numbers. It includes the amino acid sequence of number 25 or sequence number 26. In this particular embodiment, Canine antibody or antigen-binding fragment is sequence number 13, sequence number 14 or sequence number VL CDR1 containing amino acid sequence number 15 and SEQ ID NO: 16, SEQ ID NO: 17 , the amino acid combination of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 It contains both VL CDR2s, including the column.

[0032] In another such embodiment, the canine antibody or antigen-binding fragment is: 13. VL CDR1 containing the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and Sequence ID: 22, Sequence ID: 23, Sequence ID: 24, Sequence ID: 25 or Sequence ID: 2 It contains both VL CDR3 containing the amino acid sequence of 6. In this state, the canine antibody or antigen-binding fragment is sequence number 16, sequence number 17, Amino acid sequences of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 VL CDR2 and sequence numbers: 22, 23, 24, sequence number It contains both VL CDR3 containing the amino acid sequence of number 25 or sequence number 26. In yet another such embodiment, the canine antibody or antigen-binding fragment is sequence number VL CDR1 containing the amino acid sequence of number: 13, SEQ ID NO: 14 or SEQ ID NO: 15, Sequence IDs: 16, 17, 18, 19, 20 or VL CDR2 containing the amino acid sequence of SEQ ID NO: 21 and SEQ ID NO: 22, sequence number Contains the amino acid sequence of number: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 Contains VL CDR3.

[0033] The present invention further relates to the amino acid sequence of SEQ ID NO: 40 or the amino acid sequence of SEQ ID NO: 40 The amino acid sequence that is 90%, 95%, 98%, or 99% identical to sequence number 42 amino acid sequence or SEQ ID NO:42 amino acid sequence and 90%, 95%, 98%, or 99% % identical amino acid sequences, amino acid sequence of SEQ ID NO: 44, or amino acid sequence of SEQ ID NO: 44 Amino acid sequences and sequence numbers that are 90%, 95%, 98%, or 99% identical to the no-acid sequence: 46 amino acid sequence or sequence number: 46 amino acid sequence and 90%, 95%, 98% The amino acid sequences are 99% identical, the amino acid sequence of SEQ ID NO: 48, or SEQ ID NO: 4 Amino acid sequences that are 90%, 95%, 98%, or 99% identical to the amino acid sequence of 8, amino acid sequence of sequence number: 50 or sequence number: 50 amino acid sequence and 90%, 95%, 9 8% or 99% identical amino acid sequences, the amino acid sequence of sequence number 52 or sequence Amino acids that are 90%, 95%, 98%, or 99% identical to amino acid sequence number 52. Sequence, amino acid sequence of sequence number: 54 or amino acid sequence of sequence number: 54 and 90%, 9 5%, 98%, or 99% identical amino acid sequences, amino acid sequence number: 56 or is 90%, 95%, 98%, or 99% identical to the amino acid sequence of sequence number 56. Amino acid sequence, amino acid sequence of SEQ ID NO: 58 or amino acid sequence of SEQ ID NO: 58 and 9 Amino acid sequences that are 0%, 95%, 98%, or 99% identical, amino acid sequence number: 60 Acid sequence or SEQ ID NO: 60 amino acid sequence and 90%, 95%, 98%, or 99% identical The amino acid sequence that is one, the amino acid sequence of SEQ ID NO: 62, or the amino acids of SEQ ID NO: 62 Amino acid sequences that are 90%, 95%, 98%, or 99% identical to the sequence, Sequence ID: 64 The amino acid sequence or sequence number:64 amino acid sequence and 90%, 95%, 98% or 99% identical amino acid sequences, amino acid sequences of sequence number 66 or sequence number 66 An amino acid sequence that is 90%, 95%, 98%, or 99% identical to the amino acid sequence, or This provides canine antibodies containing antigen-binding fragments of these canine antibodies.

[0034] In certain embodiments, the antibody heavy chain (i) has P, A, G or S at position 239, (ii) (iii) A, G or S at 266th place, (iv) A, G or S at 298th place G, P or A, (v) T, A, G or S at position 300, (vi) A, G at position 328 S, and (vii)330 containing P, A, G or S, Sequence IDs: 40, 52, 56 or 64 (or array numbers: 40, 52, 56 or 64 and 90%, 95%, 9) It contains an amino acid sequence that is 8% or 99% identical. In other embodiments, the heavy chain of the antibody is (i) P, A, G or S in 237th place, (ii) A, G or S in 264th place, (iii) (iv) A, G, or S at 296th place, G, P, or A at 297th place, T at 298th place (vi) A, G or S in 326th place, and (vii) P in 328th place. , containing A, G or S, SEQ ID NO: 42, 54, 58 or 66 (or SEQ ID NO: 4 2, 54, 58 or 66 (identical to 90%, 95%, 98% or 99%) amino acids It contains a sequence. In yet another embodiment, the antibody heavy chain has (i) P, A, and G at position 244. or S, (ii) A, G or S in 271st place, (iii) A, G or S in 303rd place, (iv) G, P or A in 304th place, (v) T, A, G or S in 305th place, (vi) 3 A sequence containing A, G, or S at position 33, and (vii) P, A, G, or S at position 335. Number: 44, 50 or 60 (or Sequence ID: 44, 50 or 60 and 90%, 95) It contains an amino acid sequence that is 98% or 99% identical. In further embodiments, The heavy chain of the antibody has (i) P, A, G, or S at position 242, and (ii) A, G, or S, (iii) A, G or S in 301st place, (iv) G, P or A in 302nd place, (v) 303rd place: T, A, G or S, (vi) 331st place: A, G or S, and (vii) Sequence ID: 46 or 62 (or Sequence ID: 4) contains P, A, G, or S at position 333. Contains amino acid sequences that are 90%, 95%, 98%, or 99% identical to 6 or 62. In further embodiments, the antibody heavy chain has (i) P, A, G or S at position 246, ( ii) A, G, or S for 273rd place, (iii) A, G, or S for 305th place, (iv) 30 6th place: G, P or A, (v)307th place: T, A, G or S, (vi)335th place: A, Sequence ID: 48( Alternatively, use an amino acid sequence that is 90%, 95%, 98%, or 99% identical to sequence number 48. Contains.

[0035] In yet another embodiment, the antibody heavy chain has (i) P, A, G or S at position 239, (i i) A at 266th place, (iii) A at 298th place, (iv) G, P or A at 299th place, (v )T, A, G or S in 300th place, (vi)A, G or S in 328th place, and (vii )Sequence numbers 40, 52, 56 or 64 (also) containing P, A, G or S at position 330. This corresponds to sequence numbers 40, 52, 56, or 64 and 90%, 95%, 98%, or 99%. It contains the amino acid sequence (i). In other embodiments, the heavy chain of the antibody has P at position (i)237. , A, G or S, (ii) A for 264th place, (iii) A for 296th place, (iv) 297th place (v) 298th place: T, A, G, or S, (vi) 326th place: A, G (vii) 328th position containing P, A, G or S, Sequence IDs: 42, 54 , 58 or 66 (or sequence numbers: 42, 54, 58 or 66 and 90%, 95%) It contains an amino acid sequence that is 98% or 99% identical. In yet another embodiment, the antibody The heavy chain consists of (i) P, A, G, or S at position 244, (ii) A at position 271, and (iii) 3 3rd place: A, (iv) 4th place: G, P or A, (v) 5th place: T, A, G or S (vi) A, G, or S in position 333, and (vii) P, A, G, or S in position 335. Includes, SEQ ID NO: 44, 50 or 60 (or SEQ ID NO: 44, 50 or 60 and It contains amino acid sequences that are 90%, 95%, 98%, or 99% identical. In the application form, the antibody heavy chain has (i) P, A, G, or S at position 242, and (ii) at position 269. A, (iii) A in 301st place, (iv) G, P or A in 302nd place, (v) T in 303rd place , A, G or S, (vi) A, G or S in position 331, and (vii) P in position 333. , containing A, G or S, SEQ ID NO: 46 or 62 (or SEQ ID NO: 46 or 6 It contains amino acid sequences that are 90%, 95%, 98%, or 99% identical to 2. In this embodiment, the antibody heavy chain has (i) P, A, G or S at position 246, and (ii) 273 (iii) A in 305th place, (iv) G, P or A in 306th place, (v) 307th place (vi) 335th to A, G, or S, and (vii) 337th SEQ ID NO: 48 (or SEQ ID NO: 48 and 90%, 95%) It contains an amino acid sequence that is 98% or 99% identical.

[0036] In yet another embodiment, the antibody heavy chain has (i) A at position 239 and (ii) A at position 266. (iii) A at 298th place, (iv) P at 299th place, (v) A at 300th place, (vi) 32 Sequence IDs 40, 52, 56, or 6, which include G at position 8 and A at position 330. 4 (or array numbers: 40, 52, 56 or 64 and 90%, 95%, 98% or It contains an amino acid sequence that is 99% identical. In other embodiments, the heavy chain of the antibody is (i)23 (ii) A in 7th place, (iii) A in 264th place, (iv) P in 297th place, ( v) an array containing A at position 298, (vi) G at position 326, and (vii) A at position 328. Number: 42, 54, 58 or 66 (or Sequence ID: 42, 54, 58 or 66) It contains amino acid sequences that are 90%, 95%, 98%, or 99% identical. In the application form, the antibody heavy chain has (i) A at position 244, (ii) A at position 271, and (iii) 3 3rd place is A, (iv) 304th place is P, (v) 305th place is A, (vi) 333rd place is G, and (vii) containing A at position 335, sequence number: 44, 50 or 60 (or sequence number: Amino acid sequences (44, 50, or 60 are 90%, 95%, 98%, or 99% identical) It contains. In yet another embodiment, the heavy chain of the antibody has (i) A at position 242, and (ii) 2 69th place A, (iii) 301st place A, (iv) 302nd place P, (v) 303rd place A, ( vi) Sequence ID: 46 or 62, containing G at position 331 and A at position 333. (or sequence number: 46 or 62 is 90%, 95%, 98%, or 99% identical) It contains an amino acid sequence. In yet another embodiment, the heavy chain of the antibody has (i) A at position 246. (ii) A at 273rd place, (iii) A at 305th place, (iv) P at 306th place, (v) 30 Sequence ID: 4, containing A at position 7, G at position 335, and A at position 337. 8 amino acid combinations (or sequence number: 48 which is 90%, 95%, 98%, or 99% identical) It contains columns.

[0037] In yet another embodiment, the antibody heavy chain has (i) P at position 239 and (ii) A at position 266. , G, or S, (iii) A, G, or S in 298th place, (iv) G in 299th place, (v) Sequence IDs that include T at position 300, (vi)A at position 328, and (vii)P at position 330. :40, 52, 56 or 64 (or array numbers: 40, 52, 56 or 64 and 90) It contains an amino acid sequence that is 95%, 98%, or 99% identical. In other embodiments, it contains an amino acid sequence that is 95%, 98%, or 99% identical. The heavy chain of the antibody has (i) P at position 237, (ii) A, G, or S at position 264, and (iii) (iv) 297th place: A, G or S, (v) 298th place: T, (vi) 32 Sequence IDs 42, 54, 58, or 6, containing A at position 6 and P at position 328. 6 (or sequence numbers: 42, 54, 58 or 66 and 90%, 95%, 98% or It contains an amino acid sequence that is 99% identical. In yet another embodiment, the heavy chain of the antibody is (i ) P at 244th place, (ii) A, G or S at 271st place, (iii) A, G or (iv) G is 304th, (v) T is 305th, (vi) A is 333rd, and (v ii) Sequence ID: 44, 50 or 60 (or Sequence ID: 44, Contains amino acid sequences that are 50 or 60 (and 90%, 95%, 98%, or 99% identical). In further embodiments, the antibody heavy chain has (i) P at position 242 and (ii) P at position 269. (iii) A, G or S in 301st place, (iv) G in 302nd place, (v (vi) A at position 331, and (vii) P at position 333, sequence number Number: 46 or 62 (or Sequence ID: 46 or 62 and 90%, 95%, 98%) It contains an amino acid sequence that is 99% identical. In other embodiments, the heavy chain of the antibody is (i) P at 246th place, (ii) A, G or S at 273rd place, (iii) A, G at 305th place Or S, (iv) G at 306th place, (v) T at 307th place, (vi) A at 335th place, (vii) Sequence ID: 48 (or Sequence ID: 48 and 90%, 95) containing P at position 337 It contains amino acid sequences that are 98% or 99% identical.

[0038] In yet another embodiment, the antibody heavy chain has (i) P at position 239 and (ii) A at position 266. (iii) A at 298th place, (iv) G at 299th place, (v) T at 300th place, (vi) 32 Sequence IDs 40, 52, 56, or 6, which include A at position 8 and P at position 330. 4 (or array numbers: 40, 52, 56 or 64 and 90%, 95%, 98% or It contains an amino acid sequence that is 99% identical. In other embodiments, the heavy chain of the antibody is (i)23 P is in 7th place, (ii) A is in 264th place, (iii) A is in 296th place, (iv) G is in 297th place, ( v) an array containing T at position 298, (vi) A at position 326, and (vii) P at position 328. Number: 42, 54, 58 or 66 (or Sequence ID: 42, 54, 58 or 66) It contains amino acid sequences that are 90%, 95%, 98%, or 99% identical. In the application form, the antibody heavy chain has (i) P at position 244, (ii) A at position 271, and (iii) 3 3rd place A, (iv) 304th place G, (v) 305th place T, (vi) 333rd place A, o (vii) containing P at position 335, sequence number: 44, 50 or 60 (or sequence number: Amino acid sequences (44, 50, or 60 are 90%, 95%, 98%, or 99% identical) It contains. In yet another embodiment, the heavy chain of the antibody has (i) P at position 242, and (ii) 2 69th place A, (iii) 301st place A, (iv) 302nd place G, (v) 303rd place T, ( vi) Sequence ID: 46 or 62, containing A at position 331 and P at position 333. (or sequence number: 46 or 62 is 90%, 95%, 98%, or 99% identical) It contains an amino acid sequence. In yet another embodiment, the heavy chain of the antibody has (i) P at position 246. (ii) A for 273rd place, (iii) A, G or S for 305th place, (iv) G for 306th place (v) T at 307th place, (vi) A at 335th place, and (vii) P at 337th place, Sequence ID: 48 (or 90%, 95%, 98%, or 99% identical to Sequence ID: 48) It contains the following amino acid sequence.

[0039] In other embodiments, the antibody heavy chain has (i) P, A, G or S at position 239, and (ii) 2 (iii) A, G, or S at 66th place, (iv) A, G, or S at 298th place, (iv) G at 299th place (v) T at 300th place, (vi) A, G or S at 328th place, and (vii) 330th place Sequence IDs containing P, A, G, or S: 40, 52, 56, or 64 (or sequence IDs) (40, 52, 56 or 64 and 90%, 95%, 98% or 99% identical) It contains a noacid sequence. In other such embodiments, the heavy chain of the antibody has (i) P at position 237. (ii) A, G or S in 264th place, (iii) A, G in 296th place (iv) G at 297th place, (v) T at 298th place, (vi) A, G at 326th place S, and (vii) Sequence IDs 42, 54, 5, 328 containing P, A, G or S. 8 or 66 (or sequence numbers: 42, 54, 58 or 66 and 90%, 95%, 98) It contains an amino acid sequence that is % or 99% identical. In other embodiments, the weight of the antibody The chain has (i) P, A, G or S at position 244, (ii) A, G or S at position 271, (i ii) A, G or S in 303rd place, (iv) G in 304th place, (v) T in 305th place, (vi ) with A, G or S at position 333, and (vii) with P, A, G or S at position 335, Sequence ID: 44, 50 or 60 (or Sequence ID: 44, 50 or 60 and 90%) It contains an amino acid sequence that is 95%, 98%, or 99% identical. The heavy chain of the antibody has (i) P, A, G or S at position 242, and (ii) A, G at position 269. (iii) A, G or S in 301st place, (iv) G in 302nd place, (v) 303rd place (vi) T in 331st position, A, G or S, and (vii) P, A, G in 333rd position This includes S, SEQ ID NO: 46 or 62 (or SEQ ID NO: 46 or 62 and 90%) It contains an amino acid sequence that is 95%, 98%, or 99% identical. The heavy chain of the antibody has (i) P, A, G or S at position 246, and (ii) A, G at position 273. (iii) A, G or S in 305th place, (iv) G in 306th place, (v) 307th place (vi) T in 335th place, A, G or S, and (vii) P, A, G in 337th place This includes S, SEQ ID NO: 48 (or SEQ ID NO: 48 and 90%, 95%, 98%) It contains an amino acid sequence that is 99% identical.

[0040] In yet another embodiment, the antibody heavy chain has (i) P, A, G or S at position 239, (i i) A at 266th place, (iii) A at 298th place, (iv) G at 299th place, (v) at 300th place T, (vi) A, G or S in position 328, and (vii) P, A, G or S, arrays 40, 52, 56 or 64 (or arrays 40, 52, 56) Alternatively, it contains an amino acid sequence that is 64 (or 90%, 95%, 98%, or 99% identical). In other such embodiments, the antibody heavy chain (i) has P, A, G or S at position 237, (ii) A at 264th place, (iii) A at 296th place, (iv) G at 297th place, (v) 298 (vi) 326th position: T, (vii) 328th position: A, G or S, and (vii) 328th position: P, A, G or includes S, Sequence IDs: 42, 54, 58 or 66 (or Sequence IDs: 42, 54, Contains amino acid sequences that are 90%, 95%, 98%, or 99% identical to 58 or 66. In further embodiments, the antibody heavy chain (i) has P, A, G or S at position 244, (ii) A at 271st place, (iii) A at 303rd place, (iv) G at 304th place, (v) 305 (vi) 333 3rd 3rd 3rd 3rd 3rd 3rd 3rd 3rd 4th 3rd 3rd 3rd 4th 3rd 5th 3rd or includes S, SEQ ID NO: 44, 50 or 60 (or SEQ ID NO: 44 or 50 or It contains amino acid sequences that are 60 and 90%, 95%, 98%, or 99% identical. In other embodiments, the antibody heavy chain has (i) P, A, G, or S at position 242, and (ii) 26 9th place A, (iii) 301st place A, (iv) 302nd place G, (v) 303rd place T, (v i) containing A, G, or S at position 331, and (vii) containing P, A, G, or S at position 333. , Sequence ID: 46 or 62 (or Sequence ID: 46 or 62 and 90%, 95%, 9 It contains an amino acid sequence that is 8% or 99% identical. In other embodiments, the antibody The heavy chain consists of (i) P, A, G, or S at position 246, (ii) A at position 273, and (iii) 30 5th place is A, (iv) 306th place is G, (v) 307th place is T, (vi) 335th place is A, G also (vii)S, and (vii)Sequence number 48 (or Contains amino acid sequences identical to sequence number 48 (90%, 95%, 98%, or 99%). ru.

[0041] In yet another embodiment, the antibody heavy chain has (i) A at position 239 and (ii) A at position 266. (iii) A at 298th place, (iv) G at 299th place, (v) T at 300th place, (vi) 32 Sequence IDs 40, 52, 56, or 6, which include G at position 8 and A at position 330. 4 (or array numbers: 40, 52, 56 or 64 and 90%, 95%, 98% or It contains an amino acid sequence that is 99% identical. In other such embodiments, the heavy chain of the antibody is (i) A at 237th place, (ii) A at 264th place, (iii) A at 296th place, (iv) 297 (v) T at 298th place, (vi) G at 326th place, and (vii) A at 328th place. Includes, SEQ ID NOs: 42, 54, 58 or 66 (or SEQ ID NOs: 42, 54, 58 if It contains amino acid sequences that are 66 and 90%, 95%, 98%, or 99% identical. In other embodiments, the heavy chain of the antibody has (i) A at position 244, (ii) A at position 271, ( iii) A at 303rd place, (iv) G at 304th place, (v) T at 305th place, (vi) 333rd place (vii) containing G and A at position 335, sequence numbers: 44, 50 or 60 (or Sequence ID: 44, 50 or 60 (identical to 90%, 95%, 98%, or 99%) It contains a mino acid sequence. In yet another embodiment, the heavy chain of the antibody has (i) A at position 242. (ii) A at 269th place, (iii) A at 301st place, (iv) G at 302nd place, (v) 303 Sequence ID: 46, containing T at position (vi)331, G at position (vii)333, and A at position (vii)333. Or 62 (or array index: 46 or 62 and 90%, 95%, 98%, or 99) It contains the same amino acid sequence. In yet another embodiment, the heavy chain of the antibody is (i)2 (ii) A in 46th place, (ii) A in 273rd place, (iii) A in 305th place, (iv) G in 306th place, (v) T at 307th, (vi) G at 335th, and (vii) A at 337th, Column number: 48 (or array index: 48 is 90%, 95%, 98%, or 99% identical) It contains an amino acid sequence.

[0042] In addition, the present invention relates to Sequence IDs: 72, 78, 84, and 9 The amino acid sequence containing 0, SEQ ID NO: 96, SEQ ID NO: 102, or SEQ ID NO: 108 The present invention provides a canine antibody or its antigen-binding fragment further containing a Nu light chain.

[0043] Therefore, the present invention further relates to a heavy chain and sequence number comprising the amino acid sequence of SEQ ID NO: 68. No. 72: Canine antibody containing a light chain with an amino acid sequence or its antigen-binding fragment Provides a canine antibody or its antigen-binding fragment, A heavy chain containing amino acid sequence number 70 and a light chain containing amino acid sequence number 72 Contains. In another embodiment, the canine antibody or its antigen-binding fragment is SEQ ID NO: Contains a heavy chain containing the amino acid sequence of :74 and a light chain containing the amino acid sequence of SEQ ID NO: 78. In related embodiments, the canine antibody or its antigen-binding fragment is SEQ ID NO: 7 It contains a heavy chain with an amino acid sequence of 6 and a light chain with an amino acid sequence of SEQ ID NO: 78. In yet another embodiment, the canine antibody or its antigen-binding fragment is: It contains a heavy chain containing an 80-amino acid sequence and a light chain containing an amino acid sequence of SEQ ID NO: 84. In related embodiments, the canine antibody or its antigen-binding fragment is SEQ ID NO: 82 It contains a heavy chain containing the amino acid sequence of [sequence number] and a light chain containing the amino acid sequence of SEQ ID NO: 84. In yet another embodiment, the canine antibody or its antigen-binding fragment is SEQ ID NO: 8 It contains a heavy chain containing the amino acid sequence of 6 and a light chain containing the amino acid sequence of SEQ ID NO: 90. In related embodiments, the canine antibody or its antigen-binding fragment is SEQ ID NO: 88 It contains a heavy chain containing an amino acid sequence and a light chain containing the amino acid sequence of SEQ ID NO: 90. In another embodiment, the canine antibody or its antigen-binding fragment is SEQ ID NO: 92 It contains a heavy chain containing the amino acid sequence of [sequence number] and a light chain containing the amino acid sequence of SEQ ID NO: 96. In related embodiments, the canine antibody or its antigen-binding fragment is the axle of SEQ ID NO: 94 It contains a heavy chain containing amino acid sequences and a light chain containing the amino acid sequence of SEQ ID NO: 96. In another embodiment, the canine antibody or its antigen-binding fragment is SEQ ID NO: 98 It contains a heavy chain containing an amino acid sequence and a light chain containing the amino acid sequence of SEQ ID NO: 102. In related embodiments, the canine antibody or its antigen-binding fragment is SEQ ID NO: 100 It contains a heavy chain containing an amino acid sequence and a light chain containing the amino acid sequence of SEQ ID NO: 102. In yet another embodiment, the canine antibody or its antigen-binding fragment is SEQ ID NO: 1 Contains a heavy chain containing the amino acid sequence 04 and a light chain containing the amino acid sequence SEQ ID NO: 108. In related embodiments, the canine antibody or its antigen-binding fragment is SEQ ID NO: 1 Contains a heavy chain containing the amino acid sequence 06 and a light chain containing the amino acid sequence SEQ ID NO: 108. Perform.

[0044] The present invention further provides a canine antibody or an antigen-binding fragment thereof containing 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: 72. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains 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: 72. In another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and a light chain comprising the amino acid sequence of SEQ ID NO: 78. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 46 and a light chain comprising the amino acid sequence of SEQ ID NO: 78. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising the amino acid sequence of SEQ ID NO: 84. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 50 and a light chain comprising the amino acid sequence of SEQ ID NO: 84. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 52 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 54 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 96. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a light chain comprising the amino acid sequence of SEQ ID NO: 102. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 and a light chain comprising the amino acid sequence of SEQ ID NO: 108. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 114. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO: 114. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 68 and a light chain comprising the amino acid sequence of SEQ ID NO: 120. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 70 and a light chain comprising the amino acid sequence of SEQ ID NO: 120. In yet another embodiment, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 72 and a light chain comprising the amino acid sequence of SEQ ID NO: 126. In related embodiments, the canine antibody or an antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 74 and a light chain comprising the amino acid sequence of SEQ ID NO: 132. In yet another In this case,the canine antibody or antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO:58 and a light chain comprising the amino acid sequence of SEQ ID NO:96.

[0045] In yet another embodiment,the canine antibody or antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 60 and a light chain comprising the amino acid sequence of SEQ ID NO:102. In a related embodiment,the canine antibody or antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO:6 2 and a light chain comprising the amino acid sequence of SEQ ID NO:102. In yet another embodiment,the canine antibody or antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO:108. In a related embodiment,the canine antibody or antigen-binding fragment thereof contains a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO:108.

[0046] The present invention further provides a nucleic acid encoding any of the amino acid sequences of the present invention including the CDR,cFc region,hinge region of the canine antibody of the present invention,the cFc region having the heavy chain and light chain of the present invention. The present invention further provides an expression vector containing one or more of the nucleic acids of the present invention. The present invention further provides a host cell containing one or more of the expression vectors of the present invention and a method for expressing the CDR and / or cFc region and / or hinge region of the cFc region having the heavy chain and / or light chain of the canine antibody of the present invention using such host cells. The present invention also provides a polynucleotide encoding the CDR and / or The canine antibody of the present invention has a CDR and / or cFc region and / or hinge region. Genetically engineered to express the cFc region and / or heavy and / or light chain. The present invention also provides host cells that are being used. In certain embodiments, these nucleic acids and expression vectors of the present invention are also provided. Polypeptides or host cells are useful in methods for producing antibodies.

[0047] In certain embodiments, the antibody is a recombinant antibody or its antigen-binding fragment. In the linked embodiment, the variable heavy chain domain and the variable light chain domain are linked by a flexible linker. They are linked together to form a single-chain antibody.

[0048] In certain embodiments, the antibody or antigen-binding fragment is a Fab fragment.

[0049] 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 yet another embodiment, the antibody or antigen-binding fragment is a diabody. In certain embodiments, the antibody or antigen-binding fragment is a domain antibody. In the application form, the antibody or antigen-binding fragment is a camelized single-domain antibody.

[0050] In certain embodiments, the canine mouse anti-canine PD-1 antibody or antigen-binding fragment is This increases the immune response of the treated canine subjects.

[0051] In one embodiment, when binding to canine PD-1, a canine antibody or its antigen-binding fragile The components are: SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 14 1. At least one amino acid residue within the amino acid sequence of SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144 and / or SEQ ID NO: 145 binds to .

[0052] Furthermore, the present invention provides a canine PD-1 -1-inhibited antibody containing a variant of the CDR of the present invention that has the corresponding canonical structure provided herein and / or binds to the amino acid sequence of SEQ ID NO: 144. In certain embodiments of this type, the dissociation constant (Kd) for the canine-inhibited antibody-canine PD-1 binding is from 1×10 M to 1×10 M. -5 M to 1×10 -12 M. In a more specific embodiment, the canine-inhibited antibody against canine PD-1 has the corresponding canonical structure provided herein and contains a variant of the CDR of the present invention that binds to the amino acid sequence of SEQ ID NO: 145. The present invention therefore encompasses canine-inhibited antibodies and antigen-binding fragments thereof that specifically bind to canine PD-1, where when they bind to canine PD-1, the antibodies bind to at least one amino acid residue within SEQ ID NO: 144. In certain embodiments of this type, the antibody and its antigen-binding fragment bind to canine PD-1 and block its binding to canine programmed death ligand 1 (PD-L1). Therefore, in certain embodiments, when binding to canine PD-1, the canine-inhibited antibody (one or more variant CDRs, such as variants that conservatively modified and / or variants that constitute a defined canonical structure class) binds to SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144 and / or SEQ ID NO: 145 within the amino acid sequence of at least one amino acid residue. In certain embodiments of this type, the antibody and its antigen-binding fragment bind to canine PD-1 and block its binding to canine programmed death ligand 1 (PD-L1). <00已完成翻译,请确认是否符合要求。如有问题,请随时告诉我。00969>ligand 1 (PD-L1).

[0053] Thus, in certain embodiments, when binding to canine PD-1, the canine-inhibited antibody (one or more variant CDRs, such as variants that conservatively modified and / or variants that constitute a defined canonical structure class) binds to SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143 and / or at least one amino acid in one or more amino acid sequences of sequence number: 145 It binds to amino acid residues. In more specific embodiments, it binds to canine PD-1. The canine antibody or its antigen-binding fragment contains the following arginine residue: SEQ ID NO: 11 4 R 62 , R 69 , R 72 , R 75 and R 90 One or more amino acid residues It binds. In a specific embodiment, when it binds to canine PD-1, it binds to a canine antibody or The antigen-binding fragment binds to at least one amino acid residue in SEQ ID NO: 145. In a more specific embodiment, when binding to canine PD-1, the antibody or its antigen-binding agent Ragment is the R of the following arginine residue: SEQ ID NO: 114 62 , R 69 , R 72 and R 75 It binds to one or more amino acid residues. In a more specific embodiment, When binding to canine PD-1, the antibody or its antigen-binding fragment is sequence number 11. 4 R 75 Combine.

[0054] This invention is 1 × 10 -12 Lower than M (for example, 1 × 10) -13 Dissociation (M or less) A canine antibody or its antigen-binding fragment that binds to canine PD-1 at a constant (Kd) Provided to them. In a particular embodiment, the canine antibody or its antigen-binding fragment is 1 ×10 -5 M to 1x10 -12 It binds to canine PD-1 with a dissociation constant of M. In the application form, the canine antibody or its antigen-binding fragment is 1 × 10 -7 M to 1x1 0 -11 It binds to canine PD-1 with a dissociation constant of M. In more specific embodiments, canine The antibody or its antigen-binding fragment is 1 × 10 -8 M to 1x10 -11 M's Dissociation It binds to canine PD-1 at a constant rate. In more specific embodiments, a canine antibody or The antigen-binding fragment is 1 × 10 -8 M to 1x10 -10 In canine PD- with M dissociation constant Combine with 1.

[0055] This invention also includes 1 × 10 7 M -1 seconds -1 On speed exceeding (k on ) and canine PD-1 The conjugation of a canine antibody or its antigen-binding fragment is also provided. In certain embodiments, Canine antibodies or their antigen-binding fragments are 1 × 10 2 M -1 seconds -1 From 1 x 10 7 M -1 seconds -1 It binds to canine PD-1 at an on-rate. In a more specific embodiment, canine anti The body or its antigen-binding fragments are 1 × 10 3 M -1 seconds -1 From 1 x 10 6 M -1 seconds -1 It binds to canine PD-1 at an on-rate. In more specific embodiments, canine antibody or its antigen-binding fragment, 1 × 10 3 M -1 seconds -1 From 1 x 10 5 M -1 seconds - 1 It binds to canine PD-1 at an on-rate. In more specific embodiments, a canine antibody is used. The antigen-binding fragment is 1 × 10 4 M -1 seconds -1 From 1 x 10 5 M -1 seconds -1 It binds to canine PD-1 at the on-speed.

[0056] The present invention further includes 1 × 10 -7 seconds -1 A gentler off speed (k off ) with InuPD- Provides a canine antibody or its antigen-binding fragment that binds to 1. In a specific embodiment, The canine antibody or its antigen-binding fragment is 1 × 10 -3 seconds -1 From 1 x 10 - 8 seconds -1 It binds to canine PD-1 at an off-rate. In more specific embodiments, a canine antibody is used. The antigen-binding fragment is 1 × 10 -4 seconds -1 From 1 x 10 -7 seconds -1 Off speed It binds to canine PD-1 at a certain degree. In more specific embodiments, a canine antibody or its anti- The original binding fragment is 1 × 10⁻⁶ -5 seconds -1 From 1 x 10 -7 seconds -1 At the off speed, InuP Binds to D-1.

[0057] In related embodiments, the canine antibody or its antigen-binding fragment is used to target tumors or pathogens. It stimulates an antigen-specific memory response to the canine antibody or its anti- The primordial-bound fragment stimulates an antibody response in vivo. In other specific embodiments, canine transformation occurs. Antibodies or their antigen-binding fragments stimulate an immune response in animal subjects. In a specific embodiment, the animal test subject is a dog. In a related embodiment, the animal test subject is a cat. That is the case.

[0058] Therefore, any of the canine antibodies of the present invention is 1, 2, 3, 4, 5 or all of these. The characteristics, namely the dissociation constant with canine PD-1, and the characteristics relating to binding with canine PD-1. On-speed, the off-speed for dissociation from the canine antibody-canine PD-1 binding complex, tumor Or stimulation of antigen-specific memory responses to pathogens, stimulation of antibody responses in vivo, and / or it can demonstrate stimulation of the immune response in animal subjects.

[0059] In a more specific embodiment, the canine antibody of the present invention and its antigen-binding fragment are It binds to nuPD-1 and also blocks the binding of nuPD-1 to PD-L1. In certain embodiments, the canine antibody of the present invention and its antigen-binding fragment are It binds to canine PD-1, blocks the binding of canine PD-1 to PD-L1, and also It also blocks the binding of PD-1 to PD-L2.

[0060] The present invention further encodes the canine mouse anti-canine PD-1 antibody or a portion thereof. To provide nucleic acids. In related embodiments, such antibody or antigen-binding fragments are It can be used for the preparation of drugs to treat cancer in canine subjects. In addition, the present invention relates to the antibodies or antibody fragments of the present invention for diagnostic purposes. The use of any of the following is provided. Further embodiments include the canine antibody disclosed herein. Alternatively, a kit containing either an antigen-binding fragment is provided.

[0061] The present invention further relates to an anti-canine antigen antibody or a pharmaceutically acceptable carrier or diluent. The binding fragment (for example, an anti-canine PD-1 antibody or its antigen-binding fragment) The present invention also includes a pharmaceutical composition containing the present invention. Then, administer a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject (e.g., a dog) that requires it. The present invention also provides a method that includes the following: In one embodiment, this method is used for the treatment of cancer. In other embodiments, this method is used in the treatment of infection or infectious disease. In other embodiments, the canine antibody or its antigen-binding fragment of the present invention is a vaccine It is used as an adjuvant. In yet another embodiment, the canine anti-TSLP antibody is used as an adjuvant. It is administered to dogs to treat totopic dermatitis.

[0062] These and other aspects of the present invention are described in the following "Brief Description of the Drawings" and "Instructions for Carrying Out the Invention" This can be better understood by referring to "the form of the eye." [Brief explanation of the drawing]

[0063] [Figure 1] Figure 1 shows the reactivity of canine monoclonal antibodies (mAbs) to the extracellular domain of canine PD-1 as a function of OD650 / 490 to logarithmic mAb(nM). Various canine mAbs were tested by ELISA for their binding to the extracellular domain of canine PD-1. The four mAbs tested were designated as 2H9 VH4 IgGB / VL3, 3B6 VH3 IgGB / VL3, 2H9 VH4 IgGB(YZZ1062) / VL3, and 2H9 VH4 IgGB(YZZ1068) / VL3. [Figure 2]Figure 2 shows the reactivity of canine mAbs to canine PD-1 expressed on the cell surface. Various mouse mAbs were tested by CELISA for their binding to canine PD-1 expressed on CHO cells as a function of OD450 / 540 to logarithmic mAb(nM). The six mAbs tested were named 3B6 VH3 / VL4, 3B6 VH3 / VL1, 3B6 VH3 / VL3, 3B6 VH3 / VL2, 3B6 VH1 / VL1, and 3B6 mc Chimera. [Figure 3] Figure 3 shows ligand blockade of canine PD-1 by canine mAbs. Various canine mAbs were tested as a function of OD450 / 540 to log mAb(nM) for their ability to inhibit the binding of PD-1 expressed on CHO cells to PD-L1. The six mAbs tested were named 3B6 VH3 / VL4, 3B6 VH3 / VL1, 3B6 VH3 / VL3, 3B6 VH3 / VL2, 3B6 VH1 / VL1, and 3B6 mc Chimera. [Figure 4] Figure 4 shows cytokine secretion induced by canine mAbs against canine PD-1. Various canine mAbs and their variants were tested for their ability to induce cytokine secretion from PBMCs derived from healthy dogs. [Figure 5A] Figure 5A shows the binding of canine mAbs and their variants (starting at 1 μg / ml) to FcγRI. Various mAbs were tested for their ability to bind to FcRI. The antibodies are denoted as can 2H9 ADCC(1062)VH4 / VL3, can 2H9 ADCC mut1 VH4 / VL3, can 2H9 ADCC mut2 VH4 / VL3, can 2H9 IgGD VH4 / VL3, can 2H9 VH4 / VL3, and can 3B6 VH4 / VL4. [Figure 5B]Figure 5B shows the binding of canine mAbs and their variants (starting at 1 μg / ml) to FcγRI. Various mAbs were tested for their ability to bind to FcRI. The antibodies are denoted as can 2H9 ADCC(1059)VH4 / VL3, can 2H9 ADCC(1060)VH4 / VL3, can 2H9 ADCC(1061)VH4 / VL3, can 2H9 IgGB ADCC(1068)VH4 / VL3, can 2H9 VH4 / VL3, and can 3B6 VH4 / VL4. [Figure 6A] Figure 6A shows the binding of canine mAbs and their variants (starting at 1 μg / ml) to C1Q. Various mAbs were tested for their ability to bind to C1Q. The antibodies are denoted as can 2H9 VH4 IgGB ADCC(1062) / VL3, can 2H9 VH4 IgGB ADCC(mut1) / VL3, can 2H9 VH4 IgGB ADCC(mut2) / VL3, can 2H9 VH4 IgGD / VL3, can 2H9 VH4 / VL3, and can 3B6 VH4 / VL4 IgGB. [Figure 6B] Figure 6B shows the binding of canine mAbs and their variants (starting at 1 μg / ml) to C1Q. Various mAbs were tested for their ability to bind to C1Q. The antibodies are denoted as can 2H9 VH4 IgGB ADCC(1059) / VL3, can 2H9 VH4 IgGB ADCC(1060) / VL3, can 2H9 VH4 IgGB ADCC(1061) / VL3, can 2H9 VH4 IgGB ADCC(1068) / VL3, can 2H9 VH4 / VL3 IgGB, and can 3B6 VH4 / VL4 IgGB. [Figure 7A] Figure 7A shows the characterization of the interface between canine PD-1 and canine antibody 2G9. The amino acid positions refer to the PD-1 amino acid sequence without a signal sequence, i.e., SEQ ID NO: 114. Measurements were performed by chemical crosslinking, high-mass MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry. [Figure 7B]Figure 7B shows the characterization of the interface between canine PD-1 and canine antibody 3B6. The amino acid positions refer to the PD-1 amino acid sequence without a signal sequence, i.e., SEQ ID NO: 114. Measurements were performed by chemical crosslinking, high-mass MALDI mass spectrometry, and nLC-Orbitrap mass spectrometry. [Modes for carrying out the invention]

[0064] Abbreviation The following abbreviations are used throughout the "Modes for Carrying Out the Invention" and "Examples" of this invention. Use: ADCC antibody-dependent cytotoxicity CDC complement-dependent cell injury The CDR Kabat numbering system is used to define human antibodies. Furthermore, complementarity-determining regions within the immunoglobulin variable region. CHO Chinese hamster ovaries Concentration that produces an effect or binding of 50% EC50 ELISA enzyme-linked immunosorbent assay FR Antibody Framework Region: Immunoglobulin Variable Region excluding the CDR Region HRP Horseradish Peroxidase IFN (Interferon) Concentration that causes 50% inhibition of IC50 IgG (Immunoglobulin G) Kabat Elvin A.Kabat[Sequences of Prot eins of Immunological Interest,5th Ed.Pu blic Health Service,National Institutes Human developed by [of Health, Bethesda, Md. (1991)] Immunoglobulin alignment and numbering systems for antibodies mAb monoclonal antibody (also abbreviated as Mab or MAb) MES 2-(N-morpholino)ethanesulfonic acid MOA mechanism of action NHS Normal Human Serum PCR (polymerase chain reaction) PK (Pharmacokinetics) SEB (Staphylococcus enterotoxin B) TT Tetanus Toxoid V region: A segment of the human IgG chain whose sequence is variable between different antibodies. Light chain This extends to Kabat residue 109 within the chain and Kabat residue 113 within the heavy chain.

[0065] VH (Variable Region of Immunoglobulin Heavy Chain) VK immunoglobulin κ light chain variable region definition To facilitate understanding of this invention, specific technical and scientific terms are clearly defined below. Unless otherwise explicitly defined elsewhere in this document, all other terms used herein are also used. Technical and academic terms are intended to be generally understood by those skilled in the art to which this invention pertains. It has a taste.

[0066] As used herein, including in the accompanying claims, “a, an (one)” The singular form of words like "the" is used when there is no clear indication in the context that it is not singular. To the extent that it does, it includes those corresponding multiple references.

[0067] The term "activation" applied to cells or receptors is not appropriate unless the context or clearly indicates otherwise. This refers to the activation or treatment of cells or receptors by ligands. "Ligands" are naturally occurring and synthetic ligands, such as cytokines, cytokine variants, analogs, mutains and It includes antibody-derived binding compounds. "Ligands" also include small molecules, such as cytokines. This also includes peptomimetic and antibody peptomimetic. "Activation" is This refers to cell activation regulated by internal mechanisms as well as by external or environmental factors. It is possible.

[0068] Molecular "activity" includes the binding of the molecule to a ligand or receptor, catalytic activity, gene expression, and This involves the ability to stimulate cell signaling, differentiation, or maturation, antigen activity, and the activity of other molecules. It can represent or refer to regulation, etc. The "activity" of a molecule can also refer to intercellular interactions, for example, contact Activity in regulating or maintaining adhesion, or in the structure of cells, such as the cell membrane or It can also refer to activity in maintaining the cytoskeleton. "Activity" can also refer to specific activity, for example, catalytic activity. Sex / [mg protein] or [immunological activity] / [mg protein], biological compartment It can also refer to the concentration within, etc. "Activity" refers to the regulation of components of the innate or adaptive immune system. It could also refer to this.

[0069] "Administration" and "treatment" refer to animals, such as dog experimental subjects, cells, tissues, organs or living organisms. When applied to bodily fluids, it may be applied to animals, such as dogs, subjects, cells, tissues, organs, or bodily fluids. This refers to contact with causative drugs, therapeutic agents, diagnostic agents, or compositions. Cell treatment refers to contact with reagents on cells. This includes contact with the fluid and, if the fluid is in contact with the cells, contact of the reagent with that fluid. " and "treatment" also include, for example, treatment by reagents, diagnostic agents, conjugates, or by other cells. For example, this also refers to in vitro and ex vivo treatment of cells.

[0070] The term "subject" refers to any living organism, preferably an animal, more preferably a mammal (e.g., (For example, a dog, a cat, or a human), most preferably including dogs.

[0071] As used herein, the term "cat" refers to any member of the Felidae family. This refers to members of this family. Members of this family include wild, zoo, and domesticated members, for example, the Felinae ( Any member of Felinae, for example, cat, lion, tiger, puma, jaguar, hi Leopard, snow leopard, black panther, North American mountain lion, cheetah, lynx, bobcat This includes cats, caracals, or any hybrids thereof. Cats also include domestic cats and purebreds. and / or mixed breed companion cats, show cats, laboratory cats, cloned cats This also includes wild or feral cats.

[0072] When used herein, "amino acid residue" refers to another amino acid residue within an amino acid sequence. "Substitution of" is equivalent to "replacing an amino acid residue with another amino acid residue," and amino acids A specific amino acid residue at a specific position within the sequence is replaced by a different amino acid. It means (or has been replaced). For example, one such replacement (substitution) This is represented as P4A in the Fc region of the IgGB or IgGC amino acid sequence, in this case Ig Proline residue at amino acid position 4 in the amino acid sequence of the Fc region of GB or the Fc region of IgGC The group is substituted (replaced) by an alanine residue.

[0073] Therefore, such amino acid substitutions can be designed individually, i.e., intentionally For example, recombinant DNA technology can be used to convert alanine to serine at a specific position within the amino acid sequence. It can be replaced with a specific amino acid residue or a series of amino acids in the antibody. Through a more natural selection process, for example, antibodies produced by cells select their antigens. For example, the ability to bind to a given region of an epitope or a part thereof. Based on and / or the antibody that retains the same canonical structure as the substituted CDR, It can be replaced by one or more amino acid residues so as to contain the CDR. Yes, it is possible. Such substitutions / replacements result in "mutant" CDRs and / or antibodies. It is possible.

[0074] "To treat" or "to treat" refers to the binding of a therapeutic agent, such as the antibody or antigen of the present invention. A composition containing any of the fragments is used for patients with one or more disease symptoms. Alternatively, in canine subjects or patients suspected of having a disease for which the therapeutic agent is therapeutically active, This means administering it internally or externally.

[0075] Typically, therapeutic agents are used to achieve clinically measurable results in the subjects or population being treated. Each time, it induces a remission of such (one or more) symptoms or (one or more) symptoms By halting the progression of the disease, it reduces one or more disease symptoms and / or It is administered in a dose effective for improvement. It is an effective treatment for alleviating the symptoms of a particular disease. The amount of medication (also called the "therapeutic dose") depends on the state of the disease, the age of the subject (e.g., a dog), and Factors such as body weight and the ability of the pharmaceutical composition to elicit the desired response in the subject. Therefore, it can vary. Whether the symptoms of the disease have lessened or improved depends on the severity of the symptoms and To assess the progression of the disease, consult a veterinarian or other skilled medical professional. It can be evaluated by any clinical measurement typically used by the provider. An embodiment of the explicit description (e.g., in the section on treatment or manufacture) is provided in all subjects (1 or It may not be effective in alleviating symptoms of multiple target diseases, but in this field Any known statistical test, such as the Student's t-test, chi-squared test, or Mann-Whitt test. Nye's U test, Kruskal-Wallis test (H test), Yonkie-Tapstra test and In a statistically significant number of subjects determined by the Wilcoxon test, etc. (1 It should alleviate symptoms of multiple target diseases.

[0076] "Procedure" is used when applied to humans, veterinary medicine (e.g., dogs), or research subjects, and refers to treatment. This refers to the application of treatment, research, and diagnostic methods to humans, veterinary medicine (e.g., dogs), or research. "Procedures" applied to subjects or cells, tissues, or organs may be performed on dogs or other animals. The canine antibodies or antigens of the present invention that can be deposited into the body, cells, tissues, physiological compartments, or physiological fluids. It includes contact of composite fragments.

[0077] Canine PD-1 is sequence number 114 [the entire content of which is incorporated herein by reference]. [U.S. Patent Provisional Application No. 61 / 918,946, filed December 20, 2013] Amino It has been found to contain an acid sequence. In a specific embodiment, canine PD-1 is a sequence Encoded by nucleic acid containing the nucleotide sequence number 113.

[0078] Canine PD-L1 is identified as Sequence ID: 120 [U.S. Patent Provisional Application filed December 20, 2013]. It has been confirmed to contain the amino acid sequence described in [Nos. 61 / 918, 946, previously mentioned]. In a concrete embodiment, canine PD-L1 is a nucleotide sequence containing SEQ ID NO: 119. It is coded by.

[0079] The term "immune response" is used, for example, to describe cells or tissues infected with cancer cells, pathogens, or This involves selective damage to the mammalian body (e.g., dog body) by invasive pathogens, its destruction, or the feeding of mammals. Lymphocytes, antigen-presenting cells, phagocytic cells, and granules that bring about the removal of material from the body (e.g., a dog's body). This refers to the action of granulocytes and soluble polymers produced by the cells or liver.

[0080] Canine-type anti-canine antigen antibody As used herein, the term "dog" means all dogs unless otherwise specified. Domestic dog, domestic dog (Canis lupus familiaris), or canine dog It includes the genus *Canis familiaris*.

[0081] When used herein, an antibody is a canine antibody, for example, a canine PD-1 amino acid sequence It binds to polypeptides containing a portion of the sequence of canine antibodies, such as canine PD-1. If it does not bind to other canine proteins that lack that part, then the given antigen sequence (in this case) Polypeptides containing canine antibodies (e.g., a portion of the amino acid sequence of canine PD-1) are particularly useful. It is said to bind heterologously. For example, it specifically binds to polypeptides containing canine PD-1. The antibody can bind to canine PD-1 in the form of FLAG® tag, but other FL AG(registered trademark) tag does not specifically bind to canine protein. Antibodies or antigenic binding of antibodies The binding compound derived from the binding site has a higher affinity than any other canine antigen being tested. At least 10 times higher, more preferably at least 20 times higher, even more preferably less At least 100 times higher affinity for that canine antigen or its variant or mutain. If present, it binds "specifically" to the canine antigen or its variant or mutain. .

[0082] As used herein, the term "antibody" refers to any antibody exhibiting the desired biological activity. This refers to a specific type of antibody. Therefore, this term is used in its broadest sense, specifically referring to monoclonal antibodies. Antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example) (Bispecific antibodies), canine antibodies, fully canine antibodies, chimeric antibodies and camel-specific monodomyens This covers, but is not limited to, antibodies. "Parent antibody" is for the intended use. Antibody modification, for example, canine modification of an antibody for use as a therapeutic antibody for dogs, precedes antigen modification. These are antibodies obtained by exposure of the immune system to a substance.

[0083] As used herein, unless otherwise specified, the term "antibody fragment" refers to "antibody fragment" or "anti- The "proto-binding fragment" is the antigen-binding fragment of the antibody, i.e., the full-length antibody. An antibody fragment that retains the ability to specifically bind to the antigen to be bound, e.g., 1 or so This refers to fragments that retain the above-mentioned CDR region. Examples of antigen-binding fragments include: Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, Formed from single-chain antibody molecules, such as sc-Fv, nanobodies, and antibody fragments. This includes, but is not limited to, multispecific antibodies.

[0084] "Fab fragment" is a C2 fragment consisting of one light chain and one heavy chain. H 1 region and variable region It consists of the following. The heavy chain of the Fab molecule can form disulfide bonds with other heavy chain molecules. No. "Fab fragments" can be products of papain cleavage of antibodies.

[0085] The "crystalline fragment" ("Fc") region is the C of the antibody. H 2 and C H Includes 3 domains It contains two heavy chain fragments (i.e., two identical polypeptides). One heavy chain fragment is connected by two or more disulfide bonds and C H 3 degrees They are linked by the main hydrophobic interaction. In this invention, four canine Ig The amino acid sequences for each G Fc fragment are from Tang et al.[Ve t.Immunol.Immunopathol.80:259-270(2001)] Based on the identified boundaries between the CH1 and CH2 domains, as determined by [the relevant authority].

[0086] "Fab' fragment" is a single light chain and V H Domain and C H Approximately 1 domain Furthermore, interchain disulfide bonds are formed between the two heavy chains of the two Fab' fragments. C H 1 domain and C H 2 domains It contains a portion or fragment of a single heavy chain, including the region between it and the other.

[0087] The "F(ab')2 fragment" consists of two light chains and an interchain disulfide between the two heavy chains. C H 1 domain and C H 2 Part of the constant region between the domains It contains two heavy chains. The F(ab')2 fragment therefore contains two heavy chains. It consists of two Fab' fragments linked together by interchain disulfide bonds. The "F(ab')2 fragment" may be a product of pepsin cleavage of the antibody.

[0088] The "Fv region" contains variable regions derived from both heavy and light chains, but the steady region is To lack.

[0089] The term "single-stranded Fv" or "scFv" antibody refers to the V of the antibody. H and V L domain This refers to antibody fragments containing these domains, which are located within a single polypeptide chain. Generally, Fv polypeptides are formed when scFv forms the desired structure for antigen binding. V makes this possible H Domain and V L Further contains polypeptide linkers between the domains [Pluckthun, The Pharmacology of Monoclo] nal Antibodies,vol.113 Rosenburg and Moo re eds., Springer-Verlag, New York, pp.269- 315 (1994); International Publication No. 88 / 01649; and U.S. Publication No. 4,946, See U.S. Patent No. 778 and U.S. Patent No. 5,260,203.

[0090] As used herein, the term "canonical structure" refers to the superimposed structure of the heavy and light chains of an antibody. Each of the variable domains has a local conformation that it can take within the framework in which it exists. To refer to. For each hypervariable region, the amino acid sequence of the corresponding hypervariable region (especially the corresponding I Regarding the murine anti-canine PD-1 variable domain, the framework provided below is as follows: A small number of canonical structures (generally) can be predicted with high accuracy from the amino acid sequence status of the work. There exists a simple integer (represented by 1 or 2, etc.). These canonical structures are given Modification of the amino acid sequence of the CDR results in retention or loss of its ability to bind to its antigen-binding partner and can be decisive in this regard [Chothia and Le sk, Canonical Structures for the hypervar iable regions of immunoglobulins, J.Mol.B iol. 196:901-917(1987); Chothia et al., Con formation of immunoglobulin hypervaribal e regions, Nature, 34:877-883(1989); and Al- Lazikani et al., Standard Conformations f or the canonical structures of immunoglo bulins, J.Mol.Biol. 273:927-948(1997) see].

[0091] A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more V regions are covalently linked by a peptide linker to create a bivalent domain antibody. The two V H regions of the bivalent domain antibody can target the same or different antigens.

[0092]

[0093] A "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific (see below). In certain embodiments, the monoclonal antibodies herein also encompass camelized single-domain antibodies comprises. [See, e.g., Muyldermans et al., Trends Bioche m. Sci. 26:230 (2001); Reichmann et al., J. Im munol. Methods 231:25 (1999); WO 94 / 04678 ; WO 94 / 25591; U.S. Pat. No. 6,005,079]. In one em bodiment, the invention provides a single domain antibody comprising two V H domains having modifications such that a single domain antibody is formed.

[0094] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, the fragment comprising a heavy chain variable domain (V H) linked to a light chain variable domain (V L) within the same polypeptide chain (V L H-V H L or V H L-V L H). 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 on another chain, creating two antigen-binding sites. [See European Patent No. 0404097; WO 93 / 11161; and Holliger et al., Proc. Natl. A L cad. Sci. USA 90:6444-6448 (1993)]. For a review of engineered anti H body variants, see generally Holliger and Hudson Nat . Biotechnol. 23:1126-1136 (2005). 93 / 11161; and Holliger et al., Proc. Natl. A cad. Sci. USA 90:6444-6448 (1993)]. For a review of engineered anti body variants, see generally Holliger and Hudson Nat . Biotechnol. 23:1126-1136 (2005).

[0095] Typically, the antibodies or antigen-binding fragments of the invention have their canine PD-1 binding activity​​ When expressed in molar terms, at least 10% of its activity (compared to the parent antibody) To retain. Preferably, the antibody or antigen-binding fragment of the present invention is used as the parent antibody. Nu-type antibody (e.g., PD-1) binding affinity of at least 20%, 50%, 70%, 80%, It retains 90%, 95%, 100%, or more. Furthermore, the canine antibody of the present invention... Alternatively, antigen-binding fragments are either conserved or non-conservative, meaning they do not substantially alter the biological activity of the antigen. Conservative amino acid substitutions (also called "conservative variants" or "function-conserving variants" of antibodies) It is also intended that it may contain )

[0096] "Isolated antibody" refers to a purified state in which the molecule is isolated from other biological molecules, for example. For example, nucleic acids, proteins, lipids, carbohydrates or other substances, such as cell debris and growth cultures. It means that it does not substantially include land, etc. Generally, the term “isolated” means that Such substances or water, buffers or salts may be used in the experimental or therapeutic experiments of the binding compounds described herein. The complete absence of such a substance is to be limited to amounts that substantially prevent its therapeutic use. It is not intended to mean the absence of water, buffer, or salt.

[0097] As used herein, "chimeric antibody" refers to a variable domain derived from the first antibody and the second An antibody having a constant domain derived from two antibodies, wherein the first antibody and the second antibody originate from different species. It is an antibody that does the following: [U.S. Patent No. 4,816,567; and Morrison et al.] al.,Proc.Natl.Acad.Sci.USA 81:6851-6855( 1984). Typically, the variable domain is derived from antibodies from experimental animals such as rodents (" The constant domain sequence is obtained from the parent antibody, for example, from an animal test antibody, such as a dog. The resulting chimeric antibodies caused a more harmful immune response in canine subjects than the parent (e.g., rodent) antibodies. The antibodies are obtained from the animal test subjects in such a way that they are less likely to produce a response.

[0098] As used herein, the term "canine antibody" refers to canine and non-canine antibodies (for example). This refers to an antibody form that contains sequences derived from both mouse and canine antibodies. Generally, canine antibodies are , where all or substantially all of the hypervariable loop corresponds to non-canine immunoglobulins (e.g.) For example, as illustrated below, it contains six mouse anti-canine PD-1 CDRs, and at least Another is that it typically contains substantially all of two variable domains, as well as a dog frame. It contains all or substantially all of the following.

[0099] The term "complete canine antibody" refers to an antibody that contains only the canine immunoglobulin protein sequence. It refers to the body. Complete canine antibodies are found in mice, in mouse cells, or derived from mouse cells. When produced in a hybridoma, it may contain mouse glycans. Similarly, "mouse The term "antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, it refers to a complete canine antibody. The body is found in rats, in rat cells, or in hybridomas derived from rat cells. When prepared in this manner, it may contain rat glycans. Similarly, "rat antibodies" are rat immune antibodies. This refers to an antibody that contains only globulin sequences.

[0100] Each light / heavy chain pair's variable region forms an antibody binding site. Therefore, generally, intact Antibodies have two binding sites. Except in the case of bifunctional or bispecific antibodies, 2 The two bonding sites are generally the same.

[0101] Typically, both the heavy and light chain variable domains are relatively conserved frameworks. Three hypervariable regions, also known as complementary determination regions (CDRs), are located within the FR region. It contains. CDRs are usually adjacent to framework regions and to specific epitopes. Enables bonding. Generally, the variable domains of the light and heavy chains from the N-terminus to the C-terminus. Both versions use FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. It contains. The assignment of amino acids to each domain of human antibodies is generally done using Sequences. of Proteins of Immunological Interest,K abat,et al.;National Institutes of Healt h, Bethesda, Md.; 5 th ed.;NIH Publ. No.91-32 42(1991);Kabat,Adv.Prot.Chem.32:1-75(197 8);Kabat,et al.,J.Biol.Chem.252:6609-661 6(1977);Chothia,et al.,J.Mol.Biol.196:90 1-917 (1987) or Chothia, et al., Nature 342: Follow the definition in 878-883 (1989).

[0102] As used herein, the term “hypervariable region” refers to the part of an antibody involved in antigen binding. It refers to amino acid residues. The hypervariable region is the "complementarity-determining region" or "CDR" (i.e., light CDRL1, CDRL2, and CDRL3 within the chain variable domain, as well as the heavy chain variable domain It contains amino acid residues derived from CDRH1, CDRH2, and CDRH3 within the sequence. Kabat et al. define the CDR region of human antibodies using Sequence s of Proteins of Immunological Interest, 5th Ed.Public Health Service,National In See Institutes of Health, Bethesda, Md. (1991); Furthermore, Chothia and Lesk define the CDR region of antibodies based on their structure. See also J.Mol.Biol.196:901-917(1987). When used, the term "framework" or "FR" residues refers to CDR residues and This refers to variable domain residues other than the hypervariable region residues as defined herein.

[0103] As used herein, the term "canine flame" refers to the CDR residue and This refers to the amino acid sequences of the heavy and light chains of canine antibodies, excluding the hypervariable region residues defined as such. In both strands, the amino acid sequence of the natural canine CDR corresponds to the corresponding heterologous CDR (e.g., mouse). It is replaced with antibody-derived material. The heavy and / or light chains of canine antibodies are, for example, As illustrated below, in order to preserve the conformation of heterologous CDRs within canine antibodies and / or Alternatively, it may contain several heterogeneous non-CDR residues in order to modify the Fc function.

[0104] As used herein, "anti-canine PD-1 antibody" means an antibody against canine PD-1 (Mau Antibodies induced in mammals such as dogs or rabbits, and specific to canine PD-1. This refers to an antibody that specifically binds to canine PD-1, or an antibody that "specifically binds to canine PD-1," or "Sequence ID." Antibodies that specifically bind to polypeptides containing the amino acid sequence 114 are also effective against other antigens. Compared to other methods, it shows selective binding to canine PD-1, for example, "specifically" binding to canine PD-1. It is an antibody that binds. This binding does not require absolute binding specificity. Anti-canine PD-1 antibody The body determines the presence of canine PD-1 in the sample if its binding determines the presence of other canine PD-1 in the canine sample. Without excessively interfering with molecular activity, for example, in false positives in diagnostic situations or in treatment situations To alter the activity of canine PD-1 without causing undesirable consequences such as side effects. If it can do so, it is considered "specific" to canine PD-1. The degree of specificity may depend on the intended use of the antibody, and in any case, the intended purpose It is defined by its suitability for use.

[0105] Therefore, the present invention relates to a canine anti-canine PD-1 that binds to (e.g., specifically) canine PD-1. 1. An antibody or its antigen-binding fragment (including in isolated form) and such antibodies or provides the use of the fragment thereof. In a specific embodiment, it is bound to canine PD-1. To do so and of canine PD-1, at least one of its ligands, for example, canine PD-L1 Both have been shown to block binding to the mouse anti-canine PD-1 antibody, which is derived from a mouse anti-canine antibody. Us anti-canine PD-1 CDRs are provided. These CDRs are provided as illustrated herein. The modified canine frame of the present invention is inserted to produce canine-like mouse anti-canine PD-1 antibodies. It is possible.

[0106] More specifically, the "canine mouse anti-PD-1 antibody" of the present invention is a mouse anti-canine PD-1 antibody. Three heavy chain CDRs and three light chain CDRs derived from antibodies are used in canine flame or modified This refers to the antibody contained together with the canine flame. Modified canine flame is effective against canine antibodies. Further optimization, for example, by increasing, decreasing, or eliminating antibody effector function. To remove, increase its binding to canine antigens, such as canine PD-1, and / or canine The antigen, for example, canine PD-1, and its natural binding partner (for example, the antigen is canine PD-1). In some cases, this increases its ability to block binding to canine PD-L1, as illustrated herein. It contains one or more amino acid changes.

[0107] "Homologousity" refers to the relationship between two polynucleotide sequences when they are optimally aligned or between two polynucleotide sequences. This refers to sequence similarity between peptide sequences. The same base is present at a certain position in both comparison sequences. Or, if occupied by amino acid monomer subunits, for example, two DNA molecules If a certain position in each molecule is occupied by adenine, then these molecules They are homologous at the same position. The percentage of homology is the homology shared by the two sequences. It is calculated by dividing the number of positions by the total number of positions compared and multiplying by 100. For example, if there are two When the arrays are optimally aligned, 6 out of 10 positions in the two arrays coincide. If two sequences are homologous, then the two sequences are 60% homologous. In general, comparison is performed using the maximum homology pairs. This occurs when two arrays are aligned to give a cent.

[0108] "Isolated nucleic acid molecules" are isolated polynucleotides that are found in nature. Possessives that are not bound to all or part of a creotide, or are not bound in nature. Dinucleotides linked to genomes, mRNA, cDNA, or synthetic DNs A means RNA or RNA or any combination thereof. For the purposes of this disclosure, a specific nucleus It should be understood that nucleic acid molecules "containing" rheotide sequences do not contain intact chromosomes. Therefore, an isolated nucleic acid molecule "containing" the specified nucleic acid sequence contains, in addition to the specified sequence, Up to 10 or even 20 or more other proteins or parts thereof It may contain coding sequences for fragments or enumerated nucleic acid sequences It may contain an operablely linked regulatory sequence that controls the expression of the coding region, and / or Alternatively, it may contain a vector sequence.

[0109] The term "control sequence" refers to a sequence of codes that can be operably linked in a particular host organism. This refers to the DNA sequence necessary for expression. Suitable regulatory sequences for prokaryotes include, for example, p It contains a romorter, which may contain an operator sequence, and a ribosome binding site It is included. Eukaryotic cells use promoters, polyadenylation signals, and enhancers. It is common knowledge that it is used.

[0110] When nucleic acids are in a functional relationship with another nucleic acid sequence, they are "operably linked". For example, DNA related to a presequence or secretion leader may be used to secrete polypeptides. When expressed as a preprotein involved in the polypeptide chain, it acts on the DNA of the polypeptide. Possibly linked; promoter or enhancer, it is linked to the transcription of the code sequence. If it has an effect, it is operably linked to the coding sequence; or the ribosome binding site. If it is positioned to facilitate translation, it is operably linked into the code array. It is done. Generally, "operably linked" means that the linked DNA sequences are adjacent to each other. The fact that, in the case of secretion readers, that they are adjacent and within the reading phase. It means that the enhancers do not need to be adjacent. Linking is a convenient limiting element. This is achieved by ligation. If such a part does not exist, the conventional method Use the synthetic oligonucleotide adapter or linker as shown in the example.

[0111] As used herein, the terms “cell,” “cell line,” and “cell culture” are used with respect to Used interchangeably, all such designations include offspring. Thus, "traits" The terms "transformed organism" and "transformed cell" do not take into account the number of primary test cells and passages. This includes cultures derived therefrom. Also, due to intentional or accidental mutations, It is also understood that not all offspring necessarily have exactly the same DNA content. The converted cells possess the same function or biological activity as those screened. Mutant offspring are included. Any different designations will be clear from the context. .

[0112] As used herein, “germ cell line sequence” means an unreconstituted immunoglobulin. Refers to the sequence of an unreconstructed immunoglobulin sequence. Any suitable supply of unreconstructed immunoglobulin sequences. Sources can be used. Human germline sequences are used, for example, in arthritis and arthritis studies at the National Institutes of Health in the United States. National Institute of Musculoskeletal / Skin Diseases thritis and musculoskeletal and skin dis eases of the United States National Instruments JOINSOLVER (Registered Trademark) on the website of the Health Corporation (Reference) Can be obtained from germline databases. Mouse germline sequences can be found, for example, in Giud icelli et al. [Nucleic Acids Res 33:D256- It can be obtained as described in D261(2005).

[0113] Characteristics of canine antibodies In dogs, there are four IgG heavy chains, designated A, B, C, and D. The heavy chain consists of four components of canine IgG, called IgGA, IgGB, IgGC, and IgGD. This shows different subclasses. The DNA and amino acid sequences of these four heavy chains are Tang et al.[Vet.Immunol.Immunopathol.80:259- First identified by 270 (2001). The amino acids of these heavy chains DNA sequences are also available from the GenBank database. For example, IgGA The amino acid sequence of the chain has accession number AAL35301.1, and IgGB is access Accession number AAL35302.1, IgGC has accession number AAL353 It has 03.1, and IgGD has accession number (AAL35304.1). The canine antibody also contains two types of light chains, κ and λ. These light chains contain DNA and The amino acid sequences can be obtained from the GenBank database. For example, the amino acids of the κ light chain. The sequence has accession number ABY 57289.1, and the λ light chain has accession number It has ABY 55569.1. In this invention, four canine IgG Fc fragments The amino acid sequences for each were determined by Tang et al. (mentioned above) CH1 and based on the identified boundaries of the CH2 domain.

[0114] The development of therapeutic monoclonal antibodies involves a complex set of activities to generate the desired antibody. This is a complex process that requires adjustment of various factors, including antibody specificity, affinity, and functional activity. , expression levels in manipulated cell lines, long-term stability, removal of effector function, and This includes enhancement and optimization of the development of commercially viable manufacturing and refining methods. Apart from the purpose of the drug and its ability to activate immune system cells, canine PD-1 Canine-like or canine monoclonal antibodies optimally possess three additional attributes: 1. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC), etc. Lack of Effect function, 2. Relatively long half-life in vivo; and 3. Large-scale development using industry-standard techniques such as those based on protein A chromatography. It can be easily purified.

[0115] None of the naturally occurring canine IgG subclasses meet all of these criteria. For example, IgGB can be purified using protein A, but high levels of ADCC It has activity. IgGC also has considerable ADCC activity. On the other hand, IgGA is protein It weakly binds to γ-A but exhibits undesirable ADCC activity. Furthermore, IgGD is ADCC Although it does not show activity, either IgGC or IgGD is purified using the Protein A column. It cannot do so. In addition, IgGC does not bind to the canine FcRn receptor, so it does not bind to the serum half It has a short decay period. The present invention relates to canine IgG modified specifically to canine antigens, such as canine PD-1. This difficulty can be overcome by providing antibodies; such antibodies are for ADCC and CD. Lacking effector functions such as C, exhibiting a relatively long half-life, and being an industry standard protein A It can be easily purified using chromatography.

[0116] Until now, it did not have both ADCC and CDC effector functions, and in addition, Genetically modified canine IgG can be purified by rotine A chromatography. G was not described. As disclosed herein, human and mouse IgG A single substitution at a similar position within canine IgG, such as N297A or D265A, is equivalent to In the corresponding canine antibody, both ADCC and CDC effector functions are completely eliminated. That is not the case. For example, each of the substitutions N297 and D265 in human or mouse antibodies These are Fc γ This results in the elimination of binding to the receptor and C1q, but neither substitution alone is effective. We did not completely rule out the binding of the canine antibody to C1q. Instead, further disclosures are provided below. Similarly, ADCC and CD in canine antibodies of the IgGB or IgGC subclass. To remove both C, substitution of asparagine with alanine and aspartic acid It is necessary to perform a double substitution in the Fc of canine antibodies, combining both substitutions from to alanine. It was discovered that there is an effector device in human antibodies. One substitution that had been shown to reduce activity actually resulted in the corresponding canine IgG Fc γ This resulted in increased binding to R and C1q.

[0117] To create canine IgGB and IgGC mutants lacking effector function, modification These can produce modified canine IgGB or modified canine IgGC heavy chains. The total of 7 amino acid residues present in both canine crystalline fragment regions (cFc) These seven amino acid residues were identified for such possible substitutions. Regarding GB's Fc, see the amino acid sequence of SEQ ID NO: 130 and the Fc of canine IgGC. Regarding both amino acid sequences of sequence number 132: P4, D31, N63, G64, T65, A93 and P95. Therefore, the amino acid sequence of SEQ ID NO: 2 is 4, By having the aforementioned amino acid residues at positions 31, 63, 64, 65, 93, and 95, Unlike sequence number 130, they have "X" in all seven positions (and In the amino acid sequence of sequence number 130, each of the three-letter codes "Xaa" is represented as such. Leproline (P), aspartic acid (D), asparagine (N), glycine (G), tre These are onine (T), alanine (A), and proline (P), and these seven amino acid positions This indicates that it could be any of the 20 natural amino acids (see column 1 in Table 1 below). (See reference). Similarly, the amino acid sequence of sequence number 4 has "X" at all seven positions. 4, 31, 63, 64, 65, 9 (or listed as "Xaa" in 3-letter codes) Having the aforementioned amino acid residues at positions 3 and 95, it is the same as SEQ ID NO: 132 Unlike other amino acid positions, these seven amino acid positions can be any of the 20 natural amino acids. As shown, the amino acid sequence of sequence number 2 is encoded by the nucleotide sequence of sequence number 1. Therefore, the amino acid sequence of SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 3. ru.

[0118] In one embodiment, cFc is substituted with: P4(A, G or S), D31(A, G or S), N63 (A, G or S), G64 (A or P), T65 (A, G or Sequence ID: 13, which has S, A93 (G or S) and P95 (A, G or S). It contains an amino acid sequence of 0; where P4 (A, G, or S) is the proline residue at position 4. This indicates that it is replaced by one of the following residues: lanin, glycine, or serine. Similarly, G64 (P or A) is a proline or alanine residue where the glycine residue at position 64 is replaced by a proline or alanine residue. (This indicates that it is replaced by either of the above.) In certain embodiments, c Fc is a substitution for the following: P4A, D31A, N63A, G64P, T65A, A93G and It contains the amino acid sequence of Sequence ID No. 130, which has P95A.

[0119] In related embodiments, cFc is the following amino acid residues: A4, A31, A63, G64, A sequence containing seven amino acids, referred to as Xaa, having T65, G93, and A95. The amino acid sequence of number 4, namely the following five amino acid residue changes: P4A, D31A, It has N63A, A93G, and P95A, and the remaining two of the seven amino acid residues The base, G64 and T65 are retained from the amino acid sequence of Sequence ID No. 132, sequence number Contains amino acid sequence number 132.

[0120] Sequence IDs: 40, 42, 44, 46, 48 , SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 , the amino acid combination of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 and SEQ ID NO: 66 All the rows contain "X" (or "Xaa" in the 3-letter code) at the 7 amino acid positions. These seven amino acid positions are listed in column 1 of Table 1 below, representing 20 natural amino acids. This indicates that it may be any of the acids. In particular, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: Sequence numbers 64 and 66 contain the amino acid sequence of sequence number 2 within their respective sequences. Or it contains one of the amino acid sequences of SEQ ID NO: 4. These seven positions of the amino acid sequence Specific examples of amino acid residues in one or more positions are described above and below. Therefore, sequence numbers 2, 4, 40, and 42 are distributed Column number: 44, Sequence ID: 46, Sequence ID: 48, Sequence ID: 50, Sequence ID: 52, Distribution Column number: 54, Sequence ID: 56, Sequence ID: 58, Sequence ID: 60, Sequence ID: 62, Distribution Column number: 64 and Sequence ID: 66, and the individual cells in the canine antibody containing these sequences It is included in the genera of each amino acid sequence.

[0121] Table 10 below shows that if cIgGB Fc (SEQ ID NO: 130 and SEQ ID NO: 2) Furthermore, cIgGC Fc (SEQ ID NO: 132 and SEQ ID NO: 4) as disclosed herein These cFc amino acid sequences have seven amino acid positions that can be substituted in this way. Sequence IDs: 40, 42, 44, 46, 4 8, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 5 8. Contains SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, and SEQ ID NO: 66 It is specifically associated with the full-length canine heavy chain. Therefore, the full-length IgGB sequence also The actual position in IgGC is determined by using Table 10 below, and the cFc it contains. It can be easily adapted to that.

[0122] In certain embodiments, the antibody heavy chain (i) has P, A, G or S at position 239, (ii) (iii) D, A, G or S at rank 266, (iv) N, A, G or S at rank 298, 2 99th place: G, P or A, (v)300th place: T, A, G or S, (vi)328th place: A , G or S, and (vii) Sequence ID: 40 containing P, A, G or S at position 330, It contains an amino acid sequence of 52, 56, or 64. In other embodiments, the heavy chain of the antibody is ( i) P, A, G or S in 237th place, (ii) D, A, G or S in 264th place, (iii) )296th place: N, A, G or S, (iv)297th place: G, P or A, (v)298th place (vi) 326th to A, G, or S, and (vii) 328th Contains amino acid sequences of sequence numbers 42, 54, 58, or 66, which include P, A, G, or S. It has. In yet another embodiment, the antibody heavy chain has (i) P, A, G or S at position 244. (ii) D, A, G or S in 271st place, (iii) N, A, G or S in 303rd place, (iv) G, P or A in 304th place, (v) T, A, G or S in 305th place, (vi) 3 (vii) Sequence numbers containing A, G, or S at position 33, and P, A, G, or S at position 335. No.: Contains amino acid sequence 44, 50, or 60. In further embodiments, the antibody The heavy chain has (i) P, A, G, or S at position 242, and (ii) D, A, G, or S at position 269. (iii) N, A, G or S in the 301st position, (iv) G, P or A in the 302nd position, (v )303rd place: T, A, G or S, (vi)331st place: A, G or S, and (vii )Contains the amino acid sequence of SEQ ID NO: 46 or 62, which includes P, A, G, or S at position 333. In further embodiments, the antibody heavy chain (i) has P, A, G or S at position 246, (ii) D, A, G or S in 273rd place, (iii) N, A, G or S in 305th place, ( iv) G, P or A at 306th place, (v) T, A, G or S at 307th place, (vi) 33 Sequence IDs containing A, G, or S at position 5, and (vii) P, A, G, or S at position 337. It contains a sequence of 48 amino acids.

[0123] This invention also replaces the natural IgGD hinge region with IgGA, IgGB, or IgG. We also provide modified canine IgGD containing hinge regions derived from either C or The IgGD hinge region is modified by replacing serine residues with proline residues, as shown in Table 5. It can be genetically modified by such modifications. Such modifications lack fab arm replacement. It can produce nuIgGD. Modified canine IgGD is a target of recombinant DNA technology. It can be constructed using a semi-standard method [e.g., Maniatis et al., M Olecular Cloning,A Laboratory Manual(198 2) To construct these mutants, the nucleus encoding the amino acid sequence of canine IgGD was used. The acid can be modified to encode modified IgGD. Next, the modified nucleus The acid sequence is cloned into an expression plasmid for protein expression. The alternative hinge region The nucleic acids that encode canine IgGD Fc are amino acids of sequence numbers 8, 10, and 12. The nucleotide sequences of sequence numbers 7, 9, and 11, which encode the acid sequence, are exemplified by these sequences. The nucleic acid encoding canine IgGD Fc having a modified IgGD hinge region is sequence It contains the nucleotide sequence of sequence number 5, which encodes the amino acid sequence of number 6.

[0124] The present invention further provides a full-length light chain that can be matched with the corresponding light chain to produce canine antibodies. The present invention provides canine heavy chains. Therefore, the present invention further provides canine-like mouse anti-canine antigen antibodies (isolated). (Contains canine-like mouse anti-canine PD-1 antibody) and treatment of diseases, such as canine cancer. The present invention provides a method for using the antibody or its antigen-binding fragment in a setting.

[0125] Furthermore, the present invention relates to binding to canine PD-1 and to canine PD-L1. Provides canine-like mouse anti-canine PD-1 antibodies or antigen-binding fragments that block binding. In one embodiment, the canine mouse anti-canine PD-1 antibody is modified canine IgGB Fc, modified canine IgGC Fc or lacking the fab arm replacement described herein Contains modified canine IgGD.

[0126] Canine antibodies, for example, antibodies that bind to canine PD-1 or their antigen-binding fragments, The complementarity-determining regions (CDRs) 1, 2, 3, 4, and 5 of the mouse anti-canine antibodies described herein It may contain 6. 1, 2, 3, 4, 5, or 6 CDRs are provided below. It can be selected independently from the CDR sequence provided. In a further embodiment, canine PD-1 The antibody or antigen-binding fragment that binds is mouse light chain CDR-1, CDR-2. Canine antibody κ light chain containing CDR-3 and / or mouse heavy chain CDR-1, CDR- The present invention contains canine antibody heavy chain IgG containing 2 and / or CDR-3. Furthermore, it provides a full-length canine heavy chain, which is then matched with, for example, a corresponding light chain to create a canine chain. Antibodies can be produced [e.g., a set of seven sequences of mouse anti-canine PD-1 CDRs]. For example, 1B5, 2G9, 2H9, 3B6, 4D12, 5G5 and 7C9 are provided. [See Table 2 below].

[0127] In other embodiments, the present invention still exhibits desired coupling and functional characteristics, while PD It specifically binds to -1, as well as to sequence numbers: 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25 and / or 26 amino acid sequences and at least 1 to 6 sequences containing 80%, 85%, 90%, 95%, 98%, or 99% sequence identity Canine antibody κ light chains containing different CDRs and SEQ ID NOs: 27, 28, 29, 30, 3 Amino acid sequences 1, 32, 33, 34, 35, 36, 37, 38 and / or 146 1 containing at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity A canine antibody heavy chain IgG containing six different CDRs, the antibody or its antigenic chain Provides a compound fragment. In another embodiment, the antibody or antigen-binding fragment of the present invention While the to still exhibits the desired coupling and functional characteristics, 0, 1, 2, 3, 4 or 5 One of the aforementioned CDR amino acid sequences having a conserved or non-conserved amino acid substitution or It contains a canine flame consisting of a combination of a κ light chain and an IgG heavy chain sequence having more than [number] of [values].

[0128] Sequence identity means that when the two sequences are optimally aligned, the amino acids of the two polypeptides are identical. It refers to the degree to which they are the same at a particular position. As used herein, it refers to a certain amino acid sequence For the second amino acid sequence, if the amino acid residues of both sequences are identical, then 100 They are "identical". Therefore, 50% of the amino acid residues in the two amino acid sequences are identical. In this case, a certain amino acid sequence is 50% "identical" to the second amino acid sequence. Sequence comparison is , contained in a given protein, for example, a portion of the protein or polypeptide being compared This is carried out over a contiguous block of amino acid residues. In certain embodiments, If so, consider selected deletions or insertions that can alter the correspondence between two amino acid sequences. Put it in.

[0129] Sequence similarity includes identical residues and non-identical, biochemically related amino acids. Biochemically related amino acids that share similar properties and may be interchangeable are considered.

[0130] A "conservatively modified variant" or "conservative substitution" is a change in the amino acid in a protein. Similar properties (e.g., charge, side chain size, hydrophobic / hydrophilic, skeletal conformation and stiffness) Substitutions by other amino acids, which often alter the biological activity of proteins. This refers to substitutions that can be performed without transformation. Those skilled in the art will generally know that a substitution can be performed within a non-essential region of a polypeptide. It is recognized that a single amino acid substitution does not substantially alter biological activity. [e.g., Wat son et al.,Molecular Biology of the Gene ,The Benjamin / Cummings Pub.Co.,p.224(4th See Ed.;1987). In addition, substitutions of structurally or functionally similar amino acids are produced. It is less likely to disrupt the physical activity. Exemplary conservative substitutions are shown in Table I below. [Table 1]

[0131] Function-conserving variants of the antibody of the present invention are also intended by this invention. "Function-conserving variant" When used herein, one or more amino acid residues constitute the desired properties, for example (such an) an anti-antibody that has been altered without altering its antigen affinity and / or specificity. Refers to a whole or fragment. Such variants are shown in Table I above, with their conserved amino acid sets. This includes substitutions, such as substitutions of an amino acid with an amino acid having similar properties, but Not limited to this.

[0132] nucleic acid The present invention further relates to the canine mouse anti-canine PD-1 antibody and its anti- The primordial fragment contains nucleic acids encoding the immunoglobulin chain (see "Examples" below). reference).

[0133] Furthermore, when the comparison is performed using the BLAST algorithm, the C provided herein DR and / or canine cFc and / or antibody amino acid sequences and at least about 70% Identical, preferably at least about 80% identical, more preferably at least about 90% identical, most Preferably, at least about 95% are identical (for example, 95%, 96%, 97%, 98%, 99%). The nucleus encoding an immunoglobulin polypeptide containing an amino acid sequence that is 100%) Acids are also included in the present invention, where the parameters of the algorithm are the respective reference arrays The selection is made to give the greatest match between each array along the entire length. Furthermore, when comparisons were performed using the BLAST algorithm, one of the reference amino acid sequences was found to be less than At least about 70% similar, preferably at least about 80% similar, more preferably at least Approximately 90% similar, most preferably at least about 95% similar (e.g., 95%, 96%, 97%) Immunoglobulin polypeptides containing amino acid sequences that account for 98%, 99%, and 100% of the total amino acid content. A nucleic acid is provided which is encoded by the algorithm, where the parameters of the algorithm are the respective reference values. Selected to give the greatest match between each array across the entire length of the column.

[0134] As used herein, the percentage of nucleotide and amino acid sequence identity is Default parameters for alignment and identity C, MacVector(MacVector, Inc. Cary, NC 27519) , Vector NTI (Informax, Inc. MD), Oxford Mole Clustal Group PLC (1996) and Clustal W algorithm It can be used to make a decision. These commercial programs also use the same or similar data. It can also be used to determine sequence similarity using fault parameters. Or, for example... For example, GCG (Genetics Computer Gr) using default parameters oup,Program Manual for the GCG Package,V (Version 7, Madison, Wisconsin) Use a pile-up program By using this, we can perform an Advanced Blast search under the default filter conditions. It is possible.

[0135] The following references relate to the BLAST algorithm, which is often used for sequence analysis. :BLAST ALGORITHMS:Altschul,SF,et al. ,J.Mol.Biol.215:403-410(1990);Gish,W.,et al.,Nature Genet.3:266-272(1993);Madden ,T.L.,et al.,Meth.Enzymol.266:131-141(19 96);Altschul,S.F.,et al.,Nucleic Acids R es.25:3389-3402(1997);Zhang,J.,et al.,Ge nome Res.7:649-656(1997);Wootton,J.C.,et al.,Comput.Chem.17:149-163(1993);Hancoc k,J.M.et al.,Comput.Appl.Biosci.10:67-70 (1994);ALIGNMENT SCORING SYSTEMS:Dayhoff ,M.O.,et al.,「A model of evolutionary ch ange in proteins.」in Atlas of Protein Se quence and Structure,vol.5,suppl.3.M.O.D ayhoff(ed.),pp.345-352,(1978);Natl.Biome d.Res.Found.,Washington,DC;Schwartz,R.M. ,et al.,「Matrices for detecting distant relationships.」in Atlas of Protein Seque nce and Structure,vol.5,suppl.3.」(1978), M.O.Dayhoff(ed.),pp.353-358(1978),Natl.B iomed.Res.Found.,Washington,DC;Altschul, SF,J.Mol.Biol.219:555-565(1991);States ,DJ,et al.,Methods 3:66-70(1991);Henik off,S.,et al.,Proc.Natl.Acad.Sci.USA 89: 10915-10919(1992);Altschul,SF,et al.,J .Mol.Evol.36:290-300(1993);ALIGNMENT STA TISTICS:Karlin,S.,et al.,Proc.Natl.Acad. Sci.USA 87:2264-2268(1990); Karlin, S.,et al.,Proc.Natl.Acad.Sci.USA 90:5873-5877( 1993);Dembo,A.,et al.,Ann.Prob.22:2022-2 039 (1994); and Altschul, SF "Evaluating the e statistical significance of multiple d istinct local alignments.” in Theoretical and Computational Methods in Genome Res earch(S.Suhai,ed.),pp.1-14,Plenum,New Yo rk (1997).

[0136] The present invention also provides an expression vector containing the nucleic acid of the present invention (including isolated nucleic acid). And here, the nucleic acid is obtained when the host cell is transfected with a vector. It is operablely linked to a control array that is recognized as such. Furthermore, it includes the expression vector of the present invention. Host cells possessing the antibodies or antigen-binding fragments disclosed herein are formed A method for producing an expression vector that encodes an antibody or antigen-binding fragment. The process involves culturing host cells in a culture medium and transferring the antigen or its antigen-binding fragment to the host cells. Methods are also provided that include isolation from cells or culture media.

[0137] For example, canine-like mouse anti-canine PD-1 antibody can be recombinant using methods known in this field. It can be produced by the expression of the antibody or fragment disclosed herein. Mammalian cell lines that can be used as hosts are well known in this field, and the cultured cell lineages in the United States are preserved. American Type Culture Collection (ATC) This includes many immortalized cell lines available from C). These include, in particular, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster Tur kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2) This includes A549 cells, 3T3 cells, HEK-293 cells, and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, and cattle. Horse and hamster cells are included. Particularly preferred cell lines are those that exhibit high expression levels. They are selected by measuring whether they have the necessary levels. Other cell lines that can be used are insect cells. Cells, such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Heavy chain or its antigen-binding portion or fragment, light chain and / or antigen-binding fragment When introducing a recombinant expression vector encoding a signal into mammalian host cells, the host cells... This enables the expression of antibodies, or more preferably, the secretion of antibodies into the culture medium in which host cells proliferate. Antibodies are produced by culturing host cells for a sufficient period of time.

[0138] Antibodies can be recovered from the culture medium using standard protein purification methods. The expression of the antibody of the present invention (or other components derived therefrom) from the producing cell line is widely publicized. It can be enhanced using intellectual technology. For example, the glutamine synthetase gene expression system ( The GS system is a common approach to enhance expression under specific conditions. European Patent Nos. 0216846, 0256055, and 0323997 And this is considered in whole or in part in relation to European Patent Application No. 89303964.4 ru.

[0139] Generally, glycoproteins are produced in specific cell lines or transgenic animals. This refers to the characteristic glycoproteins produced in that cell line or transgenic animal. It has a cosylation pattern. Therefore, a specific glycosylation pattern of an antibody makes the antibody It depends on the specific cell line or transgenic animal used to produce it. The nucleic acid molecules provided herein or the amino acids provided herein All antibodies containing an acid sequence, regardless of the glycosylation pattern the antibody may have, This constitutes the present invention. Similarly, in certain embodiments, it comprises only non-fucosylated N-glycans. Antibodies with glycosylation patterns are typically used in vitro and in Both have been shown to exhibit a stronger effect than their fucosylated counterparts. Therefore, this can be advantageous [for example, Shinkawa et al., J. Biol. C hem.278:3466-3473(2003); US Patent No. 6,946,292 See also issues 7,214,775.

[0140] The present invention further includes the antibody flag of the canine mouse anti-canine PD-1 antibody disclosed herein. It includes the ment. Antibody fragments include, for example, those formed by the enzymatic cleavage of IgG by pepsin. This includes F(ab)2 fragments that can be generated. Fab fragments are, for example, It is produced by the reduction of F(ab)2 with thiothreitol or mercaptoethylamine. Obtained. The Fab fragment is obtained by disulfide crosslinking. H -C H1 V attached to the chain L -C L It is a chain. The F(ab)2 fragment is now connected by two disulfide bridges. These are two Fab fragments that have been added. The Fab portion of the F(ab)2 molecule is F has a disulfide bridge in between. c Includes a portion of the area. F V The fragment is V L Also is V H It is a domain.

[0141] In one embodiment, the antibody or antigen-binding fragment is a heavy chain constant region, for example, a dog Steady-state regions, e.g., IgGA, IgGB, IgGC, and IgGD canine heavy chain steady-state regions or It contains the variant. In another embodiment, the antibody or antigen-binding fragment is light chain determined. Includes a constant region, such as the canine light chain constant region, such as the λ or κ canine light chain region or its variants. It has. Not limited to, but as an example, the canine heavy chain constant region can originate from IgGB, and The constant region of the light chain may originate from κ.

[0142] Antibody Engineering The canine mouse anti-canine PD-1 antibody of the present invention, as detailed below, has characteristics such as the antibody's features To improve sex, modifications are made within the canine frame of the parent (i.e., canine) monoclonal antibody. It can be manipulated to include it.

[0143] The cross-blocked canine antibodies and their antigen-binding fragments discussed herein are standard Binding assays (for example, BIACore®, ELISA, as exemplified below) , or flow cytometry) IB5, 3B6, 4D12, 7C9, 2H9, 5 Identifying them based on their ability to cross-compete with either G5 and / or 2G9. This can be done. For example, a standard ELISA assay can be used, and in this assay, recombinant Canine PD-1 protein is immobilized on a plate, and one of the antibodies is fluorescently labeled. Evaluate the ability of unlabeled antibodies to competitively remove the binding of BIACo. In addition or selectively, The ability of cross-competitive antibodies can be evaluated using re(registered trademark) analysis. For example. Canine PDs IB5, 3B6, 4D12, 7C9, 2H9, 5G5 and / or 2G9 The ability of the test antibody to inhibit binding to -1 is such that the test antibody binds to canine PD-1. Competing with B5, 3B6, 4D12, 7C9, 2H9, 5G5 and / or 2G9 This is possible, and therefore, in some cases, IB5, 3B6, 4D12, 7C9, 2H9, 5 It was revealed that G5 and / or 2G9 can bind to the same epitopes on canine PD-1. As mentioned above, either the anti-canine PD-1 antibody or fragment of the present invention and Antibodies and fragments that bind to the same epitope also form part of the present invention.

[0144] Pharmaceutical composition and administration Pharmaceutical or sterile combination of canine mouse anti-canine PD-1 antibody or its antigen-binding fragment To prepare the product, a canine mouse anti-canine PD-1 antibody or its antigen-binding fragment is used. It can be mixed with a pharmaceutically acceptable carrier or excipient. [For example, Remin gton's Pharmaceutical Sciences and USPha rmacopeia:National Formulary,Mack Publicis hing Company, Easton, PA (1984)].

[0145] Formulations of therapeutic and diagnostic agents include, for example, lyophilized powders, slurries, aqueous solutions, or suspensions. It can be prepared by mixing with a morphologically acceptable carrier, excipient or stabilizer [for example] Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeuti cs, McGraw-Hill, New York, NY; Gennaro (2000) Remington:The Science and Practice of Ph. armacy, Lippincott, Williams, and Wilkins, N. York, NY; Avis, et al. (eds.) (1993) Pharma ceutical Dosage Forms:Parental Medicat ions,Marcel Dekker,NY;Lieberman,et al.(e ds.)(1990)Pharmaceutical Dosage Forms:Ta blets,Marcel Dekker,NY;Lieberman,et al.( eds.)(1990)Pharmaceutical Dosage Forms:D isperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie(2000)Excipient Toxicity an d Safety, Marcel Dekker, Inc., New York, NY [See]. In one embodiment, the anti-PD-1 antibody of the present invention is dissolved in a sodium acetate solution at pH 5-6. Dilute to the appropriate concentration and add NaCl or sucrose for tonicity. To achieve this, additional active ingredients such as polysorbate 20 or polysorbate 80 are added. You may do so.

[0146] The toxicity and therapeutic effects of antibody compositions administered alone or in combination with other active substances are as follows: For example, LD 50 (A lethal dose in 50% of the population) and ED 50 (50% of the population) To determine a therapeutically effective dose in a standard cell culture or experimental animal, It can be determined by standard pharmaceutical procedures. The dose-to-toxicity ratio is the therapeutic index. LD 50 / ED 50 ) In certain cases, antibodies exhibiting a high therapeutic index are desirable. The data obtained from these cell culture assays and animal studies are for use in dogs. It can be used to set the dosage range for such compounds. The dosage of such compounds is preferably close to ED with little to no toxicity 50 It is within the range of circulating concentrations, including [the specified amount]. The dosage is [the specified amount]. This range may vary depending on the dosage form and route of administration.

[0147] There may be various methods of administration. Appropriate routes of administration include oral, rectal, transmucosal, intestinal, and parenteral administration. Intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, nasal, intraocular, inhalation These include ventilation, local, cutaneous, transdermal, or intra-arterial routes.

[0148] In certain embodiments, canine mouse anti-canine PD-1 antibody or its antigen-binding fragment The drug can be administered via an invasive route such as injection. Further embodiments of the present invention So, what about mouse anti-canine PD-1 antibody or its antigen-binding fragment or its pharmaceutical composition? The substance is delivered via intravenous, subcutaneous, intramuscular, intraarterial, intratumor routes, or by inhalation or aerosol delivery. It is administered via a non-invasive route (e.g., orally; e.g., in pills, capsules, or tablets). Administration by means of ) is also within the scope of the present invention.

[0149] The pharmaceutical compositions disclosed herein may also be administered by injection. Examples of well-known implants and modules for this purpose include: drug control U.S. Patent Publication No. 4,487 discloses an implantable microinfusion pump for administering at a certain rate. U.S. Patent No. 603, disclosing a drug infusion pump for delivering drugs at a precise infusion rate. Patent No. 4,447,233, Development of a variable flow rate portable infusion device for sustained drug delivery. U.S. Patent No. 4,447,224, shows an osmotic drug delivery system with multiple chamber compartments. This includes U.S. Patent No. 4,439,196 disclosing the system. Many other such patents The plant, delivery system, and modules are well known to those skilled in the art.

[0150] Alternatively, often in depot or sustained-release formulations, for example, in arthritis of the joints or immune system. By directly injecting antibodies into pathogen-induced lesions characterized by epidemiological pathology Furthermore, canine-like mice can be administered locally rather than systemically. For example, targeting pathogen-induced lesions characterized by arthritis or immunopathology. A targeted drug delivery system, for example, delivers antibodies in liposomes coated with tissue-specific antibodies. It can be administered. Liposomes target the affected tissue and are selectively taken up by the affected tissue. .

[0151] The administration regimen depends on the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, and the therapeutic antibody Several factors, including the body's immunogenicity and the accessibility of target cells within the biological matrix. It depends on the factors. Preferably, the administration regimen should simultaneously minimize undesirable side effects. To suppress the disease while delivering sufficient therapeutic antibodies to bring about improvement at the target disease site. Therefore, the amount of biological agents delivered is divided into specific therapeutic antibodies and treatments. It depends on the severity of the condition. Guidelines are available for selecting the appropriate dose of therapeutic antibodies. [For example, Wawrzynczak Antibody Therapy, Bios Scientific Pub.Ltd,Oxfordshire,UK(1996); Kresina (ed.) Monoclonal Antibodies, Cytoki nes and Arthritis, Marcel Dekker, New York ,NY(1991);Bach(ed.) Monoclonal Antibodies and Peptide Therapy in Autoimmune Disease Ses, Marcel Dekker, New York, NY (1993);Baer t,et al.New Engl.J.Med.348:601-608(2003) ;Milgrom et al.New Engl.J.Med.341:1966-1 973(1999);Slamon et al.New Engl.J.Med.34 4:783-792(2001); Beniaminovitz et al.New Engl.J.Med.342:613-619(2000);Ghosh et al .New Engl.J.Med.348:24-32(2003);Lipsky e t al.New Engl.J.Med.343:1594-1602(2000) ].

[0152] Determining the appropriate dosage is important, for example, if it is known in the art that it may affect the treatment or have an effect. Using parameters or factors that are presumed to have an effect, veterinarians This is done by n). Generally, the dose is started at a somewhat lower amount than the optimal dose, and The dosage should be gradually increased until the desired or optimal effect is achieved compared to the negative side effects. Important diagnostic measures include, for example, measures of inflammation symptoms or levels of inflammatory cytokines produced. It includes a bell.

[0153] The antibodies or antigen-binding fragments disclosed herein are administered by continuous infusion. For example, once a day, 1-7 times a week, once a week, once every two weeks, once a month, once every two months, or every three months. It may be provided in doses administered once a week, once every six months, once a year, etc. The dose is, for example, By intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intracerebral, intrathecal, or inhalation. It may be provided as follows. The total weekly dose is generally at least 0.05 μg / kg body weight, more than Generally, at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, and 10 μg / kg are recommended. g, 100μg / kg, 0.25mg / kg, 1.0mg / kg, 2.0mg / kg, 5 0 mg / ml, 10 mg / kg, 25 mg / kg, 50 mg / kg or more [For example, Yang, et al. New Engl. J. Med. 349:427-4] 34(2003);Herold,et al.New Engl.J.Med.346 :1692-1698(2002);Liu,et al.J.Neurol.Neur osurg.Psych.67:451-456(1999);Portielji,e t al.Cancer Immunol.Immunother.52:133-14 See 4 (2003). The dose also involves canine-mutated mouse anti-canine PD-1 antibody in the subject's serum. The predetermined target concentrations, for example, 0.1, 0.3, 1, 3, 10, 30, 100 , can be provided to achieve 300 μg / ml or higher. In other embodiments, The canine mouse anti-canine PD-1 antibody of the present invention is administered once a week, once every two weeks, "once every four weeks", and monthly. Once, every other month, or every three months, on a basis of 10, 20, 50, 80, 100, 20 Administered subcutaneously or intravenously at doses of 0, 500, 1000, or 2500 mg per subject. It will be done.

[0154] As used herein, “inhibit,” “treat,” or “treat” means “impede.” This includes delaying the onset of symptoms related to such disorders and / or reducing the severity of symptoms of such disorders. These terms include improving existing uncontrolled or undesirable symptoms. To prevent additional symptoms and to improve or prevent the underlying causes of such symptoms. It further encompasses the act of having a disorder, disease, or symptom. Therefore, these terms include having a disorder, disease, or symptom. Spinal movement that causes or has the potential to manifest such disorders, diseases or symptoms This means that beneficial results are being obtained for the test subject.

[0155] As used herein, "therapeutic effective dose," "therapeutic effective amount," and "effective dose" are used in this specification. The term refers to a drug administered to cells, tissues, or subjects, either alone or in combination with additional therapeutic agents. In some cases, one or more symptoms of a disease or condition, or such disease or condition The present invention provides a canine-like mouse anti-canine PD-1 that is effective in producing a measurable improvement in the progression of the disease. This refers to the amount of antibody or its antigen-binding fragment. The therapeutically effective dose is further defined as the amount of symptoms that are least severe. Both partial improvement, for example, treatment, cure, prevention or improvement of the related medical condition, A sufficient bond to increase the rate of treatment, healing, prevention, or improvement of such conditions This refers to the amount of compound. When applied to individual active ingredients administered alone, the therapeutically effective dose is This refers to the component alone. When applied to combinations, the therapeutically effective dose is the dose taken in combination, continuously. This refers to the total amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered simultaneously or not. The effective dose of the drug is at least 10% of the diagnostic scale or parameter, usually less than that. Also 20%, preferably at least about 30%, more preferably at least 40%, most preferably It results in an improvement of at least 50%. The effective dose is also used to assess the severity of the disease. When using subjective scales, improvements in those subjective scales can also be achieved.

[0156] Other combination therapies As mentioned earlier, canine-like mouse anti-canine PD-1 antibody or its antigen-binding fragment The antibody can be administered concurrently with one or more other therapeutic agents (e.g., chemotherapy agents). It may be linked to the therapeutic agent (as an immune complex) or administered separately from other therapeutic agents. This is possible. In the latter case (separate administration), the antibody can be administered before, after, or at the same time as the other therapeutic agent. It can be administered at times or concurrently with other known therapies.

[0157] kit Furthermore, the antibody or antigen-binding fragment discussed herein, which specifically binds to PD-1. Includes (e.g., canine mouse anti-canine PD-1 antibody or its antigen-binding fragment) One or more components, not limited to these, are discussed herein as pharmaceutically acceptable. One or more attachments including, but not limited to, carriers and / or chemotherapeutic agents. A kit containing the additive components is provided. The conjugate composition and / or chemotherapeutic agent or formulated into pharmaceutical compositions as a pure composition or in combination with a pharmaceutically acceptable carrier. It is possible.

[0158] In one embodiment, the kit contains the binding composition of the present invention (for example, in one container (for example) (In a sterile glass or plastic vial) canine-mutated mouse anti-canine PD-1 antibody or The pharmaceutical composition and in another container (e.g., in a sterile glass or plastic vial) It contains the pharmaceutical composition and / or chemotherapeutic agent.

[0159] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit will administer such a drug. The kit may also include an apparatus for carrying out the above. For example, the kit may include the one or It may contain additional subcutaneous needles or other injection devices. The kit also contains the pharmaceutical composition of the kit. Package inserts containing information about the substance and dosage form may also be included. Generally, such The information is provided to pet owners and veterinarians regarding the enclosed medication. This helps to use compositions and dosage forms effectively and safely. For example, regarding combinations of the present invention. The following information may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical trials, and efficacy parameters. Indications and dosage, contraindications, warnings, precautions for use, adverse reactions, overdose, appropriate dosage and administration Instructions for supply, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0160] For convenience, the antibodies or specific conjugates disclosed herein are in the kit, i.e., diagnostic Packaged combinations of predetermined amounts of reagents, along with instructions for performing a determination or detection assay. It may be provided. If the antibody is enzyme-labeled, the kit will provide the substrate and enzyme required. It contains cofactors (e.g., substrate precursors that provide a detectable chromophore or fluorescent phore). In addition, other additives, such as stabilizers and buffers (e.g., block buffers or lysis buffers) These may include, etc. The relative amounts of various reagents substantially optimize the sensitivity of the assay. The concentration of the reagent in the solution can vary widely. In particular, the reagent can vary appropriately after dissolution. A dry powder, usually freeze-dried, containing an excipient that provides a reagent solution of a certain concentration. It may be provided as such.

[0161] [Examples] [Example 1] Canine PD-1 and PD-L1 Canine PD-1 and PD-L1: The entirety of the U.S. Patent Application Filed December 20, 2013, is incorporated herein by reference. Provisional application No. 61 / 918,946 concerns Canine PD-1 (cPD-1) of Sequence ID No. 113. The full-length nucleotide sequence for [Sequence ID: 133 includes the signal sequence]; Sequence number Corresponding translated amino acid sequence for number 114 [SEQ ID NO: 134 includes signal sequence] ]; Nucleotide sequence encoding the extracellular domain (ECD) of canine PD-1, sequence number :115; Amino acid sequence of canine PD-1 ECD, SEQ ID NO:116; SEQ ID NO:117 The Fc portion of the canine PD-1 ECD-plus GT linker and the human IgG1 Fc gene. Nucleotide sequences of minutes; as well as canine PD-1 ECD plus GT linker and human I The amino acid sequence of the Fc portion of the gG1 Fc gene, SEQ ID NO: 118 [SEQ ID NO: 137 is Provides [signal sequences].

[0162] U.S. Patent Provisional Application No. 61 / 918,946 further relates to Canine PD-L, Sequence ID: 119. Full-length nucleotide sequence for 1(cPD-L1) [SEQ ID NO: 135 is the signaling sequence] [Including the column]; the corresponding translated amino acid sequence of SEQ ID NO: 120 [SEQ ID NO: 136 is [Contains a signal sequence]; Nucleus encoding the extracellular domain (ECD) of canine PD-L1 Otid sequence, SEQ ID NO: 121; amino acid sequence of canine PD-L1 ECD, SEQ ID NO: 1 22; F of the canine PD-L1 ECD plus GT linker and human IgG1 Fc gene Nucleotide sequence of the c portion, SEQ ID NO: 123; and Canine PD-L1 ECD Plus GT linker and amino acid sequence of the Fc portion of the human IgG1 Fc gene, Sequence ID: 1 Provide 24.

[0163] [Example 2] Mouse anti-canine PD-1 antibody Production of anti-canine PD-1 monoclonal antibodies: A total of three Balb / c mice were immunized multiple times over 17 days (with 10 μg each time). The antigen to be immunized was the canine PD-1 ECD-Fc fusion protein. After immunization, each Serum was collected from mice and tested for reactivity with canine PD-1 ECD-HIS tag protein. The following was tested: Splenocytes of mice with the highest serum anti-PD-1 ECD-HIS titer. These cells were fused to the myeloma P3X63Ag8.653 cell line. Approximately two weeks after fusion, the estimated hysterosalpodium was observed. The supernatant from ibridomatous cells was used to analyze the PD-1 ECD-HIS tagged protein. The reactivity of the sample was tested by ELISA. A strong positive signal was detected in the ELISA. The hybridoma that produces canine PD-1 ECD was subcloned by limiting dilution. -The reactivity to the HIS-tagged protein was tested again.

[0164] Confirmation of the reactivity of monoclonal antibodies against canine PD-1: The reactivity of antibodies secreted by hybridomas to canine PD-1 ECD is expressed as E Confirmed by LISA. Hybridoma cells were placed in a CELLine bioreactor. The cells were cultured for 10-30 days using Integra (biosciences). First, 4 mM L-Glutamine and 10% Ultra Low I from Gibco. Hybridoma cells were maintained in DMEM supplemented with gG fetal bovine serum (FBS). Approximately 2 × 10 in 15 mL of the same medium with the FBS concentration increased to 20% 6 At a cell density of cells / mL The cells were inoculated into the cell chamber of a CELLine bioreactor. Nutrient culture was performed in the outer chamber. Fill 1L of soil (DMEM with 4mM L-glutamine and 2% standard FBS added). Hybridoma cells in the cell chamber grow to approximately 2.5 × 10⁶ over 3 to 7 days. 7 cells / m The cells were grown in L. Next, 10 mL of the cell suspension was taken from the cell chamber and mixed with fresh medium. The cells were replaced, allowed to grow again, and then harvested. This procedure was repeated as needed, and each hive An appropriate amount of mAb was obtained from the ridoma clone. The collected cell suspension was centrifuged, and the supernatant was obtained. The solution was filtered through a 0.2 micron filter membrane. For antibody purification, the supernatant of each clone was treated with P Rotein G Sepharose 4 Fast flow 5mL column (GE Purified using gravity flow with Healthcare (T E) After washing with buffer pH 8.0, knotting was performed using 0.1M glycine buffer, pH 2.7. The combined antibody was eluted, and then the pH was neutralized using 1M Tris, pH 8.0. Concentrated and filtered using Centriprep YM-10, 10kDa NMWL centrifugation filter. Use a Millipore unit to convert the buffer to phosphate-buffered saline (PBS). The sample was replaced. The antibody concentration was quantified using spectrophotometric analysis.

[0165] Reactivity of purified anti-canine PD-1 mAb with the HIS-tagged ECD domain of canine PD-1 The following tests were conducted by ELISA regarding HIS tag dog PD-1 ECD tan Dilute the protein to 10 μg / mL in coating buffer (carbonate / bicarbonate, pH 9.0), 1 Dispense 00 μl / well into a 96-well flat-bottom ELISA plate (NUNC). Incubate the plate overnight at 4°C. Then, apply 0.05% Tween (registered trademark). Wash three times with phosphate-buffered saline (PBST) containing (standard) 20. Next, block Add 200 μl of buffer solution (5% skim milk in PBST) to each well, and then heat the plate at 37°C. Incubate for 60 minutes. Then wash the plate three times with PBST. Next, b Add 100 μl of the test mAb diluted in Kick buffer to the first well of the appropriate column. Then, dilute the test mAb 2:1 in the appropriate plate location. Allow the plate to cool at 37°C for 60 minutes. After incubation for several minutes, wash the plate three times with PBST. Then, rinse with a hose. 100ml of 1:2,000 dilution of superoxidase-conjugated goat anti-mouse IgG (KPL) Add μl / well to the plate, then incubate the plate at 37°C for 60 minutes. Next, wash the plate three times with PBST and 3,3',5,5'-tetramethylbenzidine (TMB) Add 100 μl / well of substrate (from KPL) to the plate. Incubate at 37°C for 5 minutes. After allowing the color reaction to occur for 20 minutes, the absorbance is measured at 650 nm.

[0166] CHO cells expressing canine PD-1 protein: The full-length canine PD-1 gene was cloned into plasmid p96793. In Mido, PD-1 protein expression is driven by the hCMV promoter. HO DXB11 cells (dhfr-) were treated with MEM-α(G) added to 10% fetal bovine serum. CHO cells were maintained in ibco. Transfection of CHO cells by plasmid p96793 The rhythm is approximately 6 x 10 6 75 cm including cells 2 In a flask, lipofectamine (Invit This was performed by liposome-mediated gene delivery using rogen. After 48 hours, 1 Contains nucleosides with 0% FBS and 400 μg / mL hygromycin B added. Cells were subcultured in MEM-α medium (selective medium) without hygromycin resistance. dhfr+ hygromycin-resistant cells were observed. Limiting dilution cloning was performed on the sex cell pool. Clones were then subjected to immunofluorescence assays. Canine PD-1 expression was evaluated using this method. Briefly, the cell monolayer was divided into 80% acetylated cells. The cells were then fixed in a 96-well plate. Next, the fixed and dried cell monolayer was placed in a polyclonal plate. Incubated for 1 hour with goat anti-human PD-1 antibody (R&D Systems) Wash the plate with PBS, then add fluorescein-labeled rabbit anti-goat IgG antibody (KPL). The cells were incubated with ) for 1 hour. The plates were washed with PBS. The fluorescent clones were then incubated with PBS. The cells were propagated and a cell line was established.

[0167] Reactivity of mouse mAbs to canine PD-1 protein expressed on CHO cells: The reactivity of canine PD-1 on CHO cells with mouse anti-canine PD-1 mAb was investigated, and PD-1 was used to assess the reactivity of canine PD-1 on mouse cells. It was measured by a cell-based assay using CHO-expressing cells. In short, CHO cells expressing canine PD-1 were cultured in a medium (DMEM / HAM F12, 10% FBS). The cells were cultured in 50 μl to a concentration of 80-100%. Next, purified mAbs of various concentrations were added. 50 μl of the culture medium was added and incubated at 37°C for 1 hour. After washing three times with PBS-Tween, , goat anti-mouse horse radish peroxidase (H) diluted 1:1000 in culture medium RP) 100 μl was added and incubated at 37°C for 1 hour. The mixture was then washed three more times with PBS-Tween. Next, the bound mAb was visualized using a peroxidase substrate (TMB). The absorbance due to peroxidase activity at 450 nm was determined. The increase was measured using a microplate reader.

[0168] Characterization of mouse anti-canine PD-1 antibodies: The above and the entirety thereof are incorporated herein by reference, December 20, 2013. As detailed in the U.S. Provisional Patent Application No. 61 / 918,946, mouse anti-dog The PD-1 antibody was characterized by many parameters, including the following: ELISA Reactivity of PD-1 with ECD, reactivity with PD-1 expressed on the surface of CHO cells, P The ability of D-1 to block the binding of PD-L1, as well as in healthy dogs and dogs with cancer. Ability to bind to PBMC cells derived from dogs. Seven selected mouse anti-canine PD-1 antibodies ( (These are represented as IB5, 2G9, 2H9, 3B6, 4D12, 5G5, and 7C9 respectively) CDR The amino acid sequences of the two molecules exhibited substantial homology, as shown in Table 2 below. [Table 2] TIFF0007888641000003.tif95156

[0169] [Example 3] Characterization of canine transformation and canine antibodies To produce canine antibodies, the DNA sequences encoding the heavy and light chains of canine IgG are used. It needed to be identified. The nucleotide and amino acid sequences of the canine heavy chain were found in the NCBI gene. and can be obtained from protein databases. Four known I of canine IgG IgG subclasses: IgGA, IgGB, IgGC, and IgGD, as well as two types of light chains: κ and λ are present. Table 7 shows the amino acids and nucleos of the unmodified canine Fc fragment. List the CHIDO sequence numbers.

[0170] While not bound by any particular approach, the characteristics of canine and mouse sequences The process for producing variants of anti-PD-1 monoclonal antibodies having various contents is as follows: Includes a general scheme: i) Determine the nucleotide sequences of the VH and VL chains of the mouse mAb; ii) Identify the H chain and L chain CDRs of mouse mAbs; iii) Identify appropriate H and L chains of canine IgG; iv) Determine the nucleotide sequences of the canine IgG H and L chains; v) The nucleotide sequences encoding the endogenous canine H chain and L chain CDRs, respectively Replace with the nucleotide sequence that codes for UsCDR. Also, some canine flamewar The 'k' residue may be replaced with a selected residue from the mouse framework region; vi) Synthesize nucleotides from step (v) and insert them into the appropriate expression plasmid. Transfect the plasmid into appropriate cells, such as HEK293 cells. vii) Purify the expressed antibody from the HEK293 supernatant; and viii) Test the purified antibody for binding to canine PD-1.

[0171] The experimental setup was carried out according to the above steps, thereby yielding various canine and mouse sequences. A set of mutant canine antibodies containing a specific container was obtained.

[0172] Reactivity of canine mAbs to canine PD-1 protein expressed on CHO cells: The reactivity of canine-modified anti-canine PD-1 mAb with canine PD-1 on CHO cells was investigated. This was measured by a cell-based assay using CHO cells expressing -1. Briefly described below. Then, CHO cells expressing canine PD-1 were cultured in a medium (DMEM / HAM F12, 10%). The cells were cultured in 50 μl of FBS at a concentration of 80-100%. Next, purified mA cells of various concentrations were added. 50 μl of medium containing b was added and incubated at 37°C for 1 hour. Washed three times with PBS-Tween. Afterwards, goat anti-canine horseradish peroxidase diluted 1:1000 in culture medium ( 100 μl of HRP-labeled antibody was added at 37°C for 1 hour. Further 3 hours were observed using PBS-Tween. After washing, the bound mAbs were visualized with peroxidase substrate (TMB) at 450 nm. The increase in absorbance due to peroxidase activity was measured using a microplate reader.

[0173] Canine PD-1 and mouse anti-canine PD-1 mAb and canine-modified mouse anti-canine PD-1 m Ab binding test: Approximately 70 resonance units (RUs) of the canine PD-1 antigen were directly immobilized by amine coupling. Affinity measurements were performed with a binding time of 300 seconds, a dissociation time of 1200 seconds, and 50, 100, and 2 Label-free surface plasmon resonance at concentrations of 00 (×2), 400, and 800 nanomoles (nM). This was implemented using the underlying technology (e.g., Biacore® T200). 1:1 A compatible model of binding was used. The antigen (canine PD-1) was converted via amine coupling. The four antibodies shown in Table 14 below were immobilized on a sensor tip, and the analytes flowing over the antigen surface were used. It was used as such. The results showed that the anti-canine PD-1 antibody of the present invention against the canine PD-1 antigen... It exhibits strong affinity and possesses dissociation constants (Kd) at the nanomolar and even sub-nanomolar levels. Furthermore, it was revealed that mouse anti-canine PD-1 monoclonal antibody and the same The corresponding canine-like mouse anti-canine PD-1 monoclonal antibody derived from roan is extremely similar. A Kd value was generated (see Table 14 below). [Table 3]

[0174] Ligand blockade by canine-modified anti-canine PD-1 mAb Regarding canine antibodies that react with canine PD-1 (cPD-1), expressing canine PD-1 A cell-based ELISA (CELISA) assay based on CHO cell lines was used. Simply put, cPD-1 CHO cells are 4 × 10 4 Cells per well in a 96-well plate Place the cells in a container and incubate them at 37°C for 18-24 hours until the cells are 95-100% dense. The cell culture medium was removed by aspirating, and the plate was treated with PBS + 0.05% Tween (registered). Washed three times with (trademark) 20 and once with CHO medium. Starting from 30 μg / mL, CH Three-fold serial dilutions of canine anti-cPD-1 mAb were prepared in medium O, and 50 μg of each antibody dilution was prepared. L / well was added to the plate. Next, the plate was shaken for 30 minutes at 37°C with 5% CO2. The mixture was incubated under these conditions. Human PD-L1-Fc was added to CHO medium at a concentration of 4 μg / ml. Next, without removing or washing the incubated anti-PD-1 mAb, add 50 μL / The wells were incubated at 37°C with 5% CO2 for 45 minutes while shaking. The samples were washed six times with PBS + 0.05% Tween® 20. Anti-Hypertoxicating agents in CHO medium. Add 100 μl / well of Fc-HRP (Calbiochem) (1:2500). Incubated at 37°C / 5% CO2 for 30-60 minutes (anti-human Fc-HRP was used in dogs). (Does not bind to Fc). Plate 5 times with PBS + 0.05% Tween(registered trademark) 20. Washed. 100 μl / well of TMB microwell substrate was added, then left at room temperature for 10 minutes. Incubated. The reaction was stopped with 100 μl / well of 1.5 M phosphate. ELIS Measure A450~A620 with the A-leader.

[0175] Cytokine release from canine PBMCs: PBMCs were obtained from healthy dogs and dogs with cancer using Ficoll isolation. Prepared from TA blood samples. Cells were washed three times and placed in a triple well in a 96-well plate. In the case of 2.5 × 10 5 The cells were resuspended in complete tissue culture medium at a concentration of cells / well. The test antibody was activated with concanavalin A at 1 μg / ml. Test antibodies were added at various concentrations, and the culture was... The samples were incubated for 96 hours. Controls were administered with or without the antibody, or with conA. This included cells incubated with antibodies that matched unrelated isotypes. After 96 hours of incubation, the supernatant was collected and treated with commercially available canine IFN- γ ELISA kit (R&D Using IFN- γ The release was tested [see Table 4].

[0176] [Example 4] Genetically modified canine IgG To create a variant of canine IgG lacking effector function, a large number of mutant dogs were used. IgGB heavy chains were constructed. These mutants have the following single amino acid in the Fc portion of the heavy chain amino acid sequence. Or compound substitution: P4A, D31A, N63A, G64P, T65A, A93G and P It may contain one of the 95A variants (i.e., containing such amino acid substitutions). The gene was cloned into an expression plasmid and combined with a plasmid containing the gene encoding the light chain. HEK293 cells were transfected with it. The product was expressed from HEK293 cells and purified. To investigate their potential in mediating immune effector function, intact antibodies were used in Fc γBinding to RI and C1q was evaluated. Table 3 shows genetically modified canine heavy chains. List examples of plasmids encoding canine heavy chains and genetic modifications within these heavy chains. The mutant heavy chain was used to evaluate the effector function in genetically modified mAbs. The heavy chain contained CDRs derived entirely from 2H9 mouse anti-canine PD-1 antibody. [Table 4]

[0177] Fc γ RI join: Fc γ Binding to radioisotopes (RI) is a measure of the ability of an antibody to mediate ADCC. To evaluate this characteristic, the Fc of the canine antibody was used. γ An assay to measure binding to radioisotopes The procedure was performed as follows: A 96-well plate was treated with PD-1 HIS 2.5 μg / mL. Cover with 00 μl / well. Incubate overnight at 2-7°C. Turn the plate over for 15 minutes. Allow to equilibrate at room temperature. Contains 0.05% Tween(registered trademark) 20 (PBST). Wash the plate three times with nitrate-buffered saline, then add 200 μL of 5% NFDM (skim milk powder). Block the wells using / well. Incubate at 36-38°C for 60 minutes. Wash three times with PBST. Start with 1 μg / mL of antibody in 5% NFDM and prepare a 2-fold dilution of antibody. Prepare the solution. Add 100 μL / well of diluted antibody. Incubate at 36-38°C for 60 minutes. Vate. Wash three times with PBST. Recombinant human CD64 fluoride diluted to 1 μg / mL. Add 100 μL / well of protein (R&D systems). Allow to stand at 36-38°C for 6 minutes. Incubate for 0 minutes. Wash 6 times with PBST. Dilute biotin 1:3000. Add 100 μL / well of anti-CD64 antibody (R&D systems). 36~3 Incubate at 8°C for 60 minutes. Wash 6 times with PBST. Dilute to 1:7500. Add 100 μL / well of streptavidin-HRP antibody (R&D systems). Incubate at 36-38°C for 60 minutes. Wash 6 times with PBST. TMB group Add 100 μL / well of the solution. Incubate at 15-30°C for 10 minutes. ELI The plate is read at 450-540 nm using an SA plate reader.

[0178] Results: Figure 5A shows can2H9 ADCC mut-1 with the D31A gene modification. can2H with canine mAb or N63A gene modification, referred to as VH4 / VL3. 9. mAb referred to as ADCC mut-2 VH4 / VL3, Fc γ In addition to binding to RI This demonstrates a near-complete reduction. On the other hand, the combined gene modification of D31A plus N63A is shown. MAB containing can2H9 ADCC(1062)VH4 / VL3 is Fc γ It lacks detectable binding to the RI. In Figure 5A, can2H9 IgGD VH4 / VL3 is a canine antibody containing Fc derived from canine IgGD, and can3B6 V H4 / VL4 IgGB is a canine antibody that does not bind to the coated antigen (PD-1 HIS). , and canine mAb referred to as can2H9 VH4 / VL3 are unmodified IgGB Fc This is the antibody it contains. Figure 5B shows can2H9 ADCC containing the P4A gene modification. Contains a canine mAb and P95A gene modification referred to as (1059)VH4 / VL3. The mAb referred to as can2H9 ADCC(1061)VH4 / VL3 is Fc γ To RI It shows a significant reduction in binding, while can2H9 ADC containing the A93G gene modification A mAb called C(1060)VH4 / VL3 has Fc γ Shows a slight reduction in binding to radioisotopes. This presents the idea of ​​doing so. On the other hand, five gene modifications (D31A, N63A, P4A, A93G can2H9 IgGB ADCC(1068)VH4 / VL3 containing P95A mAb, which is called Fc γ It completely lacks binding to RI.

[0179] C1q binding: Binding to C1q, the first component of complement, is a measure of the ability of an antibody to mediate CDC. (Dog) To evaluate this property with respect to canine antibodies, we measure the binding of canine antibodies to C1q. The procedure was performed as follows: 96-well plates were treated with PD-1 HIS 2.5 μg / m². Cover with L. Incubate overnight at 2-7°C. Equilibrate to room temperature for 15 minutes. PB Wash three times with ST. Block with 200 μL / well using 5% BSA. 36~3 Incubate at 8°C for 60 minutes. Wash three times with PBST. 1 μg / m² in 5% BSA. Start with L and prepare a 2-fold dilution of the antibody. Add 100 μL / well of the diluted antibody. Incubate at 36-38°C for 60 minutes. Wash 6 times with PBST. 4 μg / mL Add 100 μL / well of diluted C1q protein. Incubate at 36-38°C for 60 minutes. Incubate. Wash 6 times with PBST. Dilute goat anti-C1q antibody to 1:3000. Add 100 μL / well. Incubate at 36-38°C for 60 minutes. PBST Wash 6 times. Add 100 μL / well of donkey anti-goat HRP antibody diluted 1:10000. Add. Incubate at 36-38°C for 60 minutes. Wash 6 times with PBST. ™ Add 100 μL / well of substrate B. Incubate at 15-30°C for 10 minutes. 4 Read the data using an ELISA plate reader at 50-540 nm.

[0180] Results: Figure 6A shows can2H9VH4 IgGB AD with the D31A gene modification. It has a canine mAb or N63A gene modification called CC(mut-1) / VL3. The mAb called can2H9 VH4 IgGB ADCC(mut-2) / VL3 is This demonstrates a significant reduction in binding to C1q. On the other hand, D31A plus N63A can2H9 VH4 IgGB ADCC(1062) / contains a complex gene modification. The mAb referred to as VL3 lacks detectable binding to C1q.

[0181] In Figure 6A, can2H9 VH4 IgGD / VL3 is derived from canine IgGD-derived Fc. It contains canine antibodies, and can3B6 VH4 / VL4 IgGB is a coated antigen. It is a canine antibody that does not bind to (PD-1 HIS), and can2H9 VH4 / V Canine mAbs, referred to as L3 IgGB, are antibodies containing non-mutant IgGB Fc. (Figure) 6B contains the P4A substitution can2H9 VH4 IgGB ADCC(1059) can2H9 VH4 contains a canine mAb and P95A substitution referred to as ) / VL3. A significant portion of the mAb called IgGB ADCC(1061) / VL3 binds to C1q. It shows a reduction, while can2H9 VH4 IgGB ADCC contains the A93G substitution. We present evidence that the mAb designated (1060) / VL3 enhances binding to C1q. On the other hand, the ca containing five substitutions (D31A, N63A, P4A, A93G, P95A) The mAb referred to as n2H9 VH4 IgGB ADCC(1068) / VL3 is C1q The coupling to is completely absent. In Figure 6B, can3B6 VH4 / VL4 Ig mAbs referred to as GB are canine antibodies that do not bind to coated antigens (PD-1 HIS). The canine mAb referred to as can2H9 VH4 / VL3 IgGB is a non-mutant IgGB F This is an antibody containing c. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] TIFF0007888641000014.tif221145

[0182] [Example 5] Epitope mapping of anti-canine PD-1 antibodies Introduction The interaction between antibodies and their alloprotein antigens involves the specific amino acids (epito) of the target antigen. Epitope is mediated through the binding of an epitope to a specific amino acid (paratope) of the antibody. An antigenic determinant is an antigen that elicits a specific reaction by immunoglobulins. An epitope is an antigen. It consists of a group of amino acids on its surface. The protein of interest is recognized by different antibodies. It may contain several epitopes.

[0183] Epitopes recognized by antibodies are classified as linear or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein, while Conformational epitopes consist of discontinuous (e.g., far apart) amino acids in the primary amino acid sequence. However, they come together after three-dimensional protein folding.

[0184] Epitope mapping is the process of identifying the amino acid sequence recognized by an antibody on its target antigen. This refers to the process of identifying the epitope. The identification of epitopes recognized by ) has important applications. For example, new therapeutic drugs, It can be useful in the development of diagnostic agents and vaccines. Epitope mapping is also optimized This can be helpful in selecting therapeutic mAbs and in elucidating their mechanisms of action. Epitope information can also elucidate unique cancer epitopes and determine vaccine protection or pathogenicity. The action can be defined.

[0185] Epitope mapping is performed using polyclonal or monoclonal antibodies. It is possible, and the epitope depends on the presumed properties of the epitope (i.e., linear conformation). Several methods are used for epitope identification. Mapping linear epitopes is a good approach. It is more direct and relatively easy to implement. For this purpose, linear epitope mapping Commercial services often use peptide scanning. In this case, the short segment of the target protein is scanned. A set of overlapping peptide sequences is chemically synthesized and bound to the antibody of interest. We will test their capabilities. The strategy is fast, high throughput, and relatively inexpensive to implement. It is applied. On the other hand, mapping discontinuous epitopes is technically more difficult and requires more specialized expertise. Phylocarpic techniques, such as X-ray cocrystal structure analysis of monoclonal antibodies and their target proteins, Hydrogen-deuterium (H / D) exchange and / or mass spectrometry combined with enzymatic digestion, etc. It is necessary.

[0186] PD-1 epitope using ProImmune® MicroArray Mapping: To identify the amino acids that form epitopes for anti-PD-1 mAbs, 15 It is the length of an amino acid, and consists of a total of 28 peptides in which 10 amino acids overlap. Chemically synthesized. This library of overlapping peptides is full-length canine PD- Designed to cover 1 protein. For measuring peptide-antibody binding, the antibody sample... Attached to a ProArray Ultra® peptide microarray, then This was carried out by incubation with a fluorescently labeled secondary antibody. All peptides They were synthesized separately and then combined with ProImmune® mouse IgG control for ProA The rray Ultra® is bonded to the slide surface. This optimization process involves peptide The peptide closely mimics the properties of the corresponding protein region and the inherent physicalization of the free peptide itself. To avoid scientific variations, we have created a compatible peptide and protein combination array platform. Ensure that the peptide is presented on the array in order to generate it. Test analyte (peptide Dispense the ) into separate spots onto ProArray Ultra(registered trademark) slides. A proper gal file allows for precise alignment of the resulting array features with the precipitated analyte. To enable this, use an effective blocking buffer to reduce nonspecific binding of mAbs. I blocked the ProArray Ultra(registered trademark) slides. Then I mA Incubate with sample b, then incubate with the specific fluorescently labeled secondary antibody. After several washing steps, the ProArray Ultra® array was dried and high-temperature cleaned. Scanning was performed using a high-resolution fluorescence microarray scanning system. Fluorescently labeled ProAr After scanning the ray Ultra® slide, the scanner records the image, This is the level of fluorescence intensity associated with each fluorescence spot on the scanned microarray slide. The results were evaluated using image analysis software that enables interpretation and quantification. The results showed that a portion of the canine PD-1 peptide was recognized by a portion of the mAbs evaluated. The identity of the mAbs and the amino acid sequences recognized by these mAbs are shown in Table 12. Listed below. This test is for mAb 2H9, which is represented by sequence number 138. Recognizing an epitope located in the extracellular domain of canine PD-1, which consists of an acid sequence. mAb 1A1 has the amino acid sequence and sequence number represented by SEQ ID NO: 138. Overlapping amino acid sequences represented by amino acid sequences represented by 139 This refers to recognizing an epitope containing [a specific substance].

[0187] Mapping of PD-1 epitopes using mass spectrometry: To identify potentially discontinuous epitopes recognized by anti-canine PD-1, Using a method based on scientific crosslinking and mass spectrometry detection (CovalX® Ins (trument Incorporated). Towards epitope mapping of canine PD-1. The application of this technology is to the epitopes recognized by the instruction mAbs listed in Table 13. This led to the identification of at least a portion of it. As can be seen from Table 13, mAb 3B6 is distributed Located in the extracellular domain of canine PD-1 within the amino acid sequence represented by column number: 140 It recognizes at least a portion of the epitope, and mAb 2G9 is sequence number: 14 It recognizes at least a portion of the epitope within the amino acid sequence represented by 1. On the other hand, mAb 1E4 and mAb 1B5 are represented by sequence number 142, respectively. The amino acid sequence and the small number of epitopes within the amino acid sequence represented by SEQ ID NO: 143 Even if not completely, it recognizes a part of it.

[0188] As shown in Figure 9A, chemical crosslinking, high-mass MALDI mass spectrometry and nL Measurements performed by C-Orbitrap mass spectrometry were confirmed by canine antibody 2G9. The recognized epitope on canine PD-1 is R of sequence number:114. 62 , R 69 , R 72 Oh biR 75 This indicates that it contains canine PD-1 recognized by canine antibody 3B6. A similar measurement concerning epitopes is R, sequence number: 114. 75 and R 90 It contains. Therefore, R 75 This is particularly important in one or more epitopes of canine PD-1. It appears to be an amino acid residue. Interestingly, after performing these analyses, 1A1 The amino acid sequence of the CDR was found to be identical to that of 2G9. Regions on PD-1 to which 2G9 binds and 1A1 obtained by two very different methods The agreement with what was observed in this regard is that this region is recognized by the anti-canine PD-1 antibody. This indicates that it contains amino acid residues included in the PD-1 epitope (see Table 12 below). (See also 13).

[0189] Furthermore, the amino acid composition of PD-1 recognized by the blocking antibody of the present invention tested The region in the column is located within the extracellular domain of canine PD-1. The recognized region is the following peptide. It is contained in (see Tables 12 and 13 below).

[0190] NQTDKLAAFQED R IEPGRD R RF R VM * R LPNGRDFHMSIVAA R LNDS (Sequence ID: 144) Within this peptide, there is a shorter peptide shown in bold. This shorter peptide is It was recognized by ProImmune® MicroArray (see Table 12).

[0191] D R IEPGRD R RF R VM * R LPNGR (Sequence ID: 145) Of particular note is R in sequence number 114. 62 , R 69 , R 72 and R 75 All, Both the longer peptide (SEQ ID NO: 144) and the shorter peptide (SEQ ID NO: 145) Contains R of SEQ ID NO: 114 90 These are present in longer peptides. Ruginine residues are important amino acids in one or more epitopes of canine PD-1. It appears to be a residue. As shown in Tables 6-8, the methionine residues marked with an asterisk (*) are It has also been reported to be a rheonine residue. [Table 13] [Table 14]

[0192] All references cited herein are individual publications, database entries, etc. (For example, Genbank sequences or GeneID entries), patent applications or patents, As if specifically and individually indicated to be incorporated herein by reference It is similarly incorporated herein by reference. This statement of incorporation by reference is 37 In accordance with CFR §1.57(b)(1), such citations by the applicable reference Even if they are not directly adjacent to the built-in statement, each one is 37 CFR §1.57(b )(2) Every single published document, database entry that is clearly identified in accordance with (2) A TRI (e.g., Genbank sequence or GeneID entry), patent application or patent The applicant intends for this to be relevant. The applicable statement of incorporation by reference exists. If included herein, the inclusion by reference is not in any sense of the meaning of the inclusion within this specification. This does not weaken the general statement of [the relevant]. Citations of references in this specification are made in accordance with the fact that the references are [the relevant]. This does not constitute an acknowledgment of relevant prior art, and these published documents or materials This does not constitute any endorsement of the content or date.

[0193] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, are described in the above description and accompanying drawings. Such modifications will be apparent to those skilled in the art. It is intended.

[0194] The aforementioned written specification is sufficient to enable a person skilled in the art to implement the present invention. It is thought that, in addition to what is shown and described herein, various modifications of the present invention may be made as described above. This will be apparent to those skilled in the art, and they are included within the scope of the accompanying claims.

Claims

1. An isolated canine antibody or antigen-binding fragment that specifically binds to canine programmed death receptor 1 (canine PD-1), wherein the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 44, which includes (i) P at position 244, (ii) A at position 271, (iii) A at position 303, (iv) G at position 304, (v) T at position 305, (vi) A at position 333, and (vii) P at position 335. And here, The canine antibody or the antigen-binding fragment of the canine antibody further comprises a canine light chain containing the amino acid sequence of SEQ ID NO: 78, Here, the canine antibody or its antigen-binding fragment specifically binds to the canine programmed death receptor 1 (canine PD-1), Herein, an isolated canine antibody or its antigen-binding fragment, wherein the antibody and its antigen-binding fragment bind to canine PD-1 and block the binding of canine PD-1 to canine programmed death ligand 1 (PD-L1).

2. A pharmaceutical composition comprising a canine antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier or diluent, Here, the canine antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 44, which includes (i) P at position 244, (ii) A at position 271, (iii) A at position 303, (iv) G at position 304, (v) T at position 305, (vi) A at position 333, and (vii) P at position 335. And here, The canine antibody or the antigen-binding fragment of the canine antibody further comprises a canine light chain containing the amino acid sequence of SEQ ID NO: 78, Here, the canine antibody or its antigen-binding fragment specifically binds to the canine programmed death receptor 1 (canine PD-1). The aforementioned pharmaceutical composition.

3. A method for increasing the activity of immune cells, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 2 to a canine subject in need thereof.

4. The method described above is a. Treat cancer; b. To treat an infection or infectious disease; or c. Acting as a vaccine adjuvant The method according to claim 3, used for...

5. A pair of isolated nucleic acids encoding the canine IgG heavy chain and canine light chain of a canine antibody or its antigen-binding fragment, wherein the canine antibody and its antigen-binding fragment specifically bind to canine programmed death receptor 1 (canine PD-1), Here, one nucleic acid encodes a canine IgG heavy chain, Here, the heavy chain includes a variable heavy (VH) region, and the VH region includes the following three variable heavy chain complementarity determination regions (VH CDRs): Sequence ID: Heavy chain complementarity determination region 1 (VH CDR1) having amino acid sequence 29; Sequence ID: 33, heavy chain complementarity determination region 2 (VH CDR2) containing the amino acid sequence; and, Sequence ID: Heavy chain complementarity determination region 3 (VH CDR3) containing amino acid sequence 38; And one nucleic acid codes for a light chain, which contains the following three variable light chain complementarity-determining regions (VL CDRs): Sequence ID: Light chain complementarity determination region 1 (VL CDR1) containing amino acid sequence 15; Sequence ID: Light chain complementarity determination region 2 (VL CDR2) containing 18 amino acid sequences; and Sequence ID: Light chain complementarity determination region 3 (VL CDR3) containing the amino acid sequence of 24.

6. The isolated nucleic acid according to claim 5, wherein the canine or canine IgG heavy chain comprises the amino acid sequence of SEQ ID NO: 44, where SEQ ID NO: 44 comprises a proline residue at amino acid position 244, a glycine or alanine residue at amino acid position 271, an asparagine or alanine residue at amino acid position 303, a glycine residue at amino acid position 304, a threonine residue at amino acid position 305, and an alanine residue at amino acid position 333.

7. The isolated nucleic acid according to claim 6, wherein sequence number 44 contains an alanine residue at amino acid position 271 or an alanine residue at amino acid position 303 of sequence number 44, or an alanine residue at amino acid position 271 and an alanine residue at amino acid position 303 of sequence number 44.

8. The isolated nucleic acid according to claim 7, wherein the nucleic acid sequence comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO: 43, and codes for a heavy chain having an alanine residue at amino acid position 271 or an alanine residue at amino acid position 303 of SEQ ID NO: 44, or an amino acid sequence of SEQ ID NO: 44 having an alanine residue at amino acid position 271 and an alanine residue at amino acid position 303 of SEQ ID NO:

44.

9. The isolated nucleic acid according to claim 5, wherein the canine or canine IgG heavy chain comprises the amino acid sequence of SEQ ID NO: 46, and SEQ ID NO: 46 comprises a proline residue at amino acid position 242, a glycine or alanine residue at amino acid position 269, an asparagine or alanine residue at amino acid position 301, a glycine residue at amino acid position 302, a threonine residue at amino acid position 303, and an alanine residue at amino acid position 331.

10. The isolated nucleic acid according to claim 9, wherein sequence number 46 contains an alanine residue at amino acid position 269 or an alanine residue at amino acid position 301 of sequence number 46, or contains an alanine residue at amino acid position 269 and an alanine residue at amino acid position 301 of sequence number 46.

11. The isolated nucleic acid according to claim 10, wherein the nucleic acid sequence comprises a nucleic acid sequence having at least 85% sequence identity with SEQ ID NO: 45, and codes for a heavy chain having an alanine residue at amino acid position 269 or an alanine residue at amino acid position 301 of SEQ ID NO: 46, or an amino acid sequence of SEQ ID NO: 46 having an alanine residue at amino acid position 269 and an alanine residue at amino acid position 301 of SEQ ID NO:

46.

12. The isolated nucleic acid according to claim 5, wherein the canine or canine light chain is a canine kappa light chain.

13. The isolated nucleic acid according to claim 5, wherein the canine or canine light chain comprises the amino acid sequence of SEQ ID NO: 78, or a sequence having at least 85% sequence identity with SEQ ID NO:

78.

14. The isolated nucleic acid according to claim 5, wherein the nucleic acid comprises sequence number 77 or a sequence having at least 85% sequence identity with sequence number 77.

15. An expression vector comprising a pair of nucleic acids according to claim 5 and at least one promoter operably ligated to one or both of the nucleic acid sequences according to claim 5.

16. A host cell comprising the expression vector described in claim 15.

17. The host cell according to claim 16, wherein the host cell is selected from any of the insect cell line, amphibian cell line, bacterial cell line, mammalian cell line, plant cell line, and fungal cell line.

18. The host cell according to claim 17, wherein the mammalian cell line is selected from any of the group consisting of Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

19. Methods including the following: The host cells described in claim 16 are cultured in a culture medium for a sufficient time to enable antibody expression in the host cells, and Collecting antibodies from culture media.

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