Patent Application
20250066483
The present disclosure relates to an anti-PD-L1 antibody and an anti-PD-1 antibody. More specifically, one aspect of the present disclosure relates to an anti-PD-L1 antibody comprising a variable region containing complementarity-determining regions (CDR) of a rat anti-bovine PD-L1 antibody and a constant region of an antibody of an animal other than rat. The present disclosure also relates to an anti-PD-1 antibody. Moreover, one aspect of the present disclosure relates to an anti-PD-1 antibody comprising a variable region containing complementarity-determining regions (CDRs) of a rat anti-bovine PD-1 antibody and a constant region of an antibody of an animal other than rat. The present disclosure further relates to combined use of an inhibitor targeting PD-1/PD-L1 and a COX-2 inhibitor.
Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance (Non-Patent Document No. 1: Ishida Y, Agata Y, Shibahara K, Honjo T The EMBO Journal. 1992 November; 11(11):3887-3895). Recently, it has been elucidated that these molecules are also involved in immunosuppression in tumors. In the field of human medical care, an antibody drug that inhibits the effect of PD-1 has been developed and put into practical use (Opdivo™, Ono Pharmaceutical Co., Ltd.).
To date, the present inventors have been developing an immunotherapy for animal refractory diseases targeting PD-1 or PD-L1, and have revealed that this novel immunotherapy is applicable to multiple-diseases and multiple-animals. (Non-Patent Document No. 2: Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology. 2014 August; 142(4):551-61; Non-Patent Document No. 3: Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415; Non-Patent Document No. 4: Mingala C N, Konnai S, Ikebuchi R, Ohashi K. Comp. Immunol. Microbiol. Infect. Dis. 2011 January; 34(1):55-63.). Further, the interaction between PD-1 and PD-L1 is one of the major molecular mechanisms through which tumors and infections evade immune responses. It has been reported that inhibition of the above interaction by using an antibody which specifically binds to either of those molecules can produce antitumor effects and anti-pathogenic effects.
However, the antibodies which the present inventors have prepared to date are rat antibodies, and therefore it is impossible to administer those antibodies repeatedly to animals other than rat.
It is one object of the present disclosure to provide an anti-PD-L1 antibody capable of repeated administration even to animals other than rat. It is also an object of the present disclosure to provide an anti-PD-1 antibody capable of repeated administration even to animals other than rat. It is another object of the present disclosure to provide a novel therapeutic strategy using inhibitors targeting PD-1/PD-L1.
It has been determined that the variable regions of a rat anti-bovine PD-L1 monoclonal antibody (4G12) capable of inhibiting the binding of canine PD-1 to PD-L1, and then combined genes encoding the resultant variable regions with genes encoding the constant regions of a canine immunoglobulin (IgG-D equivalent to human IgG4) to thereby obtain a chimeric antibody gene, which was introduced into Chinese hamster ovary cells (CHO cells). By culturing/proliferating the resultant CHO cells, the present inventors have succeeded in preparing a rat-canine chimeric anti-PD-L1 antibody. Further, it has been determined that the CDRs of the variable region of the rat anti-bovine PD-L1 monoclonal antibody 4G12.
Furthermore, it has been determined the variable regions of the rat anti-bovine PD-L1 monoclonal antibody 4G12 capable of inhibiting the binding of bovine PD-1 to PD-L1, and then combined genes encoding the resultant variable regions with genes encoding the constant regions of a bovine immunoglobulin (bovine IgG1, with mutations having been introduced into the putative binding sites of Fcγ receptors in CH2 domain in order to inhibit ADCC activity; see
Furthermore, toward establishment of a novel control method for canine tumors and bovine infections, it has been confirmed in in vitro tests an immunostimulatory effect induced by COX-2 inhibitors and enhancement of that effect when such inhibitors are used in combination with anti-PD-L1 antibody. The present disclosure provides what has been achieved based on these findings.
Furthermore, it has been determined the variable regions of a rat anti-bovine PD-1 monoclonal antibody (5D2) capable of inhibiting the binding of bovine PD-1 to PD-L1, and then combined genes encoding the resultant variable regions with genes encoding the constant regions of a bovine immunoglobulin (bovine IgG1, with mutations having been introduced into the putative binding sites of Fcγ receptors in CH2 domain in order to inhibit ADCC activity; see
A summary of the present disclosure according to some embodiments is as described below.
Immunostimulatory effect is enhanced by combined use of an inhibitor targeting PD-1/PD-L1 and a COX-2 inhibitor. Moreover, a novel anti-PD-1 antibody has been obtained. This antibody is applicable even to those animals other than rat.
The present specification encompasses the contents disclosed in the specifications and/or drawings of Japanese Patent Application Nos. 2017-140891, No. 2018-016074 2016-159090, and No. 2017-099615, based on which the present patent application claims priority. The present specification further incorporates by reference U.S. Patent Application Publication Nos. 2019/0185568, 2022/0227871 and 2020/0131270 in their entirety.
Hereinbelow, the present disclosure will be described in detail.
One aspect of the present disclosure provides an anti-PD-L1 antibody comprising a light chain constant region of an antibody of an animal other than rat; and a heavy chain constant region of an antibody of an animal other than rat. The present disclosure provides a pharmaceutical composition which comprises a COX-2 inhibitor and is administered before, after or simultaneously with the administration of an inhibitor targeting PD-1/PD-L1.
Cyclooxygenase 2 (COX-2) is an enzyme involved in a process of biosynthesizing prostanoids including prostaglandin E2 (PGE2). In contrast to COX-1 that is expressed constitutively, expression of COX-2 is induced by stimulation from cytokines, growth factors, etc. in inflammatory tissues. High expression of COX-2 has been reported in various tumors and infections, and it is believed that COX-2 is involved in the growth and pathogenesis of tumor cells and infected cells. Since PGE2 inhibits, in particular, the effector function of cytotoxic T-cells via receptors EP2 and EP4, PGE2 has recently been attracting attention as a humoral factor constituting an immunosuppressive tumor microenvironment. On the other hand, COX-2 inhibitors are expected to decrease PGE2 production to thereby reduce the suppression upon immune cells. In mouse models, enhancement of antitumor effect and antiviral effect by combined use of a COX-2 inhibitor (such as celecoxib) and an inhibitor targeting PD-1/PD-L1 has been recognized.
COX-2 inhibitor may be an agent that selectively inhibits COX-2. Specific examples of COX-2 inhibitor include, but are not limited to, meloxicam, piroxicam, celecoxib, firocoxib, robenacoxib, carprofen and etodolac.
PD-1 (Programmed cell death-1) is a membrane protein expressed in activated T cells and B cells. Its ligand PD-L1 is expressed in various cells such as antigen-presenting cells (monocytes, dendritic cells, etc.) and cancer cells. PD-1 and PD-L1 work as inhibitory factors which inhibit T cell activation. Certain types of cancer cells and virus-infected cells escape from host immune surveillance by expressing the ligand of PD-1 to thereby inhibit T cell activation.
As inhibitors targeting PD-1/PD-L1, substances which specifically bind to PD-1 or PD-L1 may be given. Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, nucleic acids (including natural-type and artificial nucleic acids), low molecular weight organic compounds, inorganic compounds, cell extracts, and extracts from animals, plants, soils or the like. These substances may be either natural or synthetic products.
Preferable inhibitors targeting PD-1/PD-L1 are antibodies. More preferably, antibodies such as anti-PD-1 antibody and anti-PD-L1 antibody may be given. Any type of antibody may be used as long as it has an inhibitory activity targeting PD-1/PD-L1. The antibody may be any of polyclonal antibody, monoclonal antibody, chimeric antibody, single chain antibody, humanized antibody or human-type antibody. Methods for preparing such antibodies are known. The antibody may be derived from any organisms such as human, mouse, rat, rabbit, goat, guinea pig, dog or cattle. As used herein, the term “antibody” is a concept encompassing antibodies of smaller molecular sizes such as Fab, F(ab)′2, ScFv, Diabody, VH, VL, Sc(Fv)2, Bispecific sc(Fv)2, Minibody, scFv-Fc monomer or scFv-Fc dimer.
As an example of anti-PD-L1 antibody, one comprising (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino acid sequence of WAT and CDR3 having the amino acid sequence of GQYLVYPFT (SEQ ID NO: 38) and a light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GYTFTSNF (SEQ ID NO: 39), CDR2 having the amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and CDR3 having the amino acid sequence of ASEEAVISLVY (SEQ ID NO: 41) and a heavy chain constant region of an antibody of an animal other than rat may be given.
CDR1, CDR2 and CDR3 in the light chain variable region (VL) of rat anti-bovine PD-L1 antibody 4G12 are a region comprising the amino acid sequence of QSLLYSENQKDY (SEQ ID NO: 37), a region comprising the amino acid sequence of WAT and a region comprising the amino acid sequence of GQYLVYPFT (SEQ ID NO: 38), respectively (see
Further, CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) of rat anti-bovine PD-L1 antibody 4G12 are a region comprising the amino acid sequence of GYTFTSNF (SEQ ID NO: 39), a region comprising the amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and a region comprising the amino acid sequence of ASEEAVISLVY (SEQ ID NO: 41), respectively (see
In the amino acid sequences of QSLLYSENQKDY (SEQ ID NO: 37), WAT and GQYLVYPFT (SEQ ID NO: 38), as well as the amino acid sequences of GYTFTSNF (SEQ ID NO: 39), IYPEYGNT (SEQ ID NO: 40) and ASEEAVISLVY (SEQ ID NO: 41), one, two, three, four or five amino acids may be deleted, substituted or added.
One aspect of the present disclosure provides an anti-PD-1 antibody comprising (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 164), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 165) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 166), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 167) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat.
CDR1, CDR2 and CDR3 in the light chain variable region (VL) of rat anti-bovine PD-1 antibody 5D2 (to be described later) are respectively a region consisting of the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 164), a region consisting of the amino acid sequence of GVS and a region consisting of the amino acid sequence of FQATHDPDT (SEQ ID NO: 165) (see
Further, CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) of rat anti-bovine PD-1 antibody 5D2 are respectively a region consisting of the amino acid sequence of GFSLTSYY (SEQ ID NO: 166), a region consisting of the amino acid sequence of IRSGGST (SEQ ID NO: 167) and a region consisting of the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 168) (see
In the amino acid sequences of QSLEYSDGYTY (SEQ ID NO: 164), GVS and FQATHDPDT (SEQ ID NO: 165), as well as the amino acid sequences of GFSLTSYY (SEQ ID NO: 166), IRSGGST (SEQ ID NO: 167) and ARTSSGYEGGFDY (SEQ ID NO: 168), one, two, three, four or five amino acids may be deleted, substituted or added. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function of a CDR in the light chain or heavy chain variable region of the PD-1 antibody.
In the above-described anti-PD-L1 antibody, VL and VH thereof may be derived from rat. For example, VL thereof may be the VL of a rat anti-bovine PD-L1 antibody, and VH thereof may be the VH of the rat anti-bovine PD-L1 antibody.
The amino acid sequence of the VL and the amino acid sequence of the VH of the rat anti-bovine PD-L1 antibody are shown in SEQ ID NOS: 1 and 2, respectively. The amino acid sequences as shown in SEQ ID NOS: 1 and 2 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as VL or VH of the PD-L1 antibody.
The CL and CH of an antibody of an animal other than rat may be derived from an animal which produces a PD-L1 that cross-reacts with rat anti-bovine PD-L1 antibody 4G12.
There are two types of immunoglobulin light chain, which are called Kappa chain (κ) and Lambda chain (λ). In the above-described anti-PD-L1 antibody, the light chain constant region (CL) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of either Kappa chain or Lambda chain. However, the relative abundance of Lambda chain is higher in ovine, feline, canine, equine and bovine, and that of Kappa chain is higher in mouse, rat, human and porcine. Since a chain with a higher relative abundance is considered to be preferable, an ovine, feline, canine, equine or bovine antibody preferably has the amino acid sequence of the constant region of Lambda chain whereas a mouse, rat, human or porcine antibody preferably has the amino acid sequence of the constant region of Kappa chain.
The heavy chain constant region (CH) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4. Immunoglobulin heavy chain is classified into γ chain, μ chain, α chain, δ chain and E chain depending on the difference in constant region. According to the type of heavy chain present, five classes (isotypes) of immunoglobulin are formed; they are IgG, IgM, IgA, IgD and IgE.
Immunoglobulin G (IgG) accounts for 70-75% of human immunoglobulins and is the most abundantly found monomeric antibody in plasma. IgG has a four-chain structure consisting of two light chains and two heavy chains. Human IgG1, IgG2 and IgG4 have molecular weights of about 146,000, whereas human IgG3 has a long hinge region that connects Fab region and Fc region and has a larger molecular weight of 170,000. Human IgG1 accounts for about 65%, human IgG2 about 25%, human IgG3 about 7%, and human IgG4 about 3% of human IgG. They are uniformly distributed inside and outside of blood vessels. Having a strong affinity for Fc receptors and complement factors on effector cell surfaces, human IgG1 induces antibody-dependent cell cytotoxicity (ADCC) and also activates complements to induce complement-dependent cell cytotoxicity (CDC). Human IgG2 and IgG4 are low at ADCC and CDC activities because their affinity for Fc receptors and complement factors is low.
Immunoglobulin M (IgM), which accounts for about 10% of human immunoglobulins, is a pentameric antibody consisting of five basic four-chain structures joined together. It has a molecular weight of 970,000. Usually occurring only in blood, IgM is produced against infectious microorganisms and takes charge of early stage immunity.
Immunoglobulin A (IgA) accounts for 10-15% of human immunoglobulins. It has a molecular weight of 160,000. Secreted IgA is a dimeric antibody consisting of two IgA molecules joined together. IgA1 is found in serum, nasal discharge, saliva and breast milk. In intestinal juice, IgA2 is found abundantly.
Immunoglobulin D (IgD) is a monomeric antibody accounting for no more than 1% of human immunoglobulins. IgD is found on B cell surfaces and involved in induction of antibody production.
Immunoglobulin E (IgE) is a monomeric antibody that occurs in an extremely small amount, accounting for only 0.001% or less of human immunoglobulins. Immunoglobulin E is considered to be involved in immune response to parasites but in advanced countries where parasites are rare, IgE is largely involved in bronchial asthma and allergy among other things.
With respect to canine, sequences of IgG-A (equivalent to human IgG2), IgG-B (equivalent to human IgG1), IgG-C (equivalent to human IgG3) and IgG-D (equivalent to human IgG4) have been identified as the heavy chain of IgG. In the above-described anti-PD-L1 antibody, an IgG's heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). In the case where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, one may use a constant region that has lost both ADCC activity and CDC activity as a result of introducing mutations into the relevant region of an immunoglobulin equivalent to human IgG1.
In bovine, the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, so mutations may be added at the relevant region of an immunoglobulin equivalent to human IgG1 and the resultant constant region then used. As one example, the amino acid sequence of the CH of a bovine antibody (IgG1 chain, GenBank: X62916) having mutations introduced into CH2 domain and a nucleotide sequence for such amino acid sequence (after codon optimization) are shown in SEQ ID NOS: 102 and 103, respectively.
When an animal other than rat is canine or bovine, an anti-PD-L1 antibody is more preferable in which (i) the CL of a canine or bovine antibody has the amino acid sequence of the constant region of Lambda chain and (ii) the CH of the canine or bovine antibody has the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4.
The above-described anti-PD-L1 antibody encompasses rat-canine chimeric antibodies, caninized antibodies, rat-bovine chimeric antibodies and bovinized antibodies. However, animals are not limited to canine and bovine and may be exemplified by human, porcine, simian, mouse, feline, equine, goat, sheep, water buffalo, rabbit, hamster, guinea pig, bovine and the like.
For example, the anti-PD-L1 antibody described above may be an anti-PD-L1 antibody in which the CL of a canine antibody has the amino acid sequence as shown in SEQ ID NO: 3 and the CH of the canine antibody has the amino acid sequence as shown in SEQ ID NO: 4; or an anti-PD-L1 antibody in which the CL of a bovine antibody has the amino acid sequence as shown in SEQ ID NO: 100 and the CH of the bovine antibody has the amino acid sequence as shown in SEQ ID NO: 102.
The amino acid sequences as shown in SEQ ID NOS: 3 and 4 as well as SEQ ID NOS: 100 and 102 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as CL or CH of the PD-L1 antibody.
The above-described anti-PD-L1 antibody may have a four-chain structure comprising two light chains and two heavy chains.
The above-described anti-PD-L1 antibody may be prepared as described below. Briefly, an artificial gene is synthesized which comprises (i) the identified variable region sequences of a rat anti-bovine PD-L1 antibody and (ii) the constant region sequences of an antibody of an animal other than rat (e.g., canine or bovine) (preferably, human IgG4 antibody or antibody equivalent to human IgG4 antibody). The resultant gene is inserted into a vector (e.g., plasmid), which is then introduced into a host cell (e.g., mammal cell such as CHO cell). The host cell is cultured, and the antibody of interest is collected from the resultant culture.
The amino acid sequence and the nucleotide sequence of the VL of the rat anti-bovine PD-L1 antibody identified by the present inventors are shown in SEQ ID NOS: 1 and 5, respectively. Further, nucleotide sequences after codon optimization are shown in SEQ ID NOS: 15 and 112.
The amino acid sequence and the nucleotide sequence of the VH of the rat anti-bovine PD-L1 antibody identified by the present inventors are shown in SEQ ID NOS: 2 and 6, respectively. Further, nucleotide sequences after codon optimization are shown in SEQ ID NOS: 16 and 113.
The amino acid sequence and the nucleotide sequence of the CL (Lambda chain, GenBank: E02824.1) of a canine antibody are shown in SEQ ID NOS: 3 and 7, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 17.
The amino acid sequence and the nucleotide sequence of the CH (IgG-D chain, GenBank: AF354267.1) of the canine antibody are shown in SEQ ID NOS: 4 and 8, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 18.
Further, SEQ ID NO: 9 shows the amino acid sequence of a chimeric light chain comprising the VL of the rat anti-bovine PD-L1 antibody and the CL (Lambda chain, GenBank: E02824.1) of the canine antibody. The nucleotide sequence (after codon optimization) of the chimeric light chain comprising the VL of the rat anti-bovine PD-L1 antibody and the CL (Lambda chain, GenBank: E02824.1) of the canine antibody is shown in SEQ ID NO: 19.
SEQ ID NO: 10 shows the amino acid sequence of a chimeric heavy chain comprising the VH of the rat anti-bovine PD-L1 antibody and the CH (IgG-D chain, GenBank: AF354267.1) of the canine antibody. The nucleotide sequence (after codon optimization) of the chimeric heavy chain comprising the VH of the rat anti-bovine PD-L1 antibody and the CH (IgG-D chain, GenBank: AF354267.1) of the canine antibody is shown in SEQ ID NO: 20.
The amino acid sequence and the nucleotide sequence of the CL (Lambda chain, GenBank: X62917) of a bovine antibody are shown in SEQ ID NOS: 100 and 101, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 114.
The amino acid sequence and the nucleotide sequence (after codon optimization) of the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody are shown in SEQ ID NOS: 102 and 103, respectively.
Further, SEQ ID NO: 115 shows the amino acid sequence of a chimeric light chain comprising the VL of the rat anti-bovine PD-L1 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric light chain comprising the VL of the rat anti-bovine PD-L1 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody is shown in SEQ ID NO: 117.
SEQ ID NO: 116 shows the amino acid sequence of a chimeric heavy chain comprising the VH of the rat anti-bovine PD-L1 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric heavy chain comprising the VH of the rat anti-bovine PD-L1 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody is shown in SEQ ID NO: 118.
Amino acid sequences and nucleotide sequences of CLs and CHs for various animals other than rat, canine and bovine may be obtained from known databases for use in the present disclosure.
Amino acid sequences and nucleotide sequences of CLs and CHs for canine, ovine, porcine, water buffalo, human and bovine are summarized in the table below.
Canis
lupus
familiaris)
Ovis
Sus
Bubalus
bubalis)
Homo
sapiens)
Bos
taurus)
In the anti-PD-1 antibody of the present disclosure, the VL and VH may be derived from rat. For example, the VL may be the VL of a rat anti-bovine PD-1 antibody, and the VH may be the VH of the rat anti-bovine PD-1 antibody.
The amino acid sequence of the VL and the amino acid sequence of the VH of the rat anti-bovine PD-1 antibody are shown in SEQ ID NOS: 1 and 2, respectively. The amino acid sequences as shown in SEQ ID NOS: 1 and 2 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function as VL or VH of the PD-1 antibody.
The VL and VH of an antibody of an animal other than rat may be derived from an animal which produces a PD-1 that cross-reacts with rat anti-bovine PD-1 antibody 5D2.
There are two types of immunoglobulin light chain, which are called Kappa chain (κ) and Lambda chain (λ). In the anti-PD-1 antibody of the present disclosure, the light chain constant region (CL) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of either Kappa chain or Lambda chain. However, the relative abundance of Lambda chain is higher in bovine, ovine, feline, canine and equine, and that of Kappa chain is higher in mouse, rat, human and porcine. Since a chain with a higher relative abundance is considered to be preferable, a bovine, ovine, feline, canine or equine antibody preferably has the amino acid sequence of the constant region of Lambda chain whereas a mouse, rat, human or porcine antibody preferably has the amino acid sequence of the constant region of Kappa chain.
The heavy chain constant region (CH) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4. Immunoglobulin heavy chain is classified into γ chain, μ chain, α chain, δ chain and E chain depending on the difference in constant region. According to the type of heavy chain present, five classes (isotypes) of immunoglobulin are formed; they are IgG, IgM, IgA, IgD and IgE.
Immunoglobulin G (IgG) accounts for 70-75% of human immunoglobulins and is the most abundantly found monomeric antibody in plasma. IgG has a four-chain structure consisting of two light chains and two heavy chains. Human IgG1, IgG2 and IgG4 have a molecular weight of about 146,000, whereas human IgG3 has a long hinge region that connects Fab region and Fc region and has a larger molecular weight of 170,000. Human IgG1 accounts for about 65% of human IgG, human IgG2 about 25%, human IgG3 about 7%, and human IgG4 about 3%. They are uniformly distributed inside and outside of blood vessels. Having a strong affinity for Fc receptors and complement factors on effector cell surfaces, human IgG1 induces antibody-dependent cell cytotoxicity (ADCC) and also activates complements to induce complement-dependent cell cytotoxicity (CDC). Human IgG2 and IgG4 are low at ADCC and CDC activities because their affinity for Fc receptors and complement factors is low.
Immunoglobulin M (IgM), which accounts for about 10% of human immunoglobulins, is a pentameric antibody consisting of five basic four-chain structures joined together. It has a molecular weight of 970,000. Usually occurring only in blood, IgM is first produced against infectious microorganisms and takes charge of early stage immunity.
Immunoglobulin A (IgA) accounts for 10-15% of human immunoglobulins. It has a molecular weight of 160,000. Secreted IgA is a dimeric antibody consisting of two IgA molecules joined together. IgA1 is found in serum, nasal discharge, saliva and breast milk. In intestinal juice, IgA2 is found abundantly.
Immunoglobulin D (IgD) is a monomeric antibody accounting for no more than 1% of human immunoglobulins. IgD is found on B cell surfaces and involved in induction of antibody production.
Immunoglobulin E (IgE) is a monomeric antibody that occurs in an extremely small amount, accounting for only 0.001% or less of human immunoglobulins. Immunoglobulin E is considered to be involved in immune response to parasites but in advanced countries where parasites are rare, IgE is largely involved in bronchial asthma and allergy among other things.
In canine, sequences of IgG-A (equivalent to human IgG2), IgG-B (equivalent to human IgG1), IgG-C (equivalent to human IgG3) and IgG-D (equivalent to human IgG4) have been identified as the heavy chain of IgG. In the antibody of the present disclosure, an IgG's heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). In the case where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, one may use a constant region that has lost both ADCC activity and CDC activity as a result of introducing mutations into the relevant region of an immunoglobulin equivalent to human IgG4.
In bovine, sequences of IgG1, IgG2 and IgG3 have been identified as the heavy chain of IgG. In the antibody of the present disclosure, an IgG's heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). Although the constant region of wild-type human IgG1 has ADCC activity and CDC activity, it is known that these activities can be reduced by introducing amino acid substitutions or deletions into specific sites. In bovine, the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, so mutations may be added to the relevant region of an immunoglobulin equivalent to human IgG1 and the resultant constant region then used. As one example, the amino acid sequence of the CH of a bovine antibody (IgG1 chain, GenBank: X62916) having mutations introduced into CH2 domain and a nucleotide sequence for such amino acid sequence (after codon optimization) are shown in SEQ ID NOS: 4 and 8, respectively.
An anti-PD-1 antibody is preferable in which (i) the CL of a bovine antibody has the amino acid sequence of the constant region of Lambda chain and (ii) the CH of the bovine antibody has mutations introduced thereinto that reduce ADCC activity and/or CDC activity.
The anti-PD-1 antibody of the present disclosure encompasses rat-bovine chimeric antibodies, bovinized antibodies and complete bovine-type antibodies. However, the animal is not limited to bovine and may be exemplified by human, canine, porcine, simian, mouse, feline, equine, goat, ovine, water buffalo, rabbit, hamster, guinea pig and the like.
For example, the anti-PD-1 antibody of the present disclosure may be an anti-PD-1 antibody in which the CL of a bovine antibody has the amino acid sequence as shown in SEQ ID NO: 151 and the CH of the bovine antibody has the amino acid sequence as shown in SEQ ID NO: 152.
The amino acid sequences as shown in SEQ ID NOS: 151 and 152 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function as CL or CH of the PD-1 antibody.
The anti-PD-1 antibody of the present disclosure may have a four-chain structure comprising two light chains and two heavy chains.
The anti-PD-1 antibody of the present disclosure may be prepared as described below. Briefly, an artificial gene is synthesized which comprises (i) the identified variable region sequences of a rat anti-bovine PD-1 antibody and (ii) the constant region sequences of an antibody of an animal other than rat (e.g., bovine) (preferably, an immunoglobulin equivalent to human IgG1, in which mutations have been introduced into the relevant region to reduce ADCC activity and/or CDC activity). The resultant gene is inserted into a vector (e.g., plasmid), which is then introduced into a host cell (e.g., mammal cell such as CHO cell). The host cell is cultured, and the antibody of interest is collected from the resultant culture.
The amino acid sequence and the nucleotide sequence of the VL of the rat anti-bovine PD-1 antibody identified by the present inventors are shown in SEQ ID NOS: 149 and 153, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 159.
The amino acid sequence and the nucleotide sequence of the VH of the rat anti-bovine PD-1 antibody identified by the present inventors are shown in SEQ ID NOS: 150 and 154, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 160.
The amino acid sequence and the nucleotide sequence of the CL (Lambda chain, GenBank: X62917) of a bovine antibody are shown in SEQ ID NOS: 151 and 155, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 161.
The amino acid sequence and the nucleotide sequence (after codon optimization) of the CH (IgG1 chain, modified from GenBank: X62916) of a bovine antibody are shown in SEQ ID NOS: 152 and 156, respectively.
Further, SEQ ID NO: 157 shows the amino acid sequence of a chimeric light chain consisting of the VL of the rat anti-bovine PD-1 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric light chain consisting of the VL of the rat anti-PD-1 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody is shown in SEQ ID NO: 162.
SEQ ID NO: 158 shows the amino acid sequence of a chimeric heavy chain consisting of the VH of the rat anti-bovine PD-1 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric heavy chain consisting of the VH of the rat anti-bovine PD-1 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody is shown in SEQ ID NO: 163.
Amino acid sequences and nucleotide sequences of CLs and CHs of various animals other than rat may be obtained from known databases for use in the present disclosure.
Amino acid sequences and nucleotide sequences of bovine CL and CH are summarized in the table below.
Bos
taurus)
Amino acid sequences and nucleotide sequences of ovine, water buffalo and human CL and CH are summarized in the table below.
Homo
sapiens)
The amino acid sequences as shown in SEQ ID NOS: 4, 3, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 12, 80, 82, 84-91, 100, 102, 11, 151, 169-76, 185, 187, 189, 191, 193, 196, 197, 199, 201, 203, 205 and 207 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function as a constant region of Ig heavy chain or light chain.
Although the constant region of wild-type human IgG1 has ADCC activity and CDC activity, it is known that these activities can be reduced by introducing amino acid substitutions and deletions into specific sites. In the case of animals other than human where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, mutations may be introduced into the relevant region of an immunoglobulin equivalent to human IgG1 so that the resultant constant region with reduced ADCC activity and CDC activity can be used.
Certain embodiments of the present disclosure provides an artificial genetic DNA comprising (a′) a DNA encoding a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 164), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 165) and the light chain constant region of an antibody of an animal other than rat and (b′) a DNA encoding a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 166), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 167) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 168) and the heavy chain constant region of an antibody of an animal other than rat. The present disclosure also provides a DNA encoding a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 164), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 165) and the light chain constant region of an antibody of an animal other than rat. Further, the present disclosure also provides a DNA encoding a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 166), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 167) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 168) and the heavy chain constant region of an antibody of an animal other than rat.
For (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 164), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 165) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 166), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 167) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 168) and the heavy chain constant region of an antibody of an animal other than rat, reference should be had to the foregoing description. The DNA of (a′) is a DNA (gene) encoding the light chain of (a); and the DNA of (b′) is a DNA (gene) encoding the heavy chain of (b). An artificial genetic DNA comprising the DNA of (a′) and the DNA of (′b) may be synthesized on commercial synthesizer. Restriction enzyme recognition sites, KOZAK sequences, poly-A addition signal sequences, promoter sequences, intron sequences or the like may be added to the artificial genetic DNA.
Certain embodiments of the present disclosure also provides a vector comprising the above-mentioned artificial genetic DNA.
As the vector, Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12 or pUC13); Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5 or pC194), yeast-derived plasmids (e.g., pSH19 or pSH15); bacteriophages such as λ phage; animal viruses such as retrovirus or vaccinia virus; or insect pathogen viruses such as baculovirus may be used. In the Examples described later, pDN112 (Marzi A, Yoshida R, Miyamoto H, Ishijima M, Suzuki Y, Higuchi M, Matsuyama Y, Igarashi M, Nakayama E, Kuroda M, Saijo M, Feldmann F, Brining D, Feldmann H, TakadaA. PLoS One, 7:e36192, Apr. 27, 2012; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology, 142(4):551-561, August 2014) was used.
The vector may also comprise promoters, enhancers, splicing signals, poly-A addition signals, intron sequences, selection markers, SV40 replication origins, and so forth.
The present disclosure also provides a host cell transformed by the above vector. It is possible to prepare the anti-PD-1 antibody of the invention by culturing the host cell and collecting the antibody of interest from the resultant culture. Therefore, the present disclosure also provides a method of preparing an antibody, comprising culturing the above-described host cell and collecting the anti-PD-1 antibody of the invention from the culture. In the method of the present disclosure for preparing an antibody, a vector incorporating an artificial genetic DNA comprising a DNA encoding the light chain and a DNA encoding the heavy chain may be transfected into a host cell. Alternatively, a vector incorporating a DNA encoding the light chain and a vector incorporating a DNA encoding the heavy chain may be co-transfected into a host cell.
Examples of the host cell include, but are not limited to, bacterial cells (such as Escherichia bacteria, Bacillus bacteria or Bacillus subtilis), fungal cells (such as yeast or Aspergillus), insect cells (such as S2 cells or Sf cells), animal cells (such as CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells or HEK 293 cells) and plant cells. Among these, CHO-DG44 cell (CHO-DG44(dfhr−/−)) which is a dihydrofolate reductase deficient cell is preferable.
Introduction of a recombinant vector into a host cell may be performed by the methods disclosed in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g., the calcium phosphate method, the DEAE-dextran method, transfection, microinjection, lipofection, electroporation, transduction, scrape loading, the shotgun method, etc.) or by infection.
The resultant transformant may be cultured in a medium, followed by collection of the anti-PD-1 antibody of the present disclosure from the culture. When the antibody is secreted into the medium, the medium may be recovered, followed by isolation and purification of the antibody from the medium. When the antibody is produced within the transformed cells, the cells may be lysed, followed by isolation and purification of the antibody from the cell lysate.
Examples of the medium include, but are not limited to, OptiCHO medium, Dynamis medium, CD CHO medium, ActiCHO medium, FortiCHO medium, Ex-Cell CD CHO medium, BalanCD CHO medium, ProCHO 5 medium and Cellvento CHO-100 medium.
The pH of the medium varies depending on the cell to be cultured. Generally, a pH range from 6.8 to 7.6 is used; mostly, a pH range from 7.0 to 7.4 is appropriate.
When the cell to be cultured is CHO cells, culture may be performed by methods known to those skilled in the art. For example, it is usually possible to perform culturing in a gas-phase atmosphere having a CO2 concentration of 0-40%, preferably 2-10%, at 30-39° C., preferably around 37° C.
The appropriate period of culture is usually from one day to three months, preferably from one day to three weeks.
Isolation and purification of the antibody may be performed by known methods.
Known isolation/purification methods which may be used in the present disclosure include, but are not limited to, methods using difference in solubility (such as salting-out or solvent precipitation); methods using difference in molecular weight (such as dialysis, ultrafiltration, gel filtration or SDS-polyacrylamide gel electrophoresis); methods using difference in electric charge (such as ion exchange chromatography); methods using specific affinity (such as affinity chromatography); methods using difference in hydrophobicity (such as reversed phase high performance liquid chromatography); and methods using difference in isoelectric point (such as isoelectric focusing).
The pharmaceutical composition in certain embodiments of the present disclosure may be used for prevention and/or treatment of cancers and/or infections. Examples of cancer and/or infection include, but are not limited to, neoplastic diseases (e.g., malignant melanoma, lung cancer, gastric cancer, renal cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer and the like), leukemia, Johne's disease, anaplasmosis, bacterial mastitis, mycotic mastitis, mycoplasma infections (such as mycoplasma mastitis, mycoplasma pneumonia or the like), tuberculosis, Theileria orientalis infection, cryptosporidiosis, coccidiosis, trypanosomiasis and leishmaniasis.
The pharmaceutical composition in certain embodiments of the present disclosure comprises a COX-2 inhibitor and is administered before, after or simultaneously with the administration of an inhibitor targeting PD-1/PD-L1.
In the pharmaceutical composition in certain embodiments of the present disclosure, an inhibitor targeting PD-1/PD-L1 and a COX-2 inhibitor may be used in combination or may be formulated as a single dosage.
In the pharmaceutical composition in certain embodiments of the present disclosure comprises an anti-PD-1 antibody as an antibody drug for animals or human.
When an inhibitor targeting PD-1/PD-L1 and a COX-2 inhibitor are used in combination, the inhibitor targeting PD-1/PD-L1 and the COX-2 inhibitor may be administered separately.
When an inhibitor targeting PD-1/PD-L1 and a COX-2 inhibitor are formulated as a single dosage, a combination drug containing the inhibitor targeting PD-1/PD-L1 and the COX-2 inhibitor may be prepared.
The pharmaceutical composition of the present disclosure can be administered to human or animal subjects systemically or locally by an oral or parenteral route.
The inhibitor targeting PD-1/PD-L1 and/or the anti-PD-1 antibody of the present disclosure may be dissolved in buffers such as PBS, physiological saline or sterile water, optionally filter- or otherwise sterilized before being administered to animal subjects (including human) by injection. To the solution of inhibitors targeting PD-1/PD-L1 or the solution of the anti-PD-1 antibody, additives such as coloring agents, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonizing agents, pH adjusters and the like may be added. As routes of administration, intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal administration may be selected. Transnasal or oral administration may also be selected.
The content of the inhibitor targeting PD-1/PD-L1 in a preparation varies with the type of the preparation and is usually 1-100% by weight, preferably 50-100% by weight. Such a preparation may be formulated into a unit dosage form.
The dose and the number of times and frequency of administration of the inhibitor targeting PD-1/PD-L1 (e.g., PD-L1 antibody) may vary with the symptoms, age and body weight of the human or animal subject, the method of administration, dosage form and so on. For example, 0.1-100 mg/kg body weight, preferably 1-10 mg/kg body weight, may usually be administered per adult at least once at a frequency that enables obtainment of the desired effect.
The dose and the number of times and frequency of administration of the anti-PD-1 antibody of the present disclosure may vary depending on the symptoms, age and body weight of the animal subject, the method of administration, the dosage form and so on. For example, 0.1-100 mg/kg body weight, preferably 1-10 mg/kg body weight, per adult animal may usually be administered at least once at such a frequency that enables confirmation of desired effect.
A COX-2 inhibitor may be contained in a preparation comprising an inhibitor targeting PD-1/PD-L1. Alternatively, the COX-2 inhibitor either alone or in admixture with an excipient or carrier may be formulated into tablets, capsules, powders, granules, liquids, syrups, aerosols, suppositories, injections or the like. The excipient or carrier may be of any type that is routinely used in the art and pharmaceutically acceptable, with their type and composition being appropriately changed. As a liquid carrier, for example, water, plant oil or the like may be used. As a solid carrier, saccharides such as lactose, sucrose or glucose, starches such as potato starch or corn starch, cellulose derivatives such as microcrystalline cellulose, and the like may be used. Lubricants such as magnesium stearate, binders such as gelatin or hydroxypropyl cellulose, and disintegrants such as carboxymethyl cellulose, and the like may be added. What is more, antioxidants, coloring agents, flavoring agents, preservatives, and the like may also be added.
The COX-2 inhibitor may be administered via various routes such as oral, transnasal, rectal, transdermal, subcutaneous, intravenous or intramuscular route.
The content of the COX-2 inhibitor in a preparation varies with the type of the preparation and is usually 1-100% by weight, preferably 50-100% by weight. In the case of a liquid, for example, the content of the COX-2 inhibitor in the preparation is preferably 1-100% by weight. In the case of a capsule, tablet, granule or powder, the content of the COX-2 inhibitor in the preparation is usually 10-100% by weight, preferably 50-100% by weight, with the balance being the carrier. The preparation may be formulated into a unit dosage form.
The dose and the number of times and frequency of administration of the COX-2 inhibitor may vary with the symptoms, age and body weight of the animal or human subject, the method of administration, dosage form and so on. For example, in terms of the amount of the active ingredient, 0.05 to 20 mg (or ml)/kg body weight may usually be administered per adult at least once at a frequency that enables confirmation of the desired effect.
The ratio (in mass) of inhibitor targeting PD-1/PD-L1 to COX-2 inhibitor is appropriately from 1:100 to 1000:1, preferably from 1:10 to 100:1.
The present disclosure provides a method of preventing and/or treating cancer and/or infection, comprising administering to a human or animal subject a pharmaceutically effective amount of a COX-2 inhibitor before, after or simultaneously with the administration of an inhibitor targeting PD-1/PD-L1.
Further, the present disclosure provides use of a COX-2 inhibitor for preventing and/or treating cancer and/or infection, wherein the COX-2 inhibitor is administered before, after or simultaneously with the administration of an inhibitor targeting PD-1/PD-L1.
Still further, the present disclosure provides use of a COX-2 inhibitor for use in a method of preventing and/or treating cancer and/or infection, wherein the COX-2 inhibitor is administered before, after or simultaneously with the administration of an inhibitor targeting PD-1/PD-L1.
The immunostimulatory effect of an inhibitor targeting PD-1/PD-L1 can be enhanced by using a COX-2 inhibitor in combination. Therefore, the present disclosure provides a potentiator for the immunostimulatory effect of an inhibitor targeting PD-1/PD-L1, which comprises a COX-2 inhibitor.
The potentiator may be used in combination with an inhibitor targeting PD-1/PD-L1 or formulated together with an inhibitor targeting PD-1/PD-L1 into a combination drug. Combined use of an inhibitor targeting PD-1/PD-L1 and a COX-2 inhibitor, as well as formulating them together as a single dosage are as described above. The potentiator may be used as an experimental reagent in addition to its application as a pharmaceutical.
While the pharmaceutical composition according to certain embodiments of the present disclosure may be used alone, it may be used in combination with surgical operations, radiation therapies, other immunotherapies such as cancer vaccine, or molecular target drugs.
Synergistic effect can be expected from such combinations.
Hereinbelow, the present disclosure will be described in more detail with reference to the following Examples. However, the present disclosure is not limited to these Examples.
The interaction between PD-1 and PD-L1 is one of the major molecular mechanisms through which tumors evade immune responses. It has been reported that inhibition of the above interaction by using an antibody which specifically binds to either of those molecules can produce antitumor effects. In the subject Example, toward establishment of a novel control method for canine tumors, the present inventors have confirmed in in vitro tests an immunostimulatory effect induced by a COX-2 inhibitor and enhancement of that effect when the inhibitor is used in combination with anti-PD-L1 antibody.
2.1. PGE2 Production from Canine Tumor Cell Lines
Canine melanoma-derived cell lines of CMeC and LMeC (Ohashi E, Inoue K, Kagechika H, Hong S H, Nakagawa T, et al: Effect of natural and synthetic retinoids on the proliferation and differentiation of three canine melanoma cell lines. J Vet Med Sci 64: 169-172, 2002) as well as CMM-1 and CMM-2 (Ohashi E, Hong S H, Takahashi T, Nakagawa T, Mochizuki M, et al.: Effect of retinoids on growth inhibition of two canine melanoma cell lines. J Vet Med Sci 63: 83-86, 2001) were cultured in RPMI 1640 medium (Sigma) supplemented with 2-mercaptoethanol 2×10−5 M, 10% inactivated fetal bovine serum (Valley Biomedical), antibiotics (streptomycin 100 μg/ml, penicillin 100 U/ml) (Invitrogen) and 2 mM L-glutamine (Invitrogen) at 37° C. in the presence of 5% CO2. A canine osteosarcoma-derived cell line HM-POS (Barroga E F, Kadosawa T, Okumura M, Fujinaga T: Establishment and characterization of the growth and pulmonary metastasis of a highly lung metastasizing cell line from canine osteosarcoma in nude mice. J Vet Med Sci 61: 361-367, 1999) was cultured in Dulbecco's Modified Eagle Medium (D-MEM; Invitrogen) supplemented with 10% inactivated fetal bovine serum (Valley Biomedical), antibiotics (streptomycin 100 μg/ml, penicillin 100 U/ml) (Invitrogen) and 2 mM L-glutamine (Invitrogen) at 37° C. in the presence of 5% CO2. Cells adjusted to a density of 5×105 cells/mL were cultured for 24 hours. The amount of PGE2 in the culture supernatant was quantified with Prostaglandin E2 Express EIA Kit (Cayman Chemical). As a result, CMM-1 and HM-POS showed a relatively high PGE2 production (
From the canine tumor-derived cell lines cultured as described in section 2.1. of Materials and Methods, RNA was extracted with TRI reagent (Molecular Research Center) and the concentration thereof was measured with NanoDrop8000 (Thermo Scientific). RNA samples were stored at −80° C. until use in experiments.
To 1 μg of the thus obtained RNA, DNase I Reaction buffer and 1 U DNase I Amplification Grade (Invitrogen) were added. Then, deionized distilled water was added to make a 10 μl solution, which was subjected to DNase I treatment at room temperature for 15 mn. Subsequently, 25 nmol ethylenediaminetetraacetic acid (EDTA) was added and the resultant mixture was treated at 65° C. for 10 min. Then, 200 pmol oligo-dT primer was added to the reaction mixture which was treated at 65° C. for 5 min. Thereafter, reverse transcription reaction solution [PrimeScript Buffer (TaKaRa), 7.5 nmol dNTPs, 20 U RNase Plus RNase inhibitor (Promega), 100 U PrimeScript RTase (TaKaRa)] was added to give a final volume of 20 μl. Reverse transcription reaction was carried out at 42° C. for 60 min to thereby synthesize a single-stranded cDNA.
Primers (canine COX2 rt F and canine COX2 rt R; canine HPRT1 rt F and canine HPRT1 rt R) were designed based on the nucleotide sequences of canine COX2 (NM_001003354.1) and canine HPRT1 (AY283372.1) registered at the National Center for Biotechnology Information (NCBI), and real-time PCR was performed. Using 1 μl of the cDNA of each tumor-derived cell line as a template, real-time PCR was performed with LightCycler480 System II (Roche) in a PCR reaction mixture containing 0.3 μl each of primers canine COX2 rt F and canine COX2 rt R or canine HPRT1 rt F and canine HPRT1 rt R (each of which had been adjusted to a concentration of 10 pmol/μl), 5 μl of SYBR Premix DimerEraser (TaKaRa) and 3.4 μl of DDW under the conditions described below.
The resultant COX2 mRNA expression level was divided by the expression level of internal control gene HPRT1 mRNA, and the thus obtained value was taken as COX2 expression level. As it turned out, COX2 expression level was high in CMM-1 and HM-POS, consistent with the results of PGE2 production in culture supernatant (
2.3. Effect of PGE2 on Cytokine Production from Canine Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized canine peripheral blood samples collected from healthy beagles and mixed breed dogs by density gradient centrifugation using Percoll (GE Healthcare). The resultant PBMCs were cultured in RPMI 1640 medium (Sigma), supplemented with 10% inactivated fetal bovine serum (Valley Biomedical), antibiotics (streptomycin 100 μg/ml, penicillin 100 U/ml) (Invitrogen) and 2 mM L-glutamine (Invitrogen) and further supplemented with 5 μg/ml of Staphylococcal Enterotoxin B (SEB) (Sigma) and 1 μg/ml of Anti-Canine CD28 (eBioscience), at 37° C. in the presence of 5% CO2 for 3 days. Production of interleukin 2 (IL-2) and interferon γ (IFN-γ) into the culture supernatant upon addition of prostaglandin E2 (Cayman Chemical) at a final concentration of 2.5 μM was measured with Canine IL-2 DuoSet ELISA (R&D systems) and Canine IFN-gamma DuoSet ELISA (R&D systems). PGE2 significantly decreased IL-2 and IFN-γ productions from canine PBMCs (
Canine tumor cell lines CMM-1 and HM-POS were cultured as described in section 2.1 of Materials and Methods, and meloxicam (Sigma) was added to give a final concentration of 5 μM. PGE2 production from each tumor cell line was quantified by ELISA. PGE2 production showed a tendency to decrease as a result of addition of meloxicam (
2.5. Effect of COX-2 Inhibitor on Cytokine Production from Canine Peripheral Blood Mononuclear Cells
Canine PBMCs were cultured as described in section 2.3 of Materials and Methods, and meloxicam (Sigma) was added to give a final concentration of 5 μM. Then, IL-2 concentration in the culture supernatant was quantified by ELISA. IL-2 production from canine PBMCs was increased significantly as a result of addition of meloxicam (
Canine PBMCs were cultured as described in section 2.3 of Materials and Methods. To the resultant PBMCs, rat-canine chimeric anti-PD-L1 antibody c4G12 (Maekawa et al., data in submission; see Reference Example 1 described below) and meloxicam (Sigma) were added to give final concentrations of 20 μg/mL and 5 μM, respectively. Subsequently, IL-2 concentration in the culture supernatant was quantified by ELISA. Although the PD-L1 antibody taken alone increased IL-2 production, combined use of meloxicam further increased IL-2 production (
Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance. Recently, it has been elucidated that these molecules are also involved in immunosuppression in tumors. In the subject Example, for the purpose of establishing a novel therapy for canine neoplastic diseases, a chimeric antibody gene was prepared in which a variable region gene of a rat anti-bovine PD-L1 monoclonal antibody (4G12) capable of inhibiting the binding of canine PD-1 to PD-L1 was linked to a constant region gene of a canine immunoglobulin (IgG4). The resultant chimeric antibody gene was introduced into Chinese hamster ovary cells (CHO cells), which were cultured to produce a rat-canine chimeric anti-PD-L1 antibody c4G12. The effect of this chimeric antibody was confirmed in vitro and in vivo.
The nucleotide sequence of bovine PD-L1 was identified (Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K. Vet Res. 2011 Sep. 26; 42:103). Based on the sequence information, a recombinant bovine PD-L1 was prepared. Rat was immunized with this recombinant protein to prepare a rat anti-bovine PD-L1 antibody (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology. 2014 August; 142(4):551-61; Clone 4G12 which would later serve as the variable region of the canine chimeric antibody of interest is described in this article.)
To determine the full lengths of canine PD-1 and PD-L1 cDNAs, PCR primers were first designed based on the putative nucleotide sequences of canine PD-1 and PD-L1 already registered at The National Center for Biotechnology Information (NCBI) (GenBank accession number; XM_543338 and XM_541302). Briefly, primers to amplify the inner sequence of the open reading frame (ORF) of each gene were designed (cPD-1 inner F and R, cPD-L1 inner F and R), and PCR was performed. For the amplified products, nucleotide sequences were determined with a capillary sequencer according to conventional methods. Further, to determine the nucleotide sequences of full-length PD-1 and PD-L1 cDNA, primers (cPD-1 5′ GSP and 3′ GSP; cPD-L1 5′ GSP and 3′GSP) were designed based on the canine PD-1 and PD-L1 cDNA sequences determined above. 5′-RACE and 3′-RACE were then performed using the 5′-RACE system for rapid amplification of cDNA ends and 3′-RACE system for rapid amplification of cDNA ends (Invitrogen), respectively. The resultant gene fragments of interest were sequenced as described (Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415).
For preparing canine PD-1-EGFP and PD-L1-EGFP expression plasmids, PCR was performed using a synthesized beagle PBMC-derived cDNA as a template and primers designed by adding XhoI and BamHI recognition sites (PD-1) and BglII and EcoRI recognition sites (PD-L1) on the 5′ side (cPD-1-EGFP F and R; cPD-L1-EGFP F and R). The resultant PCR products were digested with XhoI (Takara) and BamHI (Takara) (PD-1) and with BglII (New England Biolabs) and EcoRI (Takara) (PD-L1), and then purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics), followed by cloning into pEGFP-N2 vector (Clontech) treated with restriction enzymes in the same manner. The resultant expression plasmids of interest were extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmids are designated as pEGFP-N2-cPD-1 and pEGFP-N2-cPD-L1.
COS-7 cells were subcultured at a density of 5×104 cells/cm2 in 6-well plates, and then cultured overnight in RPMI 1640 medium containing 10% inactivated fetal bovine serum and 0.01% L-glutamine at 37° C. in the presence of 5% CO2. The pEGFP-N2-cPD-1, pEGFP-N2-cPD-L1 or pEGFP-N2 (negative control) was introduced into COS-7 cells at 0.4 μg/cm2 using Lipofectamine 2000 (Invitrogen). The cells were cultured for 48 hours (cPD-1-EGFP expressing cell and cPD-L1-EGFP expressing cell). In order to confirm the expression of canine PD-1 and PD-L1 in the thus prepared expressing cells, intracellular localization of enhanced green fluorescent protein (EGFP) was visualized with an inverted confocal laser microscope LSM700 (ZEISS) (Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415).
In order to amplify the extracellular regions of canine PD-1, PD-L1 and CD80 estimated from their putative amino acid sequences, primers were designed. Briefly, primers having an NheI or EcoRV recognition sequence (PD-1 and PD-L1) added on the 5′ side (cPD-1-Ig F and R; cPD-L1-Ig F and R) or having an EcoRV or KpnI (CD80) recognition sequence added on the 5′ side (cCD80-Ig F and R) were designed. PCR was performed using a synthesized beagle PBMC-derived cDNA as a template. The PCR products were digested with NheI (Takara) and EcoRV (Takara) or with EcoRV (Takara) and KpnI (New England Biolabs) and purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics). The thus purified DNAs were individually cloned into pCXN2.1-Rabbit IgG Fc vector (Niwa et al., 1991; Zettlmeissl et al., 1990; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine, and modified in the inventors' laboratory) treated with restriction enzymes in the same manner. The expression plasmids were purified with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmids are designated as pCXN2.1-cPD-1-Ig, pCXN2.1-cPD-L1-Ig and pCXN2.1-cCD80-Ig, respectively.
These expression vectors were individually transfected into Expi293F cells (Life Technologies) to obtain a culture supernatant containing a recombinant Ig fusion protein. The recombinant protein produced was purified from the supernatant with Ab Capcher Extra (Protein A mutant; ProteNova). After buffer exchange with phosphate-buffered physiological saline (PBS; pH 7.4) using PD-MidiTrap G-25 (GE Healthcare), each recombinant protein was stored at −30° C. until use in experiments (cPD-1-Ig, cPD-L1-Ig and cCD80-Ig). The concentration of each protein was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) before use in subsequent experiments.
2.5 Identification of Rat Anti-Bovine PD-L1 Monoclonal Antibody Showing Cross-Reactivity with Canine PD-L1
In order to identify rat anti-bovine PD-L1 monoclonal antibody showing cross-reactivity with canine PD-L1, flow cytometry was performed using the anti-bovine PD-L1 antibody prepared in 2.1 above. The anti-bovine PD-L1 antibody (10 μg/ml) was reacted with 2×105-1×106 cells at room temperature for 30 min. After washing, the anti-bovine PD-L1 antibody was detected with allophycocyanine-labeled anti-rat Ig goat antibody (Beckman Coulter). FACS Verse (Becton, Dickinson and Company) was used for analysis. As negative controls, rat IgG2a (κ) isotype control (BD Biosciences), rat IgG1 (κ) isotype control (BD Biosciences) and rat IgM (κ) isotype control (BD Biosciences) were used. For every washing operation and dilution of antibodies, 10% inactivated goat serum-supplemented PBS was used (MaekawaN, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, KagawaY, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415 which is an article describing the use of three bovine PD-L1 monoclonal antibodies: 4G12 (Rat IgG2a (κ)), 5A2 (Rat IgG1 (κ)) and 6G7 (Rat IgM (κ)).
Out of 10 clones of rat anti-bovine PD-L1 monoclonal antibody which showed cross-reactivity with canine PD-L1, 4G12 (Rat IgG2a (κ)), 5A2 (Rat IgG1 (κ)) and 6G7 (Rat IgM (κ)) were selected and check was made to see whether these antibodies would inhibit canine PD-1/PD-L1 binding. Briefly, canine PD-1-Ig (prepared in 2.4 above) was immobilized on flat bottomed 96-well plates and blocked with 1% BSA and 0.05% Tween 20-containing PBS. Canine PD-L1-Ig (prepared in 2.4 above) was biotinylated using Lightning-Link Biotin Conjugation Kit (Innova Bioscience) and reacted with various concentrations (0, 2.5, 5 and 10 μg/ml) of rat anti-bovine PD-L1 antibodies 4G12, 5A2 and 6G7 at 37° C. for 30 min, followed by addition to the 96-well plates. The binding of cPD-L1-Ig to cPD-1-Ig was measured by color reaction using Neutravidin-HRP (Thermo Fisher Scientific) and TMB one component substrate (Bethyl Laboratories). As a result, rat anti-bovine PD-L1 monoclonal antibodies 4G12 and 6G7 showed a good inhibitory activity against canine PD-1/PD-L1 binding, whereas 5A2 showed no binding inhibitory activity (
Using rat anti-bovine PD-L1 monoclonal antibodies 4G12 and 6G7 which showed a good inhibitory activity against canine PD-1/PD-L1 binding (
2.8 Expression of Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12
Rat-canine chimeric anti-PD-L1 antibody c4G12 expressing vector pDC6 as used in 2.7 above was transfected into CHO-DG44 cells (CHO-DG44(dfhr−/−)) which were dihydrofolate reductase deficient cells and high expression clones were selected by dot blotting. Further, gene amplification treatment was performed by adding load on cells in a medium containing 60 nM methotrexate (Mtx). Cells stably expressing rat-canine chimeric anti-PD-L1 antibody c4G12 (clone name: 4.3F1) after gene amplification were transferred to Mtx-free Opti-CHO medium and cultured under shaking for 14 days (125 rpm, 37° C., 5% CO2). Cell survival rate was calculated by trypan blue staining (
It should be noted that by exchanging the medium with Dynamis medium and doing appropriate feeding, antibody production was improved about two-fold compared to the conventional production (data not shown).
2.9 Purification of Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12
The culture supernatant provided as described above was purified with Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; PBS pH 7.4 was used as equilibration buffer and wash buffer. As elution buffer, IgG Elution Buffer (Thermo Scientific) was used. As neutralization buffer, 1 M Tris was used. The purified antibody was concentrated and buffer-exchanged with PBS by ultrafiltration using Amicon Ultra-15 (50 kDa, Millipore). The resultant antibody was passed through a 0.22 μm filter for use in respective experiments.
2.10 Confirmation of Purification of Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12 (
In order to confirm the purity of the purified antibody, antibody proteins were detected by SDS-PAGE and CBB staining. Using SuperSep Ace 5-20% (Wako) gradient gel, rat anti-bovine PD-L1 monoclonal antibody 4G12 and rat-canine chimeric anti-PD-L1 antibody c4G12 were electrophoresed under reducing conditions and non-reducing conditions. Bands were stained with Quick-CBB kit (Wako) and decolored in distilled water. Bands were observed at positions of molecular weights corresponding to antibodies. No bands of contaminant proteins were recognized visually.
2.11 Measurement of Binding Avidities to cPD-L1-His of Rat Anti-Bovine PD-L1 Monoclonal Antibody 4G12 and Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12
In order to amplify the extracellular region of canine PD-L1 estimated from its putative amino acid sequence, primers were designed. Briefly, a primer having an NheI recognition sequence added on the 5′ side (cPD-L1-His F) and a primer having an EcoRV recognition sequence and 6xHis tag sequence added on the 5′ side (cPD-L1-His R) were designed. PCR was performed using a synthesized beagle PBMC-derived cDNA as a template. The PCR products were digested with NheI (Takara) and EcoRV (Takara) and purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics). The thus purified DNA was cloned into pCXN2.1 vector (Niwa et al., 1991; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine) treated with restriction enzymes in the same manner. The expression plasmids were purified with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as CXN2.1-cPD-L1-His.
The expression vector was transfected into Expi293F cells (Life Technologies) to obtain a culture supernatant containing a recombinant protein. The recombinant protein produced was purified from the supernatant using TALON Metal Affinity Resin (Clontech), and the buffer was exchanged with PBS using Amicon Ultra-4 Ultracel-3 (Merck Millipore). The thus obtained recombinant protein was stored at 4° C. until use in experiments (cPD-L1-His). The protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) for use in subsequent experiments.
Using a biomolecular interaction analyzer (Biacore X100), the binding avidities to cPD-L1-His of rat anti-bovine PD-L1 monoclonal antibody 4G12 and rat-canine chimeric anti-PD-L1 antibody c4G12 were assessed. Briefly, anti-histidine antibody was fixed on CM5 censor chip, followed by capturing of cPD-L1-His. Subsequently, monoclonal antibodies were added as analyte to observe specific binding. Both antibodies exhibited specific binding and their avidities were almost comparable (Table 1). Further, the binding avidities of canine PD-1-Ig and CD80-Ig to cPD-L1-His were measured in the same manner and found to be clearly lower than that of rat-canine chimeric anti-PD-L1 antibody c4G12 (Table 1).
2.12 Inhibitory Activity of Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12 against Canine PD-1/PD-L1 Binding and CD80/PD-L1 Binding (
Using the canine PD-1-Ig, PD-L1-Ig and CD80-Ig (described above), anti-PD-L1 antibody was tested for its ability to inhibit canine PD-1/PD-L1 binding and CD80/PD-L1 binding. Briefly, canine PD-1-Ig or CD80-Ig was immobilized on flat-bottom 96-well plates. Canine PD-L1-Ig was reacted with various concentrations (0, 2.5, 5 and 10 μg/ml) of rat anti-bovine PD-L1 antibody 4G12 or rat-canine chimeric anti-PD-L1 antibody c4G12 according to the same procedures as described in 2.6 above, and the binding of canine PD-L1-Ig was assessed. No change in binding inhibition activity was observed due to the chimerization of antibody.
2.13. Canine Immune Cell Activating Effect of Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12 (
Canine PBMCs were cultured under stimulation with a superantigen Staphylococcal Enterotoxin B (SEB) for three days, and changes in cytokine production by addition of rat-canine chimeric anti-PD-L1 antibody c4G12 were measured by ELISA using Duoset ELISA canine IL-2 or IFN-γ (R&D systems). Rat-canine chimeric anti-PD-L1 antibody c4G12 increased the production of IL-2 and IFN-γ from canine PBMCs. Further, nucleic acid analogue EdU was added to the culture medium at day 2 of the culture under SEB stimulation. Two hours later, uptake of EdU was measured by flow cytometry using Click-iT Plus EdU flow cytometry assay kit (Life Technologies). As a result, EdU uptake in canine CD4+ and CD8+ lymphocytes was enhanced by addition of rat-canine chimeric anti-PD-L1 antibody c4G12, indications an elevated cell proliferation capacity.
Since the subject treatment is expected to manifest a higher efficacy when PD-L1 is being expressed in tumors, PD-L1 expression analysis at the tumor site of dogs was performed by immunohistochemical staining. Briefly, tumor tissue samples fixed with formaldehyde and embedded in paraffin were sliced into 4 μm thick sections with a microtome, attached to and dried on silane-coated slide glass (Matsunami Glass) and deparaffinized with xylene/alcohol. While the resultant sections were soaked in citrate buffer [citric acid (Wako Pure Chemical) 0.37 g, trisodium citrate dihydrate (Kishida Chemical) 2.4 g, distilled water 1000 ml], antigen retrieval treatment was performed for 10 min with microwave, followed by staining using a Nichirei automatic immuno-staining device. As pretreatment, sample sections were soaked in 0.3% hydrogen peroxide-containing methanol solution at room temperature for 15 min and washed with PBS. Then, anti-bovine PD-L1 monoclonal antibody was added and reaction was conducted at room temperature for 30 min. After washing with PBS, histofine simple stain MAX-PO (Rat) (Nichirei Bioscience) was added and reaction was carried at room temperature for 30 min, followed by coloring with 3,3′-diaminobenzidine tetrahydrocholride and observation with a light microscope. Dogs with oral melanoma or undifferentiated sarcoma in which tumor cells were PD-L1 positive were used in the following inoculation test (clinical trial). Anti-bovine PD-L1 monoclonal antibody was established from a rat anti-bovine PD-L1 monoclonal antibody producing hybridoma (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology. 2014 August; 142(4):551-61).
With respect to the rat-canine chimeric anti-PD-L1 antibody c4G12 to be inoculated into dogs in the clinical trial, the culture supernatant obtained by the procedures described in 2.8 above was purified by affinity chromatography using MabSelect SuRe LX (GE Healthcare) and then by hydroxyapatite chromatography using BioScale CHT20-I prepacked column (Bio-Rad) in order to remove contaminants and polymeric proteins. Aggregate-containing fractions were further purified by anion exchange chromatography using HiScreen Q-Sepharose HP prepacked column (GE Healthcare).
(1) Safety Test: The established rat-canine chimeric anti-PD-L1 antibody c4G12 was administered intravenously into a dog (beagle, spayed female, 13-year-old, about 10 kg in body weight) at 2 mg/kg, every 2 weeks, 3 times in total. There was observed no anaphylaxis or other adverse effects that would cause any trouble in clinical trials. (2) Clinical Trial 1: The established rat-canine chimeric anti-PD-L1 antibody c4G12 was administered intravenously into a PD-L1 positive dog with relapsed oral melanoma (
(3) Clinical Trial 2: Rat-canine chimeric anti-PD-L1 antibody c4G12 was administered intravenously into a dog with undifferentiated sarcoma whose primary lesion was PD-L1 positive (
(4) Clinical Trial 3: Rat-canine chimeric anti-PD-L1 antibody c4G12 was administered intravenously into a dog with oral melanoma whose primary lesion had been removed by surgery (beagle, spayed female, 11-year-old, about 10 kg in body weight) at 2 mg/kg or 5 mg/kg, every 2 weeks, 9 times in total. At week 18 after the start of treatment, a plurality of pulmonary metastatic lesions disappeared (
(5) Clinical Trial 4: Rat-canine chimeric anti-PD-L1 antibody c4G12 was administered intravenously into 4 dogs with oral melanoma with pulmonary metastasis at 2 mg/kg or 5 mg/kg, every 2 weeks. Although no clear reduction in tumor size was observed during the observation period, the duration of the treated dogs' survival after confirmation of pulmonary metastasis tended to be longer than that of a control group (antibody not administered, historical control group: n=15) (
The complementarity-determining regions (CDRs) of rat anti-bovine PD-L1 antibody 4G12 were determined using NCBI IGBLAST (http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in
Examination of Combined Effect of Anti-Bovine PD-L1 Antibody and COX-2 Inhibitor in Cattle with Johne's Disease
The interaction between PD-1 and PD-L1 is one of the major molecular mechanisms through which pathogens evade immune responses. It has been reported that inhibition of the above interaction by using an antibody which specifically binds to either of those molecules can produce anti-pathogenic effects. In the subject Example, toward establishment of a novel control method against Johne's disease, the present inventors have confirmed in in vitro tests an immunostimulatory effect induced by a COX-2 inhibitor and enhancement of that effect when the inhibitor is used in combination with anti-bovine PD-L1 antibody.
In order to examine the immunosuppressive effects of PGE2 in cattle, the present inventors evaluated how PBMCs derived from uninfected cattle stimulated with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody changed in proliferation capacity and cytokine production capacity as well as in expression levels of cytokine and transcription factor genes and PD-L1 in the presence of PGE2.
Peripheral blood mononuclear cells (PBMCs) derived from cattle not infected with Mycobacterium avium subsp. paratuberculosis (hereinafter, referred to as “uninfected cattle”) were seeded in 96-well plates (Corning) at 4×105 cells/well. The cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific), antibiotics (streptomycin 100 μg/ml, penicillin 100 U/ml) (Thermo Fisher Scientific) and 2 mM L-glutamine (Thermo Fisher Scientific) for 3 days at 37° C. in the presence of 5% CO2. The PBMCs were labeled with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE, Invitrogen). To the medium, 10-fold serially diluted PGE2 (from 2.5 nM to 2,500 nM) (Cayman Chemical) or, as a negative control, phosphate buffered physiological saline (PBS, pH 7.2, Wako Pure Chemical) was added to give a total volume of 200 μl. As stimulants for T cells, 1 μg/ml of anti-CD3 monoclonal antibody (MM1A; Washington State University Monoclonal Antibody Center) and 1 μg/ml of anti-CD28 monoclonal antibody (CC220; Bio-Rad) were added. After culturing, PBMCs were harvested and analyzed by flow cytometry. In order to prevent non-specific reactions, PBS supplemented with 10% inactivated goat serum (Thermo Fisher Scientific) was added to each well at 100 μl/well, and left stationary at room temperature for 15 min. After washing, Alexa Fluor 647-labeled anti-CD4 monoclonal antibody (CC30; Bio-Rad), peridinin-chlorophyll-protein complex/cyanin 5.5 (PerCp/Cy 5.5)-labeled anti-CD8 monoclonal antibody (CC63; Bio-Rad) and phycoerythrin/cyanin 7 (PE/Cy7)-labeled anti-IgM monoclonal antibody (IL-A30; Bio-Rad) were reacted at room temperature for 20 min. The anti-CD4 monoclonal antibody (CC30) was labeled with Alexa Fluor 647 using Zenon Mouse IgG1 labeling Kits (Thermo Fisher Scientific). The anti-CD8 monoclonal antibody (CC63) and the anti-IgM monoclonal antibody (IL-A30) were labeled with PerCp/Cy5.5 and PE/Cy7, respectively, using Lightning-Link Conjugation Kit (Innova Biosciences). After reaction, washing was performed twice. Then, cell proliferation was analyzed with FACS Verse (BD Biosciences) and FCS Express 4 (De Novo Software). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma Aldrich) was used.
PBMCs derived from uninfected cattle were seeded in 96-well plates at 4×105 cells/well and cultured in the same manner as described in (1) above for 3 days (Note: analysis of TNF-α production was performed only for the cells cultured under stimulation with 2,500 nM PGE2). After 3 days, the culture supernatant was collected. IFN-γ production was measured with ELISA for Bovine IFN-γ (MABTECH), and TNF-α production was measured with Bovine TNF alpha Do-It-Yourself ELISA (Kingfisher Biotech). For the measurement, absorbance at 450 nm was measured using a microplate reader MTP-900 (Corona Electric).
Experimental results of (1) and (2) above are shown in
(3) Changes in mRNA Expression Levels of Cytokines, etc. Induced by PGE2
PBMCs derived from uninfected cattle were seeded in 96-well plates at 1×106 cells/well and cultured for 3 days in the presence of 2,500 nM PGE2 or DMSO. Total cellular RNA was extracted from the thus cultured PBMCs using TRI reagent (Molecular Research Center), and cDNA was synthesized with PrimeScript Reverse Transcriptase (TaKaRa) and Oligo-dT primers. Using the synthesized cDNA as a template, real-time PCR was performed with LightCycler480 System II (Roche) in a 10 μl reaction solution containing SYBR Premix DimerEraser (TaKaRa) and 3 pmol each of the primers specific to individual genes. Then, changes in expression levels of individual genes were observed.
Reaction conditions of the real-time PCR were as described below.
Experimental results of (3) are shown in
PBMCs derived from uninfected cattle were seeded in 96-well plates at 1×106 cells/well and cultured for 24 hours under the same conditions as described in (1) above. Total cellular RNA was extracted from the cultured PBMCs, and cDNA was synthesized in the same manner as described in (3) above. Then, real-time PCR was performed using PDL1 specific primers. Primer (boPDL1 F): GGG GGT TTA CTG TTG CTT GA (SEQ ID NO: 137) Primer (boPDL1 R): GCC ACT CAG GAC TTG GTG AT (SEQ ID NO: 138) Further, expression of PD-L1 protein in the cultured PBMCs was analyzed by flow cytometry. Briefly, PBS supplemented with 10% inactivated goat serum (Thermo Fisher Scientific) was added to harvested PBMCs at 100 μl/well and left stationary at room temperature for 15 min. After washing, rat anti-bovine PD-L1 antibody (4G12; Rat IgG2a; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) or rat IgG2a isotype control (BD Bioscience) was added and reaction was conducted at room temperature for 20 min. After washing twice, allophycocyanin (APC)-labeled anti-rat Ig antibody (Southern Biotech) was added and reaction was conducted at room temperature for 20 min. After washing twice, expression of PD-L1 protein was analyzed with FACS Verse (BD Biosciences) and FCS Express 4 (De Novo Software). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma Aldrich) was used.
Experimental results of (3) are shown in
In order to examine immunostimulatory effects of COX-2 inhibitor in cattle, a COX-2 inhibitor meloxicam was added to a PBMC culture test under stimulation with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody, followed by evaluation of the proliferation capacity and cytokine production capacity of PBMCs derived from uninfected cattle.
PBMCs derived from uninfected cattle were seeded in 96-well plates at 4×105 cells/well and cultured for 3 days in the presence of 1,000 nM meloxicam (Sigma-Aldrich) or DMSO as a negative control. As stimulants for T cells, 1 μg/ml of anti-CD3 monoclonal antibody (MM1A; Washington State University Monoclonal Antibody Center) and 1 μg/ml of anti-CD28 monoclonal antibody (CC220; Bio-Rad) were added. After 3 days, cell proliferation capacity and cytokine production were evaluated in the same manner as described in (1) and (2) in section 2.1 above.
Experimental results are shown in
2.3. Kinetic Analysis of PGE2 in Cattle Infected with M. avium Subsp. Paratuberculosis
In order to elucidate the relation between bovine chronic infections and PGE2, the present inventors performed kinetic analysis of PGE2 in cattle infected with M. avium subsp. paratuberculosis.
First, PGE2 contained in serum derived from cattle that developed Johne's disease from natural infection and PGE2 contained in serum derived from uninfected cattle were quantified by ELISA. Briefly, the amount of PGE2 contained in serum derived from cattle that naturally developed Johne's disease (kindly provided by Dr. Yasuyuki Mori, National Institute of Animal Health, National Agriculture and Food Research Organization) was measured with Prostaglandin E2 Express ELISA Kit (Cayman Chemical). For the measurement, absorbance at 450 nm was measured with a microplate reader MTP-900 (Corona Electric).
Experimental results of (1) are shown in
(2) Changes in PGE2 Production by M. avium subsp. paratuberculosis Antigen Stimulation
In order to confirm that PGE2 production is promoted by M. avium subsp. paratuberculosis antigen, PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis and those derived from uninfected cattle were cultured with M. avium subsp. paratuberculosis antigen, and PGE2 in culture supernatants was quantified by ELISA. Briefly, PBMCs derived from experimentally infected cattle and those from uninfected cattle were seeded in 96-well plates at 4×105 cells/well and cultured for 5 days in the presence of 1 μg/ml of M. avium subsp. paratuberculosis antigen. As the M. avium subsp. paratuberculosis antigen, Johnin Purified Protein Derivative (J-PPD) was used. Further, in order to confirm that PGE2 production by antigen stimulation is inhibited by COX-2 inhibitor, 1 μg/ml of J-PPD and 1000 nM meloxicam (Signa-Aldrich) were added to the medium. After 5 days, the culture supernatant was collected, and PGE2 contained therein was quantified with Prostaglandin E2 Express ELISA Kit (Cayman Chemical).
Experimental results of (2) are shown in
PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis were seeded in 96-well plates at 1×106 cells/well and cultured in the presence of J-PPD for 24 hours. After culturing, PBMCs were harvested and total cellular RNA was extracted as described above. Then, cDNA was synthesized. Using the synthesized cDNA as a template, real-time PCR was performed with COX2 specific primers in the same manner as described above.
Experimental results of (3) are shown in
Effects of J-PPD stimulation on PD-L1 expression in cattle infected with M. avium subsp. paratuberculosis were evaluated. PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis and those derived from uninfected cattle were seeded in 96-well plates at 1×106 cells/well and cultured for 24 hours in the presence of J-PPD. Cultured PBMCs were harvested, and then PD-L1 expression on lymphocytes, CD4+ T cells, CD8+ T cells, IgM+ cells and CD14+ cells was analyzed by flow cytometry. Briefly, PBS supplemented with 10% inactivated goat serum (Thermo Fisher Scientific) was added to each well in a volume of 100 μl and cells were left stationary at room temperature for 15 min. After washing, rat anti-bovine PD-L1 antibody (4G12; Rat IgG2a; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) or rat IgG2a isotype control (BD Bioscience) was added and reaction was conducted at room temperature for 20 min. After washing twice, secondary antibodies were added and reaction was conducted at room temperature for 20 min. As secondary antibodies, phycoerythrin (PE)-labeled anti-CD3 monoclonal antibody (MM1A; Washington State University Monoclonal Antibody Center), fluorescein isothiocyanate (FITC)-labeled anti-CD4 monoclonal antibody (CC8; Bio-Rad), PerCp/Cy 5.5-labeled anti-CD8 monoclonal antibody (CC63; Bio-Rad), PE/Cy7-labeled anti-IgM monoclonal antibody (IL-A30; Bio-Rad) and APC-labeled anti-rat Ig antibody (Southern Biotech) were used for analysis of PD-L1 expression on T cells and IgM+ cells. Anti-CD3 monoclonal antibody (MM1A) was labeled with PE using Zenon Mouse IgG1 labeling Kit. For analysis of PD-L1 expression on CD14+ cells, PerCp/Cy5.5-labeld anti-CD14 monoclonal antibody (CAM36A; Washington State University Monoclonal Antibody Center) and APC-labeled anti-rat Ig antibody (Southern Biotech) were used. Anti-CD14 monoclonal antibody (CAM36A) was labeled with PerCp/Cy5.5 using Lightning-Link Conjugation Kit. After reaction, washing was conducted twice. Then, PD-L1 expression was analyzed with FACS Verse (BD Biosciences) and FCS Express 4 (De Novo Software). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma Aldrich) was used.
Experimental results are shown in
Subsequently, the present inventors performed expression analyses of PGE2, EP2 and PD-L1 in Johne's disease lesions by immunohistochemical staining. Briefly, ilium tissue blocks from cattle which naturally developed Johne's disease (#1, presenting clinical symptoms of Johne's disease such as diarrhea and severe emaciation), cattle experimentally infected with M. avium subsp. paratuberculosis (#65, clinical symptoms such as shedding of M. avium subsp. paratuberculosis and diarrhea were observed; Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S, Tanaka S, KagawaY, Murata S, Mori Y and Ohashi K., Infect Immun, 84:77-89, 2016) and uninfected control cattle (C#6) (those blocks were kindly provided by Dr. Yasuyuki Mori, National Institute of Animal Health, National Agriculture and Food Research Organization) were used for immunohistochemical staining. Samples fixed with 4% paraformaldehyde [paraformaldehyde 20 g, PBS (pH 7.4) 500 ml] and embedded in paraffin were sliced into 4 mm thick sections with a microtome, attached to and dried on silane-coated slide glass (Matsunami Glass) and deparaffinized with xylene/alcohol. While the resultant sections were soaked in citrate buffer (citric acid 0.37 g, trisodium citrate dihydrate 2.4 g, distilled water 1000 ml), antigen retrieval treatment was performed for 10 min with microwave, followed by staining using a Nichirei automatic immuno-staining device. As pretreatment, sample sections were soaked in 0.3% hydrogen peroxide-containing methanol solution at room temperature for 15 min and washed with PBS. Then, anti-PGE2-polyclonal antibody (Abcam), anti-EP2 monoclonal antibody (EPR8030(B); Abcam) or rat anti-bovine PD-L1 monoclonal antibody (6C11-3A11; Rat IgG2a; Japanese Patent Application No. 2017-61389, Konnai S, Ohashi K, Murata S, Okagawa T, Nishimori A, Maekawa N, Takagi S, Kagawa Y, Suzuki S, Nakajima C, titled “Anti-PD-L1 Antibody for Detecting PD-L1”) was added and reaction was conducted at room temperature for 30 min. After washing with PBS, histofine simple stain MAX-PO (Nichirei Bioscience) was added and reaction was carried out at room temperature for 30 min, followed by coloring with 3,3′-diaminobenzidine tetrahydrocholride and observation with a light microscope.
Experimental results are shown in
2.6. Examination of Stimulatory Effects of COX-2 Inhibitor on M. avium subsp. paratuberculosis-Specific Immune Responses
In order to confirm that COX-2 inhibitor has stimulatory effects on M. avium subsp. paratuberculosis-specific immune responses, the present inventors cultured PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis in the presence of added meloxicam and J-PPD and evaluated their proliferation capacity and cytokine production capacity. Briefly, PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis were seeded in 96-well plates at 4×105 cells/well and cultured for 5 days under stimulation in the presence of 1 μg/ml of J-PPD and 1000 nM meloxicam (Sigma-Aldrich). After culturing, cell proliferation capacity and cytokine production capacity were evaluated in the same manner as described above.
Experimental results are shown in
2.7. Examination of Immunostimulatory Effects of Rat Anti-Bovine PD-L1 Antibody in M avium subsp. paratuberculosis-Infected Cattle
In order to confirm that rat anti-bovine PD-L1 antibody also has immunostimulatory effects in M. avium subsp. paratuberculosis-infected cattle, the present inventors performed a PBMC culture test under stimulation in the presence of rat anti-bovine PD-L1 antibody and evaluated J-PPD-specific T-cell responses. Briefly, PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis were seeded in 96-well plates at 4×105 cells/well and cultured for 5 days under stimulation with 1 μg/ml of J-PPD. At the time of this stimulation culture, 1 μg/ml of rat anti-bovine PD-L1 antibody (4G12; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) as a blocking antibody or the same amount of a rat serum-derived IgG (Sigma-Aldrich) as a negative control was added. After culturing, cell proliferation capacity and cytokine production were evaluated in the same manner as described above.
Experimental results are shown in
Subsequently, the present inventors examined combined effects of COX-2 inhibitor and rat anti-bovine PD-L1 antibody on immunostimulation in cattle experimentally infected with M. avium subsp. paratuberculosis. Briefly, PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis were seeded in 96-well plated at 4×105 cells/well and cultured in the presence of J-PPD or a negative control antigen for 5 days. As the negative control antigen, Mycobacterium bovis BCG strain-derived purified protein (B-PPD) was used. To the medium, 1,000 nM meloxicam (Sigma-Aldrich) and 1 μg/ml of rat anti-bovine PD-L1 antibody (4G12; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) were added to make a total volume 200 μl. As a negative control for meloxicam, DMSO was used. As a negative control antibody, rat serum-derived IgG (Sigma-Aldrich) was used. After culturing, cell proliferation capacity and cytokine production were evaluated in the same manner as described above.
Experimental results are shown in
Finally, the present inventors examined combined effects of COX-2 inhibitor and rat-bovine chimeric anti-PD-L1 antibody on immunostimulation in cattle experimentally infected with M. avium subsp. paratuberculosis. Briefly, PBMCs derived from cattle experimentally infected with M. avium subsp. paratuberculosis were seeded in 96-well plated at 4×105 cells/well and cultured in the presence of J-PPD or a negative control antigen for 5 days. As the negative control antigen, B-PPD was used. To the medium, 1,000 nM meloxicam (Sigma-Aldrich) and 1 μg/ml of rat-bovine chimeric anti-PD-L1 antibody (ch4G12; Japanese Patent Application No. 2016-159089, Konnai S, Ohashi K, Murata S, Okagawa T, Nishimori A, Maekawa N, Suzuki S, Nakajima C; Anti-PD-L1 Antibody for Cattle) were added to make a total volume 200 μl. As a negative control for meloxicam, DMSO was used. As a negative control antibody, bovine serum-derived IgG (Sigma-Aldrich) was used. After culturing, cell proliferation capacity and cytokine production were evaluated in the same manner as described above.
Experimental results are shown in
The interaction between PD-1 and PD-L1 is one of the major molecular mechanisms through which pathogens evade immune responses. It has been reported that inhibition of the above interaction by using an antibody which specifically binds to either of those molecules can produce anti-pathogenic effects. In the subject Example, toward establishment of a novel control method against bovine leukemia virus (BLV) infection, the present inventors have confirmed in in vitro tests an immunostimulatory effect induced by a COX-2 inhibitor and enhancement of that effect when the inhibitor is used in combination with anti-bovine PD-L1 antibody.
2. Materials and Methods, as well as Experimental Results
In order to elucidate the involvement of PGE2 in the progression of pathology in BLV infection as a bovine chronic viral infection, the present inventors performed kinetic analysis of PGE2 in BLV-infected cattle.
(1) Measurement of Plasma PGE2 and Analysis of Correlation with Other Indicators
First, the amount of PGE2 contained in the plasma of BLV-infected cattle was quantified with Prostaglandin E2 Express ELISA Kit (Cayman Chemical). For the measurement, absorbance at 450 nm was measured using a microplate reader MTP-900 (Corona Electric). Further, correlation between the amount of plasma PGE2 and the number of lymphocytes in peripheral blood or PD-L1 expression rate in IgM+ cells was examined. Briefly, PBMCs isolated from BLV-infected cattle were analyzed by flow cytometry to quantify the PD-L1 expression on IgM+ cells. First, in order to block non-specific reactions of antibody, PBS supplemented with 10% inactivated goat serum (Thermo Fisher Scientific) was added to each well in an amount of 100 μl and left stationary at room temperature for 15 min. After washing, rat anti-bovine PD-L1 antibody (4G12; Rat IgG2a; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) or rat IgG2a isotype control (BD Bioscience) was added and reaction was conducted at room temperature for 20 min. After washing twice, PE/Cy7-labeled anti-IgM monoclonal antibody (IL-A30; Bio-Rad) and APC-labeled anti-rat Ig antibody (Southern Biotech) were added and reaction was conducted at room temperature for 20 min. Anti-IgM monoclonal antibody (IL-A30) was labeled with PE/Cy7 using Lightning-Link Conjugation Kit. After the reaction, washing was performed twice. Then, cells were analyzed with FACS Verse (BD Biosciences) and FCS Express 4 (De Novo Software). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma Aldrich) was used.
Experimental results of (1) are shown in
For more detailed analysis, in addition to plasma PGE2, expression levels of COX2 (i.e., involved in PGE2 synthesis) and the gene of EP4 (a PGE2 receptor that transmits immunosuppressive signals) were quantified by real-time PCR. Briefly, total cellular RNA was extracted from PBMCs, CD4+ cells, CD8+ cells, CD14+ cells and CD21+ cells derived from BLV-infected cattle and uninfected cattle in the same manner as described in (3), section 2.1 of Example 2, and cDNA was synthesized. Using the synthesized cDNA as a template, real-time PCR was performed with COX2-specific primers (already described in (3), section 2.3 of Example 2) and EP4-specific primers in the same manner as described in Example 2.
Experimental results of (2) are shown in
It has been reported that COX2 expression is increased by antigen stimulation in BLV infection (Pyeon D, Diaz F J, Splitter G A. J Virol. 74:5740-5745, 2000). From this report, it is predicted that PGE2 production by PBMCs derived from BLV-infected cattle will also be promoted by antigen stimulation. To verify this hypothesis, the present inventors cultured PBMCs with BLV antigen and quantified PGE2 in the culture supernatant by ELISA. Briefly, PBMCs derived from BLV-infected cattle and those derived from uninfected cattle were seeded in 96-wel plates at 4×105 cells/well and cultured in the presence of BLV antigen (final concentration 2%) for 6 days. As the BLV antigen, a culture supernatant of fetal lamb kidney cells persistently infected with BLV (FLK-BLV) was used after heat treatment at 65° C. In order to confirm that PGE2 production by antigen stimulation is inhibited by COX-2 inhibitor, cells were also cultured under such conditions that both BLV antigen and 1,000 nM meloxicam (Sigma Aldrich) were added to the medium. After 6 days, culture supernatant was collected and PGE2 contained in it was quantified with Prostaglandin E2 Express ELISA Kit (Cayman Chemical).
Experimental results of (3) are shown in
In various chronic infections, a possibility has been suggested that PGE2 promotes viral replication (Pyeon D, Diaz F J, Splitter G A. J Virol. 74:5740-5745, 2000; Waris D, Siddiqui A. J Virol. 79:9725-9734, 2005). Then, in order to evaluate the effect of PGE2 on viral replication in BLV infection, the present inventors cultured PBMCs with PGE2 and quantified BLV provirus load by real-time PCR. Briefly, PBMCs derived from BLV-infected cattle were seeded in 96-well plates at 1×106 cells/well and cultured in the presence of 2,500 nM PGE2 or DMSO for 3 days. After culturing, DNA was extracted from harvested PBMCs with Wizard DNA Purification kit (Promega). The concentration of the extracted DNA was quantified by measuring the absorbance (260 nm) with Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific). For measuring BLV provirus load in PBMCs, real-time PCR was performed using Cycleave PCR Reaction Mix SP (TaKaRa) and Probe/Primer/Positive control for detecting bovine leukemia virus (TaKaRa). LightCycler480 System II (Roche Diagnosis) was used for measurement.
Experimental results of (4) are shown in
Effects of BLV antigen stimulation on PD-L1 expression in BLV-infected cattle were evaluated. Briefly, PBMCs derived from BLV-infected and those from uninfected cattle were seeded in 96-well plates at 1×106 cells/well and cultured in the presence of BLV antigen (final concentration 2%) for 24 hours. After culturing, PBMCs were harvested, and PD-L1 expression on lymphocytes, CD4+ T cells, CD8+ T cells, IgM+ cells and CD14+ cells was analyzed by flow cytometry in the same manner as described in section 2.4 of Example 2.
Experimental results of 2.2 are shown in
In order to confirm that COX-2 inhibitor has stimulatory effects on BLV antigen-specific immune responses, the present inventors evaluated the proliferation capacity and cytokine production capacity of PBMCs that were cultured in the presence of meloxicam and BLV antigen. Briefly, PBMCs derived from BLV-infected cattle were seeded in 96-well plated at 4×105 cells/well and cultured under stimulation in the presence of BLV antigen (final concentration 2%) and 1,000 nM meloxicam (Sigma-Aldrich) for 6 days. After culturing, cell proliferation capacity and cytokine production capacity were evaluated in the same manner as described in Example 2.
Experimental results are shown in
In order to examine the antiviral effects of COX-2 inhibitor in vivo, the present inventors conducted a clinical application test using BLV-infected cattle. Two individuals (#1 and #2) of PL cattle of Holstein species were used in the test. The body weight and age at the beginning of the test were 736 kg and 8 years and 1 month for #1 and 749 kg and 3 years and 7 months for #2. As a COX-2 inhibitor, Metacam™ 2% injection (hereinafter, referred to as “Metacam™”; Kyoritsu Seiyaku) was inoculated subcutaneously at 0.5 mg/kg. In addition to the first inoculation, individual #1 received inoculation 7, 14, 21, 28, 35, 42, 49 and 56 days after the first inoculation; and individual #2 received inoculation 7, 14, 20, 27, 34, 41, 48 and 55 days after the first inoculation (
Experimental results are shown in
Provirus loads in BLV-infected cattle decreased upon administration of a COX-2 inhibitor (
Experimental results are shown in
Finally, the present inventors examined immunostimulatory effects due to combined use of COX-2 inhibitor and rat-bovine chimeric anti-PD-L1 antibody in BLV-infected cattle. Briefly, PBMCs derived from BLV-infected cattle were seeded in 96-well plates at 4×105 cells/well and cultured in the presence of BLV antigen or a negative control antigen for 6 days. As the negative control antigen, a culture supernatant of BLV-uninfected fetal lamb kidney cells (FLK) was used after heat treatment at 65° C. To the medium, 1,000 nM meloxicam (Sigma-Aldrich) and 1 μg/ml of rat-bovine chimeric anti-PD-L1 antibody (ch4G12; Japanese Patent Application No. 2016-159089, Konnai S, Ohashi K, Murata S, Okagawa T, Nishimori A, Maekawa N, Suzuki S, Nakajima C; Anti-PD-L1 Antibody for Cattle) were added to make a total volume of 200 μl. As a negative control for meloxicam, DMSO was used. As a negative control antibody, bovine serum-derived IgG (Sigma-Aldrich) was used. After culturing, cell proliferation capacity and cytokine production were evaluated in the same manner as described above.
Experimental results are shown in
In order to examine the in vivo antiviral effect of combined use of COX-2 inhibitor and PD-1/PD-L1 inhibitor, the present inventors conducted a clinical application test using BLV-infected cattle. Two individuals (#1719 and #2702; Holstein species) of BLV-infected cattle with a high BLV provirus load were used in the test. The body weight and age at the beginning of the test were 799 kg and 7 years and 4 months for #1719 and 799 kg and 4 years and 3 months for #2702. As a COX-2 inhibitor, Metacam™ 20 injection (hereinafter, referred to as “Metacam™”; Kyoritsu Seiyaku) was inoculated subcutaneously at 0.5 mg/kg. As a PD-1/PD-L1 inhibitor, rat-bovine chimeric anti-PD-L1 antibody (ch4G12; WO2018/034225, Konnai S, Ohashi K, Murata S, Okagawa T, Nishimori A, Maekawa N, Suzuki S, Nakajima C; Anti-PD-L1 Antibody for Cattle) was administered intravenously at 1.0 mg/kg. In addition to the first inoculation, Metacam™ was inoculated 7 and 14 days after the first inoculation. Blood collection was performed 7 days before the antibody administration (at −7 day); at the antibody administration day; at day 1, day 3 and day 7 after the antibody administration; and once weekly from day 14 to day 58 after the antibody administration. Blood collection on antibody/Metacam™ administration day (at day 0) and Metacam™ administration days (at day 7 and day 14) was carried out before administration of antibody and Metacam™. Using the collected blood samples, BLV provirus loads were quantified. Provirus loads were quantified in the same manner as described in (4), section 2.1 above.
Experimental results are shown in
Examination of Combined Effect of Anti-Bovine PD-L1 Antibody and COX-2 Inhibitor in Mycoplasma bovis-Infected Cattle
The interaction between PD-1 and PD-L1 is one of the major molecular mechanisms through which pathogens evade immune responses. It has been reported that inhibition of the above interaction by using an antibody which specifically binds to either of those molecules can produce anti-pathogenic effects. In the subject Example, toward establishment of a novel control method against bovine mycoplasma infections caused by Mycoplasma bovis, the present inventors have confirmed in in vitro tests, an immunostimulatory effect induced by COX-2 inhibitor and enhancement of that effect when the inhibitor is used in combination with anti-bovine PD-L1 antibody.
2.1. Analysis of Serum PGE2 in M. bovis-Infected Cattle
In order to elucidate the involvement of PGE2 in the disease progression of bovine mycoplasmosis caused by Mycoplasma bovis, the present inventors performed kinetic analysis of PGE2 in M. bovis-infected cattle. The amounts of PGE2 contained in the serum of M. bovis-infected cattle and M. bovis-uninfected cattle (hereinafter, referred to as “uninfected cattle”) were quantified with Prostaglandin E2 Express ELISA Kit (Cayman Chemical). For measurement, absorbance at 450 nm was measured using a microplate reader MTP-900 (Corona Electric).
Experimental results are shown in
2.2. Correlation Analysis between Plasma PGE2 and Indicators of Immune Responses
Subsequently, the present inventors examined correlation between the amount of plasma PGE2 in M. bovis-infected cattle and M. bovis-specific IFN-γ responses or PD-L1 expression rate in CD14′ cells. First, plasma was isolated from the blood of M. bovis-infected cattle, and the amount of PGE2 contained in the plasma was measured as described in 2.1 above. Subsequently, peripheral blood mononuclear cells (PBMCs) isolated from the blood of M bovis-infected cattle were suspended in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% inactivated fetal bovine serum (Thermo Fisher Scientific), antibiotics (streptomycin 100 μg/ml, penicillin 100 U/ml) (Thermo Fisher Scientific) and 2 mM L-glutamine (Thermo Fisher Scientific) and seeded in 96-well plates (Corning) at 4×105 cells/well. Then, 1.5 μg/ml of M. bovis antigen was added and cells were cultured under stimulation at 37° C. in the presence of 5% CO2 for 5 days. As M. bovis antigen, M. bovis PG45 strain (ATCC 25523; kindly provided by Prof Hidetoshi Higuchi, Rakuno Gakuen University) was used after heat treatment. After 5 days, culture supernatant was collected and IFN-γ production was measured with ELISA for Bovine IFN-γ (MABTECH). For the measurement, absorbance at 450 nm was measured using a microplate reader MTP-900 (Corona Electric).
Further, PD-L1 expression on CD14+ cells was measured by flow cytometry analysis of PBMCs derived from M. bovis-infected cattle. First, in order to block non-specific reactions of antibody, PBS supplemented with 10% inactivated goat serum (Thermo Fisher Scientific) was added to each well in an amount of 100 μl and left stationary at room temperature for 15 mn. After washing, rat anti-bovine PD-L1 antibody (4G12; Rat IgG2a; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) or rat IgG2a isotype control (BD Bioscience) was added and reaction was conducted at room temperature for 20 min. After washing twice, PerCp/Cy5.5-labeled anti-CD14 monoclonal antibody (CAM36A; Washington State University Monoclonal Antibody Center) and APC-labeled anti-rat Ig antibody (Southern Biotech) were reacted with cells. Anti-CD14 monoclonal antibody (CAM36A) was labeled with PerCp/Cy5.5 using Lightning-Link Conjugation Kit. After the reaction, washing was performed twice. Then, cells were analyzed with FACS Verse (BD Biosciences) and FCS Express 4 (De Novo Software). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma Aldrich) was used.
Experimental results are shown in
2.3. Expression Analysis of COX2 and EP4 in M. bovis-Infected Cattle
For more detailed analysis, expression levels of COX2 (involved in PGE2 synthesis) and the gene of EP4 (a PGE2 receptor that transmits immunosuppressive signals) were quantified by real-time PCR. Briefly, total cellular RNA was extracted from PBMCs derived from M. bovis-infected cattle and those from uninfected cattle in the same manner as described in (3), section 2.1 of Example 2, and cDNA was synthesized. Using the synthesized cDNA as a template, real-time PCR was performed with COX2-specific primers (described in (3), section 2.3 of Example 2) and EP4-specific primers (described in (2), section 2.1 of Example 3) according to the method described in Example 2.
Experimental results are shown in
2.4. Examination of Immunostimulatory Effects due to Combined Use of COX-2 Inhibitor and Rat Anti-Bovine PD-L1 Antibody in M. bovis-Infected Cattle
Finally, the present inventors examined immunostimulatory effects due to combined use of COX-2 inhibitor and anti-bovine PD-L1 antibody in M. bovis-infected cattle. Briefly, PBMCs derived from M. bovis-infected cattle were seeded in 96-well plates (Corning) at 4×105 cells/well and cultured in the presence of 1.5 μg/ml of M. bovis antigen (as antigen-specific stimulant) or 2 μg/ml each of anti-CD3 monoclonal antibody (MM1A; Washington State University Monoclonal Antibody Center) and anti-CD28 monoclonal antibody (CC220; Bio-Rad) (as T cell stimulants) for 5 days. To the medium, 10 μM meloxicam (Sigma-Aldrich) and 10 μg/ml of rat anti-bovine PD-L1 antibody (4G12; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561) were added. As a negative control for meloxicam, DMSO was used; and as a negative control antibody, rat serum-derived IgG (Sigma-Aldrich) was used. After culturing, cell proliferation capacity and cytokine production were evaluated in the same manner as described above.
Experimental results are shown in
Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance. Recently, it has been elucidated that these molecules are also involved in immunosuppression in tumors. In the subject Example, for the purpose of establishing a novel therapy for bovine infections, the present inventors have prepared a chimeric antibody gene by linking the variable region gene of rat anti-bovine PD-L1 monoclonal antibody (4G12) capable of inhibiting the binding of bovine PD-1 and PD-L1 to the constant region gene of a bovine immunoglobulin (IgG1 with mutations having been introduced into the putative binding sites for Fcγ receptors in CH2 domain to inhibit ADCC activity; see
The nucleotide sequences of the full length cDNAs of bovine PD-1 gene (GenBank accession number AB510901; Ikebuchi R, Konnai S, Sunden Y, Onuma M, Ohashi K. Microbiol. Immunol. 2010 May; 54(5):291-298) and bovine PD-L1 gene (GenBank accession number AB510902; Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K. Vet. Res. 2011 Sep. 26; 42:103) were determined. Based on the resultant genetic information, bovine PD-1 and bovine PD-L1 membrane expressing cells were prepared. First, for preparing bovine PD-1 or PD-L1 expressing plasmid, PCR was performed using a synthesized bovine PBMC-derived cDNA as a template and designed primers having Nod and HindIII (bovine PD-1) recognition sites and NheI and XhoI (bovine PD-L1) recognition sites on the 5′ side (boPD-1-myc F and R; boPD-L1-EGFP F and R). The PCR products were digested with Nod (Takara) and HindIII (Takara; bovine PD-1), NheI (Takara) and XhoI (Takara; bovine PD-L1), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCMV-Tag1 vector (Agilent Technologies; bovine PD-1) or pEGFP-N2 vector (Clontech; bovine PD-L1) treated with restriction enzymes in the same manner. The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCMV-Tag1-boPD-1.
Bovine PD-1 membrane expressing cells were prepared by the procedures described below. First, 2.5 μg of pCMV-Tag1-boPD-1 was introduced into 4×106 CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science), 20 ml/L GlutaMAX supplement (Life Technologies), and 18 ml/L 10% Pluronic F-68 (Life Technologies), followed by selection. The resultant expression cells were reacted with rat anti-bovine PD-1 antibody 5D2 at room temperature. After washing, the cells were further reacted with anti-rat IgG microbeads-labeled antibody (Miltenyi Biotec) at room temperature. Cells expressing bovine PD-1 at high levels were isolated with Auto MACS (Miltenyi Biotec). Subsequently, re-isolation was performed in the same manner to obtain still higher purity. The resultant expression cells were subjected to cloning by limiting dilution to thereby obtain a CHO DG44 cell clone expressing bovine PD-1 at high level (bovine PD-1 expressing cells).
Bovine PD-L1 membrane expressing cells were prepared by the procedures described below. First, 2.5 μg of pEGFP-N2-boPD-L1 or pEGFP-N2 (negative control) was introduced into 4×106 CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing G418 (Enzo Life Science) 800 μg/ml, GlutaMAX supplement (Life Technologies) 20 ml/L, and 10% Pluronic F-68 (Life Technologies) 18 ml/L, followed by selection and cloning by limiting dilution (bovine PD-L1 expressing cell clone). In order to confirm the expression of bovine PD-L1 in the thus prepared expressing cell clone, intracellular localization of EGFP was visualized with an inverted confocal laser microscope LSM700 (ZEISS).
Bovine PD-1-Ig expressing plasmid was constructed by the procedures described below. Briefly, the signal peptide and the extracellular region of bovine PD-1 (GenBank accession number AB510901) were linked to the Fc domain of the constant region of a known bovine IgG1 (GenBank accession number X62916) to prepare a gene sequence. After codons were optimized for CHO cells, gene synthesis was performed in such a manner that NotI recognition sequence, KOZAK sequence, bovine PD-1 signal peptide sequence, bovine PD-1 gene extracellular region sequence, bovine IgG1 Fc region sequence, and XbaI recognition sequence would be located in the gene in this order. It should be noted here that bovine IgG1 was mutated to inhibit ADCC activity; more specifically, mutations were introduced into the putative binding sites for Fcγ receptors of CH2 domain (sites of mutation: 185 E->P, 186 L->V, 187 P->A, 189 G->deletion, 281 A->S, 282 P->S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561; the amino acid sequence of PD-1-Ig and the sites of mutation are disclosed in
Bovine PD-L1-Ig expressing plasmid was constructed by the procedures described below. In order to amplify the signal peptide and the extracellular region of bovine PD-L1 (GenBank accession number AB510902), primers were designed that had NheI and EcoRV recognition sites added on the 5′ side (boPD-L1-Ig F and R). PCR was performed using a synthesized bovine PBMC-derived cDNA as a template. The PCR products were digested with NheI (Takara) and EcoRV (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCXN2.1-Rabbit IgG1 Fc vector (Niwa et al., 1991; Zettlmeissl et al., 1990; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine, and modified in the inventors' laboratory) treated with restriction enzymes in the same manner. The expression plasmid was purified with QIAGEN Plasmid Midi kit (Qiagen) or FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCXN2.1-boPD-L1-Ig.
Soluble bovine PD-1-Ig expressing cells were prepared by the procedures described below. Briefly, 2.5 μg of pDN11-boPD-1-Ig was introduced into 4×106 CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with OptiCHO AGT medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science) and 20 ml/L GlutaMAX supplement (Life Technologies). After cultured for 3 weeks, the cells were subjected to selection. Briefly, the concentrations of the Fc fusion recombinant protein in the culture supernatants of the resultant cell clones were measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select those cell clones that express the Fc fusion recombinant protein at high levels. The resultant highly expressing cell clone was transferred to a G418-free medium and cultured under shaking for 14 days, followed by collection of a culture supernatant. The culture supernatant containing the Fc fusion recombinants protein was ultrafiltered with Centricon Plus-70 (Millipore). Then, the Fc fusion recombinant protein was purified with Ab-Capcher Extra (ProteNova). After purification, the buffer was exchanged with phosphate-buffered physiological saline (PBS; pH 7.4) using PD-10 Desalting Column (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine PD-1-Ig). The concentration of the purified bovine PD-1-Ig was measured by ELISA using IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).
Soluble bovine PD-L1-Ig expressing cells were prepared by the procedures described below. Briefly, 30 μg of pCXN2.1-boPD-L1-Ig was introduced into 7.5×107 Expi293F cells (Life Technologies) using Expifectamine (Life Technologies). After 7-day culture under shaking, the culture supernatant was collected. The recombinant protein was purified from the supernatant using Ab-Capcher Extra (ProteNova; bovine PD-L1-Ig). After purification, the buffer was exchanged with PBS (pH 7.4) using PD MiniTrap G-25 (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine PD-L1-Ig). The concentration of the purified bovine PD-L1-Ig was measured using Rabbit IgG ELISA Quantitation Set (Bethyl). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).
Rat was immunized in the footpad with bovine PD-L1-Ig (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561; bovine PD-L1-Ig was prepared by the method disclosed in this article and used for immunization). Hybridomas were established by the iliac lymph node method to thereby obtain rat anti-bovine PD-L1 monoclonal antibody producing hybridoma 4G12. With respect to the method of establishment of rat anti-bovine PD-L1 monoclonal antibody, details are disclosed in the following non-patent document (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Vet. Res. 2013 Jul. 22; 44:59; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561).
Rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12 was established by fusing the antibody constant regions of bovine IgG1 and Igλ with rat anti-bovine PD-L1 antibody 4G12 being used as an antibody variable region.
First, the genes of heavy chain and light chain variable regions were identified from a hybridoma that would produce rat anti-bovine PD-L1 antibody 4G12. Subsequently, a gene sequence was prepared in which the heavy chain and the light chain variable regions of the antibody 4G12 were linked to known constant regions of bovine IgG1 (heavy chain; modified from GenBank Accession number X62916) and bovine Igλ (light chain; GenBank Accession number X62917), respectively, and codon optimization was carried out [rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12: SEQ ID NOS: 115 and 116 (amino acid sequences), SEQ ID NOS: 117 and 118 (nucleotide sequences after codon optimization)]. It should be noted that in order to suppress the ADCC activity of bovine IgG1, mutations were added to the putative binding sites of Fcγ receptors in CH2 domain (See
The pDC6-boPD-L1ch4G12 was transfected into CHO-DG44 cells (CHO-DG44 (dfhr−/−)) which were a dihydrofolate reductase deficient cell. Forty-eight hours later, the medium was exchanged with OptiCHO AGT medium (Life Technologies) containing 20 ml/L GlutaMAX supplement (Life Technologies). After cultured for 3 weeks, the cells were subjected to selection and cloning by limiting dilution. Subsequently, the concentrations of the chimeric antibody in the culture supernatants were measured by dot blotting and ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select high expression clones. Further, the selected clones expressing rat-bovine chimeric anti-bovine PD-L1 antibody at high levels were subjected to gene amplification treatment by adding a load with 60 nM methotrexate (Mtx)-containing medium. The thus established cell clone stably expressing rat-bovine chimeric anti-bovine PD-L1 antibody was transferred into Mtx-free Opti-CHO AGT medium and cultured under shaking for 14 days (125 rpm, 37° C., 5% CO2). Chimeric antibody production in the culture supernatant was measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric). The culture supernatant at day 14 was centrifuged at 10,000 g for 10 min to remove cells, and the centrifugal supernatant was passed through a Steritop-GP 0.22 μm filter (Millipore) for sterilization and then stored at 4° C. until it was subjected to purification.
From the culture supernatant prepared as described above, each chimeric antibody was purified using Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; PBS pH 7.4 was used as an equilibration buffer and a wash buffer. As an elution buffer, IgG Elution Buffer (Thermo Fisher Scientific) was used. As a neutralization buffer, 1M Tris (pH 9.0) was used. The purified antibody was subjected to buffer exchange with PBS (pH 7.4) using PD-10 Desalting Column (GE Healthcare) and concentrated using Amicon Ultra-15 (50 kDa, Millipore). The thus purified chimeric antibody was passed through a 0.22 μm syringe filter (Millipore) for sterilization and stored at 4° C. until use in experiments.
In order to confirm the purity of purified rat-bovine chimeric anti-bovine PD-L1 antibody, antibody proteins were detected by SDS-PAGE and CBB staining. Using 10% acrylamide gel, the purified rat-bovine chimeric antibody was electrophoresed under reducing conditions (reduction with 2-mercaptoethanol from Sigma-Aldrich) and non-reducing conditions. Bands were stained with Quick-CBB kit (Wako) and decolored in distilled water. The results are shown in
It was confirmed by flow cytometry that the rat-bovine chimeric anti-bovine PD-L1 antibody specifically binds to the bovine PD-L1 expressing cells (described above). First, rat anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12 was reacted with bovine PD-L1 expressing cells at room temperature for 30 min. After washing, APC-labeled anti-rat Ig goat antibody (Southern Biotech) or Alexa Fluor 647—labeled anti-bovine IgG (H+L) goat F(ab′)2 (Jackson ImmunoResearch) was reacted at room temperature for 30 min. As negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, each rat antibody or rat-bovine chimeric antibody bound to cell surfaces was detected by FACS Verse (BD Biosciences). For every washing operation and dilution of antibody, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.
The experimental results are shown in
Using bovine PD-L1 expressing cells (described above) and bovine PD-1-Ig (described above), bovine PD-1/PD-L1 binding inhibition by anti-bovine PD-L1 antibody was tested. First, 2×105 bovine PD-L1 expressing cells were reacted with various concentrations (0, 0.32, 0.63, 1.25, 2.5, 5 or 10 μg/ml) of rat anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12 at room temperature for 30 min. As negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, bovine PD-1-Ig labeled with biotin using Lightning-Link Type A Biotin Labeling Kit (Innova Bioscience) was added to a final concentration of 2 μg/ml, followed by reaction for another 30 min at room temperature. Subsequently, after washing, bovine PD-1-Ig bound to cell surfaces was detected with APC-labeled streptavidin (BioLegend). For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibody, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. Taking the proportion of PD-1-Ig bound cells without antibody addition as 100%, the proportion of PD-1-Ig bound cells at each antibody concentration was shown as relative value.
The experimental results are shown in
Using bovine PD-1 expressing cells (described above) and bovine PD-L1-Ig (described above), bovine pD-1/PD-L1 binding inhibition by anti-bovine PD-L1 antibody was tested. First, rat anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12 at a final concentration of 0, 0.32, 0.63, 1.25, 2.5, 5 or 10 μg/ml and bovine PD-L1-Ig at a final concentration of 1 μg/ml were placed in 96-well plates, where they were reacted at room temperature for 30 min. The resultant mixture was reacted with 2×105 bovine PD-1 expressing cells at room temperature for 30 min. As negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, Alexa Fluor 647-labeled anti-rabbit IgG (H+L) goat F(ab′)2 (Life Technologies) was reacted at room temperature for 30 min to thereby detect bovine PD-L1-Ig bound to cell surfaces. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibody, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. Taking the proportion of PD-L1-Ig bound cells without antibody addition as 100%, the proportion of PD-L1-Ig bound cells at each antibody concentration was shown as relative value.
The experimental results are shown in
In order to confirm that bovine PD-1/PD-L1 binding inhibition by rat-bovine chimeric anti-PD-L1 antibody activates lymphocytes, a biological activity test was performed using cell proliferation as an indicator. Briefly, bovine PBMCs isolated from peripheral blood of healthy cattle were suspended in PBS to give a concentration of 10×106 cells/ml, and reacted with carboxyfluorescein succinimidyl ester (CFSE) at room temperature for 20 min. After washing twice with RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), antibiotics (streptomycin 200 μg/ml, penicillin 200 U/ml) (Life Technologies) and 0.01% L-glutamine (Life Technologies), the PBMCs were reacted with anti-bovine CD3 mouse antibody (WSU Monoclonal Antibody Center) at 4° C. for 30 min. After washing, the PBMCs were reacted with anti-mouse IgG1 microbeads (Miltenyi Biotec) at 4° C. for 15 min, followed by isolation of CD3-positive T cells using autoMACS™ Pro(Miltenyi Biotec). To the isolated CD3-positive T cells, anti-bovine CD3 mouse antibody (WSU Monoclonal Antibody Center) and anti-bovine CD28 mouse antibody (Bio-Rad) were added. Then, the cells were co-cultured with bovine PD-L1 expressing cells (CD3-positive T cells: bovine PD-L1 expressing cells =10:1) in the presence or absence of 10 μg/ml of rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12. As a control for antibodies, serum-derived bovine IgG (Sigma-Aldrich) was used; as a control for PD-L1 expressing cells, EGFP expressing cells transfected with pEGFP-N2 were used. After a 6-day coculture, cells were harvested and reacted with anti-bovine CD4 mouse antibody and anti-bovine CD8 mouse antibody (Bio-Rad) at room temperature for 30 min. For the labeling of antibodies, Zenon Mouse IgG1 Labeling Kits (Life Technologies) or Lightning-Link Kit (Innova Biosciences) was used. For analysis, FACS Verse (BD Biosciences) was used. For washing operation after culturing and dilution of antibody, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.
The experimental results are shown in
In order to confirm that bovine PD-1/PD-L1 binding inhibition by rat-bovine chimeric anti-PD-L1 antibody activates lymphocytes, a biological activity test was performed using IFN-γ production as an indicator. Briefly, PBMCs isolated from peripheral blood of BLV-infected cattle were suspended in RPMI medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), antibiotics (streptomycin 200 μg/ml, penicillin 200 U/ml) (Life Technologies) and 0.01% L-glutamine (Life Technologies) to give a concentration of 4×106 cells/ml. To the PBMCs, 10 μg/ml of rat anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12, and 2% BLV-infected fetal lamp kidney cell (FLK-BLV) culture supernatant were added; culturing was then performed at 37° C. under 5% CO2 for 6 days. As control antibodies, serum-derived rat IgG (Sigma-Aldrich) and serum-derived bovine IgG (Sigma-Aldrich) were used. After a 6-day culture, a culture supernatant was collected, and IFN-γ production was measured with Bovine IFN-γ ELISA Kit (BETYL). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).
The experimental results are shown in
Established rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12 (about 260 mg; 1 mg/kg) was intravenously administered into experimentally BLV-infected calf (Holstein, male, 7 months old, 267 kg). Blood samples were collected chronologically from the infected calf, followed by isolation of PBMCs by density gradient centrifugation.
Bovine PBMCs were suspended in PBS and reacted with CFSE at room temperature for 20 min. After washing twice with RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), antibiotics (streptomycin 200 μg/ml, penicillin 200 U/ml) (Life Technologies) and 0.01% L-glutamine (Life Technologies), the cell concentration was adjusted to 4×106 cells/ml using the same medium. Culture supernatant of 2% BLV-infected fetal lamp kidney cells (FLK-BLV) was added to the PBMCs, which were then cultured at 37° C. under 5% CO2 for 6 days. As a control, culture supernatant of 2% BLV-not-infected fetal lamp kidney cells (FLK) was used. After a 6-day culture, PBMCs were collected and reacted with anti-bovine CD4 mouse antibody, anti-bovine CD8 mouse antibody and anti-bovine IgM mouse antibody (Bio-Rad) at 4° C. for 20 min. For the labeling of antibodies, Zenon Mouse IgG1 Labeling Kits (Life Technologies) or Lightning-Link Kit (Innova Biosciences) was used. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibody, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.
The experimental results are shown in
DNA was extracted from isolated bovine PBMCs using Wizard DNA Purification kit (Promega). The concentration of the extracted DNA was quantitatively determined, taking the absorbance (260 nm) measured with Nanodrop 8000 Spectrophotometer (ThermoFisher Scientific) as a reference. In order to measure the BLV provirus load in PBMCs, real time PCR was performed using Cycleave PCR Reaction Mix SP (TaKaRa) and Probe/Primer/Positive control for bovine leukemia virus detection (TaKaRa). Light Cycler 480 System II (Roche Diagnosis) was used for measurement.
The experimental results are shown in
Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance. Recently, it has been elucidated that these molecules are also involved in immunosuppression in tumors. In the subject Example, for the purpose of establishing a novel therapy for bovine infections, a chimeric antibody gene was prepared in which variable region genes of rat anti-bovine PD-1 monoclonal antibody 5D2 capable of inhibiting the binding of bovine PD-1 to PD-L1 were linked to constant region genes of bovine immunoglobulins (bovine IgG1 and Igλ, with mutations having been introduced into the putative binding sites of Fcγ receptors in bovine IgG1's CH2 domain to inhibit ADCC activity; see
The nucleotide sequences of the full length cDNAs of bovine PD-1 gene (GenBank accession number AB510901; Ikebuchi R, Konnai S, Sunden Y, Onuma M, Ohashi K. Microbiol. Immunol., 54(5):291-298; May 2010) and bovine PD-L1 gene (GenBank accession number AB510902; Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K. Vet. Res., 42:103, Sep. 26, 2011) were determined. Based on the resultant genetic information, bovine PD-1 and bovine PD-L1 expressing cells were prepared. First, for preparing bovine PD-1 or PD-L1 expressing plasmid, PCR was performed using a synthesized bovine peripheral blood mononuclear cell (PBMC)-derived cDNA as a template and designed primers having Nod and HindIII (bovine PD-1) recognition sites or NheI and XhoI (bovine PD-L1) recognition sites on the 5′ side (boPD-1-myc F and R; or boPD-L1-EGFP F and R). The PCR products were digested with Nod (Takara) and HindIII (Takara; bovine PD-1) or NheI (Takara) and XhoI (Takara; bovine PD-L1), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCMV-Tag1 vector (Agilent Technologies; bovine PD-1) or pEGFP-N2 vector (Clontech; bovine PD-L1) treated with the restriction enzymes in the same manner. The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCMV-Tag1-boPD-1 or pEGFP-N2-boPD-L1.
Bovine PD-1 expressing cells were prepared by the procedures described below. First, 2.5 μg of pCMV-Tag1-boPD-1 was introduced into 4×106 CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science), 20 ml/L GlutaMAX supplement (Life Technologies), and 18 ml/L 10% Pluronic F-68 (Life Technologies), followed by selection. The resultant expression cells were reacted with rat anti-bovine PD-1 antibody 5D2 at room temperature. After washing, the cells were further reacted with anti-rat IgG microbead-labeled antibody (Miltenyi Biotec) at room temperature. Cells expressing bovine PD-1 at high levels were isolated with Auto MACS (Miltenyi Biotec). Subsequently, re-isolation was performed in the same manner to obtain still higher purity. The resultant expression cells were subjected to cloning by limiting dilution to thereby obtain a CHO DG44 cell clone expressing bovine PD-1 at high level (bovine PD-1 expressing cells).
Bovine PD-L1 membrane expressing cells were prepared by the procedures described below. First, 2.5 μg of pEGFP-N2-boPD-L1 or pEGFP-N2 (negative control) was introduced into 4×106 CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science), 20 ml/L GlutaMAX supplement (Life Technologies) and 18 ml/L 10% Pluronic F-68 (Life Technologies), followed by selection and cloning by limiting dilution (bovine PD-L1 expressing cell clone). In order to confirm the expression of bovine PD-L1 in the thus prepared cell clone, intracellular localization of EGFP was visualized with an inverted confocal laser microscope LSM700 (ZEISS).
Bovine PD-1-Ig expressing plasmid was constructed by the procedures described below. Briefly, the signal peptide and the extracellular region of bovine PD-1 (GenBank accession number AB510901) were linked to the constant region of a known bovine IgG1 (GenBank accession number X62916) to prepare a gene sequence. After codons were optimized for CHO cells, gene synthesis was performed in such a manner that Nod recognition sequence, KOZAK sequence, bovine PD-1 signal peptide sequence, bovine PD-1 gene extracellular region sequence, bovine IgG1 Fc region sequence, and XbaI recognition sequence would be located in the gene in this order. It should be noted here that bovine IgG1 was mutated to inhibit ADCC activity; more specifically, mutations were introduced into the putative binding sites for Fcγ receptors of CH2 domain (sites of mutation: 185 E->P, 186 L-V, 187 P->A, 189 G->deletion, 281 A->S, 282 P->S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology, 142(4):551-561, August 204; the amino acid sequence of PD-1-Ig and the sites of mutation are disclosed in
Bovine PD-1-His expressing plasmid was prepared by the procedures described below. Briefly, for the purpose of amplifying the signal peptide and the extracellular region of bovine PD-1 (GenBank accession number AB510901), primers were designed in which NotI and XhoI recognition sites were added on the 5′ side (boPD-1-His F and R). A genetic sequence encoding a 6xHis tag was added to the reverse primer. PCR was performed using a synthesized bovine PBMC-derived cDNA as a template. The respective PCR products were digested with Nod (Takara) and XhoI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCXN2.1(+) vector (Niwa H, Yamamura K, Miyazaki J. Gene, 108(2):193-199; Dec. 15, 1991; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine) treated with the restriction enzymes in the same manner. The resultant expression plasmid was purified with FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCXN2.1-boPD-1-His.
Soluble bovine PD-1-Ig expressing cells were prepared by the procedures described below. Briefly, 2.5 μg of pDN11-boPD-1-Ig was introduced into 4×106 CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD OptiCHO medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science) and 20 ml/L GlutaMAX supplement (Life Technologies). After cultured for 3 weeks, the cells were subjected to selection. Briefly, the concentrations of the Fc fusion recombinant protein in the culture supernatants of the resultant cell clones were measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select those cell clones that express the Fc fusion recombinant protein at high levels. The resultant highly expressing cell clone was transferred to a G418-free medium and cultured under shaking for 14 days, followed by collection of a culture supernatant. The culture supernatant containing the Fc fusion recombinants protein was ultrafiltered with Centricon Plus-70 (Millipore). Then, the Fc fusion recombinant protein was purified with Ab-Capcher Extra (ProteNova). After purification, the buffer was exchanged with phosphate-buffered physiological saline (PBS; pH 7.4) using PD-10 Desalting Column (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine PD-1-Ig). The concentration of the purified bovine PD-1-Ig was measured by ELISA using IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).
Soluble bovine PD-1-His expressing cells were prepared by the procedures described below. Briefly, 30 μg of pCXN2.1-boPD-1-His was introduced into 7.5×107 Expi293F cells (Life Technologies) using Expifectamine (Life Technologies). After a 7-day culture under shaking, the culture supernatant was collected. The recombinant protein of interest was purified from the culture supernatant using TALON Metal Affinity Resin (Clontech; bovine PD-1-His). After purification, the buffer was exchanged with PBS (pH 7.4) using PD MiniTrap G-25 (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine PD-1-His). The concentration of purified bovine PD-1-His was quantitatively determined in terms of the absorbance (280 nm) measured with Nanodrop8000 Spectrophotometer (Thermo Fisher Scientific).
Rat was immunized in the footpad with bovine PD-1-Ig (described above). Hybridomas were established by the iliac lymph node method to thereby obtain rat anti-bovine PD-1 monoclonal antibody producing hybridoma 5D2. With respect to the method of establishment of rat anti-bovine PD-1 monoclonal antibody, details are disclosed in the following non-patent document (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Vet. Res. 44:59; Jul. 22, 2013).
Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was established by fusing the antibody constant regions of bovine IgG1 and Igλ, with rat anti-bovine PD-1 antibody 5D2 being used as antibody variable regions.
First, the genes of heavy chain and light chain variable regions were identified by the RACE method from a hybridoma that would produce rat anti-bovine PD-1 antibody 5D2. Subsequently, a gene sequence was prepared in which the heavy chain and the light chain variable regions of the rat anti-bovine PD-1 antibody 5D2 were linked to known constant regions of bovine IgG1 (heavy chain, modified from GenBank Accession number X62916) and bovine Igλ (light chain; GenBank Accession number X62917), respectively. Then, codon optimization was carried out (SEQ ID NOS: 157 and 158 (amino acid sequences); SEQ ID NOS: 162 and 163 (nucleotide sequences after codon optimization)). It should be noted that bovine IgG1 had mutations added to the putative binding sites of Fcγ receptors in CH2 domain in order to suppress ADCC activity (See
The pDN112-boPD-1ch5D2 prepared above was transfected into CHO-DG44 cells (CHO-DG44(dfhr−/−)) which were a dihydrofolate reductase deficient cell. Forty-eight hours later, the medium was exchanged with CD OptiCHO medium (Life Technologies) containing 2 mM GlutaMAX supplement (Life Technologies) and 800 μg/ml G418 sulfate (Enzo Life Science). After cultured for 3 weeks, the expression cells were subjected to selection and cloning by limiting dilution. Subsequently, the concentrations of the chimeric antibody in the culture supernatants were measured by dot blotting and ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select high expression clones. Further, to the selected clones expressing rat-bovine chimeric anti-bovine PD-1 antibody at high levels were subjected to gene amplification treatment by adding a load with 60 nM methotrexate (Mtx; Wako)-containing medium. The thus established cell clone stably expressing rat-bovine chimeric anti-bovine PD-1 antibody was transferred into Mtx-free CD Opti-CHO medium and cultured under shaking for 14 days (125 rpm, 37° C., 5% CO2). Chimeric antibody production in the culture supernatant was measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric). The culture supernatant at day 14 was centrifuged at 10,000 g for 10 min to remove cells, and the centrifugal supernatant was passed through a Steritop-GP 0.22 μm filter (Millipore) for sterilization and then stored at 4° C. until it was subjected to purification.
The results are shown in
From the culture supernatant prepared as described above, each chimeric antibody was purified using Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; 1.5 M Glycine/3 M NaCl (pH 8.0) was used as equilibration buffer and wash buffer. As elution buffer, 0.1 M Glycine-HCl (pH 2.8) was used. As neutralization buffer, 1M Tris (pH 9.0) was used. The purified antibody was subjected to buffer exchange with PBS (pH 7.4) using PD-10 Desalting Column (GE Healthcare) and concentrated using Amicon Ultra-15 (50 kDa, Millipore). The thus purified chimeric antibody was passed through a 0.22 μm syringe filter (Pall Life Sciences) for sterilization and stored at 4° C. until use in experiments.
In order to confirm the purity of purified rat-bovine chimeric anti-bovine PD-1 antibody, antibody proteins were detected by SDS-PAGE and CBB staining. Purified rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was suspended in Laemmli Sample Buffer (Bio-Rad) and denatured at 95° C. for 5 min under reducing conditions (reduced with 2-mercaptoethaanol; Sigma-Aldrich) or under non-reducing conditions. The thus prepared samples were electrophoresed using 10% polyacrylamide gel. As molecular weight markers, Precision Plus Protein All Blue Standards (Bio-Rad) were used. After electrophoresis, the gel was stained with Quick-CBB kit (Wako) and subsequently decolored in distilled water.
The results are shown in
It was confirmed by flow cytometry that rat-bovine chimeric anti-bovine PD-1 antibody specifically binds to bovine PD-1 expressing cells (described above). First, rat anti-bovine PD-1 antibody 5D2 or rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was reacted with bovine PD-1 expressing cells at room temperature for 30 min. After washing, Allophycocyanine (APC)-labeled anti-rat Ig goat antibody (Southern Biotech) or Alexa Fluor 647-labeled anti-bovine IgG (H+L) goat F(ab′)2 (Jackson ImmunoResearch) was reacted at room temperature for 30 min. As negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, each rat antibody or rat-bovine chimeric antibody bound to cell surfaces was detected by FACS Verse (BD Biosciences). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.
The experimental results are shown in
The binding avidities to bovine PD-1 of rat anti-bovine PD-1 antibody 5D2 and rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 were measured by surface plasmon resonance using a biomolecular interaction analyzer (Biacore X100). Briefly, bovine PD-1-His (described above) was immobilized on a CM5 sensor chip (GE Healthcare) as a ligand. Subsequently, rat anti-bovine PD-1 antibody 5D2 or rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was reacted as an analyte, followed by single kinetics analysis. The experiment was repeated 3 times under the same conditions. Binding constant (kd value) and dissociation constant (ka value) were determined in each experiment, and binding avidity (KD value) was obtained.
The experimental results are shown in the table below. The binding avidity of rat-bovine chimeric anti-bovine PD-1 antibody for PD-1 protein was similar to that of rat anti-bovine PD-1 antibody 5D2, with no statistical difference observed (p>0.05; Welch's t-test).
Using bovine PD-L1 expressing cells (described above) and bovine PD-1-Ig (described above), bovine PD-1/PD-L1 binding inhibition by anti-PD-1 antibodies was tested. First, rat anti-bovine PD-1 antibody 5D2 or rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 (final concentration: 0, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25 or 50 μg/ml) and bovine PD-1-Ig (final concentration: 5 μg/ml) labeled with biotin using Lightning-Link Type A Biotin Labeling Kit (Innova Biosciences) were added to 96-well plates, followed by reaction at 37° C. for 30 min. The resultant mixture was reacted with 1×105 bovine PD-L1 expressing cells at 37° C. for 30 mn. After washing, the reaction mixture was reacted with APC-labeled streptavidin (BioLegend) at room temperature for 30 min to thereby detect bovine PD-1-Ig bound to cell surfaces. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. Taking the proportion of bovine PD-1-Ig-bound cells without addition of antibodies as 100%, the proportion of bovine PD-1-Ig-bound cells at each antibody concentration was shown as a relative value.
The experimental results are shown in
The complementarity-determining regions (CDRs) of rat anti-bovine PD-1 antibody 5D2 were determined using NCBI IGBLAST (http://www.ncbi.nlm.nih.2ov/i2blast/). The results are shown in
2.12. Inoculation Test on Cattle with Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody
Established rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 (14 mg; 0.08 mg/kg) was intravenously administrated into an experimentally BLV-infected calf (Holstein, male, 4 months old, 173.5 kg). Blood samples were collected chronologically from the infected calf, followed by collection of blood (with heparin sodium (Ajinomoto) used as anticoagulant) and serum. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation using Percoll (GE Healthcare).
Bovine PD-1-His (described above) was immobilized on ELISA plates (H type, Sumitomo Bakelite) at a final concentration of 10 μg/ml at 4° C. overnight. Subsequently, each well was washed with 200 μl of 0.05% Tween 20-supplemented Tris-buffered saline (TBS-T) five times, followed by blocking with 1% skim milk-supplemented TBS-T at room temperature for 1 hr. Another washing was carried out in the same manner. The serum collected from the test calf was added to each well and reacted at room temperature for 1 hr. After washing, horseradish peroxidase-labeled anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) was reacted at room temperature for 1 hr. Each well was washed again and then TMB One Component Substrate (Bethyl) was added for coloring. The enzyme reaction was terminated with 0.18 M dilute sulfuric acid. Absorbance (450 nm) was measured with Microplate Reader MTP-650FA (Corona Electric). For every plate washing operation, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used.
The experimental results are shown in
Bovine PBMCs were suspended in PBS and reacted with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) at room temperature for 20 min for labeling. After washing twice with RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies), cell concentration was adjusted to 4×106 cells/ml using the same medium. To the PBMCs, culture supernatant of 2% BLV-infected fetal lamb kidney cell (FLK-BLV), culture supernatant of fetal lamb kidney cell (FLK) not infected with 2% BLV, or BLV gp51 peptide mix 0.1 μg/ml or 1 μg/ml was added, followed by a 6-day culture at 37° C. under 5% CO2. After 6 days, PBMCs were recovered and reacted with Alexa Fluor 647-labeled mouse anti-bovine CD4 antibody (CC30, AbD Serotec), Peridinin-chlorophyll-protein complex/cyanin 5.5-labeled mouse anti-bovine CD8 antibody (CC63, AbD Serotec) and R-Phycoerythrin/cyanin 7 (PE/Cy7)-labeled anti-bovine IgM mouse antibody (IL-A30, AbD Serotec) at 4° C. for 20 min. For labeling antibodies, Zenon Mouse IgG1 Labeling Kits (Life Technologies) or Lightning-Link Kits (Innova Biosciences) was used. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. With respect to the proportion of proliferated T cells (CFSElow cells), statistical test was performed using the method of Dunnett.
The experimental results are shown in
DNA was extracted from isolated bovine PBMCs using Wizard DNA Purification kit (Promega). The concentration of the extracted DNA was quantitatively determined based on the absorbance (260 nm) measured with Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific). For measuring BLV proviral load in PBMCs, real time PCR was performed using Cycleave PCR Reaction Mix SP (Takara) and Probe/Primer/Positive control (Takara) for bovine leukemia virus detection. LightCycler480 System II (Roche Diagnosis) was used for the measurement. With respect to the measured proviral load, statistical test was performed by the method of Dunnett.
The experimental results are shown in
In order to determine the full-lengths of the coding sequences (CDSs) of ovine and water buffalo PD-1 cDNAs, primers for amplifying the full lengths of CDSs were first designed (ovPD-1 CDS F and R; buPD-1 CDS F1, R1, F2 and R2) based on the nucleotide sequences of ovine and water buffalo PD-1 genes (GenBank accession numbers BC123854 and XM_012176227), and then PCR was performed using a synthesized ovine or water buffalo PBMC-derived cDNA as a template. For the resultant amplified products, nucleotide sequences were determined with a capillary sequencer according to conventional methods (Mingala C N, Konnai S, Ikebuchi R, Ohashi K. Comp. Immunol. Microbiol. Infect. Dis., 34(1):55-63; January 2011; Water buffalo PD-1 gene was identified in this article).
In order to prepare ovine PD-1 expression plasmid, PCR was performed using a synthesized ovine PBMC-derived cDNA as a template and primers designed by adding BglII and SmaI recognition sites on the 5′ side (ovPD-1-EGFP F and R). The resultant PCR products were digested with BglII (Takara) and SmaI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pEGFP-N2 vector (Clontech) treated with the restriction enzymes in the same manner. The expression plasmid of interest was extracted using FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared plasmid is designated as pEGFP-N2-ovPD-1.
COS-7 cells were subcultured at a density of 5×104 cells/cm2 in 6-well plates, and then cultured overnight in RPMI 1640 medium containing 10% inactivated fetal bovine serum (Invitrogen) and 0.01% L-glutamine (Life Technologies) at 37° C. in the presence of 5% CO2. The pEGFP-N2-ovPD-1 or pEGFP-N2 (negative control) was introduced into COS-7 cells at 0.4 μg/cm2 using Lipofectamine 2000 (Invitrogen). The cells were cultured for 48 hours (ovPD-1-EGFP expressing cells). In order to confirm the expression of ovine PD-1 in the thus prepared expressing cells, intracellular localization of EGFP was visualized with an all-in-one fluorescence microscope BZ-9000 (KEYENCE).
1.3. Reactivity of Rat Anti-Bovine PD-1 Antibody 5D2 with Ovine PD-1 (
It was confirmed by flow cytometry that rat anti-bovine PD-1 monoclonal antibody cross-reacts with ovine PD-1. Ovine PD-1-EGFP expressing COS-7 cells were blocked with 10% inactivated goat serum (Invitrogen)-supplemented PBS at room temperature for 15 min and reacted with 10 μg/ml of rat anti-bovine PD-1 antibody 5D2 at room temperature for 30 min. After washing, the cells were reacted with APC-labeled anti-rat Ig goat antibody (Beckman Coulter) at room temperature for 30 min. As a negative control antibody, rat IgG2a (κ) isotype control (BD Bioscience) was used. For analysis, FACS Verse (BD Bioscience) was used. For every washing operation and dilution of antibodies, 1% bovine serum albumin (Sigma-Aldrich)-supplemented PBS was used.
The experimental results are shown in
1.4. Reactivity of Rat Anti-Bovine PD-1 Antibody 5D2 with Water Buffalo Lymphocytes (
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of water buffalo (Bubalus ubalis; Asian water buffalo) by density gradient centrifugation using Percoll C Healthcare). The isolated water buffalo PBMCs were suspended in RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies). Cell density was adjusted to 2×106 cells/ml. To these PBMCs, phorbol 12-myristate acetate (PMA) 20 ng/ml and ionomycin 1 μg/ml (Sigma-Aldrich) were added, followed by a 2-day culture at 37° C. under 5% CO2. Cultured PBMCs were harvested and blocked with 10% inactivated goat serum (Invitrogen)-supplemented PBS at room temperature for 15 min. Then, rat anti-bovine PD-1 antibody 5D2 and mouse anti-bovine CD8 antibody (38.65, AbD Serotec) were reacted at room temperature for 30 min. As a negative control, rat IgG2a (κ) isotype control (BD Bioscience) was used. After washing, APC-labeled goat anti-rat Ig antibody (Beckman Coulter) and PE-labeled goat anti mouse IgG antibody (Beckman Coulter) were reacted at room temperature for 30 min. After further washing, Alexa Flour488-labeled mouse anti-bovine CD4 antibody (CC30, AbD Serotec) and PE/Cy7-labeled anti-bovine IgM mouse antibody (IL-A30, AbD Serotec) were reacted at room temperature for 30 min. For antibody labeling, Zenon Mouse IgG1 Labeling Kits (Life Technologies) or Lightning-Link Kits (Innova Biosciences) was used. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, 10% inactivated goat serum (Invitrogen)-supplemented PBS was used.
The experimental results are shown in
The present inventors have established a rat-bovine chimeric anti-bovine PD-1 antibody in Example 5 with a view to establishing a novel therapy for bovine infections. In the process, mutations were added to putative binding sites for Fcγ receptors in bovine IgG1 CH2 domain in order to suppress ADCC activity mediated by the chimeric antibody (
Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 having wild-type bovine IgG1 (IgG1 WT) or mutated bovine IgG1 (IgG1 ADCC—described above) was established.
An expression plasmid encoding rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 having mutated bovine IgG1 (IgG1 ADCC—) was prepared according to the procedures described in Example 5 (SEQ ID NOS: 157 and 158 (amino acid sequences), SEQ ID NOS: 162 and 163 (nucleotide sequences after codon optimization)). It should be noted that in order to suppress ADCC activity, the bovine IgG1 used in ch5D2 IgG1 ADCC—had mutations added to the putative binding sites for Fcγ receptors in CH2 domain (see
An expression plasmid encoding rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 having wild-type IgG1 (IgG1 WT) was prepared according to the procedures described below. First, in order to amplify the gene encoding the constant region of wild-type bovine IgG1 (GenBank accession number X62916), PCR was performed using a synthesized bovine PBMC-derived cDNA as a template and designed primers that have NheI and XbaI recognition sites added on the 5′ side (boIgG1 CH1 F and boIgG1 CH3 R). The amplified gene strand was digested with NheI (Takara) and XbaI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics), and cloned into pDN112-boPD-1ch5D2 IgG1 ADCC—that had been treated with the restriction enzymes in the same manner. Further, the resultant plasmid was purified with QIAGEN Plasmid Midi kit (Qiagen) and digested with Nod (Takara) and XbaI (Takara) to thereby obtain an expression cassette for ch5D2's light chain (SEQ ID NO: 157 (amino acid sequence), SEQ ID NO: 162 (nucleotide sequence)) and heavy chain (IgG1 WT) (SEQ ID NO: 223 (amino acid sequence), SEQ ID NO: 224 (nucleotide sequence)). This gene fragment was purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into the cloning site (Nod and XbaI restriction enzyme recognition sequences downstream of PCMV and between INRBG and PABGH) of expression vector pDC6 (kindly provided by Prof. S. Suzuki, Hokkaido University Research Center for Zoonosis Control) (
Thirty micrograms of pDC6-boPD-1ch5D2 IgG1 WT or pDN112-boPD-1ch5D2 IgG1 ADCC—was introduced into 7.5×107 Expi293F cells (Life Technologies) using Expifectamine (Life Technologies) and the transfected cells were then cultured under shaking for 5 to 7 days, followed by collection of a culture supernatant. Each chimeric antibody was purified from the culture supernatant using Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; 1.5 M Glycine/3 M NaCl (pH 8.0) was used as equilibration buffer and wash buffer. As elution buffer, 0.1 M Glycine-HCl (pH 2.8) was used. As neutralization buffer, 1M Tris (pH 9.0) was used. The purified antibody was subjected to buffer exchange with PBS (pH 7.4) using PD-10 Desalting Column (GE Healthcare) and concentrated using Amicon Ultra-15 (50 kDa, Millipore). The thus purified chimeric antibody was passed through a 0.22 μm syringe filter (Pall Life Science) for sterilization and stored at 4° C. until use in experiments. The concentration of each chimeric antibody as purified was quantitatively determined with the absorbance (280 nm) measured with Nanodrop8000 Spectrophotometer (Thermo Fisher Scientific).
In order to confirm the purity of purified rat-bovine chimeric anti-bovine PD-1 antibodies (ch5D2 IgG1 WT and ch5D2 IgG1 ADCC—), antibody proteins were detected by SDS-PAGE and CBB staining. Each chimeric antibody purified was suspended in Laemmli Sample Buffer (Bio-Rad) and denatured at 95° C. for 5 min under reducing conditions (reduced with 2-mercaptoethaanol; Sigma-Aldrich) or under non-reducing conditions. The thus prepared samples were electrophoresed using SuperSep Ace 5%-20% gradient polyacrylamide gel (Wako). As molecular weight markers, Precision Plus Protein All Blue Standards (Bio-Rad) were used. After electrophoresis, the gel was stained with Quick-CBB kit (Wako) and decolored in distilled water.
The results are shown in
Bovine FcγRI-His, FcγRII-His, FcγRIII-His and Fcγ2R-His expressing plasmids were constructed according to the procedures described below. In order to amplify the signal peptide and the extracellular region of bovine FcγRI, FcγRII, FcγRIII and Fcγ2R (GenBank accession numbers NM_174538, NM_174539, NM_001077402 and NM_001001138), primers were designed which had NotI and XhoI recognition sites added on the 5′ side (boFγyRI-His F and R; boFγyRIII-His F and R; or boFcγ2R-His F and R) or NheI and EcoRV recognition sites added on the 5′ side (boFcγRIII-His F and R). A gene sequence encoding a 6xHis tag was added to reverse primers. PCR was performed using a synthesized bovine PBMC-derived cDNA as a template. The respective PCR products were digested with Nod (Takara) and XhoI (Takara) (FcγRI-His, FcγRIII-His and Fcγ2R-His) or NheI (Takara) and EcoRV (Takara) (FcγRII-His), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCXN2.1(+) vector (Niwa H, Yamamura K, Miyazaki J. Gene, 108(2):193-199; Dec. 15, 1991; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine). The resultant expression plasmids were purified with FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCXN2.1-boFcγRI-His, pCXN2.1-boFcγRII-His, pCXN2.1-boFcγRIII-His or pCXN2.1-boFcγ2R-His.
Soluble bovine FcγRI-His, FcγRII-His, FcγRIII-His and Fcγ2R-His expressing cells were prepared according to the procedures described below. Briefly, 30 μg of pCXN2.1-boFcγRI-His, pCXN2.1-boFcγRII-His, pCXN2.1-boFcγRIII-His or pCXN2.1-boFcγ2R-His was introduced into 7.5×107 Expi293F cells (Life Technologies) using Expifectamine (Life Technologies) and the transfected cells were then cultured under shaking for 5 to 7 days, followed by collection of a culture supernatant. Recombinant proteins were purified from the culture supernatant using TALON Metal Affinity Resin (Clontech). After purification, the buffer was exchanged with PBS (pH 7.4) using Amicon Ultra-15 Centrifugal Filter Unit (10 kDa, Millipore), and the recombinant proteins were stored at −30° C. until use in experiments (bovine PD-1-His). The concentrations of purified bovine FcγRI-His, FcγRII-His, FcγRIII-His and Fcγ2R-His were quantitatively determined in terms of the absorbance (280 nm) measured with Nanodrop8000 Spectrophotometer (Thermo Fisher Scientific).
2.5. Binding to Bovine FcγRs of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody ch5D2 IgG1 WT and IgG1 ADCC—(
Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 IgG1 WT or IgG1 ADCC—was immobilized on Nunc MaxiSorp ELISA plates (Nunc) at a final concentration of 50, 25, 12.5, 6.25, 3.12 or 1.5610 nM at 37° C. for 2 hr. Subsequently, each well was washed with 200 μl of 0.05% Tween 20-supplemented PBS (PBS-T) five times, followed by blocking with SuperBlock (PBS) Blocking Buffer (Thermo Fisher Scientific) at 37° C. for 30 min. Each well was washed again in the same manner. Then, bovine FcγRI-His, FcγRII-His, FcγRIII-His or Fcγ2R-His was added to each well at a final concentration of 10 μg/ml and reacted at 37° C. for 1 hr. After washing, anti-polyhistidine tag mouse monoclonal antibody (Abcam) was reacted at 37° C. for 30 min. Subsequently, each well was washed, and horseradish peroxidase-labeled anti-mouse IgG goat polyclonal antibody (MP Biomedicals) was reacted at 37° C. for 30 min. Each well was washed again, and then TMB One Component Substrate (Bethyl) was added for coloring. Thereafter, the enzyme reaction was terminated with 0.18 M dilute sulfuric acid, and absorbance (450 nm) was measured with Microplate Reader MTP-900 (Corona Electric). For every plate washing operation, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used.
The experimental results are shown in
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
The anti-PD-L1 antibody of the present disclosure is applicable to prevention and/or treatment of cancers and infections in animals. Further, the pharmaceutical composition of the present disclosure comprising a COX-2 inhibitor and an inhibitor targeting PD-1/PD-L1 is applicable to prevention and/or treatment of cancer and/or infection.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-159088 | Aug 2016 | JP | national |
| 2016-159089 | Aug 2016 | JP | national |
| 2017-061454 | Mar 2017 | JP | national |
| 2017-110723 | Jun 2017 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17579517 | Jan 2022 | US |
| Child | 18914727 | US | |
| Parent | 16325040 | Feb 2019 | US |
| Child | 17579517 | US |
