Patent Grant
11312773
This application is a U.S. National Stage Filing under 35 U.S.C. 371 from International Application No. PCT/JP2017/029055, filed on Aug. 10, 2017, and published as WO2018/034225 on Feb. 22, 2018, which claims the benefit of priority to Japanese Application No. 2017-110723, filed on Jun. 5, 2017 and to Japanese Application No. 2017-061454, filed on Mar. 27, 2017 and to Japanese Application No. 2016-159089, filed on Aug. 15, 2016 and to Japanese Application No. 2016-159088, filed on Aug. 15, 2016; the benefit of priority of each of which is hereby claimed herein, and which applications and publication are hereby incorporated herein by reference in their entirety.
The present invention relates to an anti-PD-L1 antibody. More specifically, the present invention 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.
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(1):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, Nakjima 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.)
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 an object of the present invention to provide an anti-PD-L1 antibody capable of repeated administration even to animals other than rat.
The present inventors have determined 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, the present inventors have determined the CDRs of the variable region of the rat anti-bovine PD-L1 monoclonal antibody 4G12.
Furthermore, the present inventors have 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
A summary of the present invention is as described below.
The present specification encompasses the contents disclosed in the specifications and/or drawings of Japanese Patent Applications No. 2016-159088, No. 2016-159089, No. 2017-110723 and No. 2017-61454 based on which the present patent application claims priority.
According to the present invention, a novel anti-PD-L1 antibody has been obtained. This antibody is applicable even to those animals other than rat.
Hereinbelow, the present invention will be described in detail.
The present invention provides an anti-PD-L1 antibody 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.
CDR1, CDR2 and CDR3 in the light chain variable region (VL) of rat anti-bovine PD-L1 antibody 4G12 are a region consisting of the amino acid sequence of QSLLYSENQKDY (SEQ ID NO: 37), a region consisting of the amino acid sequence of WAT and a region consisting of 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 consisting of the amino acid sequence of GYTFTSNF (SEQ ID NO: 39), a region consisting of the amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and a region consisting of 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.
As used herein, the term “antibody” is a concept encompassing not only full-length antibodies but also 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 and scFv-Fc dimer.
In the anti-PD-L1 antibody of the present invention, 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 VL and VH 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 Q). In the anti-PD-L1 antibody of the present invention, 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 ε 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 antibody of the present invention, 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.
With respect to bovine, sequences of IgG1, IgG2 and IgG3 have been identified as the heavy chain of IgG. In the antibody of the present invention, 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 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 102, respectively.
When an animal other than rat is canine, an anti-PD-L1 antibody is more preferable in which (i) the CL of a canine antibody has the amino acid sequence of the constant region of Lambda chain and (ii) the CH of the canine antibody has the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4.
When an animal other than rat is bovine, an anti-PD-L1 antibody is more 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-L1 antibody of the present invention encompasses rat-canine chimeric antibodies, caninized antibodies, complete canine-type antibodies, rat-bovine chimeric antibodies, bovinized antibodies and complete bovine-type 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 and the like.
For example, the anti-PD-L1 antibody of the present invention 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.
As a further example, the anti-PD-L1 antibody of the present invention may be 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 anti-PD-L1 antibody of the present invention may have a four-chain structure comprising two light chains and two heavy chains.
The anti-PD-L1 antibody of the present invention 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; antibody equivalent to human IgG4 antibody; or 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-L1 antibody identified by the present inventors are shown in SEQ ID NOS: 1 and 5, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 15.
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, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 16.
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 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: 104.
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.
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: 9 shows the amino acid sequence of a chimeric light chain consisting of 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 consisting of 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.
Further, SEQ ID NO: 105 shows the amino acid sequence of a chimeric light chain consisting of 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 consisting of 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: 107.
SEQ ID NO: 10 shows the amino acid sequence of a chimeric heavy chain consisting of 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 consisting of 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.
SEQ ID NO: 106 shows the amino acid sequence of a chimeric heavy chain consisting of 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 consisting of 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: 108.
Amino acid sequences and nucleotide sequences of CLs and CHs for various animals other than the above may be obtained from known databases for use in the present invention.
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.
Table.
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 and 11 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 the 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.
The present invention provides an artificial genetic DNA comprising (a′) a DNA encoding alight chain comprising a light chain variable region (VL) 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 the light chain constant region (CL) of an antibody of an animal other than rat and (b′) a DNA encoding a heavy chain comprising a heavy chain variable region (VH) 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 the heavy chain constant region (CH) of an antibody of an animal other than rat. The present invention also provides a DNA encoding a light chain comprising a VL 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 the CL of an antibody of an animal other than rat (i.e., the DNA of (a′) above). Further, the present invention also provides a DNA encoding a heavy chain comprising a VH containing CDR1 having 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 the CH of an antibody of an animal other than rat (i.e., the DNA of (b′) above).
For (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 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 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 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.
The present invention 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, pDC6 (Japanese Patent No. 5704753, U.S. Pat. No. 9,096,878, EU Patent 2385115, Hong Kong (China) patent HK1163739 and Australia Patent 2009331326) 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 invention also provides a host cell transformed by the above vector. It is possible to prepare the anti-PD-L1 antibody of the invention by culturing the host cell and collecting the antibody of interest from the resultant culture. Therefore, the present invention also provides a method of preparing an antibody, comprising culturing the above-described host cell and collecting the anti-PD-L1 antibody of the invention from the culture. In the method of the present invention 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-L1 antibody of the present invention 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 invention 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 anti-PD-L1 antibody of the present invention may be used as an antibody drug for animals or human. Therefore, the present invention provides a pharmaceutical composition comprising the above-described anti-PD-L1 antibody as an active ingredient.
The pharmaceutical composition of the present invention may be used for prevention and/or treatment of cancers and/or infections. Examples of cancers and/or infections 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 anti-PD-L1 antibody of the present invention may be dissolved in buffers such as PBS, physiological saline or sterile water, optionally filter-sterilized with a filter or the like, and then administered to animal subjects (including human) by injection. To the solution of this 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 and the like may be selected. Transnasal or oral administration may also be used.
The dose and the number of times and frequency of administration of the anti-PD-L1 antibody of the present invention 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 the desired effect.
While the pharmaceutical composition of the present invention 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 invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples.
1. Introduction
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.
2. Materials and Methods
2.1 Bovine PD-L1 Monoclonal Antibody Producing Cells
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.)
2.2 Identification of Full-Length Canine PD-1 and PD-L1 Genes
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).
Primer (cPD-1 inner F): AGGATGGCTCCTAGACTCCC (SEQ ID NO: 21)
Primer (cPD-1 inner R): AGACGATGGTGGCATACTCG (SEQ ID NO: 22)
Primer (cPD-L1 inner F): ATGAGAATGTTTAGTGTCTT (SEQ ID NO: 23)
Primer (cPD-L1 inner R): TTATGTCTCTTCAAATTGTATATC (SEQ ID NO: 24)
Primer (cPD-1 5′GSP): GTTGATCTGTGTGTTG (SEQ ID NO: 25)
Primer (cPD-1 3′GSP): CGGGACTTCCACATGAGCAT (SEQ ID NO: 26)
Primer (cPD-L1 5′GSP): TTTTAGACAGAAAGTGA (SEQ ID NO: 27)
Primer (cPD-L1 3′GSP): GACCAGCTCTTCTTGGGGAA (SEQ ID NO: 28)
2.3 Construction of Canine PD-1 and PD-L1 Expressing COS-7 Cells
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.
Primer (cPD-1-EGFP F): CCGCTCGAGATGGGGAGCCGGCGGGGGCC (SEQ ID NO: 29)
Primer (cPD-1-EGFP R): CGCGGATCCTGAGGGGCCACAGGCCGGGTC (SEQ ID NO: 30)
Primer (cPD-L1-EGFP F): GAAGATCTATGAGAATGTTTAGTGTC (SEQ ID NO: 31)
Primer (cPD-L1-EGFP R): GGAATTCTGTCTCTTCAAATTGTATATC (SEQ ID NO: 32)
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).
2.4 Construction of Recombinant Canine PD-1, PD-L1 and CD80
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 EcRV (Takara) or with EcoRV (Takara) and KpnT (New England Biolabs) and purified with FastGene GeV/PCR Extraction Kit (NIPPON Genetics). The thus purified DNAs were individually cloned into pCXN2.1-Rabbit IgG Fc vector (Niwa et al., 1991; Zettimeissl 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 (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 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)).
2.6 Selection Test of Variable Region for Establishment of Rat-Canine Chimeric Anti-PD-L1 Antibody
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 607 (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 (
2.7 Preparation of Rat-Canine Chimeric Anti-PD-L1 Antibody Expressing Vector (
Using rat anti-bovine PD-L1 monoclonal antibodies 4G12 and 6G7 which showed a good inhibitory activity against canine PD-1/PD-L1 binding (
Briefly, heavy chain and light chain variable region genes were identified from hybridomas producing rat anti-bovine PD-L1 monoclonal antibodies 4G12 and 6G7. Further, the heavy chain and light chain variable region genes of the above rat antibodies were linked to the constant region of heavy chain IgG4 and the constant region of light chain Lambda of a known canine antibody, respectively, to prepare nucleotide sequences, followed by codon optimization (SEQ ID NOS: 9 and 10 (amino acid sequences), SEQ ID NOS: 19 and 20 (nucleotide sequences after codon optimization). Then, synthesis of genes was performed so that NotI restriction enzyme recognition site. KOZAK sequence, chimeric antibody's light chain sequence, poly-A addition signal sequence (PABGH), promoter sequence (PCMV), SacI restriction enzyme recognition site, intron sequence (INRBG), KOZAK sequence, chimeric antibody's heavy chain sequence and XbaI restriction enzyme recognition site would be located in this order. The synthesized gene strands were individually incorporated into the cloning site (NotI 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) using restriction enzyme recognition sequences so that the above-listed sequences would be located in the above-mentioned order (
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 6×His 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 mid is designated as pCXN2.1-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 CMS 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 c4012 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 super-antigen 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, indicating an elevated cell proliferation capacity.
2.14 Selection of Tumor-Affected Dogs to be Used in Canine Inoculation Test
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).
2.15 Inoculation Test on Dogs
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) (
2.16 CDR Analysis of Anti-PD-L1 Antibody
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
1.1 Identification of Ovine, Porcine and Water Buffalo PD-L1 Genes
In order to determine the full-lengths of the coding sequences (CDSs) of ovine, porcine and water buffalo PD-L1 cDNAs, primers for amplifying the full lengths of CDSs from the nucleotide sequences of ovine, porcine and water buffalo PD-L1 genes (GenBank accession number, XM_004004362, NM_001025221 and XM_613366) were first designed (ovPD-L1 CDS F and R; poPD-L1 CDS F and R; buPD-L1 CDS F1, R1, F2 and R2), and then PCR was performed. 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. 2011 January; 34(1):55-63; Water buffalo PD-L1 gene was identified in this article).
1.2 Construction of Ovine PD-1, Ovine PD-L1, Porcine PD-1 and Porcine PD-L1 Expressing COS-7 Cells
In order to prepare ovine PD-1, ovine PD-L1, porcine PD-1 and porcine PD-L1 expressing plasmids, PCR was performed using a synthesized ovine or porcine PBMC-derived cDNA as a template and primers designed by adding BglII and SmaI (ovine PD-1), HindIII and SmaI (porcine PD-1), or XhoI and SmaI (ovine and porcine PD-L1) recognition sites on the 5′ side (ovPD-1-EGFP F and R; ovPD-L1-EGFP F and R; poPD-1-EGFP F and R; or poPD-L1-EGFP F and R). The resultant PCR products were digested with BglII (Takara) and SmaI (Takara) (ovine PD-1), HindIII (Takara) and SmaI (Takara) (porcine PD-1), and XhoI (Takara) and SmaI (Takara) (ovine and porcine PD-L1), then purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pEGFP-N2 vector (Clontech) treated with restriction enzymes in the same manner. Expression plasmids were 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, pEGFP-N2-ovPD-L1, pEGFP-N2-poPD-1 or pEGFP-N2-poPD-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-ovPD-1, pEGFP-N2-ovPD-L1, pEGFP-N2-poPD-1, pEGFP-N2-poPD-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 (ovPD-1-EGFP expressing cell, ovPD-L1-EGFP expressing cell, poPD-1-EGFP expressing cell, and poPD-L1-EGFP expressing cell). In order to confirm the expression of ovine PD-1, ovine PD-L1, porcine PD-1 and porcine PD-L1 in the thus prepared expressing cells, intracellular localization of EGFP was visualized with an inverted confocal laser microscope LSM700 (ZEISS) or an all-in-one fluorescence microscope BZ-9000 (KEYENCE).
1.3 Construction of Recombinant Ovine PD-L1 and Porcine PD-L1
In order to amplify the extracellular regions of ovine PD-L1 or porcine PD-L1 estimated from their putative amino acid sequences, primers were designed. Briefly, primers having an NheI or EcoRV recognition sequence added on the 5′ side (ovPD-L1-Ig F and R, or poPD-L1-Ig F and R) were designed. PCR was performed using a synthesized ovine or porcine 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 DNAs were individually cloned into pCXN2.1-Rabbit IgG Fc vector (Niwa et al., 1991; Zettlmeissi 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 FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmids are designated as pCXN2.1-ovPD-L1-Ig and pCXN2.1-poPD-L1-Ig, respectively.
Thirty micrograms of pCXN2.1-ovPD-L1-Ig or pCXN2.1-poPD-L1-Ig was introduced into 7.5×107 Expi293F cells (Life Technologies) using Expifectamin (Life Technologies). After 6-day shaking culture, a culture supernatant was collected. The culture supernatant contained an Fc fusion recombinant protein. The produced Fc recombinant protein was purified from the supernatant using Ab-Capcher Extra (ProteNova). After purification, the buffer was exchanged with PBS (pH 7.4) using PD-10 Desalting Column (GE Healthcare). The resultant recombinant protein was stored at −30° C. until use in Experiment (ovine PD-L1-Ig). Concentrations of purified ovine PD-L1-Ig and porcine PD-L1-Ig were measured with Rabbit IgG ELISA Quantitation Set (BETHYL). For each washing operation in ELISA, Auto Palte Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).
1.4 Reactivity of Rat Anti-Bovine PD-L1 Antibody 4G12 with Ovine and Porcine PD-L1
It was confirmed by flow cytometry that rat anti-bovine PD-L1 monoclonal antibody cross-reacts with ovine and porcine PD-LL. Ovine or Porcine PD-L1-EGFP expressing COS-7 cells were blocked with 10% inactivated goat serum supplemented PBS at room temperature for 15 min and reacted with 10 μg/ml of rat anti-bovine PD-L1 antibody 4G12 at room temperature for 30 min. After washing, the cells were reacted with allophycocyanine-labeled anti-rat Ig goat antibody (Beckman Coulter) at room temperature for 30 min. For analysis, FACS Verse (BD Bioscience) was used. As a negative control antibody, rat IgG2a (κ) isotype control (BD Bioscience) was used. For every washing operation and dilution of antibodies, 1% bovine serum albumin supplemented PBS was used.
Experimental results are shown in
1.5 Reactivity of Rat Anti-Bovine PD-L1 Antibody 4G12 with Water Buffalo Leukocytes
Peripheral blood of water buffalo (Bubalus bubalis; Asian water buffalo) was hemolyzed with ACK buffer to isolate leukocytes. After blocking with 10% inactivated goat serum supplemented PBS at room temperature for 15 min, reaction with rat anti-bovine PD-L1 antibody 4G12, peridinin-chlorophyll-protein complex/cyanin 5.5-labeled anti-bovine CD14 antibody (mouse IgG1, CAM36A, VMRD) and anti-bovine CD11b antibody (mouse IgG2b, CC126, AbD Serotec) was conducted at room temperature for 30 min. After washing, reaction with allophycocyanine-labelled anti-rat Ig goat antibody (Beckman Coulter) and fluorescein isothiocyanate-labeled anti-mouse IgG2 goat antibody (Beckman Coulter) was conducted at room temperature for 30 min. For analysis, FACS Calibur (BD Biosciences) was used. As a negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) was used. For every washing operation and dilution of antibodies, 10% inactivated goat serum supplemented PBS was used.
Experimental results are shown in
1.6 Inhibition Test on Ovine or Porcine PD-1/PD-L1 Binding with Rat Anti-Bovine PD-L1 Antibody 4G12
Using ovine PD-1-EGFP expressing COS-7 cells and ovine PD-L1-Ig recombinant protein, or porcine PD-1-EGFP expressing COS-7 cells and porcine PD-L1-Ig recombinant protein, inhibition of ovine or porcine PD-1/PD-L1 binding by rat anti-bovine PD-L1 antibody (4012) was tested. Briefly, rat anti-bovine PD-L1 antibody 4G12 of various concentrations (0, 1, 5, 10, 20, 50 μg/ml) was reacted in advance with ovine PD-L1-Ig (final concentration 1 μg/ml) or porcine PD-L1-Ig (final concentration 5 μg/ml) at 37° C. for 30 min. Subsequently, the antibody 4G12 was reacted with 2×105 ovine PD-1-EGFP expressing COS-7 cells or porcine PD-1-EGFP expressing COS-7 cells at 37° C. for 30 min. After washing, ovine PD-L1-Ig or porcine PD-L1-Ig bound to cell surfaces was detected with Alexa Fluor 647-labeled anti-rabbit IgG (H+L) goat F(ab′)2 (Life Technologies). For analysis, FACS Verse (BD Biosciences) was used. As a negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) 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 results revealed that rat anti-bovine PD-L1 antibody 4G12 is capable of inhibiting ovine PD-1/PD-L1 and porcine PD-1/PD-L1 binding in a concentration dependent manner (
1. Introduction
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
2. Materials and Methods
Construction of Bovine PD-1 and PD-L1 Expressing Cells
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 NotI and HindIII (bovine PD-1) recognition sites and NheI and XhoI (bovine PD-L1) recognition sites on the 5′ side (boPD-L1-myc F and R; boPD-L1-EGFP F and R). The PCR products were digested with NotI (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).
Construction of Soluble Bovine PD-1 and PD-L1
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 Fe 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 Noll 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 Fe vector (Niwa et al., 1991; Zettdmeissl 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).
Preparation of Rat Anti-Bovine PD-L1 Monoclonal Antibody Producing Cells 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).
Preparation of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody Expressing Vector 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 4012 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: 105 and 106 (amino acid sequences), SEQ ID NOS: 107 and 108 (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
Expression of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody 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.
Purification of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody
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, IM 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.
Confirmation of the Purity of Purified Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody (
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
Binding Specificity of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody (
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 (k) 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
Inhibitory Activity of Rat-Bovine Chimeric Anti-PD-L1 Antibody against Bovine PD-1/PD-L1 Binding
(1) Binding Inhibition Test on Bovine PD-L1 Expressing Cells and Soluble Bovine PD-1 (
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 (w) 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
(2) Binding Inhibition Test on Bovine PD-1 Expressing Cells and Soluble Bovine PD-L1 (
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
Biological Activity Test Using Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody
(1) Effect on Cell Proliferation (
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 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
(2) Effect on IFN-γ Production (
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
CDR Analysis of Rat Anti-Bovine PD-L1 Antibody
The complementarity-determining regions (CDRs) of rat anti-bovine PD-L1 antibody 4012 were determined using NCBI IGBLAST (http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in
Inoculation Test on Cattle
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.
(1) Cell Proliferation Response of T Cells to BLV Antigen (
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
(2) Changes in the BLV Provirus Load (
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 (Thermo Fisher 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
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
The anti-PD-L1 antibody of the present invention is applicable to prevention and/or treatment of cancers and infections in animals.
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