Patent Application
20220002389
This international patent application claims the priority based on Japanese Patent Application No. 2018-203114 filed on Oct. 29, 2018, and Japanese Patent Application No. 2019-166040 filed on Sep. 12, 2019, the entire contents of which are hereby incorporated by reference.
The present invention relates to a method for efficiently producing an anti-HIV antibody. More specifically, the present invention relates to a method for producing an anti-HIV antibody with high efficiency by utilizing silkworms.
An attempt has been made to place an antibody gene downstream of the sericin promoter of silkworms to express an antibody molecule in the cocoons, thereby producing a therapeutic antibody with silkworms (for example, Non-Patent Documents 1 and 2). In particular, an antibody expressed in the cocoons of silkworms, which contains no core fucose in the glycans, has been known to exhibit an excellent ADCC activity (Patent Documents 1 and 2).
Also, in the therapeutic strategy of HIV, an antibody has been expected that enables to control HIV over a long period of time with one or several times of administration of the antibody. For example, Non-Patent Document 3 reports that, among four rhesus macaques to which PGT121 has been administered once, the viral RNA load is suppressed to the detection limit or lower over about 70 days in one rhesus macaque.
Further, KD247, which is a humanized antibody recognizing the V3 loop of gp120 which is envelope protein of HIV, has been developed and a clinical test thereof has been conducted. In a patient to which KD247 has been administered three times, suppression of the viral load is confirmed. However, suppression of the viral load to the detection limit or lower for a long period of time is not achieved. As described above, an antibody that can suppress the viral load to the detection limit or lower with a high probability for a long period of time with one to several times of single-agent administration has not been developed.
In view of the above circumstances, development of a 1C10 antibody, which is a human antibody having an antigen recognition site same as KD247 has proceeded. The 1C10 antibody is an antibody that is isolated from a patient having antibodies capable of neutralizing a wide range of HIV strains, whose symptom has been suppressed without any treatment for a long period of more than 25 years. The 1C10 antibody has been expected to exhibit a high therapeutic effect by being administered to an HIV-infected patient as a therapeutic antibody (Patent Document 2 and Non-Patent Document 4).
The present inventors have conducted intensive studies on an antibody which highly probably controls HIV in an administration group over a long period of time with one or several times of single-agent administration. As a result, the inventors have surprisingly found that, small number of times of administration of SW-1C10 antibody, which is obtained by producing an antibody from a gene reported as 1C10 in silkworms, not only suppressed the viral load in the blood below the detection limit at an early stage in all of administered individuals, but also the viral RNA load in the blood is maintained below the detection limit for such a long time as 12 weeks. Further, the inventors have compared an antibody produced in silkworms and an antibody produced in CHO cells to confirm that the structural difference between these antibodies exists in their glycan structures. In view of the above, the present inventors have concluded that removal of fucose from glycans make the 1C10 antibody to have an extremely excellent activity in suppressing the viral load for a long period of time in all administered cases. It has not been reported in the past that viral replication can be suppressed over a long period of time in all administered cases. The inventors, for the first time, have succeeded in discovering an antibody that can widely and stably suppresses the viral load in an administered subject with a few times of single-agent administration.
Further, the yield of the antibody in silkworms was approximately several hundreds μg per cocoon, or several μg per 1 mg of cocoon in the past, but no study have been conducted to increase the productivity to yield more than this level. The inventors have conducted studies to find an anti-HIV antibody having a higher yield in silk-spinning insects among a large number of anti-HIV antibodies. As a result, the inventors have found that anti-HIV antibodies, 1C10 antibody and 1D9 antibody, are produced in silk threads of silk-spinning insects at a higher yield than the previously obtained yield, and thus have completed the present invention.
Accordingly, in an embodiment, the present invention relates to an antibody, wherein the antibody has a binding ability to HIV; glycans bound to the antibody contain no fucose; and the antibody has an activity of suppressing HIV load in the blood of an HIV-infected patient below a detection limit at a probability of 90% or more with one or several times of administration to the HIV-infected patient. A method for examining whether a given antibody has a binding ability to HIV has been widely known in the technical field. The ability can be confirmed by, for example, bringing the antibody into contact with a carrier to which HIV is immobilized and detecting the antibody bound to the carrier.
In an embodiment, the present invention relates to an IgG antibody, wherein the antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 7, and a light chain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 9; the antibody has a binding ability to HIV; and glycans bound to the antibody contain no fucose.
Preferably, in the antibody of the present invention, the heavy chain has the three CDR sequences in the heavy chain amino acid sequence of SEQ ID NO: 7, and the light chain has the three CDR sequences in the amino acid sequence of SEQ ID NO: 9. A determination of the CDR sequence can be conducted by referring to the numbering system of Kabat et al. (Kabat, E. et al., U.S. Department of Health and Human Services, (1983) and later versions), the numbering system of Chothia et al. (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)), the numbering system of Honegger et al. (Honegger, A et al., J. Mol. Biol. 309: 657-670 (2001)), the contact determination method (MacCallum et al., J Mol Biol. 262(5): 732-745 (1996)), the numbering system according to the IMGT database (http://www.imgt.org/), or alignment to known sequence databases. The CDR sequence of the antibody of the present invention can be, for example, CDRH1: GFMFSNYA (SEQ ID NO: 14); CDRH2: ISNDGSDK (SEQ ID NO: 15); and CDRH3: CARDLDQTIPDLTAPAFEV (SEQ ID NO: 16), and CDRL1: QSLLHSDGNN (SEQ ID NO: 17); CDRL2: LTS (SEQ ID NO: 18); and CDRL3: MQSLQTWT (SEQ ID NO: 19). More preferably, the antibody of the present invention has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence of SEQ ID NO: 9.
In the antibody of the present invention, no fucose is contained in glycans attached to the antibody. The antibody of the present invention may have a glycan structure selected from the following formulas, for example.
In an embodiment, the present invention relates to an expression cassette containing any one polynucleotide selected from the following (i) to (x) which is functionally linked to downstream of a silk gland-specific gene promoter:
(i) a polynucleotide having the nucleotide sequence of SEQ ID NO: 6 and/or the nucleotide sequence of SEQ ID NO: 8;
(ii) a polynucleotide having a nucleotide sequence which hybridizes with the nucleotide sequence of SEQ ID NO: 6 under a stringent condition and/or a nucleotide sequence which hybridizes with the nucleotide sequence of SEQ ID NO: 8 under a stringent condition;
(iii) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 7, and/or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 9;
(iv) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 7, and/or a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 9;
(v) a polynucleotide encoding an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 7, and/or a polynucleotide encoding an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 9;
(vi) a polynucleotide having the nucleotide sequence of SEQ ID NO: 10 and/or the nucleotide sequence of SEQ ID NO: 12;
(vii) a polynucleotide having a nucleotide sequence which hybridizes with the nucleotide sequence of SEQ ID NO: 10 under a stringent condition and/or a nucleotide sequence which hybridizes with the nucleotide sequence of SEQ ID NO: 12 under a stringent condition;
(viii) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 11, and/or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 13;
(ix) a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 11, and/or a polynucleotide encoding an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 13; and
(x) a polynucleotide encoding an amino acid sequence in which several amino acids are substituted, deleted, added, or inserted in the amino acid sequence of SEQ ID NO: 11, and/or a polynucleotide encoding an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 13.
In the present specification, the 1C10 antibody is an antibody having a heavy chain consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain consisting of the amino acid sequence of SEQ ID NO: 9. In the amino acid sequence of SEQ ID NO: 7, the positions 1 to 19 constitute of a signal sequence. The heavy chain and/or light chain of the antibody described in the present specification may not include the signal sequence described in the present specification. Alternatively, the heavy chain and/or light chain may include a signal sequence other than the signal sequence described in the present specification. Accordingly, in the present specification, the wording “heavy chain consisting of the amino acid sequence of SEQ ID NO: 7” may be interchangeably read as “heavy chain consisting of the amino acid sequence at positions 20 to 474 in the amino acid sequence of SEQ ID NO: 7”. Similarly, in the amino acid sequence of SEQ ID NO: 9, the positions 1 to 20 constitute of a signal sequence. Accordingly, in the present specification, the wording “light chain consisting of the amino acid sequence of SEQ ID NO; 9” may be interchangeably read as “light chain consisting of the amino acid sequence at positions 21 to 238 in the amino acid sequence of SEQ ID NO: 9”. In the present specification, the 1D9 antibody is an antibody having a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11 and a light chain consisting of the amino acid sequence of SEQ ID NO: 13. In the amino acid sequence of SEQ ID NO: 11, the positions 1 to 19 constitute of a signal sequence. Accordingly, in the present specification, the wording “heavy chain consisting of the amino acid sequence of SEQ ID NO: 11” may be interchangeably read as “heavy chain consisting of the amino acid sequence at positions 20 to 472 in the amino acid sequence of SEQ ID NO: 11”. Similarly, in the amino acid sequence of SEQ ID NO: 13, the positions 1 to 20 constitute of a signal sequence. Accordingly, in the present specification, the wording “light chain consisting of the amino acid sequence of SEQ ID NO: 13” may be interchangeably read as “a light chain consisting of the amino acid sequence at positions 21 to 238 in the amino acid sequence of SEQ ID NO: 9”.
A DNA sequence encoding the 1C10 antibody includes, for example, the nucleotide sequence (heavy chain) of SEQ ID NO: 6 and the nucleotide sequence (light chain) of SEQ ID NO: 8. As described above, in the nucleotide sequence (heavy chain) of SEQ ID NO: 6, 57 bases encode a signal sequence. Accordingly, in the present specification, the wording “nucleotide sequence of SEQ ID NO; 6” may be interchangeably read as “nucleotide sequence at positions 58 to 1425 in the nucleotide sequence of SEQ ID NO: 6”. Similarly, in the nucleotide sequence (light chain) of SEQ ID NO: 8, 60 bases encode a signal sequence. Accordingly, in the present specification, the wording “nucleotide sequence of SEQ ID NO: 8” may be appropriately interchangeably read as “nucleotide sequence at positions 61 to 717 in the nucleotide sequence of SEQ ID NO: 8”. Also, a DNA sequence encoding the 1D9 antibody includes, for example, the nucleotide sequence (heavy chain) of SEQ ID NO: 10 and the nucleotide sequence (light chain) of SEQ ID NO: 12. As described above, in the nucleotide sequence (heavy chain) of SEQ ID NO: 10, 57 bases encode a signal sequence. Accordingly, in the present specification, the wording “nucleotide sequence of SEQ ID NO: 10” may be appropriately interchangeably read as “nucleotide sequence at position 58 to 1419 in the nucleotide sequence of SEQ ID NO: 10”. Similarly, in the nucleotide sequence (light chain) of SEQ ID NO: 12, 60 bases encode a signal sequence. Accordingly, in the present specification, the wording “nucleotide sequence of SEQ ID NO: 12” may be appropriately interchangeably read as “nucleotide sequence at positions 61 to 717 in the nucleotide sequence of SEQ ID NO: 12”.
In the present specification, the expression “hybridize under a stringent condition” means hybridizing under a hybridization condition ordinarily used by those skilled in the art. For example, whether to hybridize can be determined according to the method described in Molecular Cloning, a Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012). Current Protocols in Molecular Biology, Wiley Online Library, or the like. For example, the hybridization condition may be a condition where hybridization is carried out with 6×SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6×SSPE (3M NaCl, 0.2 M NaH2PO4, 20 mM EDTA-2Na, pH 7.4) at 42° C. and then washed with 0.5×SSC at 42° C.
In an embodiment, the present invention relates to a polynucleotide encoding an amino acid sequence having 80% or more identity with each of the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence of SEQ ID NO: 11 and the amino acid sequence of SEQ ID NO: 13 (referred to as “the amino acid sequences of SEQ ID NO: 7 and the like” in this paragraph). The identity of the amino acid sequence means the proportion (%) of the number of the same type of amino acids in a range of the amino acid sequence to be compared in two types of proteins. The identity of the amino acid sequence can be determined by, for example, a publicly known program such as BLAST or FASTA. The above identity may be a higher identity than an identity of 80% or higher, for example, an identity of 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. Alternatively, the polynucleotide of the present invention may also be a polynucleotide encoding an amino acid sequence exhibiting a homology score of 200 or more with the amino acid sequence of SEQ ID NO: 7 and the like in Blast. The homology score can be obtained by aligning each of the amino acid sequences of SEQ ID NO: 7 and the like and the amino acid sequence of a candidate protein, then determining the score of each amino acid to be compared from a score matrix, and calculating the total score thereof as the homology score (see, http://www.gsic.titech.ac.jp/supercon/supercon2004-e/alignmentE.html). The homology score can be determined by, for example a publicly known BLAST program. As the score matrix, BLOSUM62, PAM32, and the like have been known, and BLOSUM62 is preferable in the present specification.
The number of amino acids which are substituted, deleted, added, and/or inserted are not particularly limited as long as it is a number that does not affect the biological activity of the antibody, but can be, for example, 1 to 10, 1 to 5, 1 to 4, or 1 to 3. The substitution of the amino acid is preferably substitution between conservative amino acids (see, Molecular Biology of the Cell, Garland Science; 6th edition).
The term “silk gland-specific gene promoter” is a promoter of a gene which is expressed specifically in the silk glands of the silk-spinning insects. Examples of such a promoter include silk protein gene promoters such as a posterior silk gland-specific gene promoter and a middle silk gland-specific gene promoter. Specific examples include sericin gene promoters (sericin 1 gene promoter, sericin 2 gene promoter, and sericin 3 gene promoter) or fibroin gene promoters (fibroin heavy chain gene promoter, fibroin light chain gene promoter, and fibrohexamerin gene promoter). Preferably, the silk gland-specific gene promoter is a sericin 1 gene promoter, a sericin 2 gene promoter, and sericin 3 gene promoter, and includes an MSG promoter and a PSG promoter (WO 2017135452 A1). The expression “functionally linked to downstream of the promoter” means being linked in the manner where the gene can be expressed by activation of the promoter.
In another embodiment, the present invention relates to a plasmid vector containing the above expression cassette. The plasmid vector is not particularly limited as long as it is a vector that can be introduced into cells of silk-spinning insects and maintained. Examples thereof include a plasmid vector into which DNA type transposon piggyBac derived from Trichoplusia ni is incorporated (Tamura, T et al. Nature Biotechnology, 18: 81-84 (2000)). Examples of the vector include pPIGA3GFP (Tamura, T et al., Nature Biotechnology, 18: 81-84 (2000)) and pBac [3xP3-DsRed/pA] (Nature Biotechnology, 21: 52-56 (2003)). The vector of the present invention can also be produced by, for example, inserting the above expression cassette into a cleavage site for the restriction enzyme of a vector (pMSG1.IMG) produced by the method described in US patent application publication No. 2008/0301823. Alternatively, the vector may also be a vector for silk-spinning insect transformation, pMSG3.IMG (Japanese Unexamined Patent Application Publication No. 2012-182995), for example.
In an embodiment, the present invention relates to a transgenic silk-spinning insect wherein the above expression cassette is incorporated into a chromosome thereof. In the present specification, the “silk-spinning insect” is not particularly limited as long as it is an insect that has silk glands and can spin silk threads. The “silk-spinning insect” means a Lepidoptera insect, a Hymenoptera insect, a Neuroptera insect, a Trichoptera insect, and the like that spin threads for nesting, cocooning, or moving mainly at the larva stage. The silk-spinning insect is preferably a Lepidoptera insect which can spin a large amount of silk threads, and specific examples thereof include species belonging to Bombycidae, Saturniidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Arctiidae, Noctuidae, and the like. The silk-spinning insect includes a silkworm, Bombyx mandarina, Samia cynthia, Samia cynthia ricini, Antheraea yamamai, Antheraea pernyi, Saturnia jonasii, and Actias artemis. The transgenic silk-spinning insect of the present invention produces (secretes) the above antibody in silk threads (cocoon) (preferably, a sericin layer).
In another embodiment, the present invention relates to an antibody produced by the transgenic silk-spinning insect. The antibody may include an antibody including a heavy chain having the amino acid sequence of SEQ ID NO: 7 and/or a light chain having the amino acid sequence of SEQ ID NO: 9, or may include a heavy chain having the amino acid sequence of SEQ ID NO: 11 and/or a light chain having the amino acid sequence of SEQ ID NO: 13. The antibody may also include a heavy chain and/or a light chain comprising an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 9, respectively, or the amino acid sequence of SEQ ID NO: 11 and/or SEQ ID NO: 13, respectively. The antibody of the present invention may also have a heavy chain and/or a light chain comprising an amino acid sequence exhibiting a homology score of 200 or more with the amino acid sequence of SEQ ID NO: 7 and/or SEQ ID NO: 9, respectively, or the amino acid sequence of SEQ ID NO: 11 and/or SEQ ID NO: 13, respectively, in Blast. The antibody of the present invention may also have a heavy chain and a light chain consisting of amino acid sequences in which several amino acids are substituted, deleted, added, and/or inserted in SEQ ID NO: 7 and SEQ ID NO: 9, respectively, or SEQ ID NO: 11 and SEQ ID NO: 13, respectively. Further, in the present specification, the term “antibody” may be a part or a fragment of an antibody, or a modified antibody as long as it includes Fc and an antigen binding site. For example, the antibody may be a single-chain antibody (for example, a heavy chain antibody) or a bispecific antibody.
It is known that no fucose is bound as glycans in the antibody produced in the cocoon of silkworms. Also, an antibody to which fucose is not attached has been widely known to have excellent ADCC activity in the technical field. There has been no report on relationship between virus suppression by administration of the antibody and the ADCC activity so far. However, such an effect of the present invention may be brought by the ADCC activity. Accordingly, in an embodiment, the present invention may include an anti-HIV antibody composition containing an IgG antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 7, and a light chain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence in which several amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO: 9; having a binding ability to HIV; and having the Fc region modified for improving ADCC activity, wherein the ADCC activity is higher than wild-type antibodies produced in CHO cells; and wherein the composition has an activity of suppressing HIV in the blood of an HIV-infected patient under detection limit at a probability of 90% or more with one or several times of administration to the HIV-infected patient.
Further, it has been indicated that the relationship between Fc and FcγR affects the ADCC activity, and it has been reported that the affinity with FcγR can be increased by amino acid substitution in the Fc region. In particular, a Fc which strongly binds to an activated form of FcγR such as FcγRIIIa, but weakly binds to an inhibitory FcγR such as FcγRIIb has been known to have excellent effector function. As such mutations, F158V, A330L, S239D, and 1332E (Greg ALazar et al., PNAS (2006) 103(11): 4005-4010): and F243L, D270E, R292P, S298N, Y300L, V305I, A330V, and P396L (Cancer Res (2007) 67(18): 8882-8890) have been reported. The Fc region of the antibody of the present invention may have such mutations, and may have 1 to 10 mutations selected from A330L, S239D, 1332E, F243L, D270E, R292P, S298N, Y300L, V305I, A330V, and P396L, for example.
In another embodiment, the present invention relates to a composition containing IgG antibodies having a binding ability to HIV, wherein 80% or more of the IgG antibodies does not have fucose in glycans binding to the antibody. In other words, in the composition of the present invention, it is not necessary that fucose is not included in the all anti-HIV antibodies (100%), and fucose may not include in at least 80% or more (preferably, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more) of the anti-HIV antibody. Preferably, the composition has an activity of suppressing HIV load in the blood of an HIV-infected patient below detection limit at a probability of 90% or more by one or several times of administration to the HIV-infected patient. The characteristics of the sequence and the like of the anti-HIV antibody contained in the composition are the same as those of the anti-HIV antibody of the present invention.
In the present specification, whether “an antibody has an activity of suppressing HIV load in the blood of an HIV-infected patient below detection limit at a probability of 90% or more (preferably, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) by one or several times of administration to the HIV-infected patient” can be confirmed by administering an test antibody to a plurality of HIV-infected patients, and then detecting HIV in the blood thereof in accordance with an ordinary method, for example, a method utilizing PCR. When HIV in the blood is not detected in 90% or more of the HIV-infected patients to which the test antibody has been administered, it can be determined to have an activity of suppressing HIV in the blood of an HIV-infected patient below detection limit at a probability of 90% or more by one or several times of administration to the HIV-infected patient. The determination whether the antibody suppresses HIV load in the blood below detection limit is desirably conducted at a time after all of the administrations of the antibody have been completed. The determination is conducted preferably, at 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, 40 weeks, 1 year, 2 years, 3 years, 4 years, or 5 years after completion of administration.
Further, in another embodiment, the present invention relates to a therapeutic agent or a prophylactic agent (pharmaceutical composition) for HIV infection, containing the above antibody as an active ingredient. The pharmaceutical composition of the present invention may be in any formulation for oral or parenteral administration as long as it is a formulation that can be administered to a patient. Examples of the composition for parenteral administration include an injection, a nasal drop, a suppository, a patch, and an ointment. The composition is preferably an injection. The dosage form of the pharmaceutical composition of the present invention include a liquid formulation, or a lyophilized formulation. When the pharmaceutical composition of the present invention is used as an injection, excipients can be added as necessary that include a solubilizing agent such as propylene glycol and ethylenediamine: a buffer such as phosphate: a tonicity agent such as sodium chloride and glycerin; a stabilizer such as sulfite; a preservative such as phenol; and a soothing agent such as lidocaine (see “Japanese Pharmaceutical Excipients” Yakuji Nippo Limited. “Handbook of Pharmaceutical Excipients Fifth Edition” APhA Publications). When the pharmaceutical composition of the present invention is used as an injection, a storage container therefor may be an ampoule, a vial, a prefilled syringe, a cartridge for pen type syringes, a bag for infusion, and the like.
The antibody of the present invention can be produced in silkworms with high efficiency, and therefore a therapeutic antibody for HIV infection with lower production cost can be provided.
1. Antibody
In an embodiment, the present invention includes a method for producing the antibody of the present invention, including expressing the antibody gene of the present invention in silkworms, as well as an antibody produced by the method. The production method of the present invention can be specifically performed by the following method.
(Production of Expression Cassette)
The expression cassette of the present invention containing an antibody gene functionally linked to a promoter region causing gene expression in silk gland cells in the silkworm can be produced by binding a polynucleotide encoding a promoter region causing gene expression in silk gland cells in the silkworm to any one polynucleotide selected from the above (i) to (x) utilizing a gene recombination technology known to those skilled in the art. The polynucleotide of a promoter region causing gene expression in silk gland cells in the silkworm can be obtained by, for example, performing PCR using a genomic DNA extracted from silkworm cells as a template and using a promoter corresponding to a desired promoter. For example, US patent application publication No. 2008/0301823 describes a method for acquiring a sericin 1 gene promoter.
When the expression cassette of the present invention includes an enhancer, a polynucleotide encoding a −1860 to −1127 region and/or a −5000 to −3848 region of fibroin heavy chain gene, a polynucleotide encoding the baculovirus homologous region, a polynucleotide constituting the 5′ untranslated region of baculovirus polyhedrin, and/or a polynucleotide encoding baculovirus IE1, and the like, the expression cassette can be obtained by binding these polynucleotides to an antibody gene functionally linked to a promoter region causing gene expression in silk gland cells of silkworms, in accordance with a method known to those skilled in the art (for example, by utilizing a cleavage site for the restriction enzyme). For example, the expression cassette of the present invention can be produced in accordance with the methods described in Japanese patent application publication No. 2004-344123, US patent application publication No. 2008/0301823, Japanese patent application publication No. 2008-125366, and the like.
(Production of Vector)
A plasmid vector containing the above expression cassette can be obtained by incorporating the above expression cassette or its constituent into a desired vector. The vector is not particularly limited as long as it is a plasmid vector that can produce a transgenic silkworm. The vector can be produced by, for example, inserting the above expression cassette into a cleavage site for the restriction enzyme of the above vector.
(Production of Transgenic Silkworm)
In an embodiment, the present invention relates to a method for producing a transgenic silkworm which produces (secretes) the antibody in silk threads (cocoon) (preferably, in a sericin layer) comprising, inserting the above plasmid vector into eggs of a silk-spinning insect. Specifically, the method for producing the transgenic silkworm of the present invention includes injecting the plasmid vector into silkworm eggs (silkworm embryos) 2 to 8 hours after egg laying, interbreeding hatched silkworm imagoes to obtain G1 egg masses, and screening transgenic silkworms into which the expression cassette of the present invention is incorporated by using expression of a marker gene and the like as an indicator.
As an example, after purification of the obtained plasmid, the plasmid is mixed with a helper plasmid pHA3PIG (Nat. Biotechnol. 18, 81-84 (2000)) in the amount ratio of 1:1, which is subjected to ethanol precipitation and dissolved in an injection buffer (0.5 mM phosphate buffer: pH 7.0, 5 mM KCl) so that the DNA concentration is 10 to 1,000 μg/ml. This vector mixed solution is injected into silkworm eggs (silkworm embryos) at 2 to 8 hours after egg laying at the pre-blastoderm stage in a trace liquid amount of about 1 to 200 nl per egg. The eggs injected by the trace amount of vector DNA are incubated at about 25° C., and the hatched silkworms are reared. The obtained fertile imagoes are interbred to obtain broods of eggs at G1 generation. Eggs of a transgenic silkworm emitting green fluorescence from the eyes and the nervous system thereof are selected from the G1 egg broods on Day 3 to Day 10 from the day of egg laying, and then hatched, thereby establishing a transgenic silkworm into which antibody cDNA is incorporated.
Further, in the transgenic silkworm of the present invention, a polynucleotide for enhancing the gene expression may be introduced separately from the expression cassette of the present invention. For example, a polynucleotide for enhancing gene expression may be inserted into a plasmid vector different from the plasmid vector of the present invention, and injected into silkworm eggs at the same time with or separately from the plasmid vector of the present invention. Alternatively, a transgenic silkworm introduced with the expression cassette of the present invention and the polynucleotide for enhancing gene expression can be obtained by interbreeding a transgenic silkworm into which the expression cassette of the present invention has been introduced by the above method with a transgenic silkworm introduced with a polynucleotide for enhancing gene expression. For example, the above obtained transgenic silkworm can be interbred with a silkworm expressing the ie1 gene which is a trans-activator derived from BmNPV (Japanese patent application publication 2012-182995), and then silkworms having both the antibody cDNA and the ie1 gene can be selected from the obtained G2 generation silkworms.
(Production Method of Antibody)
In another embodiment, the present invention relates to a method for producing an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 7 and a light chain having the amino acid sequence of SEQ ID NO: 9, or an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 11 and a light chain having the amino acid sequence of SEQ ID NO: 13, comprising extracting the antibody from silk threads produced from the transgenic silk-spinning insect.
For example, in the production of the antibody, the above silkworms are reared to spin cocoons. The cocoons of the silkworms are immersed in an extraction buffer (PBS (final concentration of NaCl: 0.5 M), and 0.1% Triton X-100) and stirred at room temperature for 1 hour to prepare a cocoon extract. The extract is filtrated with a 0.45-μm filter, and loaded to a protein G column (Protein G Sepharose 4 Fast Flow, GE Healthcare). The elute obtained with 0.1 M glycine (pH 2.7) is neutralized by adding 1 M Tris-HCl (pH 9.0), and then finally dialyzed against PBS, which would give the antibody.
Also, a fragment of the antibody can be prepared by various methods known to those skilled in the art. The fragment can be obtained by, for example, subjecting the antibody obtained by the above method to papain treatment.
In addition to the above method of producing an antibody in the cocoon of silkworms, the preparation methods of antibody without fucose binding are known such as a method of producing an antibody by cells exhibiting reduced fucosylation which are selected by killing cells having high fucose in CHO cells treated with an agent (US patent application publication No. 2010/0081150), a method of producing an antibody by using CHO cell lines in which fucosylation is reduced due to spontaneous mutation in Fx protein and control of fucose supply from the outer source (US patent application publication No. 2010/0304436), a method of producing an antibody by using GMD knockout cells in which genome corresponding to GMD exon 5, 6 and 7 regions are deleted and FUT8 knockout host cell lines (KANDA, Y. et al. (2007) J. BIOTECHNOL; 130(3): 300-10), a method of producing an antibody by using four types of lectin-resistant CHO mutant cells which have been obtained by being incubated with N-methyl-N-nitrosoguanidine (RIPKA, J. et al. (1986) SOMAT CELL MOL GENET; 12(1): 51-62), a method of producing an antibody by using Lec13 (fucose-deficient CHO) cell lines (SHIELDS, R. L. et al. (2002) J BIOL CHEM; 277(30): 26733-40), a method of producing an antibody by using CHO cells which have been obtained by contacting a population of methotrexate (MTX) treated CHO cells with a non-toxic fucose binder of Aleuria aurantia lectin (AAL) or Aspergillus oryzae 1-fucose-specific lectin (AOL) to remove cells bound to the fucose binder (WO2012/120500), a method of cleaving the glycan from the antibody, and then attaching a non-fucose containing glycan to the antibody (Japanese patent application publication 2016-082962), and a method of producing an antibody by using cells expressing acetylglucosaminyltransferase III (U.S. Pat. No. 6,602,684). The antibody of the present invention may be produced by any of these methods, or other publicly known antibody production methods not described herein, in which the amount of fucose bound is reduced.
2. Pharmaceutical Composition
The antibody of the present invention can be used as a pharmaceutical composition in the form of oral administration, or in the form of parenteral administration such as an injection or a drip infusion. When the pharmaceutical composition is administered to mammals and the like, the pharmaceutical composition can be in the form of oral administration such as a tablet, a powder, a granule, or a syrup, or can be in the form of parenteral administration such as an injection or a drip infusion.
The pharmaceutical composition of the present invention can be formulated by using a normal pharmaceutically acceptable carrier by an ordinary method. In preparing a solid formulation for oral administration, an excipient, further, as necessary, a binder, a disintegrant, a lubricant, and the like are added to the base, and then this is formulated into a solution, a granule, a powder, a capsule, and the like by an ordinary method. In preparing an injection, a pH adjusting agent, a buffer, a stabilizer, a solubilizer, and the like are added to the base as necessary, and this can be used as an injection for subcutaneous or intravenous administration by an ordinary method.
In another embodiment, the present invention relates to a method for treatment or prevention of HIV infectious disease, comprising administering an effective amount of the antibody of the present invention to a patient in need thereof. Alternatively, the present invention relates to a use of the antibody of the present invention for manufacturing a therapeutic composition or a prophylactic composition for HIV infection. The dose when the antibody of the present invention is administered to mammals and the like varies depending on symptom, age, gender, body weight, and type of administration form. When the antibody of the present invention is intravenously administered to an adult, for example, the dose per administration can be usually 0.1 to 10,000 mg. The administration method is preferably a method that can maintain HIV load in the blood at the detection limit or lower over a long period of time after administration (for example, 12 weeks or more, 16 weeks or more, 20 weeks or more, 24 weeks or more, 28 weeks or more, 32 weeks or more, 36 weeks or more, 40 weeks or more, 1 year or more, 2 years or more, 3 years or more, 4 years or more, or 5 years or more). In order to check whether HIV load in the blood is maintained at the detection limit or lower after administration, the amount of HIV load in the blood (for example, HIV RNA amount) may be monitored as necessary during or after the administration period. The antibody of the present invention can be administered to a patient infected with HIV, for example, 1 to 10 times, 1 to 8 times, 1 to 5 times, 1 to 4 times, 1 to 3 times, 1 to 2 times, 1 time, 2 times, or 3 times in total. The administration interval can be 3 to 30 days, 3 to 15 days, 4 to 10 days, 5 to 9 days, 6 to 8 days, or 7 days.
Although the present invention will be hereinafter described in detail with reference to examples, the present invention is not intended to be limited thereto. The all documents cited throughout the present specification are incorporated as it is into the present specification by reference. The present application claims the priority based on Japanese Patent Application No. 2018-203114 filed on Oct. 29, 2018. The entire contents of Japanese Patent Application No. 2018-203114 to which the present application claims the priority are incorporated herein by reference.
(1) Production of Vector
cDNA of a 1C10 heavy chain was amplified by performing PCR using, as a template, a plasmid (pMPE-1C10) into which cDNAs of a heavy chain (SEQ ID NO: 6) and a light chain (SEQ ID NO: 8) of the 1C10 antibody are incorporated; as a forward primer, a mixed solution of a primer containing a restriction enzyme NruI recognized sequence and the 5′ untranslated region sequence of BmNPV polyhedrin (NruI-BmNPV-ATG) (Japanese Unexamined Patent Application Publication No. 2008-125366) and a primer (Hc-F) consisting of a sequence of the 5′ end of the 1C10 heavy chain; and as a reverse primer, a primer consisting of a sequence of the 3′ end of the heavy chain and restriction enzyme NruI and XhoI recognized sequences (C-hIgG1-NruI-XhoI).
The amplified fragments were treated with XhoI, and incorporated into a cloning vector (pCR-MCS) treated with EcoRV and XhoI. Similarly, cDNA of a 1C10 light chain was amplified by performing PCR using, as a template, a plasmid (pKVA2-1C10) into which cDNA of the 1C10 light chain is incorporated; as a forward primer, a mixed solution of NruI-BmNPV-ATG and a primer (LcK-F) consisting of a sequence of the 5′ end of the light chain; and as a reverse primer, a primer (LcK-R) consisting of a sequence of the 3′ end of the light chain and restriction enzyme NruI and XhoI recognized sequences, and then the obtained fragments are inserted into the pCR-MCS.
The pCR-MCS into which the cDNA of the 1C10 light chain has been incorporated was cut with NruI to cut out the cDNA of the light chain. Then, the cut cDNA of the light chain was incorporated into a vector for producing transgenic silkworms treated with Aor51HI (pMSG3.1MG, Japanese Unexamined Patent Application Publication No. 2012-182995). Next, this vector was digested with NruI, and ligated to the cDNA of the heavy chain cut out with NruI from the pCR-MCS into which the cDNA of the 1C10 heavy chain has been incorporated. In the obtained vector (1C10/pMSG3.1MG), the cDNAs of 1C10 heavy chain and light chain were each incorporated downstream of the sericin 1 promoter.
Vectors for producing a transgenic silkworm, wherein the heavy chain and the light chain of 1D9 (cDNA sequence of the heavy chain: SEQ ID NO: 10; cDNA sequence of the light chain: SEQ ID NO: 12) and 49G2 which are anti-HIV human antibodies derived from the same patient as 1C10 as well as VRC01 (Science. 329, 856-61 (2010)) and PGT121 (Nature. 477, 466-470 (2011)) which are human antibodies derived from another HIV patient, were incorporated into the pMSG3.1MG, were prepared by the same method.
(2) Preparation of Transgenic Silkworm
The 1C10/pMSG3.IMG was purified with Plasmid Midi Kit (QIAGEN), and then mixed with pHA3PIG (Nat. Biotechnol.; 18: 81-84 (2000)) as a helper plasmid so that the amount ratio of plasmid was 1:1. The mixture was subjected to ethanol precipitation and dissolved in an injection buffer (0.5 mM phosphate buffer: pH 7.0, 5 mM KCl) so that the DNA concentration was 200 μg/mL. This vector mixed solution was micro-injected into 383 silkworm eggs (silkworm embryos) 2 to 8 hours after egg laying at the pre-blastoderm stage in a liquid amount of about 15 to 20 nl per egg.
The eggs, into which a vector DNA was micro-injected, were incubated at 25° C. and 85% of the eggs were hatched. These silkworm larvae were reared, the grown imagoes were interbred, and G1 generation eggs of 72 broods (egg masses laid by female imagoes) were thus obtained. The G1 egg masses on Day 5 to Day 6 from the day of egg laying were observed with a fluorescence stereoscopic microscope, and 31 broods of egg containing eggs of the transgenic silkworm emitting green fluorescence from the eyes and the nervous system thereof were obtained. The obtained eggs were hatched and reared to establish 48 transgenic silkworms. The cocoon protein of each of these silkworms was extracted and analyzed by SDS-PAGE. Further, genomic DNA was extracted from each of the imagoes, and Southern blotting was performed. Based on these analyses, 6 lines of transgenic silkworms in which the 1C10 antibody was expressed in the cocoon thereof, and which have a single copy of recombinant gene in their genomes were selected.
The above transgenic silkworms were interbred with silkworms expressing the ie1 gene which is a trans-activator derived from BmNPV (Japanese Unexamined Patent Application Publication No. 2012-182995). The IE1 protein synthesized from the ie1 gene is known to act on the hr3 enhancer derived from BmNPV included in the pMSG3.1MG and the sericin 1 promoter to increase the expression level of the recombinant protein in the middle silk gland (Biotechol. Bioeng.; 106:860-870 (2010)). Silkworms having both 1C10 cDNA and ie1 gene (hereinafter, denoted as “1C10 production line”) were selected from the interbred G2 generation silkworms, and these silkworms were reared to spin cocoons.
Similarly, a vector, into which cDNA of each of 1D9 and 49G2 derived from the same patient as that of 1C10, or each of VRC01 and PGT121 derived from another patient has been incorporated, was micro-injected into silkworm eggs to produce each transgenic silkworm. The produced transgenic silkworm was interbred with the silkworm expressing the ie1 gene, and the interbred silkworm was caused to spin a cocoon containing each antibody.
The weight of the cocoon layer of the obtained cocoon for each one line of silkworm into which each gene was incorporated was shown (Table 1). As for 1C10, 1D9, VRC01, and PGT121, a cocoon with an average weight was formed, but no cocoon was formed as for 49G2.
(3) Analysis of Expression Level
The expression level of the antibody for each of 1C10, 1D9, VRC01, and PGT121, in which the cocoon was obtained, was examined. 10 mg of cocoon of each silkworm was immersed in 1 mL of 8 M urea, 50 mM Tris buffer (pH 8.0), and 0.1 M DTT, and then heated at 80° C. for 5 minutes to solubilize all the protein contained in the sericin layer of the silk threads (total proteins). Then, SDS-PAGE was performed under the reduced conditions, followed by CBB staining, and the expression levels of the antibodies were compared.
The results are shown in
Next, the amount of extracted antibody in a neutral pH buffered solution was analyzed. 10 mg of cocoon was immersed in 1 mL of PBS (final concentration of NaCl: 0.5 M) containing 0.1% Triton X-100, and then stirred at room temperature for 1 hour, followed by centrifugation, to recover the supernatant. When the protein in the extract was analyzed by SDS-PAGE, the amount of extracted antibody was the largest in 1C10, and the extraction ratios of 1D9 and PGT121, which were relatively high in the expression level, were considerably lower than that of 1C10. The amount of antibody contained in the extract was determined by using an HPLC system (Alliance HPLC System, Waters) equipped with a protein A column (HiTrap MabSelect SuRe column (0.7×2.5 cm: 1 mL). GE Healthcare). 300 μL of extract prepared from each silkworm cocoon was applied to the protein A column, followed by washing with PBS. Thereafter, the bound antibody was eluted with 100 mM citric acid (pH 3.0). The concentration of the antibody was then determined from the area of the elution peak. Further, the amount of antibody extracted per cocoon was calculated from this result. Table 1 shows that the amount of antibody that can be extracted per cocoon of 1C10 was 1.48 mg, and an amount of antibody about 3.2 times the amount of 1D9 derived from the same HIV-infected patient can be extracted.
(4) Purification of 1C10 Antibody
The cocoon of the 1C10 production line was immersed in PBS (final concentration of NaCl: 0.5 M) containing 0.1% Triton X-100, and stirred at room temperature for 1 hour to prepare a cocoon extract. The extract was filtrated with a 0.45-μm filter, and applied to a protein G column (Protein G Sepharose 4 Fast Flow, GE Healthcare). A 0.1 M glycine-HCl buffer (pH 2.7) was used for elution of the antibody from the column. The eluted antibody solution was neutralized by adding 1 M Tris-HCl (pH 9.0) thereto, and the resulting solution was finally dialyzed against PBS. The purified antibody was used as SW-1C10 for the following experiments.
(5) Preparation of 1C10 Antibodies Derived from Different Origins
In order to examine the difference in the binding activity and neutralizing activity due to the origin of the antibody, 1C10 antibodies derived from different origins, namely, Bcell-1C10 (Virology.; 475: 187-203 (2015)), 293A-1C10 (Virology.; 475: 187-203 (2015)), and CHO-1C10 were prepared.
The CHO-AC10 was produced as follows. Transfection of a plasmid (pMPE-1C10) in which cDNA of 1C10 was incorporated into ExpiCHO-S cells (attached to a kit) was performed by using an ExpiCHO Expression System Kit (ThermoFisher Scientific). The culture supernatant was collected 12 to 14 days after transfection, then filtrated with a 0.2-μm filter, and allowed to be bound to a protein A column (HiTrap rProtein A FF, GE Healthcare). A 50 mM glycine-HCl buffer (pH 2.39) was used for elution of the antibody from the column. The eluted antibody solution was neutralized by adding 1 M Tris-HCl (pH 9.0) thereto, and the resulting solution was dialyzed against PBS. The antibody solution was concentrated by using PEG 6,000 (Wako), and then the solution was again dialyzed twice against PBS.
(6) Measurement of Binding Activity to HIV-A BaL Strain
Comparison of the binding activity to the HIV-1 BaL strain (Science.; 253: 71-4 (1991)) between SW-1C10 and 293A-1C10 was performed. First. BaL virus infected cells were prepared. A suspension of CEM.NKR-CCR5 (NKR24) cells (J Virol.; 86: 12039-52 (2012)) (1×106 cells/50 μL) and 50 μL of suspension of the BaL virus cells were mixed in a 1.5-mL tube, and centrifuged at 1,200×g for 2 hours at room temperature. An R10 medium (J Virol.; 86: 12039-52 (2012)) was added thereto, and then culturing was started on a 24-well plate at 37° C. under 5% C02. A Luciferase gene controlled by an LTR promoter of HIV-1 is introduced into the NKR24 cells (J Virol.; 86: 12039-52 (2012)). The Luciferase activity produced in the NKR24 cells was measured by the neolite Reporter Gene Assay System (Perkin Elmer), and the state of virus infection was checked as appropriate.
FACS analysis samples were prepared at the stage in which BaL-infected NKR24 cells and noninfected (Normal) NKR24 cells were prepared (0.2% BSA/PBS was used as a reaction solution). 50 μL of suspension of cells prepared by suspending cells in the reaction solution at 2.5×106 cells/mL was added to a 96-well plate (25×104 cells/tube). An equivalent amount of antibody solution (concentration was adjusted with D-PBS(−)) was added thereto at final concentrations of 0.032/0.16/0.8/4/20/100 μg/mL. The cells were incubated for 30 to 40 minutes at room temperature, and washed twice with a reaction solution. Then, 50 μL of APC-labeled anti-human IgG (Jackson ImmunoResearch) diluted 200 times with a reaction solution was added thereto (thereafter, operation was carried out with light shielding). The resulting cells were incubated for 15 minutes at room temperature, washed twice with a reaction solution, and then 100 μL of 10% Formalin/PBS was added thereto. The cells were incubated on ice for 15 minutes, and then analyzed by BD FACS Canto II (BD Biosciences). The binding activity was examined from the mean fluorescence intensity (MFI) of the APC.
The results show that SW-1C10 and 293A-1C10 are both bound specifically to the BaL-infected NKR24 cells, and further, exhibit the binding activity dependent on the antibody concentration (Table 2 and
(7) Measurement of Neutralizing Activity to HIV-1 BaL Strain
Comparison of the neutralizing activity to the HIV-1 BaL strain among SW-1C10, Bcell-1C10, 293A-1C10, and CHO-1C10 was performed. 5-fold antibody dilution series in 8 stages with 4 μg/mL being a maximum concentration were prepared on a 96-well plate (100 μL/well). Then, 50 IL of BaL virus prepared to 4,000 TCID 50/mL was added thereto (final 200 TCID50). After incubation at 37° C. under 5% CO2 for 1 hour, TZM-bl cells (AIDS; 23: 897-906 (2009)) were prepared to 1×105 cells/mL (+37.5 μg/mL DEAE dextran), and added thereto in an amount of 100 μL. Wells including a VC (virus control; only virus and cells) as a positive control, and a CC (cell control; only cells) as a negative control were prepared at the same time. After culturing at 37° C. under 5% CO2 for 2 days, the cells were washed with PBS, and 30 μL of Luciferase Cell Lysis Buffer (Promega) was added to each well, followed by stirring for 15 minutes. 50 μL of Luciferase Assay Reagent was added to a white plate for detection (Coster), and then 10 μL of cell lysate after stirring was added thereto. The RLU (relative luminescence unit) was measured by a luminometer. The infection inhibition ratio (% inhibition)={(RLU in VC)-(RLU in each antibody concentration)}/(RLU in VC) was calculated as the RLU in CC being the background, and IC50 (50% inhibitory concentration) was determined.
Table 3 shows IC50, and
(8) Analysis of Glycan Structure
The glycan structure of each of SW-1C10 and CHO-1C10 was analyzed in accordance with a literature (Mol. Cellular Proteom.; 6: 1437-1445 (2007)) by the following operation. 50 μg of each purified 1C10 was subjected to reductive alkylation and trypsin digestion in the presence of a surfactant, and then subjected to enzyme digestion with PNGaseA, thus releasing N-glycan. Subsequently, 50 pmol of internal standard substance was added, and the Glycoblotting method was performed (in this process, capturing of N-glycan, methylation of a carboxyl group, and BOA labeling were performed). This was subjected to mass analysis (MALDI-TOF-MS: Ultraflex III, positive mode). The obtained spectrum was compared with GlycoMod Tool (http://web.expasy.org/glycomod/) to estimate the structure of N-glycan. Further, each peak area was normalized by using the peak area with the internal standard substance added in advance.
The principal glycan structures estimated from the obtained mass spectrum and the abundances thereof were shown in
(9) Measurement of ADCC Activity
KD-247, which is a humanized antibody recognizing the V3 loop of gp120, was prepared in addition to SW-1C10, 293A-1C10, and CHO-1C10. Comparison of the ADCC activity to the HIV-1 BaL strain was performed among these antibodies.
BaL-infected NKR24 cells were prepared by a method as in the case of binding activity measurement. Samples for ADCC activity measurement were prepared at the stage in which BaL-infected NKR24 cells and noninfected (Normal) NKR24 cells were prepared (R10 medium IOU/ml IL-2 was used as a reaction solution and an antibody diluent). 40 μL of NKR24 cells, which were washed three times with the reaction solution and prepared to 2.5×105 cells/mL, were added to a 96-well plate (1×104 cells/well). 40 μL of human CD16+KHYG-1 cells being a natural killer cell line ((N6 cells; J Virol.; 86: 12039-52 (2012)), which were washed once with the reaction solution and prepared to 2.5×106 cells/mL, were added as the effector cell (10×104 cells/well). Thereafter, 20 μL of each prepared antibody was added at a final concentration of 0.2, 2, or 20 μg/mL. Wells including a VC (virus control; only BaL-infected NKR24 cells and N6 cells) as a positive control, and a CC (cell control; only noninfected NKR24 cells and N6 cells) as a negative control were prepared at the same time, and incubation was performed at 37° C. under 5% CO2 for 6 hours.
A neolite Reporter Gene Assay System was used for ADCC activity measurement. 40 μL of neolite reagent was added to a white plate for detection (Perkin Elmer), then the reaction solution after incubation was suspended, and 40 μl of the suspension was added thereto. The RLU (relative luminescence unit) was measured by a luminometer. The virus killing ratio (% killing; (RLU in VC-RLU in each antibody concentration)/RLU in VC) was calculated as the RLU in CC being the background, and the calculation result was taken as the ADCC activity (
(10) Mass Production of 1C10 for Animal Experiment
Approximately 30,000 1C10 production lines were reared with an artificial diet (Silkmate PS, Nosan Corp.) throughout the entire instars to produce cocoons. The cocoons were each cut with scissors, and pupas were taken out. About 1.1 kg of cocoon shell was obtained. Extraction and purification of SW-1C10 were performed using 1.0 kg of this cocoon shell.
1 kg of cocoon was immersed in 100 L of extraction buffer (50 mM acetic acid buffer solution, pH 5.3, 30 mM NaCl, 0.2% Triton X-100, 0.01% polydimethylsiloxane), and then squeezed at 25° C. for 2 hours to extract protein. The protein was filtrated with a 10 μm-industrial filter (SMC), and then applied to a STREAMLINE 200 Column (GE Healthcare) filled with 5 L of cation exchange carrier (STREAMLINE SP (GE Healthcare)). After washing with a SP washing buffer (50 mM acetic acid buffer solution, pH 5.3, 30 mM NaCl, 0.2% Triton X-100), elution was performed with a SP elution buffer (50 mM acetic acid buffer solution pH 5.3, 300 mM NaCl) to recover a 1C10 antibody. Further, the recovered antibody was concentrated with an ultrafiltration membrane (Biomax-100 TF (Millipore)), and then the solvent was exchanged with PBS. This was applied to a column filled with a protein A carrier (MabSelect SuRe (GE Healthcare)), followed by washing with PBS. Then, a 1C10 antibody was eluted with a 100 mM citric acid buffer solution (pH 3.0). Finally, the eluted antibody was concentrated with an ultrafiltration membrane, and the solvent was exchanged with a preserving solution (10 mM acetic acid buffer solution pH 5.5, 50 mM NaCl, 100 mM arginine hydrochloride). About 7.9 g of purified SW-1C10 with a purity of 99.0% or more was prepared by the above operation.
(11) Administration of 1C10 to HIV-Infected Cynomolgus Monkey
In order to evaluate the effect of SW-1C10 in the acute infection phase, 50,000 TCID50 of strong toxic SHIV89.6P (Reimann K. A. et al., J. Virol. 70, 6922-6928), which is a chimeric virus in which Env derived from the HIV89.6 strain is incorporated into SIV, was inoculated into the rectum of seven cynomolgus monkeys to establish systemic infection. For the group constitution of the cynomolgus monkey, an untreated group as a control consists of four cynomolgus monkeys, and a group administered with SW-1C10 consists of three cynomolgus monkeys. Administration of SW-1C10 was performed via vein on Day 3, Day 10, and Day 17 after virus inoculation, and an effect of suppressing virus in the blood was observed.
The peripheral blood (EDTA was added) was collected from anesthetized monkeys over time, normally, every 7 days until Week 8 after virus inoculation, and thereafter, once every 4 weeks. The plasma was recovered from the collected blood by centrifugation. Then, the hemocyte was diluted with PBS, and overlaid onto Percoll with a specific gravity of 1.070, followed by centrifugation, to separate PBMCs (peripheral blood mononuclear cells). Viral RNA was extracted from the plasma, and the gag region of SIVmac239 was amplified by quantitative RT-PCR. The number of copies of viral RNA in the plasma was calculated from the concentration of the products thereof. The viral RNA in the plasma was extracted and purified by utilizing a MagNA PureCompact Nucleic Acid isolation kit (Roche Diagnosticks).
Calculation of the RNA amount was performed by designing a primer and probe targeting the gag region of SIVmac239, and using a LightCycler 480 thermocycler (Roche Diagnostics). The viral RNA was amplified and detected by using a QuantiTec Probe RT-PCR kit (Qiagen). The followings were designed as a primer and template. That is, as a forward primer, “5′-GCAGAGGAGGAAATTACCCAGTAC-3′/SEQ ID NO: 14”: as a reverse primer, “5′-CAATTTTACCCAGGCATTTAATGTT-3′/SEQ ID NO: 15”; and as a probe, “5′-FAM-TGTCCACCTGCCATTAAGTCCCGA-TAMRA-3′/SEQ ID NO: 16” were respectively used.
Fluorescent detection was performed on the RT-PCR product by a LightCycler 480 thermocycler to determine the amount thereof. The amount of virus in the plasma was determined by performing measurement twice in duplicate and performing conversion using a calibration curve created by serial dilution of SIV RNA of known concentration. Also, the template DNA and other DNA mixed were treated with DNAaseI. The sensitivity of this measurement system was 100 copy/ml.
In the untreated monkey serving as a control, the number of copies of viral RNA in 1 ml of plasma on Week 2 of virus inoculation was raised to several tens of millions to several hundreds of millions of copies, then brought into a stationary state referred to as virological setpoint on Week 8 or later, and shifted by several tens of thousands to several hundreds of thousands copies/ml. The number of CD4+T cells which are an infection target of HIV was rapidly decreased in Week 2 to Week 4, and thereafter, the number of cells was sifted at a lower level. Meanwhile, in the group administered with SW-1C10, the virus amount reached 1 hundred thousand to 1 million copies/ml as a peak on Week 4 of virus inoculation, and then decreased. No virus was detected on Week 12 or later in all the three monkeys. Also, the number of CD4+T cells did not significantly decrease, and the level before virus administration was maintained (
From these results, surprisingly, the viral load was suppressed to the detection limit or lower in the early stage in all the individuals administered with SW-1C10. The viral RNA load was controlled to the detection limit or lower over a long period of 12 weeks. It has not been no reported in the past that such a control of suppressing viral replication over a long period of time in all administration examples is possible. The present inventors have succeeded in finding, for the first time, an antibody that widely and stably controls the virus relative to administration subject.
Sequence Listing
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-203114 | Oct 2018 | JP | national |
| 2019-166040 | Sep 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP19/42184 | 10/28/2019 | WO | 00 |
