US20210171611A1

US20210171611A1 - Antibody that binds to envelope glycoprotein of sever fever with thrombocytopenia syndrome virus and use for same

Info

Publication number: US20210171611A1
Application number: US17/171,044
Authority: US
Inventors: Junho Chung; Ki-Hyun Kim; Hyori KIM; Myoung-don OH; Wan Beom PARK; Seungtaek KIM; Jinhee Kim; Ji-Young Min; Meehyun Ko
Original assignee: Seoul National University R&DB Foundation; Institut Pasteur Korea
Current assignee: SNU R&DB Foundation; Institut Pasteur Korea
Priority date: 2016-03-23
Filing date: 2021-02-09
Publication date: 2021-06-10
Also published as: AU2020202948A1; KR20210019491A; AU2017237543A1; CN109071637A; WO2017164678A2; KR102201391B1; KR20190123816A; KR20180122456A; JP2019516348A; JP2021184710A; AU2017237543B2; JP7235256B2; EP3434690A4; EP3434690A2; CN109071637B; WO2017164678A3; JP7328479B2; KR102039189B1; US20190112360A1; MY196095A

Abstract

The present invention relates to an antibody which specifically binds to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus (SFTSV), the pathogen of severe fever with thrombocytopenia syndrome (SFTS), and is used in order to effectively detect or diagnosis SFTSV and treat SFTS.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser. No. 16/086,761, filed on Sep. 20, 2018, which is a national phase application of PCT Application No. PCT/KR2017/003156, filed on Mar. 23, 2017, which claims the benefit and priority to Korean patent application No. 10-2016-0034727, filed on Mar. 23, 2016. The entire disclosures of the applications identified in this paragraph are incorporated herein by references.

TECHNICAL FIELD

The present invention relates to an antibody which specifically binds to the envelope glycoprotein of severe fever with thrombocytopenia syndrome virus (SFTSV), the pathogen of severe fever with thrombocytopenia syndrome (SFTS), and is used in order to detect or diagnosis SFTSV and treat SFTS.

BACKGROUND

Severe Fever with Thrombocytopenia Syndrome (SFTS) is a new kind of mite-mediated infectious disease, and is mostly occurred by Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) mediated by Haemaphysalis longicornis or Amblyomma testudinarium. SFTS was firstly reported in China in 2009, and the disease and virus was reported in Japan and Korea in 2012. The main symptoms of SFTS are fever, abdominal pain, nausea, vomiting, thrombocytopenia or leukopenia, etc., and in case of serious case, multiple organ failure may occur and result in death. SFTS has consistently occurred in China, Japan or Korea every year, and the fatality rate caused thereby is very high, and it mostly occurs in the period between spring and summer. A black-stripped field mouse is probable as the wild host of SFTSV, and it was presumed that domestic animals can play a role of host, since the serum antibody was found at the high ratio in domestic animals such as goat, cow, dog or chicken, etc. in the major outbreak areas of China. It has been reported that the infection from person to person occurred by mediating a body fluid of an infected person, but there is no approved therapeutic agent or prevention method to effectively treat SFTS until now.
There is a method of confirming an anti-SFTSV antibody titer in blood to confirm SFTS infection. Then the anti-SFTSV antibody titer is mostly measured with an antibody for N protein of SFTSV. The antibody is an antibody for SFTSV internal protein exposed when SFTSV becomes extinct. Thus, the conventional diagnosis by confirming the anti-SFTSV antibody titer has limitation that the existence of virus which is alive and actively acts cannot be accurately figured out. As another method of diagnosing SFTS, the method for detecting the RNA sequence of SFTSV in a subject derived from a human body has been known as having high accuracy, but it has a difficulty to isolate virus RNA of good quality from the subject.
On the other hand, International patent publication No. 2015/053455 (WO2015/053455A1) discloses the method for detecting an antibody for SFTSV, but specifically it does not disclose to which antigen of SFTSV the antibody binds and the neutralization activity of the antibody at all.
Thus, the development of an antibody or method which can effectively detect, isolate or purify SFTSV by recovering limitations of an inaccurate virus titer measurement method of conventional enzyme immunoreaction diagnosis method detecting the amount of killed SFTSV protein, or conventional low purity of virus RNA isolation method in blood is need.

DISCLOSURE

Technical Problem

The problem to be solved by the present invention is to provide an antibody which can effectively detect or diagnose SFTSV and treat SFTS. In addition, the other problem to be solved by the present invention is to provide an antibody which specifically binds to SFTSV, particularly an envelope glycoprotein of SFTSV.

Technical Solution

To solve the technical problems, the present invention provides a novel antibody which specifically binds to SFTSV, particularly its envelope glycoprotein. In addition, the present invention provides a method for effectively detecting, isolating or purifying SFTSV using the antibody. In addition, the present invention, a method for effectively preventing or treating SFTS using the antibody.
As the result that the present inventors have made extensive efforts to overcome the limitations of conventional diagnosis methods of SFTSV, they found a novel antibody which specifically binds to an envelope glycoprotein of SFTSV, particularly Gc or Gn, and found that SFTSV can be effectively detected using it, to complete the present invention.
SFTSV is a minus single strand RNA virus, and belongs to Bunyaviridae family, phlebovirus species. The virus is a globular virus of 80-100 nm diameter and uses Haemaphysalis longicornis as a mediator. The genome of the virus consists of large (L), Medium (M) and small (S) segments, and these encode 6 proteins of RNA dependent RNA polymerase (RdRp), glycoprotein precursor (M), glycoprotein N (Gn), glycoprotein C (Gc), nucleocapsid protein (NP) and non-structural protein (NSs).
In the present invention, an “antibody” may include whole antibodies and any antigen binding portion or single chains thereof. A naturally occurring “antibody” is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDR), interspersed with regions that are more conserved, referred to as framework regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
The present invention provides an antibody which specifically binds to an envelope glycoprotein of SFTSV, particularly an envelope glycoprotein of SFTSV, Gc or Gn. Preferably, the antibody may be comprise a specific amino acid sequence as follows or consists of them. In addition, certain modifications which are obvious in constant regions of heavy chains and light chains are included in the scope of the present invention in the range having same or similar binding specificity. Furthermore, as each of those antibodies can bind to the envelope glycoprotein of SFTSV, an antibody binding to other envelope glycoproteins of SFTSV of the present invention can be produced by mixing and matching VH, VL, full length light chain and full length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences).
In one example, the amino acid sequences of antibody clones (Ab1-5) which binds to Gc envelope glycoprotein of the present invention are shown in the following Tables 1-8.
Amino acid sequences of light chains and heavy chains binding to Gc envelope glycoprotein.

TABLE 1

SEQ
ID	Antibody
NO	and site	Sequence

1	light chain	ELTLTQSPATLSLSPGETATLSCGASQSVSTNYLA
	of Ab1	WYQQKPGLAPRLLIY DASSRAT
		GIPDRFSGSGSGTDFTLTISRLAPEDSAVYYC QQYGSSPLT
		FGGGTKLEIK

2	light chain	ELVVTQPPSVSGAPGQRVTISC SGSSSNIGNNTVN
	of Ab2	WYQQLPGTAPKLLIY SNNQRPS
		GVPDRFSGSKSGTSASLAITGLQADDEADYYC QSFDSSLNDWV
		FGGGTKLTVL


3	light chain	ELELTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH
	of Ab3	WYQQLPGTAPKLLIY GNSNRPS
		GVPDRFSGSKSDTSASLAISGLRSEDEADYYC AAWDDSLNGQVV
		FGGGTKLTVL

4	light chain	ELVLTQPPSASGTPGQRVTISC SGSSSNIGSNTVN
	of Ab4	WYQQLPGTAPKLLIY SNNQRPP
		GVPDRFSGSKSGTSASLAISGLQSEDEADYYC QSYDSSLSYV
		FGTGTKVTVL

5	light chain	ELVVTQEPSLTVPPGGTVTLTC GSSTGPVTTTQYPY
	of Ab5	WFQQKPGQAPRTLIY DTNNRHP
		WTPARFSGSLLGGKAALTLSGAQPEDDA-YYC LLTSASAPWV
		FGGGTKLTVL

6	heavy chain	QVQLVQSGPEVKKPGSSVKVSCKAS GGTFSTYAIS
	of Ab1	WVRQAPGQGLEWMG GIIPISGTANYAQKFQG
		RVTITADESTSTAYMELSSLRSEDTAVYYCA VPV---------
		VPAASGPFDYWG QGTLVTVSS

7	heavy chain	EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN
	of Ab2	WVRQAPGKGLEWVS SISSSSRYIFYADSVKG
		RFTISRDNAKNSLYLQMNSLRAEDTAVYYCA
		SLGYCSGGSCYGFPEGGNAFDIWG QGTMVTVSS

8	heavy chain	QVQLQESGPGLVKPSETLSLTCTVS GGSFSGYYWS
	of Ab3	WIRQPPGKGLEWIG EIIHSGSTNYNPSLKS
		RVTISVDTSKNQFSLKLSSVTAADTAVYYCA RGDYYD---------
		SSGAFDYWG QGTLVTVSS

9	heavy chain	EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYSMN
	of Ab4	WVRQAPGKGLEWVS SISSSSRYIFYADSVKG
		RFTISRDNAKNSLYLQMNSLRAEDTAVYY--
		SLGYCSGGSCYGFPEGGNAFDIWG QGTMVTVSS

10	heavy chain	QVQLVQSGGGLVQPGGSLRLSCSAS GFTFSSYAMH
	of Ab5	WVRQAPGKGLEYVS AISSDGGSTYYADSVKG
		RFTISRDNSKNTLYLQMSSLRAEDTAVYYCV NDG------------
		SSNHFDYWG QGTLVTVSS

Amino acid sequences of light chain or heavy chain framework region 1 (LFR1 or HFR1) of the antibody binding to Gc envelope glycoprotein.

TABLE 2

SEQ ID	Antibody
NO	and site	Sequence

11	LFR1 of Ab1	ELTLTQSPATLSLSPGETATLSC

12	LFR1 of Ab2	ELVVTQPPSVSGAPGQRVTISC

13	LFR1 of Ab3	ELELTQPPSVSGAPGQRVTISC

14	LFR1 of Ab4	ELVLTQPPSASGTPGQRVTISC

15	LFR1 of Ab5	ELVVTQEPSLTVPPGGTVTLTC

16	HFR1 of Ab1	QVQLVQSGPEVKKPGSSVKVSCKAS

17	HFR1 of Ab2	EVQLVESGGGLVKPGGSLRLSCAAS

18	HFR1 of Ab3	QVQLQESGPGLVKPSETLSLTCTVS

19	HFR1 of Ab4	EVQLVESGGGLVQPGGSLRLSCAAS

20	HFR1 of Ab5	QVQLVQSGGGLVQPGGSLRLSCSAS

Amino acid sequences of light chain or heavy chain complementarity determining region 1 (LCDR1 or HCDR1) of the antibody binding to Gc envelope glycoprotein.

TABLE 3

SEQ ID	Antibody and
NO	site	Sequence

21	LCDR1 of Ab1	GASQSVSTNYLA

22	LCDR1 of Ab2	SGSSSNIGNNTVN

23	LCDR1 of Ab3	TGSSSNIGAGYDVH

24	LCDR1 of Ab4	SGSSSNIGSNTVN

25	LCDR1 of Ab5	GSSTGPVTTTQYPY

26	HCDR1 of Ab1	GGTFSTYAIS

27	HCDR1 of Ab2	GFTFSSYSMN

28	HCDR1 of Ab3	GGSFSGYYWS

29	HCDR1 of Ab4	GFTFSSYSMN

30	HCDR1 of Ab5	GFTFSSYAMH

Amino acid sequences of light chain or heavy chain framework region 2 (LFR2 or HFR2) of the antibody binding to Gc envelope glycoprotein.

TABLE 4

SEQ ID	Antibody and
NO	site	Sequence

31	LFR2 of Ab1	WYQQKPGLAPRLLIY

32	LFR2 of Ab2	WYQQLPGTAPKLLIY

33	LFR2 of Ab3	WYQQLPGTAPKLLIY

34	LFR2 of Ab4	WYQQLPGTAPKLLIY

35	LFR2 of Ab5	WFQQKPGQAPRTLIY

36	HFR2 of Ab1	WVRQAPGQGLEWMG

37	HFR2 of Ab2	WVRQAPGKGLEWVS

38	HFR2 of Ab3	WIRQPPGKGLEWIG

39	HFR2 of Ab4	WVRQAPGKGLEWVS

40	HFR2 of Ab5	WVRQAPGKGLEYVS

Amino acid sequences of light chain or heavy chain complementarity determining region 2 (LCDR2 or HCDR2) of the antibody binding to Gc envelope glycoprotein.

TABLE 5

SEQ ID	Antibody and
NO	site	Sequence

41	LCDR2 of Ab1	DASSRAT

42	LCDR2 of Ab2	SNNQRPS

43	LCDR2 of Ab3	GNSNRPS

44	LCDR2 of Ab4	SNNQRPP

45	LCDR2 of Ab5	DTNNRHP

46	HCDR2 of Ab1	GIIPISGTANYAQKFQG

47	HCDR2 of Ab2	SISSSSRYIFYADSVKG

48	HCDR2 of Ab3	EIIHSGSTNYNPSLKS

49	HCDR2 of Ab4	SISSSSRYIFYADSVKG

50	HCDR2 of Ab5	AISSDGGSTYYADSVKG

Amino acid sequences of light chain or heavy chain framework region 3 (LFR3 or HFR3) of the antibody binding to Gc envelope glycoprotein.

TABLE 6

SEQ
ID	Antibody
NO	and site	Sequence

51	LFR3 of Ab1	GIPDRFSGSGSGTDFTLTISRLAPEDSAVYYC

52	LFR3 of Ab2	GVPDRFSGSKSGTSASLAITGLQADDEADYYC

53	LFR3 of Ab3	GVPDRFSGSKSDTSASLAISGLRSEDEADYYC

54	LFR3 of Ab4	GVPDRFSGSKSGTSASLAISGLQSEDEADYYC

55	LFR3 of Ab5	WTPARFSGSLLGGKAALTLSGAQPEDDA-YYC

56	HFR3 of Ab1	RVTITADESTSTAYMELSSLRSEDTAVYYCA

57	HFR3 of Ab2	RFTISRDNAKNSLYLQMNSLRAEDTAVYYCA

58	HFR3 of Ab3	RVTISVDTSKNQFSLKLSSVTAADTAVYYCA

59	HFR3 of Ab4	RFTISRDNAKNSLYLQMNSLRAEDTAVYY--

60	HFR3 of Ab5	RFTISRDNSKNTLYLQMSSLRAEDTAVYYCV

Amino acid sequences of light chain or heavy chain complementarity determining region 3 (LCDR3 or HCDR3) of the antibody binding to Gc envelope glycoprotein.

TABLE 7

SEQ ID	Antibody and
NO	site	Sequence

61	LCDR3 of Ab1	QQYGSSPLT

62	LCDR3 of Ab2	QSFDSSLNDWV

63	LCDR3 of Ab3	AAWDDSLNGQVV

64	LCDR3 of Ab4	QSYDSSLSYV

65	LCDR3 of Ab5	LLTSASAPWV

66	HCDR3 of Ab1	VPV---------VPAASGPFDYWG

67	HCDR3 of Ab2	SLGYCSGGSCYGFPEGGNAFDIWG

68	HCDR3 of Ab3	RGDYYD---------SSGAFDYWG

69	HCDR3 of Ab4	SLGYCSGGSCYGFPEGGNAFDIWG

70	HCDR3 of Ab5	NDG------------SSNHFDYWG

Amino acid sequences of light chain or heavy chain framework region 4 (LFR4 or HFR4) of the antibody binding to Gc envelope glycoprotein.

TABLE 8

SEQ ID	Antibody and
NO	site	Sequence

71	LFR4 of Ab1	FGGGTKLEIK

72	LFR4 of Ab2	FGGGTKLTVL

73	LFR4 of Ab3	FGGGTKLTVL

74	LFR4 of Ab4	FGTGTKVTVL

75	LFR4 of Ab5	FGGGTKLTVL

76	HFR4 of Ab1	QGTLVTVSS

77	HFR4 of Ab2	QGTMVTVSS

78	HFR4 of Ab3	QGTLVTVSS

79	HFR4 of Ab4	QGTMVTVSS

80	HFR4 of Ab5	QGTLVTVSS

In some exemplary embodiments, the antibody specifically binding to the envelope glycoprotein of SFTSV, Gc may comprise a light chain comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 1, 2, 3, 4 and 5, and a heavy chain comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 6, 7, 8, 9 and 10. The antibody consisting of these specific sequences can specifically and effectively bind to the envelope glycoprotein, Gc, and thus can be very usefully used for detection of SFTSV.
In another exemplary embodiment, preferably, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gc of the present invention can be provided as an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 1 and a heavy chain comprising an amino acid of SEQ ID NO 6, an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 2 and a heavy chain comprising an amino acid of SEQ ID NO 7, an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 3 and a heavy chain comprising an amino acid of SEQ ID NO 8, an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 4 and a heavy chain comprising an amino acid of SEQ ID NO 9, and an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 5 and a heavy chain comprising an amino acid of SEQ ID NO 10.
In another exemplary embodiment, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gc of the present invention can comprise a light chain complementarity determining region 1 (LCDR1) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 21, 22, 23, 24 and 25, a light chain complementarity determining region 2 (LCDR2) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 41, 42, 43, 44 and 45, a light chain complementarity determining region 3 (LCDR3) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 61, 62, 63, 64 and 65, a heavy chain complementarity determining region 1 (HCDR1) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 26, 27, 28, 29 and 30, a heavy chain complementarity determining region 2 (HCDR2) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 46, 47, 48, 49 and 50, and a heavy chain complementarity determining region 3 (HCDR3) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 66, 67, 68, 69 and 70.
In another exemplary embodiment, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gc of the present invention can be provided as an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 21, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 41, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 61, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 26, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 46, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 66; an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 22, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 42, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 62, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 27, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 47, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 67; an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 23, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 43, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 63, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 28, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 48, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 68; an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 24, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 44, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 64, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 29, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 49, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 69; or an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 25, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 45, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 65, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 30, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 50, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 70.
In one example, the amino acid sequences of antibody clones (Ab6-10) which binds to Gn envelope glycoprotein of the present invention are shown in the following Tables 9-16.
Amino acid sequences of light chains and heavy chains binding to Gn envelope glycoprotein.

TABLE 9

SEQ
ID	Antibody
NO	and site	Sequence

81	light chain	ELALTQPPSVSVAPGKTAKITC GGDDIGSKTVQ
	of Ab6	WYQQTSGQAPVLVVY DDSDRPS
		GIPERFSGANSGNTATLTISRVEAGDEADYYC QVWDGRSDHVV
		FGGGTKLTVL

82	light chain	ELVLTQPPSVSAAPGQKVTISC SGSSSNIGNNVVS
	of Ab7	WYQQLPGTAPKLLIY DDNRRPS
		GIPDRFSGSKSGTSATLDITGLQTGDEADYYC
		ATWDGSLTAGRVL FGSGTKLTVL

83	light chain	ELALTQPPSVSVAPAMTAKITC GGDDIGSTTVQ
	of Ab8	WYQQTSGQAPVLVVY DDSDRPS
		GIPERFSGANSGNTATLTISRVEAGDEADYYC QVWDGRSDHVV
		FGGGTKLTVL

84	light chain	ELELTQPPSVSGTPGKRVSMSC SGSRSNIGGNVVN
	of Ab9	WYQQLPGKAPKLFIY NNDQRPS
		GVPDRVSGSKSGTSVSVAISGLQPEDEADYYC AAWDDILNGVV
		FGGGTQLTVL

85	light chain	ELVMTQSPSSLSASVGDTVTITC RASQSIYTYLN
	of Ab10	WYHQTPGKAPKLLIS AASSLQS
		GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYADVPVT
		FGGGTKLEIK

86	heavy chain	QVQLVQSGAEVKKPGESLKISCKGS GYIFTNYWIG
	of Ab6	WVRQMPGKGLEWM GIIYPGDSDTRYSPSFQG
		QVTISADRSISTAYLQWSSLKASDTAMYYCA
		RLKLRGFSGGYGSGRRYFDYWG QGTLVTVSS

87	heavy chain	QVQLVQSGAEVKKPGESLKISCKGS GYSFTSYWIG
	of Ab7	WVRQMPGKGLEWM GIIYPGDSDTRYSPSFQG
		QVTISADKSISTAYLQWSSLKASDTAMYYCA
		RLKLRGFSGGYGSGSRYFDYWG QGTLVTVSS

88	heavy chain	QVQLVQSGAEVKKPGESLKISCKGS GYIFTNYWIG
	of Ab8	WVRQMPGKGLEWM GIIYPGDSDTRYSPSFQG
		QVTISADRSISTANLQWSSLKASDTALYYCA
		RLKLRGFSGGYGSGRRYFDYWG QGTLVTVSS

89	heavy chain	QVQLVQSGAEVKKPGESLKISCKGS GYNFTNYWIG
	of Ab9	WVRQLPGKGLEWM GIIYPGDSDTRYSPSFQG
		QVTISADKSISTAYLQWSSLKASDTAMYYCA RIRVIGFYD--
		SSPPPLFDYWG QGTLVTVSS

90	heavy chain	EVQLVESGGGVVQPGRSLRLSCAAS GFTFSGYGIH
	of Ab10	WVRQAPGKGLEWV ALISYDGSNKYYADSVKG
		RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KDR-----DYFGSG--
		FFDYWG QGTLVTVSS

Amino acid sequences of light chain or heavy chain framework region 1 (LFR1 or HFR1) of the antibody binding to Gn envelope glycoprotein.

TABLE 10

SEQ ID	Antibody and
NO	site	Sequence

91	LFR1 of Ab6	ELALTQPPSVSVAPGKTAKITC

92	LFR1 of Ab7	ELVLTQPPSVSAAPGQKVTISC

93	LFR1 of Ab8	ELALTQPPSVSVAPAMTAKITC

94	LFR1 of Ab9	ELELTQPPSVSGTPGKRVSMSC

95	LFR1 of Ab10	ELVMTQSPSSLSASVGDTVTITC

96	HFR1 of Ab6	QVQLVQSGAEVKKPGESLKISCKGS

97	HFR1 of Ab7	QVQLVQSGAEVKKPGESLKISCKGS

98	HFR1 of Ab8	QVQLVQSGAEVKKPGESLKISCKGS

99	HFR1 of Ab9	QVQLVQSGAEVKKPGESLKISCKGS

100	HFR1 of Ab10	EVQLVESGGGVVQPGRSLRLSCAAS

Amino acid sequences of light chain or heavy chain complementarity determining region 1 (LCDR1 or HCDR1) of the antibody binding to Gn envelope glycoprotein.

TABLE 11

SEQ ID	Antibody and
NO	site	Sequence

101	LCDR1 of Ab6	GGDDIGSKTVQ

102	LCDR1 of Ab7	SGSSSNIGNNVVS

103	LCDR1 of Ab8	GGDDIGSTTVQ

104	LCDR1 of Ab9	SGSRSNIGGNVVN

105	LCDR1 of Ab10	RASQSIYTYLN

106	HCDR1 of Ab6	GYIFTNYWIG

107	HCDR1 of Ab7	GYSFTSYWIG

108	HCDR1 of Ab8	GYIFTNYWIG

109	HCDR1 of Ab9	GYNFTNYWIG

110	HCDR1 of Ab10	GFTFSGYGIH

Amino acid sequences of light chain or heavy chain framework region 2 (LFR2 or HFR2) of the antibody binding to Gn envelope glycoprotein.

TABLE 12

SEQ ID	Antibody and
NO	site	Sequence

111	LFR2 of Ab6	WYQQTSGQAPVLVVY

112	LFR2 of Ab7	WYQQLPGTAPKLLIY

113	LFR2 of Ab8	WYQQTSGQAPVLVVY

114	LFR2 of Ab9	WYQQLPGKAPKLFIY

115	LFR2 of Ab10	WYHQTPGKAPKLLIS

116	HFR2 of Ab6	WVRQMPGKGLEWM

117	HFR2 of Ab7	WVRQMPGKGLEWM

118	HFR2 of Ab8	WVRQMPGKGLEWM

119	HFR2 of Ab9	WVRQLPGKGLEWM

120	HFR2 of Ab10	WVRQAPGKGLEWV

Amino acid sequences of light chain or heavy chain complementarity determining region 2 (LCDR2 or HCDR2) of the antibody binding to Gn envelope glycoprotein.

TABLE 13

SEQ ID	Antibody and
NO	site	Sequence

121	LCDR2 of Ab6	DDSDRPS

122	LCDR2 of Ab7	DDNRRPS

123	LCDR2 of Ab8	DDSDRPS

124	LCDR2 of Ab9	NNDQRPS

125	LCDR2 of Ab10	AASSLQS

126	HCDR2 of Ab6	GIIYPGDSDTRYSPSFQG

127	HCDR2 of Ab7	GIIYPGDSDTRYSPSFQG

128	HCDR2 of Ab8	GIIYPGDSDTRYSPSFQG

129	HCDR2 of Ab9	GIIYPGDSDTRYSPSFQG

130	HCDR2 of Ab10	ALISYDGSNKYYADSVKG

Amino acid sequences of light chain or heavy chain framework region 3 (LFR3 or HFR3) of the antibody binding to Gn envelope glycoprotein.

TABLE 14

SEQ
ID	Antibody
NO	and site	Sequence

131	LFR3 of Ab6	GIPERFSGANSGNTATLTISRVEAGDEADYYC

132	LFR3 of Ab7	GIPDRFSGSKSGTSATLDITGLQTGDEADYYC

133	LFR3 of Ab8	GIPERFSGANSGNTATLTISRVEAGDEADYYC

134	LFR3 of Ab9	GVPDRVSGSKSGTSVSVAISGLQPEDEADYYC

135	LFR3 of Ab10	GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

136	HFR3 of Ab6	QVTISADRSISTAYLQWSSLKASDTAMYYCA

137	HFR3 of Ab7	QVTISADKSISTAYLQWSSLKASDTAMYYCA

138	HFR3 of Ab8	QVTISADRSISTANLQWSSLKASDTALYYCA

139	HFR3 of Ab9	QVTISADKSISTAYLQWSSLKASDTAMYYCA

140	HFR3 of Ab10	RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

Amino acid sequences of light chain or heavy chain complementarity determining region 3 (LCDR3 or HCDR3) of the antibody binding to Gn envelope glycoprotein.

TABLE 15

SEQ ID	Antibody and
NO	site	Sequence

141	LCDR3 of Ab6	QVWDGRSDHVV

142	LCDR3 of Ab7	ATWDGSLTAGRVL

143	LCDR3 of Ab8	QVWDGRSDHVV

144	LCDR3 of Ab9	AAWDDILNGVV

145	LCDR3 of Ab10	QQYADVPVT

146	HCDR3 of Ab6	RLKLRGFSGGYGSGRRYFDYWG

147	HCDR3 of Ab7	RLKLRGFSGGYGSGSRYFDYWG

148	HCDR3 of Ab8	RLKLRGFSGGYGSGRRYFDYWG

149	HCDR3 of Ab9	RIRVIGFYD--SSPPPLFDYWG

150	HCDR3 of Ab10	KDR-----DYFGSG--FFDYWG

Amino acid sequences of light chain or heavy chain framework region 4 (LFR4 or HFR4) of the antibody binding to Gn envelope glycoprotein.

TABLE 16

SEQ ID	Antibody and
NO	site	Sequence

151	LFR4 of Ab6	FGGGTKLTVL

152	LFR4 of Ab7	FGSGTKLTVL

153	LFR4 of Ab8	FGGGTKLTVL

154	LFR4 of Ab9	FGGGTQLTVL

155	LFR4 of Ab10	FGGGTKLEIK

156	HFR4 of Ab6	QGTLVTVSS

157	HFR4 of Ab7	QGTLVTVSS

158	HFR4 of Ab8	QGTLVTVSS

159	HFR4 of Ab9	QGTLVTVSS

160	HFR4 of Ab10	QGTLVTVSS

In one exemplary embodiment, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gn of the present invention may comprise a light chain comprising any one of amino acid sequences selected from the group consisting of SEQ ID NO 81, 82, 83, 84 and 85, and a heavy chain comprising any one of amino acid sequences selected from the group consisting of SEQ ID NO 86, 87, 88, 89 and 90. The antibody consisting of these specific sequences can specifically and effectively bind to the envelope glycoprotein, Gn, and thus can be very usefully used for detection of SFTSV.
In another exemplary embodiment, preferably, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gn of the present invention can be provided as an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 81 and a heavy chain comprising an amino acid of SEQ ID NO 86, an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 82 and a heavy chain comprising an amino acid of SEQ ID NO 87, an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 83 and a heavy chain comprising an amino acid of SEQ ID NO 88, an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 84 and a heavy chain comprising an amino acid of SEQ ID NO 89, and an antibody comprising a light chain comprising an amino acid sequence of SEQ ID NO 85 and a heavy chain comprising an amino acid of SEQ ID NO 90.
In another exemplary embodiment, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gn of the present invention can comprise a light chain complementarity determining region 1 (LCDR1) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 101, 102, 103, 104 and 105, a light chain complementarity determining region 2 (LCDR2) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 121, 122, 123, 124 and 125, a light chain complementarity determining region 3 (LCDR3) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 141, 142, 143, 144 and 145, a heavy chain complementarity determining region 1 (HCDR1) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 106, 107, 108, 109 and 110, a heavy chain complementarity determining region 2 (HCDR2) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 126, 127, 128, 129 and 130, and a heavy chain complementarity determining region 3 (HCDR3) comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 146, 147, 148, 149 and 150.
In another exemplary embodiment, the antibody which specifically binds to the envelope glycoprotein of SFTSV, Gn of the present invention can be provided as an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 101, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 121, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 141, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 106, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 126, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 146; an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 102, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 122, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 142, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 107, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 127, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 147; an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 103, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 123, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 143, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 108, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 128, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 148; an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 104, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 124, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 144, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 109, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 129, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 149; or an antibody comprising a light chain complementarity determining region 1 (LCDR1) of SEQ ID NO 105, a light chain complementarity determining region 2 (LCDR2) of SEQ ID NO 125, a light chain complementarity determining region 3 (LCDR3) of SEQ ID NO 145, a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO 110, a heavy chain complementarity determining region 2 (HCDR2) of SEQ ID NO 130, and a heavy chain complementarity determining region 3 (HCDR3) of SEQ ID NO 150.
In one exemplary embodiment, the antibody of the present invention may include an antibody comprising an amino acid which is a homologue of an antibody comprising heavy chains and light chains described in the above Table 1 or Table 9. In addition, the antibody of the present invention may comprise a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, and at least one of these CDR sequences may have the antibody disclosed herein or a specific amino acid sequence based on its conservative modification. In addition, the antibody of the present invention may be an antibody possessing functional properties of antibody binding to the envelope glycoprotein of SFTSV, Gc or Gn, and may be an antibody which binds to a same epitope as an antibody comprising heavy chains and light chains disclosed in Table 1 or Table 9. Furthermore, the antibody of the present invention may be prepared using an antibody having one or more kinds of light chains or antibody sequences suggested herein as a starting material for engineering the modified antibody, and comprise all the antibodies having partially modified properties from the starting antibody.
In the present invention, the antibody may comprise a modification to the framework region in the light chain or heavy chain in order to improve properties of the antibody. In addition, the antibody may have at least 1×10⁷M⁻¹, 1×10⁸M⁻¹, 1×10⁹M⁻¹, 1×10¹⁰M⁻¹or 1×10¹¹M⁻¹of affinity constant (KA) for the envelope glycoprotein of SFTSV.
In addition, the antibody of the present invention may be a complete human antibody which specifically binds to the SFTSV envelope glycoprotein. This can have further reduced antigenicity when administered into a human subject, compared with chimera antibody, etc. The human antibody may comprise a heavy chain or light chain variable region, or a full length of heavy chain or light chain that are products of or one derived from a specific germline sequence, when it is collected from a system using a variable region or full length chain human germ line immunoglobulin gene. Moreover, the antibody of the present invention may be a De-immunized antibody having antigenicity.
In addition, in the present invention, the antigen may be a bispecific or a multispecific antibody. The antibody or its antigen-binding fragment of the present invention may be a bispecific molecule binding to two or more of different binding sites or target molecules.
In some exemplary embodiments, the antibody of the present invention may be a monoclonal antibody which specifically binds to the envelope glycoprotein of SFTSV. For example, the antibody of the present invention may be a human or humanized monoclonal antibody or chimera antibody which specifically binds to the envelope glycoprotein of SFTSV, and the antibody of the present invention may comprise a human heavy chain constant region and a human light chain constant region. In addition, the antibody of the present invention may be a single chain antibody, and the antibody of the present invention may be a Fab fragment, and may be a scFv (Single-chain variable fragment), and may be an IgG isotype. Preferably, the antibody of the present invention may be the scFv.
In the present invention, the monoclonal antibody may be produced by common monoclonal antibody methods, and the synthesized antibody genes can be expressed and purified by inserting them into a vector for antibody expression, preferably pcDNA, pCI, pCMV or pCEP4. In addition, viral or carcinogenic transformation of B lymphocytes may be used, and it may be prepared on the basis of the sequence of murine monoclonal antibody prepared using a murine system. For example, using a standard molecule biology technology, a DNA encoding heavy chain and light chain immunoglobulins is obtained from a murine hybridoma, and a non-murine immunoglobulin sequence can be contained with it.
In some exemplary embodiments, the present invention provides an antibody comprising a framework in which an amino acid is substituted with an antibody framework from each human VH or VL germline sequence, or its antigen binding fragment.
In another exemplary embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a light chain comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 1, 2, 3, 4 and 5, and a polypeptide comprising a heavy chain comprising any one of amino acid sequences selected from the group consisting of SEQ ID NOs 6, 7, 8, 9 and 10. In one embodiment, the nucleic acid may be any one of nucleic acid sequences selected from the group consisting of SEQ ID NOs 161, 162, 163, 164, 165, 166, 167, 168, 169 and 170, and this is shown in the following Table 17 (The bolded parts are light chain variable regions (VL), and the underlined parts are heavy chain variable regions (VH)).

TABLE 17

SEQ ID
NO	Antibody	Nucleic acid sequence

161	Ab1	GAGCTCACACTCACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAG
	scFv	GGGAAACAGCCACCCTCTCCTGCGGGGCCAGTCAGAGTGTTAGCA
		CCAACTACTTAGCCTGGTACCAGCAGAAACCTGGCCTGGCGCCCA
		GGCTCCTCATCTATGATGCATCCAGCAGGGCCACTGGCATCCCAG
		ACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCA
		TCAGCAGACTGGCGCCTGAAGATTCTGCGGTGTATTACTGTCAGC
		AATATGGTAGCTCACCTCTCACTTTCGGCGGAGGGACCAAGCTGG
		AGATCAAAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGG
		CTCGGGCGGTGGTGGGCAGGTGCAGCTGGTGCAGTCTGGGCCTGA
		GGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCT
		GGAGGCACCTTCAGCACCTATGCTATCAGCTGGGTGCGACAGGCC
		CCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTCTG
		GTACAGCAAACTACGCACAGAAATTCCAGGGCAGAGTCACCATTAC
		CGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCT
		GAGATCTGAGGACACGGCCGTGTATTACTGTGCGGTACCAGTAGTA
		CCAGCTGCCAGCGGCCCTTTTGACTACTGGGGCCAGGGAACCCTG
		GTCACCGTCTCCTCAGCC

162	Ab2	GAGCTCGTGGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGG
	scFv	GCAGAGGGTCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGG
		AAATAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCC
		CAAACTCCTCATCTATAGTAATAATCAGCGGCCCTCAGGGGTCCCT
		GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC
		ATCACTGGGCTCCAGGCTGACGATGAGGCTGATTATTACTGCCAG
		TCCTTTGACAGCAGCCTGAATGATTGGGTGTTCGGCGGGGGCACC
		AAGCTGACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTG
		GTGGCGGTGGCTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAG
		TCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCC
		TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGT
		CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAG
		TAGTAGTAGTCGTTACATATTCTACGCAGACTCAGTGAAGGGCCGAT
		TCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATG
		AACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGC
		CTAGGATATTGTAGTGGTGGTAGCTGCTACGGGTTCCCGGAAGGTG
		GGAATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTC
		TTCA

163	Ab3	GAGCTCGAGCTGACTCAGCCACCCTCAGTGTCTGGGGCCCCAGG
	scFv	GCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGG
		GGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGC
		CCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGT
		CCCTGACCGATTCTCTGGCTCCAAGTCTGACACCTCAGCCTCCCTG
		GCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGT
		GCAGCATGGGATGACAGCCTGAATGGCCAGGTGGTATTCGGCGG
		AGGCACCAAGCTGACCGTCCTAGGCGGTGGTTCCTCTAGATCTTC
		CTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCT
		GCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTC
		CCTCACCTGCACTGTCTCTGGTGGGTCCTTCAGTGGTTACTACTGG
		AGCTGGATCCGCCAGCCCCCAGGAAAGGGGCTGGAGTGGATTGGG
		GAAATCATTCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGA
		GTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAATTCTCCCTG
		AAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTG
		CGAGAGGTGATTATTATGATAGTAGTGGTGCCTTTGACTACTGGGG
		CCAGGGAACCCTGGTCACCGTCTCCTCA

164	Ab4	GAGCTCGTGCTGACTCAGCCACCTTCAGCGTCTGGGACCCCCGGG
	scFv	CAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGA
		AGTAATACTGTAAACTGGTACCAGCAGCTCCCCGGAACGGCCCCC
		AAACTCCTCATCTATAGTAATAATCAGCGGCCCCCAGGGGTCCCT
		GACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC
		ATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGCCAG
		TCCTATGACAGCAGCCTGAGTTATGTCTTCGGAACTGGCACCAAG
		GTGACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTG
		GCGGTGGCTCGGGCGGTGGTGGGGAGGTGCAGCTGGTGGAGTCT
		GGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGT
		GCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCC
		GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTA
		GTAGTAGTCGTTACATATTCTACGCAGACTCAGTGAAGGGCCGATTC
		ACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAA
		CAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGCCTA
		GGATATTGTAGTGGTGGTAGCTGCTACGGGTTCCCGGAAGGTGGG
		AATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTC
		A

165	Ab5	GAGCTCGTGGTGACCCAGGAGCCCTCACTGACTGTGCCCCCAGGA
	scFv	GGGACAGTCACTCTCACCTGTGGCTCCAGCACTGGACCTGTCACC
		ACTACTCAGTATCCCTACTGGTTCCAGCAGAAGCCTGGCCAGGCC
		CCCAGGACACTCATTTATGATACCAACAACAGACACCCCTGGACA
		CCTGCCCGCTTCTCAGGCTCCCTCCTTGGGGGCAAGGCTGCCCTG
		ACCCTTTCGGGAGCGCAGCCTGAGGATGACGCTTAGTATTATTGCT
		TGCTCACCTCTGCTAGCGCTCCTTGGGTGTTCGGCGGAGGCACCA
		AGCTGACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGG
		TGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGGTGCAGT
		CTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCT
		GTTCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTC
		CGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGCTATTAGTA
		GTGATGGGGGTAGCACATACTACGCAGACTCCGTGAAGGGCAGATT
		CACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGA
		GCAGTCTGAGAGCTGAGGACACGGCTGTATATTACTGTGTGAACGA
		TGGCAGCTCGAACCATTTTGACTACTGGGGCCAGGGAACCCTGGTC
		ACCGTCTCCTCA

166	Ab6	GAGCTCGCCCTGACTCAGCCTCCCTCCGTGTCAGTGGCCCCAGGA
	scFv	AAGACGGCCAAGATTACCTGTGGGGGTGACGACATTGGAAGTAAA
		ACTGTGCAATGGTACCAACAGACCTCAGGCCAGGCCCCTGTGCTG
		GTCGTCTATGACGATAGCGACCGGCCCTCAGGGATCCCTGAGCGA
		TTCTCCGGCGCCAACTCTGGGAACACGGCCACCCTGACCATCAGC
		AGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTG
		GGACGGCAGAAGTGATCATGTGGTTTTCGGCGGAGGGACCAAGCT
		GACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGC
		GGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGGTGCAGTCTGG
		AGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAG
		GGTTCTGGATACATCTTTACCAACTACTGGATCGGCTGGGTGCGCC
		AGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTG
		GTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCAC
		CATCTCAGCCGACAGGTCCATCAGCACCGCCTACCTGCAGTGGAGC
		AGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACTAA
		AGCTCCGGGGGTTTTCGGGCGGCTATGGTTCAGGGAGACGCTACT
		TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

167	Ab7	GAGCTCGTGCTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGG
	scFv	ACTGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCTAACATTGG
		GAATAATGTTGTATCCTGGTACCAGCAACTCCCAGGAACAGCCCC
		CAAACTCCTCATTTATGACGATAACCGGCGACCCTCAGGGATTCCT
		GACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGAC
		ATCACCGGACTCCAGACTGGGGACGAGGCCGATTACTACTGCGCA
		ACATGGGATGGCAGCCTGACTGCTGGCCGTGTGTTGTTCGGCAGT
		GGCACCAAGCTGACCGTCCTAGGTGGTGGTTCCTCTAGATCTTCCT
		CCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTG
		GTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAG
		ATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGG
		CTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGAT
		CATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAG
		GCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT
		GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGT
		GCGAGACTAAAGCTCCGGGGGTTTTCGGGCGGCTATGGTTCAGGG
		AGCCGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCT
		CCTCA

168	Ab8	GAGCTCGCCCTGACTCAGCCTCCCTCCGTGTCAGTGGCCCCAGCA
	scFv	ATGACGGCCAAGATTACCTGTGGGGGTGACGACATTGGAAGTACT
		ACTGTGCAATGGTACCAACAGACCTCAGGCCAGGCCCCTGTGCTG
		GTCGTCTATGACGATAGCGACCGGCCCTCAGGGATCCCTGAGCGA
		TTCTCCGGCGCCAACTCTGGGAACACGGCCACCCTGACCATCAGC
		AGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTG
		GGACGGCAGAAGTGATCATGTGGTTTTCGGCGGAGGGACCAAGCT
		GACCGTCCTAGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGC
		GGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGGTGCAGTCTGG
		AGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAG
		GGTTCTGGATACATCTTTACCAACTACTGGATCGGCTGGGTGCGCC
		AGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTG
		GTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCAC
		CATCTCAGCCGACAGGTCCATCAGCACCGCCAACCTGCAGTGGAG
		CAGCCTGAAGGCCTCGGACACCGCCCTGTATTACTGTGCGAGACTA
		AAGCTCCGGGGGTTTTCGGGCGGCTATGGTTCAGGGAGACGCTAC
		TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

169	Ab9	GAGCTCGAGCTGACTCAGCCACCCTCAGTGTCTGGGACCCCCGGG
	scFv	AAGAGGGTCAGTATGTCTTGTTCTGGAAGTAGGTCCAACATCGGA
		GGTAATGTTGTGAACTGGTACCAGCAGCTCCCAGGAAAGGCCCCC
		AAACTCTTCATCTACAATAATGATCAGCGGCCCTCAGGGGTCCCTG
		ACCGAGTCTCTGGCTCCAAGTCAGGCACCTCAGTCTCCGTGGCCA
		TCAGTGGGCTCCAGCCTGAAGATGAGGCTGATTATTACTGTGCAG
		CTTGGGATGACATCCTGAATGGTGTGGTCTTCGGCGGAGGGACCC
		AGCTGACCGTCCTCGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGG
		TGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGGTGCAGT
		CTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCT
		GTAAGGGTTCTGGATACAACTTCACCAACTACTGGATCGGGTGGGT
		GCGCCAGCTGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTA
		TCCTGGTGACTCCGACACCAGATATAGCCCGTCCTTCCAAGGCCAG
		GTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGT
		GGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGA
		GAATTCGAGTTATCGGATTCTATGATAGTAGCCCCCCGCCCTTATTT
		GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

170	Ab10	GAGCTCGTGATGACTCAGTCTCCATCTTCCCTGTCCGCATCTGTGG
	scFv	GAGACACAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTACA
		CCTATTTAAATTGGTATCACCAGACACCAGGGAAAGCCCCTAAACT
		CCTGATTTCTGCTGCATCTAGTTTGCAAAGTGGTGTCCCATCAAGG
		TTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGC
		AGTCTGCAACCTGAGGATTTTGCAACGTACTACTGTCAACAGTATG
		CGGATGTCCCGGTCACTTTCGGCGGAGGGACCAAGCTGGAGATCA
		AAGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGG
		CGGTGGTGGGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGG
		TCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATT
		CACCTTCAGTGGCTATGGCATACACTGGGTCCGCCAGGCTCCAGGC
		AAGGGGCTGGAGTGGGTGGCACTTATATCATATGATGGAAGTAATA
		AATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA
		CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCT
		GAGGACACGGCTGTGTATTACTGTGCGAAAGATCGGGATTACTTTG
		GTTCAGGGTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT
		CTCCTCA

In another exemplary embodiment, the antibody of the present invention may comprise an amino acid sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity with any one of amino acid sequences disclosed in the above Tables 1-16, within the range that the antibody specificity to the envelope glycoprotein of SFTSV is maintained. In addition, a nucleic acid which can express the antibody of the present invention may comprise a nucleic acid having at least 90%, 95%, 97%, 98% or 99% sequence identity with any one of nucleic acid sequences disclosed in the above Table 17.
In addition, the present invention provides a vector and a host cell comprising the nucleic acid. The vector of the present invention may comprise a nucleic acid encoding an amino acid sequence of the antibody binding to the envelope glycoprotein of SFTSV, Gc, or a nucleic acid encoding an amino acid of the antibody binding to Gn. Otherwise, the vector of the present invention may express a bispecific antibody, by comprising all the two kinds of nucleic acids.
In one exemplary embodiment, the present invention provides (1) a first recombinant DNA fragment encoding a heavy chain of the antibody of the present invention, and (2) a second recombinant DNA fragment encoding a light chain of the antibody of the present invention. In another exemplary embodiment, the present invention provides a host cell comprising a recombinant DNA fragment encoding a heavy chain and a light chain of the present invention, respectively. In some exemplary embodiments, the antibody or its antigen binding fragment is a human monoclonal antibody or its antigen binding fragment.
To express a polynucleotide encoding the antibody binding to the envelope glycoprotein of SFTSV of the present invention, various expression vectors can be used. To produce an antibody in a mammalian host cell, both of virus-based or non-viral expression vector may be used. For example, vectors such as pcDNA, pCI, pCMV or pCEP4, and the like and host cells such as HEK293, CHO or CHO-DG44, and the like may be used.
The host cell possessing and expressing the antibody of the present invention may be a prokaryotic or eukaryotic cell. For example, the host cell may be E. coli, preferably, E. coli ER2738. HB2151, BL21 and the like, and they may be useful for cloning and expressing the polynucleotide of the present invention. In addition, as other microbial hosts, Bacillus, for example, Bacillus subtilis or other intestinal bacteria, for example, Salmonella or Serratia, or various Pseudomonas species may be used. To express the antibody of the present invention, other microorganisms, for example, yeasts can be used, and an insect cell combined with a baculovirus vector may be also used.
In some preferable exemplary embodiments, a mammalian host cell may be used for expressing and preparing the SFTSV envelope glycoprotein binding polypeptide of the present invention. For example, it may be a hybridoma cell line expressing an endogenous immunoglobulin gene or a mammalian cell line possessing an exogenous expression vector. Further, it may comprise for example, CHO cell line, Cos cell line, HeLa cell, myeloma cell line, HEK cell line, transformed B-cell and hybridoma, as any animal or human cell. In addition, numerous appropriate host cell lines which can secret an immunoglobulin can be used, and preferably, HEK293, CHO or CHO-DG44 may be used.
In addition, the present invention provides a composition for diagnosing SFTSV comprising one or more kinds of SFTSV envelope glycoprotein binding molecules (for example, Gc or Gn binding antibody or its antigen binding fragment). The composition for diagnosis of the present invention may be usefully used for detection, isolation or purification of SFTSV. Moreover, the composition may further comprise one or more kinds of other agents appropriate for diagnosing SFTSV. In addition, the present invention provides a method for diagnosing SFTSV using the antibody of the present invention. The method may be used for quantitative or qualitative detection or diagnosis of SFTSV. Specifically, the diagnosis method may comprise a diagnosis examination to determine the expression of envelope glycoprotein and/or nucleic acid of SFTSV and the function of envelope glycoprotein of SFTSV from a biological sample (for example, blood, serum, cell or tissue) or a subject who is suffering from or at risk of developing SFTS. In the present invention, the detection includes quantitative and/or qualitative analysis, and includes detection of existence and absence and detection of virus titer, and this method has been known in the art, and those skilled in the art may select a proper method to conduct the present invention.
In the present invention, the detection of diagnosis or diagnosis of SFTSV may be detected by radio immunoassay, western blot, ELISA (Enzyme linked immunosorbent assay) or immune fluorescence assay, etc. which detects an antigen-antibody complex. In the present invention, an antigen may be labeled with a label such as a radioactive material, enzyme or fluorescent material, etc.
In one embodiment, the method of diagnosis of the present invention may use a complex in which the antibody to the envelope glycoprotein of SFTSV is conjugated to magnetic beads. Specifically, the method can more effectively detect, isolate or purify SFTSV, using the complex in which the antibody specific to the envelope glycoprotein of SFTSV, Gc or Gn is combined to magnetic beads. The antibody to the SFTSV envelope glycoprotein-magnetic bead complex combines with SFTSV existed in a subject using properties of the antibody and at that time, when the magnetic beads are pulled by magnetic power, viruses and other materials in the subject are separated, thereby effectively purifying the virus. The virus purified in this way is relatively useful for RNA isolation, as impurities are removed, and through this, purification result data of good quality can be obtained. In addition, an immunochemical response using another antibody can be processed for the virus attached to magnetic beads, and through this, SFTSV existed in the subject can be rapidly confirmed. The schematic figure of the diagnosis method was shown in FIG. 4.
In addition, the present invention provides a kit for diagnosing SFTSV comprising an antibody binding to an envelope glycoprotein of SFTSV. The kit may comprise any one or more aforementioned antibodies and a reagent for detecting an antigen-antibody complex. As the reagent for detecting an antigen-antibody complex, reagents used for radio immunoassay, ELISA (Enzyme linked immunosorbent assay) or immune fluorescence assay and the like may be used.
For example, for the detection of the immunoreaction, the detection reagent may be labeled directly or indirectly in the form of sandwich. In case of direct labeling method, a serum sample used for array, etc. may be labeled by a fluorescence label such as Cy3 or Cy5. In case of sandwich method, the detection may be performed by combining a target protein with a labeled detection antibody, after combining a non-labeled serum sample with an array in which a detection reagent is attached in advance. In case of sandwich method, as the sensitivity and specificity can be increased, the detection in the level of pg/mL is possible. Besides that, a radioactive material, a color material, a magnetic particle or a dense electron particle and the like may be used as a labeling material. A confocal microscope may be used for the fluorescence strength, and for example, may be obtained from Affymetrix, Inc. or Agilent Technologies, Inc, etc.
The kit of the present invention may further comprise one or more additional components needed for binding analysis, and for example, may further comprise a binding buffer, a reagent needed for sample preparation, a syringe for blood collection or negative and/or positive control. The kit of the present invention which can comprise various detection reagents may be provided for ELISA analysis, dip stick rapid kit analysis, microarray, gene amplification, or immunoassay, etc. according to analysis aspects, and proper detection reagents may be sorted according to the analysis aspects.
In addition, the present invention provides a pharmaceutical composition comprising the antibody binding to SFTSV envelope glycoprotein of the present invention. Preferably, the pharmaceutical composition may be used for prevention or treatment of SFTS. The antibody of the present invention can effectively prevent or treat SFTS, by neutralizing SFTSV and blocking proliferation of virus.
In the present invention, the composition may further contain one or more kinds of other agents appropriate for treating or preventing an SFTSV related disease. The carrier which can be used for the pharmaceutical composition may enhance the effect of composition, or stabilize the composition, or make preparation of the composition easy. The pharmaceutically acceptable carrier may comprise a physiologically acceptable solvent, a dispersive medium, a coating agent, an anti-bacterial agent, an anti-fungal agent, an isotonic agent or an absorption delaying agent and the like.
In the present invention, the pharmaceutical composition may be administered by a variety of methods known in the art, and the administration route and/or method may vary depending on the desired result. The pharmaceutical composition may be administered by administration methods, for example, intravenous, intramuscular, intraperitoneal or subcutaneous, and the like. According to the administration route, the active compound, antibody may be coated with a material protecting the compound from the action of acids and other natural conditions which may inactivate the compound.
In the present invention, the composition may be a sterile fluid. To maintain a proper fluidity, for example, a coating material such as lecithin or a surfactant may be used. In addition, the composition may comprise an isotonic agent (for example, sugar, polyalcohol, mannitol, sorbitol, and sodium chloride, etc.) or an absorption delaying agent (aluminum monostearate or gelatin, etc.).
In the present invention, the pharmaceutical composition may be prepared according to methods known in the art and commonly conducted, and preferably, may be prepared under GMP condition. The pharmaceutical composition may comprise a therapeutically effective dose or efficacious dose of the SFTSV envelope glycoprotein binding antibody. In addition, the dosage level of active ingredients in the pharmaceutical composition may be enough to achieve a therapeutic effect without toxicity to a patient.
In the present invention, the treatment dosage may be titrated to optimize safety and efficacy. When the antibody of the present invention is administered systemically, the range of dosage may be about 0.0001 to 100 mg, more commonly 0.01 to 15 mg per 1 kg of the host body weight. An exemplary treatment method entails systemic administration once per two weeks, or once per one month, or once per three months to 6 months. In some methods of systemic administration, the dosage is, and in some methods, the dosage may be adjusted to achieve the serum antibody concentration of 1 to 1000 μg/mL in some methods of systemic administration and 25 to 500 μg/mL in some methods. Otherwise, when less frequent administration is required, the antibody may be administered by a time-release agent. The dosage and frequency may be differed according to the half-life of the antibody in a patient. In prophylactic purposes, the relatively low dosage may be administered at relatively infrequent intervals for a long period of time.
In addition, the present invention provides a method for preventing or treating SFTS using the pharmaceutical composition. The prevention or treatment method may comprise administering the composition comprising the antibody of the present invention in an therapeutically effective amount. The “therapeutically effective amount” indicates an amount of the antibody of the present invention or the composition comprising thereof which is effective for prevention or treatment of SFTS diseases.
In addition, the present invention provides a use of an SFTSV envelope glycoprotein binding antibody for preparation of a composition for diagnosis of SFTSV. For the preparation of the composition for diagnosis, the antibody or composition comprising thereof of the present invention may comprise additional components such as an acceptable carrier, etc.
Furthermore, the present invention provides a use of an SFTSV envelope glycoprotein binding antibody. The antibody which specifically binds to SFTSV of the present invention may be used for SFTSV diagnosis, and may be used as a diagnosis use determining expression of the envelope glycoprotein and/or nucleic acid of SFTSV and the function of the protein from a subject who is suffering from or at risk of developing SFTS. In addition, the antibody of the present invention may be used as a use of prevention or treatment of SFTS occurred by SFTSV for a who is at risk of developing or suffering from SFTS.

Advantageous Effects

The antibody of the present invention can specifically bind to envelope glycoprotein of SFTSV, Gc or Gn, and thus SFTSV can be effectively detected or diagnosed and SFTS can be treated, using the antibody of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the amino acid sequences of antibody clones Ab1 to Ab10.

FIG. 2 shows the ELISA analysis result of scFv fragment antibody purified for SFTSV envelope glycoprotein Gc and Gn. These data show mean±S.D of 3 times repeated samples.

FIGS. 3A and 3B is (A) the immune fluorescence analysis result and (B) the fluorescence strength measurement of SFTSV infection. In the immune fluorescence analysis result, it was shown that Vero cells infected by SFTSV reacted with the antibody to Gn, and it was shown that Ab10 inhibited the virus infection dose-dependently. Ab10 was significantly excellent in inhibiting virus invasion compared with MAb 4-5.

FIG. 4 is a schematic figure showing the method for detecting SFTSV using an antibody-magnetic bead complex.

DETAILED DESCRIPTION

Hereinafter, examples, etc. will be described in detail to facilitate understanding of the present invention. However, the examples according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to describe the present invention more completely to those skilled in the art.

Example 1: Preparation of Cells

Vero cells derived from African green monkey kidneys were purchased from Korean Cell Line Bank, and cultured at 37° C. under 5% carbon dioxide circumstance with Roswell Park Memorial Institute (RPMI)-1640 medium (Welgene) supplemented with 2% heat inactivated fetal bovine serum (Gibco) and penicillin-streptomycin (Gibco).

Example 2: Preparation of Virus Strains

The SFTS virus used in the present experiment was KF358691 which was isolated from a serum sample of 63-year-old female patient who was hospitalized in Seoul National University hospital and dead in 2012 [Kim K H, Yi J, Kim G, Choi S J, Jun K I, Kim N H, et al. Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerging infectious diseases. 2013; 19(11):1892-4]. The isolated virus was inoculated into a single layer of Vero cells and cultured at 37° C. under 5% carbon dioxide circumstance. The virus was proliferated in Vero cells and all the experiments were performed at the third viral passage of virus culturing. Using Reed-Muench method, 50% tissue culture infection dose (TCID50) was titrated in Vero cells.

Example 3: Preparation of Recombinant SFTS Virus Glycoprotein and Single Chain Variable Fragment Antibody Fusion Protein

The amino acid sequence of SFTS virus glycoprotein used in the present experiment was previously reported [Kim K H, Yi J, Kim G, Choi S J, Jun K I, Kim N H, et al. Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerging infectious diseases. 2013; 19(11):1892-4]. To get a DNA strand encoding the SFTS virus glycoprotein, a human codon optimized DNA sequence corresponding to the amino acid sequence of SFTS virus glycoprotein of SEQ ID NO 171 (GenBank Accession No: AGT98506, amino acids 20-452 for Gn glycoprotein, amino acids 563-1035 for Gc glycoprotein) was synthesized (GenScript).
To overexpress recombinant SFTS virus glycoprotein Gc and Gn which were fused to human immunoglobulin G1 (IgG1) Fc region (Gc-Fc, Gn-Fc) or fused to human Ig k-chain constant region (Gc-Ck, Gn-Ck), the SFTS glycoprotein-encoding gene was prepared according to the method disclosed in [Park S, Lee D H, Park J G, Lee Y T, Chung J. A sensitive enzyme immunoassay for measuring cotinine in passive smokers. Clinica chimica acta; international journal of clinical chemistry 2010; 411(17-18): 1238-42], [Lee Y, Kim H, Chung J. An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding. Experimental & molecular medicine. 2014; 46:e114].
First of all, a DNA sequence obtained by amplifying the Fc region of human IgG1 using 2 kinds of primers (5′-GAGCCCAAATCTTGTGACAAAACTCAC-3′) and (5′-GGATCCTCATTTACCCGGGGACAGGGAG-3′) from human marrow-derived cDNA library (Clontech Laboratories), or the synthesized constant region of human Ig k-chain (UniProtKB/Swiss-Prot: P01834.1) was modified to be positioned at the DNA 3′ side of gene sequence to be added. The gene sequence to be added was cloned in a modified pCEP4 vector (Invitrogen) to enable gene addition by Sfil restriction enzyme.
The antibody clone was produced in the form of single chain variable fragment-human IgG1 Fc region fusion protein (scFv-Fc) using scFv coding DNA of each clone. Then, the vector was transfected into HEK293F cell (Invitrogen) using polyethyleneimine (Polysciences), and the transfected cell was cultured in FreeStyle™ 293 expression medium containing 100 U/L penicillin-streptomycin. The overexpressed recombinant SFTS virus glycoprotein fusion protein was purified through an affinity chromatography using A/KappaSelect column and AKTA pure chromatography system (GE Healthcare).

Example 4: Antibody Library Construction and Biopanning

Peripheral blood monocytes of patient recovered from SFTS were collected using Ficoll-Paque solution (GE Healthcare). The total RNAs were separated using TRIzol reagent (Invitrogen), and cDNA was synthesized from the total RNAs using SuperScript III first strand cDNA synthesis kit with oligo(dT) priming. Using the cDNA, the phage-display library of human single chain variable fragment (scFv) was constructed using pComb3XSS phagemid vector. In addition, to select scFv clone from the library, as disclosed in [Barbas C F, Burton D R, Scott J K, Silverman G J. Phage display: a laboratory manual: CSHL Press; 2004.], 4 rounds of biopanning were performed. 3 μg of recombinant SFTS virus glycoprotein Gc or Gn human IgG1 Fc region fusion protein (Gc-Fc, Gn-Fc) was used for coating 5×106 of magnetic Dynabeads M-270 epoxy beads (Invitrogen) according to the manufacturer's instruction for each round of biopanning. And then the beads bound with proteins were used for biopanning procedures.

Example 5: Screening of Single Chain Variable Fragment Antibody to SFTS Virus

To select an individual antibody clone which bound to SFTS virus glycoproteins, the phage clone was selected form the last round of biopanning, and scFv-display phage was prepared for phage enzyme immunoassay. Microtiter plate (Corning) was coated with 100 ng of recombinant Gc, Gn human Ig k-chain constant region fusion proteins (Gc-Ck, Gn-Ck) per well at 4° C. overnight. The well was blocked with 3% (w/v) BSA in 100 μl of PBS at 37° C. for 1 hour, and cultured with 50 μl of culture supernatant containing phage at 37° C. for 2 hours, and washed with 0.05% (v/v) Tween20 in 150 μl of PBS three times. Then, 50 ml of horseradish peroxidase (HRP)-bound anti-M13 antibody distilled in a blocking buffer (1:5000) was added to each well, and then the plate was cultured at 37° C. for 1 hour. After washing with 150 μl of 0.05% PBST, 50 μl of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) substrate solution (Pierce) was added to each well, and cultured at the room temperature for 30 minutes. And then the absorbance of each well was measured at 405 nm using a microplate reader (Labsystems).

Example 6: Neutralization Analysis

The SFTS virus specific scFv-Fc fusion antibody (100 μl/ml) was serially diluted to be decreased 10 folds each by 0.01 μl/ml. scFvs of each concentration was mixed in an equivalent volume of 100 TCID50 SFTS virus (strain KF358691) and cultured at 37° C. for 1 hours. Then, the virus-antibody mixture was transferred to the single layer of Vero cells in an 8-well confocal microscope chamber and cultured at 37° C. for 1 hour. After removing the virus-antibody mixture, samples were cultured in RPMI-1640 medium containing 2% FBS and antibiotics at 37° C. under 5% carbon dioxide circumstance. Vero cells in the 8-well confocal microscope chamber were used for immune fluorescence assay (IFA). All the experiments were performed three times and the relative neutralization effect was measured by comparing with MAb 4-5 [Xiling Guo et al. A human antibody neutralizing SFTS virus, an emerging hemorrhagic fever virus, 2013. Clin. Vaccine Immunol. 2013; 20(9):1426-32).] as a positive control and anti-newcastle disease virus (NDV) antibody as a negative control

Example 7: Immune Fluorescence Analysis (IFA) and Fluorescence Intensity Measurement

The relative neutralization effect was measured using immune fluorescence assay (IFA). Cells with or without treatment with virus-antibody mixture having or not having Ab10, MAb 4-5 (positive control), anti-NDV (negative control) were cultured for 2 days. The cells were fixed with 4% paraformaldehyde in phosphate-buffer saline (PBS) for 1 hour. After blocking and penetrating slides with 0.1% triton X-100 in 1% fetal bovine serum (BSA), they were cultured together with anti-SFTS virus glycoprotein Gn clone Ab6 antibody (5 μl/ml) at 4° C. overnight. The cells were washed and cultured with fluorescein isothiocyanate (FITC)-bound anti-human IgG (Pierce) at the room temperature for 1 hour. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used for dying a nucleus. Samples were experimented with a confocal microscope (Leica, Buffalo Grove, Ill., USA). Fluorescence signal strength was measured using computer assisted Leica application suite advanced fluorescence (LAS AF). The microscope photographs were taken in 5 regions of each slide using ×10/0.3 lens, and 3 median values were used for analysis. DAPI signal was set with 405 nm blue diode laser and Alexa 488 was adjusted with an argon ion laser.

Example 8: Production of scFv Antibody to SFTS Virus

Human scFv library was biopanned for the recombinant SFTS virus glycoprotein. After 4 rounds of panning, the antibody clone was screened by enzyme-linked immunosorbent assay analysis (ELISA). It was shown that 10 clones (Ab1 to 5 for Gc and Ab6 to 10 for Gn) recognized the SFTS virus through ELISA. The ELISA analysis result was shown in FIG. 2, and the amino acid sequences of each antibody clone were shown in FIG. 1.

Example 9: Neutralization Activity of Antibody to SFTS Virus

The neutralization activity of scFv-hFc antibody purified for the SFTS virus was experimented in Vero cells. Among 10 clones (Ab1 to Ab10) experimented, Ab10 exhibited the strongest neutralization activity. The Ab10 scFv-hFc antibody (100 μl/ml) was diluted 10 folds and titrated for 100 TCID50 SFTS virus (KF358691 strain). The immune fluorescence analysis result and fluorescence strength measurement result of SFTSV infection were shown in FIG. 3.
In the immune fluorescence analysis (IFA), the cell treated with Ab10(100 μl/ml) exhibited the least virus infection and its neutralization activity was dose-dependent. In other words, the more the amount of MAb 10 to be treated was, the smaller the number of cells infected by SFTS virus was. Compared with MAb 4-5 (positive control), Ab10 showed significantly high neutralization activity. The negative control antibody did not exhibit the neutralization activity at all.
This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e).

Claims

1.-19. (canceled)

20. An antibody which specifically binds to Gc that is an envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, wherein the antibody comprises

a) a light chain complementarity determining region 1 (LCDR1) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24 and 25,

b) a light chain complementarity determining region 2 (LCDR2) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44 and 45,

c) a light chain complementarity determining region 3 (LCDR3) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 61, 62, 63, 64 and 65,

d) a heavy chain complementarity determining region 1 (HCDR1) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29 and 30,

e) a heavy chain complementarity determining region 2 (HCDR2) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49 and 50, and

f) a heavy chain complementarity determining region 3 (HCDR3) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 66, 67, 68, 69 and 70.

21. The antibody according to claim 20, wherein the antibody comprises

a light chain comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and 5, and

a heavy chain comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9 and 10.

22. The antibody according to claim 20, wherein the antibody comprises

a light chain comprising a sequence having 95% or more of sequence identity to any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and 5, and

a heavy chain comprising a sequence having 95% or more of sequence identity to any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9 and 10.

23. An antibody which specifically binds to Gn that is an envelope glycoprotein of severe fever with thrombocytopenia syndrome virus, wherein the antibody comprises

a) a light chain complementarity determining region 1 (LCDR1) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104 and 105,

b) a light chain complementarity determining region 2 (LCDR2) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 121, 122, 123, 124 and 125,

c) a light chain complementarity determining region 3 (LCDR3) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 141, 142, 143, 144 and 145,

d) a heavy chain complementarity determining region 1 (HCDR1) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 106, 107, 108, 109 and 110,

e) a heavy chain complementarity determining region 2 (HCDR2) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 126, 127, 128, 129 and 130, and

f) a heavy chain complementarity determining region 3 (HCDR3) comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 146, 147, 148, 149 and 150.

24. The antibody according to claim 23, wherein the antibody comprises

a light chain comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84 and 85, and

a heavy chain comprising any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 86, 87, 88, 89 and 90.

25. The antibody according to claim 23, wherein the antibody comprises

a light chain comprising a sequence having 95% or more of sequence identity to any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84 and 85, and

a heavy chain comprising a sequence having 95% or more of sequence identity to any one of amino acids sequence selected from the group consisting of SEQ ID NOs: 86, 87, 88, 89 and 90.

26. A nucleic acid comprising at least one of

a) a nucleotide sequence encoding a polypeptide comprising a sequence having 95% or more of sequence identity to any one of amino acids selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and 5, and a nucleotide sequence encoding a polypeptide comprising a sequence having 95% or more of sequence identity to any one of amino acids selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9 and 10; and

b) a nucleotide sequence encoding a polypeptide comprising a sequence having 95% or more of sequence identity to any one of amino acids selected from the group consisting of SEQ ID NOs: 81, 82, 83, 84 and 85, and a nucleotide sequence encoding a polypeptide comprising a sequence having 95% or more of sequence identity to any one of amino acids selected from the group consisting of SEQ ID NOs: 86, 87, 88, 89 and 90.

27. A vector comprising the nucleic acid of claim 26.

28. A host cell comprising the vector of claim 27.

29. A composition for diagnosing or detecting SFTSV comprising the antibody of claim 20.

30. A kit for diagnosing or detecting SFTSV comprising the antibody of claim 20.

31. A method for diagnosing or detecting SFTSV using the antibody of claim 20.

32. The method according to claim 31, wherein the method uses a complex in which the antibody and a magnetic bead are bound.

33. A pharmaceutical composition comprising the antibody of claim 20.

34. A method for preventing or treating SFTS using the antibody of claim claim 20.