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`CN 111647076 A
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`ML Fl B He B
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`CN 111647076 A
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`ie ARO
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`

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`CN 111647076 A
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`it
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`CN 111647076 A
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`
`
`
`

`

`CN 111647076 A
`
`it
`
`AR
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`
`4/7 wt
`
`agcctctgatttcectttctcttcttatgaaatgagctegetcceccagectccagggaaggecctagagtgegatt
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`geegaaatccatcatagteggagcacctactacaacccetccctcaagagtctagtcaccatctccagagacaatt
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`ccaagaacacectgtatctgcaaatgaacagcctgagagccgaggacacagccetatattactgtetgaaagattt
`
`cgggcacctcggtcaaatggcctcctggggccagggaaccctggtcaccgtctcctca.
`[0049]
`#t{44D5,SEQ ID NO:2,
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`gagetecagctettggagtctgegrereragecttggetacagcctggagegetccctgagactctcctgtgc
`
`agcctcttctttcgatttctcttcttatgaaatgagctgggstcceccagectccagggaageccctggagtggatt
`
`ggegaaatccatcatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatctccagagacaatt
`
`ccaagaacacectgetatctgcaaatgaacagcctgagagccgaggacacagccatgetattactgetgetgaaggattt
`
`ggegetttgcggaccactgggeccagggaaccctggtcaccgtctcctca.
`[0051]
`#t444A10,SEQ ID NO:3,
`[0052]
`gagetecagctettggagtctegeggagecttgetacagcctggagggtccctgagactctcctgtgec
`
`agcectleltettlcgalttcetclgallalgaaalgagclegegtcceccagyc lecagggaagge lclagagtlggalt
`
`geegaaatccatcatagtgeggagcacctactacaacccgetccctcaagagtcgagtcaccatctccagagacaatt
`
`ccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccacgtattactgtgtgaaagattt
`
`tgtagtgggagaaaccecggagttttcetattggggccagggaaccctggtcaccgtctcctca.
`[0053]
`ftf44C5,SEQ ID NO:4,
`[0054]
`gagetecagctettggagtcteseepeapecttgetacagcctggagegtccctgagactctcctgtec
`
`agcctctgatttctatttcectgattatgaaatgagctgegetcceccagectccaggrgaaggeerctagagtegatt
`
`gegeeagtatctatcatagteggagcacctactacaacccetccctcaagagtcgagtcaccatctccagagacaatt
`
`ccaagaacacgectgtatctgcaaatgaacagcctgagagccgaggacacagccatgtattactgtgcaagagaateg
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`gcatagtggctatgactactggggccagggaaccctggtcaccetctcctcao
`
`
`
`
`
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`aq FERRY SI :
`
`
`[0056]
`#t444A12,SEQ ID NO:5,
`[0057]
`EVQLLESGGGLVQPGGSLRLSCAASDFAFSSYEMSWV RQAPGKGLEW LGE LHHSGSTYYNPSLKSLVT
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`ISRDNSKNTLYLQMNSLRAEDTAVYYCVKDFGHLGQMASWGQGTLVTVSS.
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`[0058]
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`[0060]
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`#7if44D5,SEQ ID NO:6,
`EVQLLESGGGLVQPGGSLRLSCAASSFDFSSYEMSWVRQAPGKALEW IGE LHHSGSTYYNPSLKSRVT
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`ISRDNSKNTLYLQMNSLRAEDTAMYYCVKDLGFADHWGQGTLVTVSS.
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`(2% A EWS eH, AL WS RE 26-32 (BUSPDI'SSY) A BE RECDRI , AEB RE L8-67
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`[0062]
`#tf44A10,SEQ ID NO:7,
`[0063]
`EVQLLESGGGLVQPGGSLRLSCAASSFDFSDYEMSWVRQAPGKGLEW IGE LHHSGSTYYNPSLKSRVT
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`ISRDNSKNTLYLQMNSLRAEDTATYYCVKDFVVGETAEFSYWGQGTLVTVSS ©
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`CN 111647076 A
`
`it
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`AR
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`OB
`
`5/7 wi
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`CDR3.
`
`[0065]
`[0066]
`
`#t{44C5,SEQ TD NO:8,
`EVQLLESGGGLVQPGGSLRLSCAASDFYFADYEMSWVRQAPGKGLEWIGSIYHSGSTYYNPSLKSRVT
`
`
`
`
`
`
`
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`TSRDNSKNTLYLQMNSLRAEDTAMYYCAREWHSGYDYWGQGTLVTVSS.
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`
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`jeSERB eh, SL RE 26-32 (BI DFYFADY) (hy HH RECDRI , BEB RIE 48-67
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`(ED IGSLYHSGSTYYNPSLKSRV) 2y Bf SECDR2 , A FE MRR ILOC-105 (EJ AREWHSGYDY) y HE 4ECDR3 ©
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`[0068]
`SE fife2. BELArL AitE
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`[0069]
`ARSE ite GE FQELI SARS WU SE iti Hl] 1 YY 4A1 2. 4D5 4104 COW BA fs -G SARS—Cov-2-
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`RBD-hFcs A AY 26 Gti Ol.
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`EL fA EPEC: 3} 5) EH Sue /ml AY SARS—Cov—1-RBD-hFe , SARS-Cov-2-RBD-hFe ,
`SARS-Cov-2-RBD mut—hFe4 °C 6% (J2 Mit Ks AAA 3% Mik HEWi , 0.05% Tween-20f4
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`PBSYA YR 25 TRS PA SJE BR LT s 2 BIG 4A12,4D5,4A10 , 4C5 (AS WE Bei ASASt a EY SE
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`BRA (50n1/FL) » Sie A LZ IND s HO. 05% Tween—20f) PBSIAWRUEA 31R (340u1/FL) sx
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`Au Rl 2A aS, 4 ER EAS 1A1 2,41D5 1A10. 1054904s PE LE GISARS—Cov—-2-RBD-hF cB
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`aj HE SK Yates Al) Li 1A PY EA A Jt STA XK 148 pFUSE-4.A12—-hFC, pFUSE-4D5-hFC,
`pFUSE-4A10-hFC, pFUSE-4C5-hFC (Invivigen,San Diego,CA) , QUA3ATAR. ZE293TANI BS
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`Al] FAPET#4%10ug pFUSE-4A12-hFc , pFUSE-4D5—hFc , pFUSE-4A10—hFc , pFUSE-4C5—hFc Jit FE #4
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`SET WA, AUIA ARAL DD oP AT
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`A] FRELISASE 3%, WE 4A12.4D5.4A10.4C5 dit 4 4G SARS—Cov—2-RBDhis& 1 (J 38 Fl
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`

`CN 111647076 A
`
`it
`
`AR
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`OB
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`6/7 ut
`
`
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`[0078] A {ATHEA: 18 5ue/mlAYSARS-Cov-2-RBD his&AT1C BR QERK: AA
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`AG 3.% Hi EW} 0.05% Tween—20 FY PRS IZ Ye SS TELATl SeA ZYE41 2-hPc AD5-HPc ,
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`4A10-hFe ,4C5-hFc2& A 413% ARH Wy . 0.05% Tween-201 PBSIAWK > FI) Fs FE BK 20, 10,
`5,2.5,1.25,0.625,0.3125,0. 15625,0.078125,0.0390625 ,0.01953125,0.009765625ug/
`
`ml (ASCE) OAS PAE EAR GOu1/FL) . SE LZs FO. 05 % Tween-20f4)
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`PBSIA RYE IEA BIK (340u1/FL) goat anti-human Fe y -HRPLA1: 2000Ek fj -5 3% fit
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`8 Wy} .0.05% Tween— 208) PBSIA WIE > . IABER UTAY SE AEA (50ul/4L) . S27BL)
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`AY ; AYO .05 % Tween-20 A PBSIFWRI Fe JEIK sTMB Sie FEI A See RP OL 00u1/4L) .
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`SEE 2.347 4h as, DAO. SMaIE EE (LOOUL /FL) 5 1 FRee SHE 45 Ony¥ HE
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`“FS CASE FUSe A 9 HE AR 4 HFA 2-hFc , 4D5—-hF ec , 4A10-hF cc, 4C5—hFc #248 45 Se Fl
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`[0079]
`
`FAS} A AV2. 69nM, 2. 20nM,1.65nM,1.58nM.
`[0080]
`SE jit {15 BF Ia Sy Ae} SARS—Co v—2-RBD-y ACE2-CHO 4H Hg 205 Zo AY BELT
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`#2 WiP4F4A12-hFc ,4D5-hFc ,4A10-hFc, 4C5—hFc, Ctrl , M3964 4 CHK -ASARS-Cov-1
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`cA AEP tas) 4 SARS-Cov—2-RBD-hF c 2 A 43 a UE FL a»10°ACE2-CHO ZH Ni UA
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`BIE ATR AAP, OkZINE. PBSYET AM 1: 20000 AGoat anti-human PE, vk _E 9% A 1
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`FY . PBSG7K Zid , BD FACS Calibur EL ALR2 UUSARS—Cov-2-RBD-hFc 49 ACE2-CHO4H iid 24 A
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`SL te HIG EET FUSE SS
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`
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`7F Pera re LE St PS VSV-GSs A ZEA) AR ANSARS-Cov-2spikez& Al, GpLVX-
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`EGFP-Luci ferase sk Fi 2k Al E48 Be 293 TA te (ROLY (aa eee BE) UCR 48) EN eg Be LEH Ls
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`[0085]=QUI 7 Aras 45 A ASA 4A12,.4D5.4A10.4C5 Sr BE Oe Se SMT Hl Ce FE HTACE 2-CHOZH
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`AAR , 1Cs0tp BUA: 0.19Ne/m1,1.13ug/ml,0.66ne/ml ,2.32ue/m1 YE: BPAY 31A2¢E 46
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`[0086] AAMASBAe id, 80, KG he i. Bh 1 BOEas ie
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`ANAC HAAS 7 BR aRAs BEEZ A/V IISARS-Cov-2-RBD hisdt J,fSARS-Cov—-
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`2-RBD hi sk AA REE ASE AUD BES Fy 8 HTT Hill SARS—Cov—2.A 42 2A» FLAG DK ACOVID- 19 Fi
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`[0088]
`BR_ LA SE te ISS, ANAC AR A A A ft SK Tite FoA ST] 9 le AB FT
`PME ERAN TTSE SOIREABAC A BERN PRP I
`[0089] Be 5cHK
`[0090]
`[1].Zhu,N.,et al.,A Novel Coronavirus from Patients with Pneumonia in
`China,2019.N Engl J Med, 2020. 382 (8) :727-733.
`
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`

`

`CN 111647076 A
`
`it
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`AR
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`OB
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`7/7 UK
`
`[2].Zhou,P.,et al.,A pneumonia outbreak associated with a new
`[0091]
`coronavirus of probable bat origin.Nature, 2020.579 (7798) :270-273.
`[0092]
`[3].Lan,J.,et al.,Structure of the SARS-CoV-2spike receptor-binding
`
`domain bound to the ACE2 receptor.Nature, 2020.
`[0093]
`[4] .Zou,X.,et al.,Single-cell RNA-seg data analysis on the receptor
`
`ACE2 expression reveals the potential risk of different human organs
`
`vulnerable to 2019-nCoV infection.Front Med,2020.
`[0094]
`[5].Hoffmann,M.,et al.,SARS-CoV-2Cell Entry Depends on ACE2 and
`
`TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell,
`
`2020.181 @) :271-280e278.
`[0095]
`[6].Walls,A.C.,et al.,Structure,Function,and Antigenicity of the
`SARS-CoV-2Spike Glycoprotein. Cell ,2020.181 (2) : 281-292. e286.
`[0096]
`[7] .Muyldermans,S.,Nanobodies:natural single-domain antibodies. Annu
`
`Rev Biochem, 2013.82:775-797.
`[0097]
`[8].Ingram,J.R.,F.1.Schmidt,and H.L.Ploegh,Exploiting Nanobodies’
`
`Singular Traits.Annu Rev Immunol ,2018.36:695-715.
`
`

`

`CN 111647076 A
`
`FF
`
`wl)
`
`Fe
`
`1/4 i
`
`FRIAR
`
`<110>
`
`<120>
`
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`
`<l60> 8
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`
`SlPOSequenceListing 1.0
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`<210> 1
`
`<211> 354
`
`<212> DNA
`
`<213>
`
`ALFArtificial Sequence)
`
`<400> 1
`
`gagetegcage
`
`tettggagtc
`
`tgggggaggc
`
`ttggtacagc
`
`ctggagggtc
`
`cctgagactc
`
`tectgtgcag
`
`cectctgattt
`
`egcetttctct
`
`tcttatgaaa
`
`tgagctgget
`
`cegccaggct
`
`CCARBBAARE
`
`ecclagagle
`
`galleggegaa
`
`atccatlcata
`
`glgggagcac
`
`claclacaac
`
`ecgetccctca
`
`agagtctagt
`
`caccatctcc
`
`agagacaatt
`
`CCaagaacac
`
`gectetatcteg
`
`caaatgaaca
`
`gcctgagagce
`
`cgaggacaca
`
`gccetatatt
`
`actgtgtgaa
`
`agatttcggg
`
`cacctcggtc
`
`aaatgecctc
`
`ctggggccag
`
`geaaccctgg
`
`tcaccegtctc
`
`ctca 354
`
`<210> 2
`
`<211> 345
`
`<212> DNA
`
`<213> AT FRSI Artificial Sequence)
`
`<400> 2
`
`gaggtgcage
`
`tettggagtc
`
`tgggggaggc
`
`ttgegtacage
`
`ctggagggtc
`
`cctgagactc
`
`ectcttettt
`
`60
`
`120
`
`180
`
`240
`
`300
`
`60
`
`120
`
`tectgtgcag
`
`cgatttctct
`
`tettatgaaa
`
`tgagctgget
`
`cegcecaggct
`
`ccagggaags
`
`ccctggagtg
`
`gattgegggaa
`
`atccatcata
`
`gtgggagcac
`
`ctactacaac
`
`180
`
`ccgtccctca
`
`agagtcgagt
`
`caccatctcc
`
`agagacaatt
`
`ccaagaacac
`
`gctgetatctg
`
`caaalgaaca
`
`gcclgagage
`
`cgaggacaca
`
`gccalglattl
`
`aclgtgtgaa
`
`ggalllggeg
`
`240
`
`300
`
`tttecggacc
`
`actgggecca
`
`geeaaccctg
`
`gtcaccetct
`
`cectca 345
`
`<210> 3
`
`<211> 360
`
`<212> DNA
`
`<213> A TFSI Artificial Sequence)
`
`<A00> 3
`
`gaggtgcage
`
`tettggagtc
`
`tgggggaggec
`
`ttggtacagc
`
`ctggagggtc
`
`cctgagactc
`
`tectgtgcag
`
`ectcttcttt
`
`cgatttctct
`
`gattatgaaa
`
`tgagctgget
`
`ccgccagect
`
`ccagggaage
`
`gtctagagtg
`
`gattggggaa
`
`atccatcata
`
`gtgggagcac
`
`ctactacaac
`
`ecgtcectca
`
`agagtcgagt
`
`caccatctcc
`
`agagacaatt
`
`ccaagaacac
`
`ectgtatctg
`
`caaatgaaca
`
`ecctgagage
`
`cgaggacaca
`
`gccacetatt
`
`actgtgetgaa
`
`agattttgta
`
`egtgggagaaa
`
`ccgcggagtt
`
`ttcgtatteg
`
`gssccagggaa
`
`ccctgetcac
`
`cetctcctca <
`
`10
`
`

`

`CN 111647076 A
`
`Fe FW
`
`2/4 i
`
`<210> 4
`
`<211> 348
`
`¢<212> DNA
`
`<213> ATFPSI Artificial Sequence)
`
`<400> 4
`
`gagetecagc tettggagtc tegegggagec ttegtacage ctggaggegtc cctgagactc
`
`tectgtgcag cctctgattt ctatttcgct gattatgaaa tgagcteget ccgccaggct
`
`ccagggaage ggctagagtge gattgggagt atctatcata gtgggagcac ctactacaac
`
`ecgtccectca agagtcgagt caccatctcc agagacaatt ccaagaacac gctgtatctg
`
`caaatgaaca gcectgagagc cgaggacaca gcecatgtatt actgtgcaag agaatggcat
`
`60
`
`120
`
`180
`
`240
`
`300
`
`agtegectatg actactggge ccagggaacc ctgegtcacce tctectca 348
`
`<210> 5
`
`<211> 118
`
`<212> PRT
`
`<213> AFRArtificial Sequence)
`
`<400> 5
`
`Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
`
`1
`
`5
`
`10
`
`15
`
`Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe Ala Phe Ser Ser Tyr
`
`20
`
`20
`
`30
`
`Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
`
`35
`
`40
`
`45
`
`Gly Glu Ile His His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
`
`50
`
`55
`
`60
`
`Ser Leu Val Thr lle Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
`
`65
`
`70
`
`75
`
`80
`
`Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val
`
`85
`
`90
`
`95
`
`Lys Asp Phe Gly His Leu Gly Gln Met Ala Ser Trp Gly Gln Gly Thr
`
`100
`
`105
`
`110
`
`Leu Val Thr Val Ser Ser
`
`115
`
`<210> 6
`
`<211> 115
`
`<212> PRT
`
`<213> ATFPSI (Artificial Sequence)
`
`<A00> 6
`
`Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
`
`1
`
`5
`
`10
`
`11
`
`15
`
`

`

`CN 111647076 A
`
`FF
`
`a
`
`a
`
`3/4
`
`Ser Leu Arg
`
`Leu
`
`ser
`
`Cys
`
`Ala
`
`Ala
`
`Ser Ser Phe
`
`Asp
`
`Phe
`
`Ser
`
`Ser
`
`Tyr
`
`20
`
`20
`
`30
`
`Glu
`
`Met
`
`Ser
`
`Trp
`
`Val
`
`Are
`
`Gln
`
`Ala
`
`Pro
`
`Gly
`
`Lys
`
`Ala
`
`Leu
`
`Glu
`
`Trp
`
`Ile
`
`35
`
`40
`
`45
`
`Gly
`
`Glu
`
`Ile
`
`His
`
`His
`
`Ser
`
`Gly
`
`55
`
`Ser
`
`Thr
`
`Tyr
`
`Tyr
`
`Asn
`
`Pro
`
`Ser
`
`Leu
`
`Lys
`
`60
`
`50
`
`Ser
`
`Ile
`
`Ser
`
`Asn
`
`Ser
`
`Asn
`
`Thr
`
`Leu
`
`Leu
`
`Arg
`
`Val
`
`Thr
`
`Arg
`
`Asp
`
`65
`
`70
`
`Lys
`
`75
`
`Tyr
`
`80
`
`Gln
`
`Met
`
`Asn
`
`ser
`
`Leu
`
`Arg
`
`Ala
`
`Glu
`
`Asp
`
`Thr
`
`Ala
`
`Met
`
`Tyr
`
`Tyr
`
`Cys
`
`Val
`
`85
`
`90
`
`95
`
`Phe
`
`Ala
`
`Asp
`
`His
`
`Trp
`
`Gly
`
`Gln
`
`Gly
`
`Thr
`
`Leu
`
`Val
`
`Thr
`
`105
`
`110
`
`Lys
`
`Asp
`
`Leu
`
`Gly
`
`100
`
`Val
`
`Ser
`
`Ser
`
`115
`
`<210> 7
`
`<211> 120
`
`<212> PRT
`
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`© TXPMTCEA
`
`CN111647076A 20200911
`
`EPO
`
`CN111647076A
`
`FP3
`
`SUBJECT OF THE INVENTION
`Neutralizing single domain antibody against new coronavirus SARS-Cov-2 andits application
`
`[0001]
`
`Technicalfield
`
`[0002]
`
`The invention relates to a neutralizing single domain antibody against the new coronavirus SARS-
`Cov-2 and its application, and belongs to the technical field of biomedicine.
`
`[0003]
`
`Background technique
`
`[0004]
`
`2019The numberof new coronavirus infectious diseases (COVID-19) in the world in 2015 has
`exceeded 2 million, with a fatality rate of 6% (https://covid19.who.int/ ), which seriously endangers
`human health. COVID-19 is induced by a new type of coronavirus (Severe Acute Respiratory
`Syndrome Coronavirus 2, SARS-CoV-2) infection. SARS-CoV-2 is a positive-stranded single-
`stranded RNA virus with a diameter of about 80-120nm. It is a member of the Betacoronavirus
`
`It belongs to the Sarbecovirus subgenus with SARS-CoV, and its nucleic acid homology is
`[1].
`79.5%. [2]. At present, the source and natural host of the SARS-CoV-2 virus are not yet clear, and
`effective measures cannot be taken to prevent and stop the spreadof this virus.
`
`[0005]
`
`Existing studies have shownthat, like other coronaviruses, the host receptor of SARS-Cov-2is
`angiotensin-converting enzyme 2 (ACE2)[3], which is abundantly expressed in lung tissue and
`small intestine tissue [4]. When SARS-CoV-2 infects the human body, it binds to ACE2 through
`the receptor binding domain (RBD) of the S1 subunit of the Spike protein on the virus shell, and
`induces changesin the structure of the S2 subunit, thereby promoting the virus and hostcell
`membrane The fusion of the virus mediates the invasion of the host cell by the virus [5].
`
`[0006]
`
`The Spike protein is expressed on the virus shell as a trimer, and each monomerstructure consists
`of an S1 subunit and an S2 subunit. Studies have found that the RBD of the S1 protein on the
`Spike protein trimer has different states of "closed" and "open". Whenin the "open"state, one of
`the RBDsin the trimer is in a stretched state [6]. This fine change in conformation mediates the
`recognition and binding of Spike protein to ACE2.
`
`[0007]
`
`Single domain antibody (single domain antibody, sdAb), namely heavy chain single domain
`antibody VHH, includes only the variable region of the antibody heavy chain. Single domain
`
`Page 1
`
`23.09.2024 06:34:22
`
`

`

`CN111647076A
`
`antibody has the advantages of small molecular weight, high protein expression level, weak
`immunogenicity, and easy production and preparation [7]. More importantly, single-domain
`antibodies can recognize the concealedfine structure on the surface of the antigen, can precisely
`target and capture the target point, and specifically bind to the target molecule [8].
`
`[0008]
`
`At present, there is no SARS-CoV-2 virus specific vaccine and neutralizing antibody usedin clinical
`practice. Therefore, it is an urgent need for the prevention and treatment of COVID-19 to obtain
`a neutralizing human monoclonal antibody through rapid and efficient SARS-Cov-2 neutralizing
`antibody screening.
`
`[0009]
`
`Summaryof the invention
`
`[0010]
`
`The main purposeof the present invention is to overcome the problems existing in the prior art and
`provide a neutralizing single domain antibody against the new coronavirus SARS-Cov-2, which has
`high antiviral ability against the new coronavirus SARS-Cov-2 . At the same time, the application of
`the antibodyis also provided.
`
`[0011]
`
`The technical solutions of the present invention to solve its technical problems are as follows:
`
`[0012]
`
`A neutralizing single-domain antibody against the new coronavirus SARS-Cov-2, the single-domain
`antibody is composedof a heavy chain, and is characterized in that the antibody has at least one
`of the following technical characteristics:
`
`[0013]
`
`i. The heavy chain includes heavy chain CDR1, whose amino acid sequenceis: X1-F-X2-F-X3-xX4-
`Y, wherein X1 is D or S, X2 is A, Dor Y, and X3 is S or A, X4 is S or D;
`
`[0014]
`
`ii. The heavy chain includes heavy chain CDR2, whose amino acid sequenceis: I|-G-X5-|-X6-H-S-
`G-S-T-Y-Y-N-P-S-L-K-S-X7-V, wherein X5 is E or S, X6 is H or Y, and X7 is L or R;
`
`[0015]
`
`iii. The heavy chain includes heavy chain CDR3, the amino acid sequenceof whichis:
`VKDFGHLGQMAS, VKDLGFADH, VKDFVVGETAEFSY, or AREWHSGYDY.
`
`[0016]
`
`Preferably, the amino acid sequence of the heavy chain CDR1 is: DFAFSSY, SFDFSSY,
`SFDFSDY, or DFYFADY;
`
`[0017]
`
`The amino acid sequence of the heavy chain CDR2is: IGEIHHSGSTYYNPSLKSLYV,
`IGEIHHSGSTYYNPSLKSRV, IGEIHHSGSTYYNPSLKSRYV, or IGSIYHSGSTYYNPSLKSRV.
`
`Page 2
`
`23.09.2024 06:34:22
`
`

`

`CN111647076A
`
`[0018]
`
`Preferably, the heavy chain includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain
`CDR3;
`
`[0019]
`
`Whenthe heavy chain CDR1 is DFAFSSY,the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSLV,
`and the heavy chain CDR3 is VKDFGHLGQMAS;
`
`[0020]
`
`When the heavy chain CDR1 is SFDFSSY,the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSRV,
`and the heavy chain CDR3 is VKDLGFADH;
`
`[0021]
`
`Whenthe heavy chain CDR1 is SFDFSDY, the heavy chain CDR2 is IGEIHHSGSTYYNPSLKSRV,
`and the heavy chain CDR3 is VADFVVGETAEFSY;
`
`[0022]
`
`When the heavy chain CDR1 is DFYFADY, the heavy chain CDR2 is IGSIYHSGSTYYNPSLKSRV,
`and the heavy chain CDR3 is AREWHSGYDY.
`
`[0023]
`
`Preferably, the amino acid sequenceof the single domain antibody is shownin one of SEQ ID NO:
`5 to SEQ ID NO: 8.
`
`[0024]
`
`Preferably, the heavy chain has a label, and the label includes a fluorescent label, an enzyme
`label, and a radioactive label.
`
`[0025]
`
`The invention also provides:
`
`[0026]
`
`The nucleic acid encoding the neutralizing single domain antibody against the new coronavirus
`SARS-Cov-2 described above.
`
`[0027]
`
`Preferably, the sequence of the nucleic acid is as shown in one of SEQ ID NO:1 to SEQ ID NO:4.
`
`[0028]
`
`The invention also provides:
`
`[0029]
`
`The aforementioned neutralizing single-domain antibody against the new coronavirus SARS-Cov-2
`is used for preparing diagnostic reagents or diagnostic kits, drugs or pharmaceutical compositions.
`
`[0030]
`
`The aforementioned nucleic acid is used to prepare neutralizing single-domain antibodies, drugs or
`pharmaceutical compositions against the new coronavirus SARS-Cov-2.
`
`[0031]
`
`Wherein, the medicine or pharmaceutical composition has a neutralizing antiviral effect against the
`new coronavirus SARS-Cov-2.
`
`Page 3
`
`23.09.2024 06:34:22
`
`

`

`CN111647076A
`
`[0032]
`
`The present invention obtains a neutralizing single-domain antibody against the new coronavirus
`SARS-Cov-2 through phage display technology, which can block the binding of SARS-Cov-2-RBD
`to ACE2-positive cells, and has a positive effect on SARS-Cov-2 Pseudovirus has a significant
`virus neutralization effect, provides an effective candidate antibody drug for the prevention and
`treatment of COVID-19, and has potential clinical application prospects.
`
`[0033