U.S. patent application number 17/034840 was filed with the patent office on 2021-01-14 for antibodies that bind zika virus envelope protein and uses thereof.
The applicant listed for this patent is Massachusetts Institute of Technology, National University of Singapore. Invention is credited to Kuan Rong CHAN, Eng Eong OOI, Ram SASISEKHARAN, Kannan THARAKARAMAN, Subhash G. VASUDEVAN, Satoru WATANABE.
Application Number | 20210009663 17/034840 |
Document ID | / |
Family ID | 1000005120728 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009663 |
Kind Code |
A1 |
SASISEKHARAN; Ram ; et
al. |
January 14, 2021 |
ANTIBODIES THAT BIND ZIKA VIRUS ENVELOPE PROTEIN AND USES
THEREOF
Abstract
Isolated monoclonal antibodies which bind to Zika virus envelope
protein and related antibody-based compositions and molecules are
disclosed. Also disclosed are therapeutic and diagnostic methods
for using the antibodies.
Inventors: |
SASISEKHARAN; Ram;
(Lexington, MA) ; THARAKARAMAN; Kannan; (Woburn,
MA) ; CHAN; Kuan Rong; (Singapore, SG) ;
WATANABE; Satoru; (Singapore, SG) ; VASUDEVAN;
Subhash G.; (Singapore, SG) ; OOI; Eng Eong;
(Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology
National University of Singapore |
Cambridge
Singapore |
MA |
US
SG |
|
|
Family ID: |
1000005120728 |
Appl. No.: |
17/034840 |
Filed: |
September 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15783655 |
Oct 13, 2017 |
10829545 |
|
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17034840 |
|
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|
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62408020 |
Oct 13, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
C12N 15/63 20130101; Y02A 50/30 20180101; C07K 2317/21 20130101;
A61P 31/12 20180101; C07K 2317/33 20130101; A61P 31/14 20180101;
A61K 39/42 20130101; C07K 16/10 20130101; A61K 39/12 20130101; C07K
14/1825 20130101; C07K 16/1081 20130101; C07K 2317/92 20130101;
C12N 2770/24122 20130101; C07K 2317/76 20130101; A61K 2039/505
20130101; A61K 39/395 20130101 |
International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 39/395 20060101 A61K039/395; A61K 39/12 20060101
A61K039/12; C07K 14/18 20060101 C07K014/18; A61P 31/12 20060101
A61P031/12; A61K 39/42 20060101 A61K039/42; A61P 31/14 20060101
A61P031/14; C12N 15/63 20060101 C12N015/63 |
Claims
1. A method for treating or preventing Zika virus infection in a
subject, comprising administering to the subject in need thereof,
an effective amount of an isolated monoclonal antibody which
specifically binds Zika virus envelope protein, or antigen binding
portion thereof, comprising heavy and light chain CDRs, wherein (i)
heavy chain CDR1 comprises GFSFSTY (SEQ ID NO: 21); (ii) heavy
chain CDR2 comprises SGEGDS (SEQ ID NO: 27); (iii) heavy chain CDR3
comprises GYSNFYYYYTMDA (SEQ ID NO: 32); (iv) light chain CDR1
comprises RATQSISTFLA (SEQ ID NO: 38); (v) light chain CDR2
comprises DASTRAS (SEQ ID NO: 44); and (vi) light chain CDR3
comprises QQRYNWPPYS (SEQ ID NO: 50).
2. The method of claim 1, wherein the antibody or antigen binding
portion thereof has neutralizing activity against Zika virus.
3. The method of claim 1, wherein the antibody or antigen binding
portion thereof is selected from the group consisting of an IgG1,
an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgD, and an
IgE antibody.
4. The method of claim 1, wherein the antibody or antigen binding
portion thereof comprises a heavy chain variable region, wherein
the heavy chain variable region comprises a lysine at position 82B,
numbering according to Chothia.
5. The method of claim 4, wherein the antibody or antigen binding
portion thereof is selected from the group consisting of an IgG1,
an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgD, and an
IgE antibody.
6. The method of claim 1, wherein the antibody or antigen binding
portion thereof comprises heavy and light chain variable regions
comprising the amino acid sequences of SEQ ID NOs: 6 and 15,
respectively.
7. The method claim 6, wherein the antibody or antigen binding
portion thereof is selected from the group consisting of an IgG1,
an IgG2, an IgG3, an IgG4, an IgM, an IgA1, an IgA2, an IgD, and an
IgE antibody.
8. The method of claim 1, wherein the antibody or antigen binding
portion thereof is formulated as a pharmaceutical composition.
9. A method for treating, preventing, reducing, or reducing the
risk of vertical Zika virus infection to a fetus in a pregnant
subject, comprising administering to the subject an effective
amount of an isolated monoclonal antibody which specifically binds
Zika virus envelope protein, or antigen binding portion thereof,
comprising heavy and light chain CDRs, wherein (i) heavy chain CDR1
comprises GFSFSTY (SEQ ID NO: 21); (ii) heavy chain CDR2 comprises
SGEGDS (SEQ ID NO: 27); (iii) heavy chain CDR3 comprises
GYSNFYYYYTMDA (SEQ ID NO: 32); (iv) light chain CDR1 comprises
RATQSISTFLA (SEQ ID NO: 38); (v) light chain CDR2 comprises DASTRAS
(SEQ ID NO: 44); and (vi) light chain CDR3 comprises QQRYNWPPYS
(SEQ ID NO: 50).
10. The method of claim 9, wherein the pregnant subject is infected
with a Zika virus or the pregnant subject is at risk of Zika virus
infection.
11. A method for treating, preventing, reducing, or reducing the
risk of fetal Zika virus infection, comprising administering to a
pregnant subject in need thereof, an effective amount of an
isolated monoclonal antibody which specifically binds Zika virus
envelope protein, or antigen binding portion thereof, comprising
heavy and light chain CDRs, wherein (i) heavy chain CDR1 comprises
GFSFSTY (SEQ ID NO: 21); (ii) heavy chain CDR2 comprises SGEGDS
(SEQ ID NO: 27); (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA
(SEQ ID NO: 32); (iv) light chain CDR1 comprises RATQSISTFLA (SEQ
ID NO: 38); (v) light chain CDR2 comprises DASTRAS (SEQ ID NO: 44);
and (vi) light chain CDR3 comprises QQRYNWPPYS (SEQ ID NO: 50).
12. The method of claim 11, wherein the pregnant subject is
infected with Zika virus or the pregnant subject is at risk of
being infected with Zika virus.
13. A method for treating, preventing, reducing, or reducing the
risk of fetal mortality in a pregnant subject, comprising
administering to the subject an effective amount of an isolated
monoclonal antibody which specifically binds Zika virus envelope
protein, or antigen binding portion thereof, comprising heavy and
light chain CDRs, wherein (i) heavy chain CDR1 comprises GFSFSTY
(SEQ ID NO: 21); (ii) heavy chain CDR2 comprises SGEGDS (SEQ ID NO:
27); (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA (SEQ ID NO:
32); (iv) light chain CDR1 comprises RATQSISTFLA (SEQ ID NO: 38);
(v) light chain CDR2 comprises DASTRAS (SEQ ID NO: 44); and (vi)
light chain CDR3 comprises QQRYNWPPYS (SEQ ID NO: 50).
14. The method of claim 13, wherein the pregnant subject is
infected with Zika virus or the pregnant subject is at risk of
being infected with Zika virus.
15. A method for treating, preventing, reducing, or reducing the
risk of placental Zika virus infection, comprising administering to
a pregnant subject in need thereof, an effective amount of an
isolated monoclonal antibody which specifically binds Zika virus
envelope protein, or antigen binding portion thereof, comprising
heavy and light chain CDRs, wherein (i) heavy chain CDR1 comprises
GFSFSTY (SEQ ID NO: 21); (ii) heavy chain CDR2 comprises SGEGDS
(SEQ ID NO: 27); (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA
(SEQ ID NO: 32); (iv) light chain CDR1 comprises RATQSISTFLA (SEQ
ID NO: 38); (v) light chain CDR2 comprises DASTRAS (SEQ ID NO: 44);
and (vi) light chain CDR3 comprises QQRYNWPPYS (SEQ ID NO: 50).
16. The method of claim 15, wherein the pregnant subject is
infected with Zika virus or the pregnant subject is at risk of
being infected with Zika virus.
17. A nucleic acid comprising a nucleotide sequence encoding the
light chain, heavy chain, or both light and heavy chains of an
isolated monoclonal antibody which specifically binds Zika virus
envelope protein, or antigen binding portion thereof, comprising
heavy and light chain CDRs, wherein (i) heavy chain CDR1 comprises
GFSFSTY (SEQ ID NO: 21); (ii) heavy chain CDR2 comprises SGEGDS
(SEQ ID NO: 27); (iii) heavy chain CDR3 comprises GYSNFYYYYTMDA
(SEQ ID NO: 32); (iv) light chain CDR1 comprises RATQSISTFLA (SEQ
ID NO: 38); (v) light chain CDR2 comprises DASTRAS (SEQ ID NO: 44);
and (vi) light chain CDR3 comprises QQRYNWPPYS (SEQ ID NO: 50).
18. An expression vector comprising the nucleic acid of claim
17.
19. A cell transformed with an expression vector of claim 18.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/783,655, filed on Oct. 13, 2017, pending, which claims
the benefits of the priority date of U.S. Provisional Application
No. 62/408,020, which was filed on Oct. 13, 2016. The entire
contents of the above-referenced applications are incorporated
herein by this reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format via EFS-Web and
is hereby incorporated by reference in its entirety. Said ASCII
copy, created Sep. 28, 2020, is named
"MITN-037DV_Sequence-Listing.txt" and is 31529 Kilobytes in
size.
BACKGROUND
[0003] Zika virus (ZIKV) is a vector-borne arbovirus transmitted by
Aedes aegypti mosquito. ZIKV infection typically causes mild
symptoms, including fever, rash, joint pain, and/or conjunctivitis.
However, recent research indicates a link between infection and
microcephaly in newborn babies, along with a link between infection
and Guillain-barre syndrome in adults. There is an urgent need for
effective counter measures as there are no approved vaccines or
therapies against ZIKV.
[0004] Little is known about the virus, structure, or biology of
ZIKV. ZIKV is a member of the virus family Flaviviridae, which
includes Dengue (DV), West Nile, Japanese Encephalitis, Tick-born
Encephalitis, and Yellow Fever virus. The envelope (F) protein of
flavivirus mediates host cell entry and immune evasion. The E
protein consists of three structural and functional domains.
Antibodies against E protein domain III (E-DIII) have shown
prophylactic and therapeutic effects in animal models infected with
DV (Robinson, L., et al., Cell Vol. 162: 493-504, 2015). Therefore,
antibody-based agents against ZIKV envelope protein provide a
promising option for combating ZIKV outbreaks in humans.
SUMMARY
[0005] The present disclosure pertains to antibodies directed
towards Zika virus envelope protein (EP). A systematic analysis of
the Zika virus surface guided by the residue interatomic
interactions network (or SIN) led to the identification of fusion
loop epitope proximal (FLEP) region as being structurally
constrained. A structure based computational approach yielded a set
of promising scaffolds with potential to interact with the FLEP
region. Following this, an anti-TDRD3 (Tudor Domain Containing 3)
antibody was investigated due to its ability to interface with the
FLEP region. A framework to compute the inter-residue atomic
interaction between interacting amino acid pairs of the
antigen-antibody interface was utilized, and interactions were
rendered in a 2D graph format to analyze the connectivity network.
Mutations in the CDRs and/or framework regions that contributed to
more favorable contacts, as evaluated by the structural analysis
and connectivity network, were identified and various amino acid
residues which potentially mediate new or improved contacts were
analyzed to identify CDR and/or framework mutations that would
result in binding to Zika virus EP. The identified antibodies were
found to treat and prevent Zika virus infection in a subject, as
well as prevent vertical infection and fetal mortality in a
pregnant subject.
[0006] Accordingly, the present disclosure relates to antibodies
that bind Zika virus EP. Also provided herein are host cells and
methods for treating Zika virus with these antibodies.
[0007] In some aspects, the isolated monoclonal antibody which
specifically binds Zika virus envelope protein, or antigen binding
portion thereof, comprises heavy and light chain CDRs, wherein
[0008] (i) heavy chain CDR1 comprises GFX.sub.1FSTY (SEQ ID NO:
54), wherein X.sub.1 may or may not be present, and if present is a
polar amino acid residue;
[0009] (ii) heavy chain CDR2 comprises X.sub.2GEGDS (SEQ ID NO:
55), wherein X.sub.2 is a polar amino acid residue;
[0010] (iii) heavy chain CDR3 comprises GYX.sub.3NFYYYYTMDX.sub.4
(SEQ ID NO: 56), wherein X.sub.3 is a polar amino acid residue and
X.sub.4 is a nonpolar amino acid residue;
[0011] (iv) light chain CDR1 comprises RAX.sub.5QSIX.sub.6TFLA (SEQ
ID NO: 57), wherein X.sub.5 is a polar amino acid residue and
X.sub.6 is a polar amino acid residue or a hydrophobic amino acid
residue;
[0012] (v) light chain CDR2 comprises DASTX.sub.7AX.sub.8 (SEQ ID
NO: 58), wherein X.sub.7 and X.sub.8 are polar amino acids; and
[0013] (vi) light chain CDR3 comprises QQRYNWPPYX.sub.9 (SEQ ID NO:
59), wherein X.sub.9 is a polar amino acid.
[0014] In other aspects, provided herein is an isolated monoclonal
antibody, or antigen binding portion thereof, comprising heavy and
light chain CDRs wherein
[0015] (i) heavy chain CDR1 comprises GFX.sub.1FSTY (SEQ ID NO:
54), wherein X.sub.1 is selected from S and T;
[0016] (ii) heavy chain CDR2 comprises X.sub.2GEGDS (SEQ ID NO:
55), wherein X.sub.2 is selected from S and T;
[0017] (iii) heavy chain CDR3 comprises GYX.sub.3NFYYYYTMDX.sub.4
(SEQ ID NO: 56), wherein X.sub.3 is selected from S and T and
X.sub.4 is selected from A and V;
[0018] (iv) light chain CDR1 comprises RAX.sub.5QSIX.sub.6TFLA (SEQ
ID NO: 57), wherein X.sub.5 is selected from S and T and X.sub.6 is
selected from S and V;
[0019] (v) light chain CDR2 comprises DASTX.sub.7AX.sub.8 (SEQ ID
NO: 58), wherein X.sub.7 is selected from R and N and X.sub.8 is
selected from S and T; and
[0020] (vi) light chain CDR3 comprises QQRYNWPPYX.sub.9 (SEQ ID NO:
59), wherein X.sub.9 is selected from S and T.
[0021] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0022] (i) heavy chain CDR1 comprising GFX.sub.1FSTY (SEQ ID NO:
54), wherein X.sub.1 is not present;
[0023] (ii) heavy chain CDR2 comprising X.sub.2GEGDS (SEQ ID NO:
55), wherein X.sub.2 is selected from S and T;
[0024] (iii) heavy chain CDR3 comprising GYX.sub.3NFYYYYTMDX.sub.4
(SEQ ID NO: 56), wherein X.sub.3 is selected from S and T and
X.sub.4 is selected from A and V;
[0025] (iv) light chain CDR1 comprising RAX.sub.5QSIX.sub.6TFLA
(SEQ ID NO: 57), wherein X.sub.5 is selected from S and T and
X.sub.6 is selected from S and V;
[0026] (v) light chain CDR2 comprising DASTX.sub.7AX.sub.8 (SEQ ID
NO: 58), wherein X.sub.7 is selected from R and N and X.sub.8 is
selected from S and T; and
[0027] (vi) light chain CDR3 comprising QQRYNWPPYX.sub.9 (SEQ ID
NO: 59), wherein X.sub.9 is selected from S and T.
[0028] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises,
[0029] (i) heavy chain CDR1 comprising GFSFSTY (SEQ ID NO: 21);
[0030] (ii) heavy chain CDR2 comprising SGEGDS (SEQ ID NO: 27);
and
[0031] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDA (SEQ ID NO:
32).
[0032] In other aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0033] (i) heavy chain CDR1 comprising GFSFSTY (SEQ ID NO: 21);
[0034] (ii) heavy chain CDR2 comprising TGEGDS (SEQ ID NO: 28);
and
[0035] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDA (SEQ ID NO:
32).
[0036] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0037] (i) heavy chain CDR1 comprising GFTFSTY (SEQ ID NO: 22);
[0038] (ii) heavy chain CDR2 comprising TGEGDS (SEQ ID NO: 28);
and
[0039] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDV (SEQ ID NO:
33).
[0040] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0041] (i) heavy chain CDR1 comprising GFFSTY (SEQ ID NO: 23);
[0042] (ii) heavy chain CDR2 comprising TGEGDS (SEQ ID NO: 28);
and
[0043] (iii) heavy chain CDR3 comprising GYTNFYYYYTMDA (SEQ ID NO:
34).
[0044] In other aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0045] (i) heavy chain CDR1 comprising GFSFSTY (SEQ ID NO: 21);
[0046] (ii) heavy chain CDR2 comprising TGEGDS (SEQ ID NO: 28);
and
[0047] (iii) heavy chain CDR3 comprising GYTNFYYYYTMDA (SEQ ID NO:
34).
[0048] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0049] (i) light chain CDR1 comprising RATQSISTFLA (SEQ ID NO:
38);
[0050] (ii) light chain CDR2 comprising DASTRAS (SEQ ID NO: 44);
and
[0051] (iii) light chain CDR3 comprising QQRYNWPPYS (SEQ ID NO:
50).
[0052] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0053] (i) light chain CDR1 comprising RASQSISTFLA (SEQ ID NO:
39);
[0054] (ii) light chain CDR2 comprising DASTRAT (SEQ ID NO: 45);
and
[0055] (iii) light chain CDR3 comprising QQRYNWPPYT (SEQ ID NO:
51).
[0056] In other aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0057] (i) light chain CDR1 comprising RATQSIVTFLA (SEQ ID NO:
40);
[0058] (ii) light chain CDR2 comprising DASTNAS (SEQ ID NO: 46);
and
[0059] (iii) light chain CDR3 comprising QQRYNWPPYS (SEQ ID NO:
50).
[0060] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0061] (i) heavy chain CDR1 comprising GFSFSTY (SEQ ID NO: 21);
[0062] (ii) heavy chain CDR2 comprising SGEGDS (SEQ ID NO: 27);
[0063] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDA (SEQ ID NO:
32);
[0064] (iv) light chain CDR1 comprising RATQSISTFLA (SEQ ID NO:
38);
[0065] (v) light chain CDR2 comprising DASTRAS (SEQ ID NO: 44);
and
[0066] (vi) light chain CDR3 comprising QQRYNWPPYS (SEQ ID NO:
50).
[0067] In other aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0068] (i) heavy chain CDR1 comprising GFSFSTY (SEQ ID NO: 21);
[0069] (ii) heavy chain CDR2 comprising SGEGDS (SEQ ID NO: 27);
[0070] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDA (SEQ ID NO:
32);
[0071] (iv) light chain CDR1 comprising RATQSIVTFLA (SEQ ID NO:
40);
[0072] (v) light chain CDR2 comprising DASTNAS (SEQ ID NO: 46);
and
[0073] (vi) light chain CDR3 comprising QQRYNWPPYS (SEQ ID NO:
50).
[0074] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0075] (i) heavy chain CDR1 comprising GFSFSTY (SEQ ID NO: 21);
[0076] (ii) heavy chain CDR2 comprising TGEGDS (SEQ ID NO: 28);
[0077] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDA (SEQ ID NO:
32);
[0078] (iv) light chain CDR1 comprising RATQSISTFLA (SEQ ID NO:
38);
[0079] (v) light chain CDR2 comprising DASTRAS (SEQ ID NO: 44);
and
[0080] (vi) light chain CDR3 comprising QQRYNWPPYS (SEQ ID NO:
50).
[0081] In other aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises
[0082] (i) heavy chain CDR1 comprising GFTFSTY (SEQ ID NO: 22);
[0083] (ii) heavy chain CDR2 comprising TGEGDS (SEQ ID NO: 28);
[0084] (iii) heavy chain CDR3 comprising GYSNFYYYYTMDV (SEQ ID NO:
33);
[0085] (iv) light chain CDR1 comprising RASQSISTFLA (SEQ ID NO:
39);
[0086] (v) light chain CDR2 comprising DASTRAT (SEQ ID NO: 45);
and
[0087] (vi) light chain CDR3 comprising QQRYNWPPYT (SEQ ID NO:
51).
[0088] In some aspects, the disclosure provides an isolated
monoclonal antibody which specifically binds Zika virus envelope
protein, or antigen binding portion thereof, comprising a heavy
chain variable region as set forth in SEQ ID NO: 4, wherein
[0089] (i) heavy chain CDR1 comprises an amino acid substitution or
deletion at N28, and amino acid substitutions at L29, S31, S32;
[0090] (ii) heavy chain CDR2 comprises amino acid substitutions at
S52, S52A, 553, Y54, G55;
[0091] (iii) heavy chain CDR3 comprises an amino acid deletion at
S99 and amino acid substitutions at K100, K100A, P100B, Y100C,
F100D, S100E, G100F, W100G, A100H, and Y102; and
[0092] wherein the heavy chain variable region comprises at least
one amino acid deletion at R94, and at CDR3 residues T95, V96, and
R97, and combinations thereof, numbering according to Chothia. In
some aspects, the heavy chain variable region further comprises at
least one amino acid substitution at V5, Y33, A49, 550, T57, A71,
T73, A78, S82B, L82C, A93, L108 and combinations thereof, numbering
according to Chothia. In some aspects, the heavy chain variable
region further comprises an amino acid substitution at T68,
numbering according to Chothia. In some aspects, the heavy chain
variable region further comprises at least one amino acid
substitution at A23, W47, Y58, L80, Q81, A84, and combinations
thereof, numbering according to Chothia. In some aspects, the heavy
chain variable region further comprises at least one amino acid
substitution at E1, A23, R38, W47, Y58, L80, Q81, A84, and
combinations thereof, numbering according to Chothia. In some
aspects, the heavy chain variable region further comprises at least
one amino acid substitution at E1, A23, R38, W47, Y58, T68, L80,
Q81, A84, and combinations thereof, numbering according to
Chothia.
[0093] In any of the foregoing aspects, the isolated monoclonal
antibody, or antigen binding portion thereof, described herein,
comprising a heavy chain variable region as set forth in SEQ ID NO:
4, comprises
[0094] (i) heavy chain CDR1 comprising N28S or N28T, L29F, S31T,
S32Y;
[0095] (ii) heavy chain CDR2 comprising S52T, S52AG, S53E, Y54G,
G55D;
[0096] (iii) heavy chain CDR3 comprising K100Y, K100AS or K100AT,
P100BN, Y100CF, F100DY, S100EY, G100FY, W100GY, A100HT, and Y102A
or Y102V.
[0097] In some aspects, the heavy chain variable region comprises
amino acid deletions at R94, and at CDR3 residues T95, V96, and
R97, numbering according to Chothia.
[0098] In any of the foregoing aspects, the isolated monoclonal
antibody, or antigen binding portion thereof, described herein,
comprises a light chain variable region comprising an amino acid
sequence set forth in SEQ ID NO: 5, wherein
[0099] (i) light chain CDR1 comprises amino acid substitutions at
S26, V29, S31, A32, and V33;
[0100] (ii) light chain CDR2 comprises amino acid substitutions at
S50, S53, L54, Y55 and optionally S56; and
[0101] (iii) light chain CDR3 comprises amino acid substitutions at
H91, P93, F94, Y95, L95B, F96, and T97 and an amino acid deletion
at G92.
[0102] In any of the foregoing aspects, the isolated monoclonal
antibody, or antigen binding portion thereof, described herein,
comprises
[0103] (i) light chain CDR1 comprising S26T, V29I, S31V, A32F, and
V33L;
[0104] (ii) light chain CDR2 comprising S50D, S53T, L54R or L54N,
Y55A and, optionally S56T; and
[0105] (iii) light chain CDR3 comprising amino acid substitutions
at H91R, P93Y, F94N, Y95W, L95BP, F96Y, and T97S.
[0106] In any of the foregoing aspects, the light chain variable
region further comprises at least one amino acid substitution at
D1, Q3, M4, S9, A13, V15, D17, V19, I21, Y22, Q38, K42, K45, S60,
Q79, T85, and combinations thereof, numbering according to Chothia.
In some aspects, the light chain variable region further comprises
at least one amino acid substitution at S10, V58, S76, S77, V104,
and combinations thereof, numbering according to Chothia.
[0107] In some aspects, the disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof, which
binds to Zika virus envelope protein and comprises heavy and light
chain variable regions, wherein the heavy and light chain amino
acid sequences are selected from the group consisting of:
[0108] (a) SEQ ID NOs: 4 and 14, respectively;
[0109] (b) SEQ ID NOs: 4 and 15, respectively;
[0110] (c) SEQ ID NOs: 9 and 16, respectively;
[0111] (d) SEQ ID NOs: 4 and 16, respectively;
[0112] (e) SEQ ID NOs: 4 and 17, respectively;
[0113] (f) SEQ ID NOs: 8 and 14, respectively;
[0114] (g) SEQ ID NOs: 7 and 17, respectively;
[0115] (h) SEQ ID NOs: 6 and 15, respectively;
[0116] (i) SEQ ID NOs: 6 and 5, respectively;
[0117] (j) SEQ ID NOs: 7 and 5, respectively;
[0118] (k) SEQ ID NOs: 8 and 5, respectively;
[0119] (l) SEQ ID NOs: 9 and 5, respectively;
[0120] (m) SEQ ID NOs: 10 and 5, respectively;
[0121] (n) SEQ ID NOs: 11 and 5, respectively;
[0122] (o) SEQ ID NOs: 6 and 14, respectively;
[0123] (p) SEQ ID NOs: 6 and 16, respectively;
[0124] (q) SEQ ID NOs: 6 and 17, respectively;
[0125] (r) SEQ ID NOs: 7 and 14, respectively;
[0126] (s) SEQ ID NOs: 7 and 15, respectively;
[0127] (t) SEQ ID NOs: 7 and 16, respectively;
[0128] (u) SEQ ID NOs: 8 and 15, respectively;
[0129] (v) SEQ ID NOs: 8 and 16, respectively;
[0130] (w) SEQ ID NOs: 8 and 17, respectively;
[0131] (x) SEQ ID NOs: 9 and 14, respectively;
[0132] (y) SEQ ID NOs: 9 and 15, respectively;
[0133] (z) SEQ ID NOs: 9 and 17, respectively;
[0134] (aa) SEQ ID NOs: 10 and 14, respectively;
[0135] (bb) SEQ ID NOs: 10 and 15, respectively;
[0136] (cc) SEQ ID NOs: 10 and 16, respectively;
[0137] (dd) SEQ ID NOs: 10 and 17, respectively;
[0138] (ee) SEQ ID NOs: 11 and 14, respectively;
[0139] (ff) SEQ ID NOs: 11 and 15, respectively;
[0140] (gg) SEQ ID NOs: 11 and 16, respectively; and
[0141] (hh) SEQ ID NOs: 11 and 17, respectively.
[0142] In other aspects, the disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof, which
binds to Zika virus envelope protein, comprising heavy and light
chain CDRs selected from the group consisting of:
[0143] (a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 20, 26 and 31, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,
respectively;
[0144] (b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,
respectively;
[0145] (c) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 20, 26 and 31, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,
respectively;
[0146] (d) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 20, 26 and 31, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,
respectively;
[0147] (e) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,
respectively;
[0148] (f) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,
respectively;
[0149] (g) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,
respectively;
[0150] (h) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,
respectively;
[0151] (i) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,
respectively;
[0152] (j) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,
respectively;
[0153] (k) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,
respectively;
[0154] (1) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 37, 43 and 49,
respectively;
[0155] (m) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 27 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,
respectively;
[0156] (n) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,
respectively;
[0157] (o) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 32, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,
respectively;
[0158] (p) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,
respectively;
[0159] (q) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 22, 28 and 33, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,
respectively;
[0160] (r) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,
respectively;
[0161] (s) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,
respectively;
[0162] (t) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 23, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,
respectively;
[0163] (u) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 38, 44 and 50,
respectively;
[0164] (v) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 39, 45 and 51,
respectively; and
[0165] (w) heavy chain CDR1, CDR2 and CDR3 sequences set forth in
SEQ ID NOs: 21, 28 and 34, respectively, and light chain CDR1, CDR2
and CDR3 sequences set forth in SEQ ID NOs: 40, 46 and 50,
respectively.
[0166] In some aspects, the disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof, which
binds to Zika virus envelope protein and comprises heavy and light
chain variable regions, wherein the heavy chain variable region
comprises an amino acid sequence which is at least 90% identical to
the amino acid sequence selected from the group consisting of SEQ
ID NOs: 4, 6, 7, 8, 9, 10 and 11; and wherein the light chain
variable region comprises an amino acid sequence which is at least
90% identical to the amino acid sequence selected from the group
consisting of SEQ ID NOs: 5, 14, 15, 16, and 17, provided that the
monoclonal antibody does not comprise SEQ ID NOs: 4 and 5.
[0167] In some aspects, the isolated monoclonal antibody, or
antigen binding portion thereof, described herein, comprises heavy
chain and light chain sequences having at least 90% identity to the
heavy and light chain amino acid sequences selected from the group
consisting of:
[0168] (a) SEQ ID NOs: 4 and 14, respectively;
[0169] (b) SEQ ID NOs: 4 and 15, respectively;
[0170] (c) SEQ ID NOs: 9 and 16, respectively;
[0171] (d) SEQ ID NOs: 4 and 16, respectively;
[0172] (e) SEQ ID NOs: 4 and 17, respectively;
[0173] (f) SEQ ID NOs: 8 and 14, respectively;
[0174] (g) SEQ ID NOs: 7 and 17, respectively;
[0175] (h) SEQ ID NOs: 6 and 15, respectively;
[0176] (i) SEQ ID NOs: 6 and 5, respectively;
[0177] (j) SEQ ID NOs: 7 and 5, respectively;
[0178] (k) SEQ ID NOs: 8 and 5, respectively;
[0179] (l) SEQ ID NOs: 9 and 5, respectively;
[0180] (m) SEQ ID NOs: 10 and 5, respectively;
[0181] (n) SEQ ID NOs: 11 and 5, respectively;
[0182] (o) SEQ ID NOs: 6 and 14, respectively;
[0183] (p) SEQ ID NOs: 6 and 16, respectively;
[0184] (q) SEQ ID NOs: 6 and 17, respectively;
[0185] (r) SEQ ID NOs: 7 and 14, respectively;
[0186] (s) SEQ ID NOs: 7 and 15, respectively;
[0187] (t) SEQ ID NOs: 7 and 16, respectively;
[0188] (u) SEQ ID NOs: 8 and 15, respectively;
[0189] (v) SEQ ID NOs: 8 and 16, respectively;
[0190] (w) SEQ ID NOs: 8 and 17, respectively;
[0191] (x) SEQ ID NOs: 9 and 14, respectively;
[0192] (y) SEQ ID NOs: 9 and 15, respectively;
[0193] (z) SEQ ID NOs: 9 and 17, respectively;
[0194] (aa) SEQ ID NOs: 10 and 14, respectively;
[0195] (bb) SEQ ID NOs: 10 and 15, respectively;
[0196] (cc) SEQ ID NOs: 10 and 16, respectively;
[0197] (dd) SEQ ID NOs: 10 and 17, respectively;
[0198] (ee) SEQ ID NOs: 11 and 14, respectively;
[0199] (ff) SEQ ID NOs: 11 and 15, respectively;
[0200] (gg) SEQ ID NOs: 11 and 16, respectively; and
[0201] (hh) SEQ ID NOs: 11 and 17, respectively.
[0202] Some aspects of the disclosure relate to any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, in which the antibody is a humanized
antibody.
[0203] Other aspects of the disclosure relate to any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, in which the antibody has neutralizing activity
against Zika virus.
[0204] Some aspects of the disclosure relate to any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, in which the antibody is selected from the group
consisting of an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1,
an IgA2, an IgD, and an IgE antibody. In some aspects of the
disclosure, any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, is an IgG1 antibody.
[0205] Other aspects of the disclosure relate to a pharmaceutical
composition comprising any of the preceding isolated monoclonal
antibodies, or antigen binding portions thereof, and a
pharmaceutically acceptable carrier.
[0206] Another aspect of the disclosure relates to a method for
treating Zika virus infection in a subject in need thereof,
comprising administering an effective amount of any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, or a pharmaceutical composition comprising any of
the preceding isolated monoclonal antibodies, or antigen binding
portions thereof, and a pharmaceutically acceptable carrier.
[0207] Another aspect of the disclosure relates to a method for
preventing Zika virus infection in a subject, comprising
administering an effective amount of any of the preceding isolated
monoclonal antibodies, or antigen binding portions thereof, or a
pharmaceutical composition comprising any of the preceding isolated
monoclonal antibodies, or antigen binding portions thereof, and a
pharmaceutically acceptable carrier.
[0208] In other aspects, the disclosure relates to methods for
treating or preventing vertical infection to a fetus in a pregnant
subject, comprising administering an effective amount of any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, or a pharmaceutical composition comprising any of
the preceding isolated monoclonal antibodies, or antigen binding
portions thereof, and a pharmaceutically acceptable carrier. In
some aspects, the disclosure relates to methods for reducing or
reducing the risk of vertical infection to a fetus in a pregnant
subject, comprising administering an effective amount of any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, or a pharmaceutical composition comprising any of
the preceding isolated monoclonal antibodies, or antigen binding
portions thereof, and a pharmaceutically acceptable carrier.
[0209] In another aspect, the disclosure relates to methods for
treating or preventing fetal Zika virus infection, comprising
administering to a pregnant subject in need thereof, an effective
amount of any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, or a pharmaceutical composition
comprising any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, and a pharmaceutically acceptable
carrier. In some aspects, the disclosure relates to methods for
reducing or reducing the risk of fetal Zika virus infection,
comprising administering to a pregnant subject an effective amount
of any of the preceding isolated monoclonal antibodies, or antigen
binding portions thereof, or a pharmaceutical composition
comprising any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, and a pharmaceutically acceptable
carrier.
[0210] Another aspect of the disclosure relates to methods for
treating or preventing fetal mortality in a pregnant subject,
comprising administering to the subject an effective amount of any
of the preceding isolated monoclonal antibodies, or antigen binding
portions thereof, or a pharmaceutical composition comprising any of
the preceding isolated monoclonal antibodies, or antigen binding
portions thereof, and a pharmaceutically acceptable carrier. In
some aspects, the disclosure relates to methods for reducing or
reducing the risk of fetal mortality in a pregnant subject,
comprising administering an effective amount of any of the
preceding isolated monoclonal antibodies, or antigen binding
portions thereof, or a pharmaceutical composition comprising any of
the preceding isolated monoclonal antibodies, or antigen binding
portions thereof, and a pharmaceutically acceptable carrier.
[0211] In other aspects, the disclosure relates to methods for
treating or preventing placental Zika virus infection, comprising
administering to a pregnant subject in need thereof, an effective
amount of any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, or a pharmaceutical composition
comprising any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, and a pharmaceutically acceptable
carrier. In some aspects, the disclosure relates to methods for
reducing or reducing the risk of placental Zika virus infection,
comprising administering to a pregnant subject an effective amount
of any of the preceding isolated monoclonal antibodies, or antigen
binding portions thereof, or a pharmaceutical composition
comprising any of the preceding isolated monoclonal antibodies, or
antigen binding portions thereof, and a pharmaceutically acceptable
carrier.
[0212] In any of the foregoing methods, the pregnant subject is
infected with Zika virus. In some aspects, the pregnant subject is
at risk of being infected with Zika virus.
[0213] Some aspects of the disclosure relate to a nucleic acid
comprising a nucleotide sequence encoding the light chain, heavy
chain, or both light and heavy chains of any of the preceding
isolated monoclonal antibodies, or antigen binding portions
thereof. In some aspects, the disclosure relates to an expression
vector comprising the nucleic acid. In further aspects, the
disclosure relates to a cell transformed with the expression
vector.
BRIEF DESCRIPTION OF THE FIGURES
[0214] FIG. 1A shows the heavy chain sequence for anti-TDRD3
antibody (top) and the heavy chain sequences for the anti-Zika
antibodies generated (bottom). Modifications are bold and
underlined.
[0215] FIG. 1B shows the light chain sequence for anti-TDRD3
antibody (top) and the light chain sequences for the anti-Zika
antibodies generated (bottom). Modifications are bold and
underlined.
[0216] FIG. 2 shows binding data of anti-Zika antibodies to
different strains of Zika virus as measured by a sandwich
ELISA.
[0217] FIGS. 3A and 3B are graphs showing the percent
neutralization by anti-Zika antibodies determined in a plaque
reduction neutralization test using the Zika virus ILM strain
(Brazil Paraiba 2015) (3A) or H/PF/2013 strain (3B).
[0218] FIGS. 4A-4D are graphs showing body weight over time (4A and
4B) and survival over time (4C and 4D) in mice with Zika virus
infection (H/PF/2013 strain) treated either prophylactically (4A
and 4C) or therapeutically (4B and 4D) with anti-Zika
antibodies.
[0219] FIG. 5 is graphs depicting viral load over time in mice with
Zika virus infection (H/PF/2013 strain) treated either
prophylactically (top) or therapeutically (bottom) with anti-Zika
antibodies.
[0220] FIG. 6A is a line graph showing the weight, in grams, of
mice over time treated with varying doses of mAb 8 administered a
day after Zika virus infection (H/PF/2013 strain).
[0221] FIG. 6B is a line graph depicting viral load over time in
mice treated with varying doses of mAb 8 administered a day after
Zika virus infection (H/PF/2013 strain).
[0222] FIG. 6C is a Kaplan-Meier graph showing survival of mice
treated with varying doses of mAb 8 administered a day after Zika
virus infection (H/PF/2013 strain).
[0223] FIG. 7A is a graph depicting antibody-dependent enhancement
activity of various antibodies in THP-1 cells infected with Zika
virus (H/PF/2013 strain). Plaque titers resulting from opsonization
with different concentrations of antibodies is shown.
[0224] FIG. 7B provides a comparison of peak enhancement titers
(viral titer at point of greatest enhancement observed) resulting
from the various antibodies as indicated. *<0.05
(non-parametric, two-tailed student's T-test).
[0225] FIG. 8A is a schematic providing the study design for
assessing vertical infection and fetal mortality in pregnant A129
mice infected with Zika virus (H/PF/2013 strain).
[0226] FIGS. 8B-8F provide graphs showing the results of the study
described in FIG. 8A. Specifically, levels of viral RNA in the
mothers at day 2 (8B) or day 7 (8C), percent fetus survival as
measured by percent lethality (8D), and viral RNA in fetuses (8E)
or placenta (8F) from mice treated with an isotype control IgG or
mAb 8 are shown.
DETAILED DESCRIPTION
Definitions
[0227] Terms used in the claims and specification are defined as
set forth below unless otherwise specified.
[0228] The term "Zika virus", refers to members of the family
Flaviviridae, and is normally associated with mild symptoms
including fever, rash joint pain or conjunctivitis, but has
recently been linked to microcephaly in newborn babies by
mother-to-child transmission and Guillain-Barre syndrome. The
genome of Zika virus consists of a single strand of positive sense
RNA that is approximately 11 kb in length. Zika virions, like
virions of other falviviruses, contain 10 functionally distinct
proteins, including three structural proteins incorporated into the
virus particle.
[0229] The term "Zika virus envelope protein ("E" or "EP")" refers
to the protein responsible for viral entry and virion budding. It
is composed of three distinct domains. E Domain I (E-DI) is the
central domain that organizes the entire E protein structure. E
Domain II (E-DI) is formed from two extended loops projecting from
E-DI and lies in a pocket at the E-DI and E Domain III (E-DIII).
E-DIII is an immunoglobulin-like domain that forms small
protrusions on the surface of an otherwise smooth spherical mature
virus particle, and is thought to interact with cellular receptors
on target cells. At the distal end of E-DlI is a glycine-rich,
hydrophobic sequences referred to as the "fusion loop." The fusion
loop encompasses residues 98 to 110 and is highly conserved among
flaviviruses. In certain embodiments, "Zika virus envelope protein"
refers to the fusion loop. In certain embodiments, the fusion loop
has the amino acid sequence set forth in SEQ ID NO: 3.
[0230] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chain thereof. An "antibody" refers, in certain
embodiments, to a glycoprotein comprising at least two heavy (H)
chains and two light (L) chains inter-connected by disulfide bonds,
or an antigen binding portion thereof. Each heavy chain is
comprised of a heavy chain variable region (abbreviated herein as
V.sub.H) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CH1, CH2 and CH3.
Each light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L) and a light chain constant region.
The light chain constant region is comprised of one domain, CL. The
V.sub.H and V.sub.L regions can be further subdivided into regions
of hypervariability, termed complementarity determining regions
(CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each V.sub.H and V.sub.L is composed 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
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells) and the first component
(C1q) of the classical complement system.
[0231] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., Zika virus EP). Such "fragments" are, for
example between about 8 and about 1500 amino acids in length,
suitably between about 8 and about 745 amino acids in length,
suitably about 8 to about 300, for example about 8 to about 200
amino acids, or about 10 to about 50 or 100 amino acids in length.
It has been shown that the antigen-binding function of an antibody
can be performed by fragments of a full-length antibody. Examples
of binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, CL and CH1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a Fd fragment consisting of the V.sub.H and CH1 domains; (iv)
a Fv fragment consisting of the V.sub.L and V.sub.H domains of a
single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)
Nature 341:544-546), which consists of a VH domain; and (vi) an
isolated complementarity determining region (CDR) or (vii) a
combination of two or more isolated CDRs which may optionally be
joined by a synthetic linker. Furthermore, although the two domains
of the Fv fragment, V.sub.L and V.sub.H, are coded for by separate
genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are screened for utility in the same manner as
are intact antibodies. Antigen-binding portions can be produced by
recombinant DNA techniques, or by enzymatic or chemical cleavage of
intact immunoglobulins.
[0232] The term "monoclonal antibody," as used herein, refers to an
antibody which displays a single binding specificity and affinity
for a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to an antibody which displays a single binding
specificity and which has variable and optional constant regions
derived from human germline immunoglobulin sequences. In one
embodiment, human monoclonal antibodies are produced by a hybridoma
which includes a B cell obtained from a transgenic non-human
animal, e.g., a transgenic mouse, having a genome comprising a
human heavy chain transgene and a light chain transgene fused to an
immortalized cell.
[0233] The term "recombinant human antibody," as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom, (b) antibodies isolated from a host cell
transformed to express the antibody, e.g., from a transfectoma, (c)
antibodies isolated from a recombinant, combinatorial human
antibody library, and (d) antibodies prepared, expressed, created
or isolated by any other means that involve splicing of human
immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies comprise variable and constant regions
that utilize particular human germline immunoglobulin sequences are
encoded by the germline genes, but include subsequent
rearrangements and mutations which occur, for example, during
antibody maturation. As known in the art (see, e.g., Lonberg (2005)
Nature Biotech. 23(9):1117-1125), the variable region contains the
antigen binding domain, which is encoded by various genes that
rearrange to form an antibody specific for a foreign antigen. In
addition to rearrangement, the variable region can be further
modified by multiple single amino acid changes (referred to as
somatic mutation or hypermutation) to increase the affinity of the
antibody to the foreign antigen. The constant region will change in
further response to an antigen (i.e., isotype switch). Therefore,
the rearranged and somatically mutated nucleic acid molecules that
encode the light chain and heavy chain immunoglobulin polypeptides
in response to an antigen may not have sequence identity with the
original nucleic acid molecules, but instead will be substantially
identical or similar (i.e., have at least 80% identity).
[0234] The term "human antibody" includes antibodies having
variable and constant regions (if present) of human germline
immunoglobulin sequences. Human antibodies of the disclosure can
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo)
(see, Lonberg, N. et al. (1994) Nature 368(6474): 856-859);
Lonberg, N. (1994) Handbook of Experimental Pharmacology
113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad. Sci 764:536-546). However, the term "human antibody" does not
include antibodies in which CDR sequences derived from the germline
of another mammalian species, such as a mouse, have been grafted
onto human framework sequences (i.e., humanized antibodies).
[0235] As used herein, a "heterologous antibody" is defined in
relation to the transgenic non-human organism producing such an
antibody. This term refers to an antibody having an amino acid
sequence or an encoding nucleic acid sequence corresponding to that
found in an organism not consisting of the transgenic non-human
animal, and generally from a species other than that of the
transgenic non-human animal.
[0236] As used herein, "neutralizing antibody" refers to an
antibody, for example, a monoclonal antibody, capable of disrupting
a formed viral particle or inhibiting formatting of a viral
particle or prevention of binding to or infection of mammalian
cells by a viral particle. In some embodiments, the antibodies
described herein neutralize Zika virus.
[0237] As used herein, "diagnostic antibody" or "detection
antibody" or "detecting antibody" refers to an antibody, for
example, a monoclonal antibody, capable of detecting the presence
of an antigenic target within a sample. As will be appreciated by
one of skill in the art, such diagnostic antibodies preferably have
high specificity for their antigenic target.
[0238] The term "humanized immunoglobulin" or "humanized antibody"
refers to an immunoglobulin or antibody that includes at least one
humanized immunoglobulin or antibody chain (i.e., at least one
humanized light or heavy chain). The term "humanized immunoglobulin
chain" or "humanized antibody chain" (i.e., a "humanized
immunoglobulin light chain" or "humanized immunoglobulin heavy
chain") refers to an immunoglobulin or antibody chain (i.e., a
light or heavy chain, respectively) having a variable region that
includes a variable framework region substantially from a human
immunoglobulin or antibody and complementarity determining regions
(CDRs) (e.g., at least one CDR, preferably two CDRs, more
preferably three CDRs) substantially from a non-human
immunoglobulin or antibody, and further includes constant regions
(e.g., at least one constant region or portion thereof, in the case
of a light chain, and preferably three constant regions in the case
of a heavy chain). The term "humanized variable region" (e.g.,
"humanized light chain variable region" or "humanized heavy chain
variable region") refers to a variable region that includes a
variable framework region substantially from a human immunoglobulin
or antibody and complementarity determining regions (CDRs)
substantially from a non-human immunoglobulin or antibody.
[0239] The phrase "substantially from a human immunoglobulin or
antibody" or "substantially human" means that, when aligned to a
human immunoglobulin or antibody amino acid sequence for comparison
purposes, the region shares at least 80-90%, preferably at least
90-95%, more preferably at least 95-99% identity (i.e., local
sequence identity) with the human framework or constant region
sequence, allowing, for example, for conservative substitutions,
consensus sequence substitutions, germline substitutions,
back-mutations, and the like. The introduction of conservative
substitutions, consensus sequence substitutions, germline
substitutions, back-mutations, and the like, is often referred to
as "optimization" of a humanized antibody or chain. The phrase
"substantially from a non-human immunoglobulin or antibody" or
"substantially non-human" means having an immunoglobulin or
antibody sequence at least 80-95%, preferably at least 90-95%, more
preferably, 96%, 97%, 98%, or 99% identical to that of a non-human
organism, e.g., a non-human mammal.
[0240] Referring to the well-recognized nomenclature for amino
acids, the three letter code, or one letter code, is used,
including the codes "Xaa" or "X" to indicate any amino acid
residue. Thus, Xaa or X may typically represent any of the 20
naturally occurring amino acids.
[0241] Preferably, residue positions which are not identical differ
by conservative amino acid substitutions. For purposes of
classifying amino acids substitutions as conservative or
nonconservative, amino acids are grouped as follows: Group I
(hydrophobic sidechains): leu, met, ala, val, leu, ile; Group II
(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic
side chains): asp, glu; Group IV (basic side chains): asn, gln,
his, lys, arg; Group V (residues influencing chain orientation):
gly, pro; and Group VI (aromatic side chains): trp, tyr, phe.
Conservative substitutions involve substitutions between amino
acids in the same class. Non-conservative substitutions constitute
exchanging a member of one of these classes for a member of
another.
[0242] A mutation (e.g., a back-mutation) is said to substantially
affect the ability of a heavy or light chain to direct antigen
binding if it affects (e.g., decreases) the binding affinity of an
intact immunoglobulin or antibody (or antigen binding fragment
thereof) comprising said chain by at least an order of magnitude
compared to that of the antibody (or antigen binding fragment
thereof) comprising an equivalent chain lacking said mutation. A
mutation "does not substantially affect (e.g., decrease) the
ability of a chain to direct antigen binding" if it affects (e.g.,
decreases) the binding affinity of an intact immunoglobulin or
antibody (or antigen binding fragment thereof) comprising said
chain by only a factor of two, three, or four of that of the
antibody (or antigen binding fragment thereof) comprising an
equivalent chain lacking said mutation.
[0243] In certain embodiments, humanized immunoglobulins or
antibodies bind antigen with an affinity that is within a factor of
three, four, or five of that of the corresponding nonhumanized
antibody. For example, if the nonhumanized antibody has a binding
affinity of 10.sup.9 M.sup.-1, humanized antibodies will have a
binding affinity of at least 3 times 10.sup.9 M.sup.-1, 4 times
10.sup.9 M.sup.-1 or 10.sup.9 M.sup.-1. When describing the binding
properties of an immunoglobulin or antibody chain, the chain can be
described based on its ability to "direct antigen (e.g., Zika virus
EP) binding". A chain is said to "direct antigen binding" when it
confers upon an intact immunoglobulin or antibody (or antigen
binding fragment thereof) a specific binding property or binding
affinity.
[0244] The term "chimeric immunoglobulin" or antibody refers to an
immunoglobulin or antibody whose variable regions derive from a
first species and whose constant regions derive from a second
species. Chimeric immunoglobulins or antibodies can be constructed,
for example by genetic engineering, from immunoglobulin gene
segments belonging to different species. The terms "humanized
immunoglobulin" or "humanized antibody" are not intended to
encompass chimeric immunoglobulins or antibodies, as defined infra.
Although humanized immunoglobulins or antibodies are chimeric in
their construction (i.e., comprise regions from more than one
species of protein), they include additional features (i.e.,
variable regions comprising donor CDR residues and acceptor
framework residues) not found in chimeric immunoglobulins or
antibodies, as defined herein.
[0245] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to Zika virus EP is substantially
free of antibodies that specifically bind antigens other than Zika
virus EP). An isolated antibody is typically substantially free of
other cellular material and/or chemicals. In certain embodiments of
the disclosure, a combination of "isolated" antibodies having
different Zika virus EP specificities is combined in a well-defined
composition.
[0246] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods for determining what epitopes are
bound by a given antibody (i.e., epitope mapping) are well known in
the art and include, for example, immunoblotting and
immunoprecipitation assays, wherein overlapping or contiguous
peptides from Zika virus EP are tested for reactivity with the
given anti-EP antibody. Methods of determining spatial conformation
of epitopes include techniques in the art and those described
herein, for example, x-ray crystallography and 2-dimensional
nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in
Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
(1996)).
[0247] Antibodies that recognize the same epitope can be identified
in a simple immunoassay showing the ability of one antibody to
block the binding of another antibody to a target antigen, i.e., a
competitive binding assay. Competitive binding is determined in an
assay in which the immunoglobulin under test inhibits specific
binding of a reference antibody to a common antigen. Numerous types
of competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label RIA using 1-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer
et al., Scand. J. Immunol. 32:77 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0248] The term "epitope mapping" refers to the process of
identification of the molecular determinants for antibody-antigen
recognition. Numerous methods for epitope mapping are known in the
art, such as x-ray analysis, protease mapping, hydrogen/deuterium
exchange mass spectrometry (HDX-MS), 2D nuclear magnetic resonance,
alanine scanning, and deep mutational scanning.
[0249] To facilitate engineering of antibodies that target the Zika
virus envelope protein (EP), epitope hotspots were determined by
analyzing the percent buried surface area of interface residues. In
some embodiments, the anti-Zika virus antibodies described herein
bind an epitope on the fusion loop comprising residues D98, R99 and
W101 (SEQ ID NO: 3).
[0250] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd). For
example, the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM,
40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or
stronger. Affinity can be measured by common methods known in the
art, including those described herein. Low-affinity antibodies
generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and
tend to remain bound longer. A variety of methods of measuring
binding affinity are known in the art, any of which can be used for
purposes of the present disclosure.
[0251] As used herein, the terms "specific binding," "selective
binding," "selectively binds," and "specifically binds," refer to
antibody binding to an epitope on a predetermined antigen.
Typically, the antibody binds with an equilibrium dissociation
constant (K.sub.D) of approximately less than 10.sup.-7 M, such as
approximately less than 10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or
even lower when determined by surface plasmon resonance (SPR)
technology in a BIACORE 2000 instrument using recombinant Zika
virus EP as the analyte and the antibody as the ligand and binds to
the predetermined antigen with an affinity that is at least
two-fold greater than its affinity for binding to a non-specific
antigen (e.g., BSA, casein) other than the predetermined antigen or
a closely-related antigen. The phrases "an antibody recognizing an
antigen" and "an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen."
[0252] The term "K.sub.D," as used herein, is intended to refer to
the dissociation equilibrium constant of a particular
antibody-antigen interaction.
[0253] The term "kd" as used herein, is intended to refer to the
off rate constant for the dissociation of an antibody from the
antibody/antigen complex.
[0254] The term "ka" as used herein, is intended to refer to the on
rate constant for the association of an antibody with the
antigen.
[0255] The term "EC50," as used herein, refers to the concentration
of an antibody or an antigen-binding portion thereof, which induces
a response, either in an in vitro or an in vivo assay, which is 50%
of the maximal response, i.e., halfway between the maximal response
and the baseline.
[0256] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes. In one embodiment, a human monoclonal antibody of the
disclosure is of the IgG1 isotype. In certain embodiments, the
human IgG1 has a heavy chain constant domain sequence as set forth
in SEQ ID NO: 1 and a light chain constant domain sequence as set
forth in SEQ ID NO: 2.
[0257] The term "binds to Zika virus EP," refers to the ability of
an antibody described herein to bind to Zika virus EP, for example,
expressed on the surface of a cell or which is attached to a solid
support.
[0258] The term "nucleic acid molecule," as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0259] The present disclosure also encompasses "conservative
sequence modifications" of the sequences set forth in SEQ ID NOs:
4-53 i.e., amino acid sequence modifications which do not abrogate
the binding of the antibody encoded by the nucleotide sequence or
containing the amino acid sequence, to the antigen. Such
conservative sequence modifications include conservative nucleotide
and amino acid substitutions, as well as, nucleotide and amino acid
additions and deletions. For example, modifications can be
introduced into SEQ ID NOs: 4-53 by standard techniques known in
the art, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Conservative amino acid substitutions include ones in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in an anti-EP antibody is
preferably replaced with another amino acid residue from the same
side chain family. Methods of identifying nucleotide and amino acid
conservative substitutions which do not eliminate antigen binding
are well-known in the art (see, e.g., Brummell et al., Biochem.
32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884
(1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417
(1997)).
[0260] Alternatively, in certain embodiments, mutations can be
introduced randomly along all or part of an anti-EP antibody coding
sequence, such as by saturation mutagenesis, and the resulting
modified anti-EP antibodies can be screened for binding
activity.
[0261] For nucleic acids, the term "substantial homology" indicates
that two nucleic acids, or designated sequences thereof, when
optimally aligned and compared, are identical, with appropriate
nucleotide insertions or deletions, in at least about 80% of the
nucleotides, usually at least about 90% to 95%, and more preferably
at least about 98% to 99.5% of the nucleotides. Alternatively,
substantial homology exists when the segments will hybridize under
selective hybridization conditions, to the complement of the
strand.
[0262] The percent identity between two sequences is a function of
the number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions x 100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, as described in the non-limiting examples
below.
[0263] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences can also be determined using the algorithm of E.
Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. In addition, the percent identity between two amino acid
sequences can be determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0264] The nucleic acid and protein sequences of the present
disclosure can further be used as a "query sequence" to perform a
search against public databases to, for example, identify related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
disclosure. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the protein molecules of the disclosure. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
[0265] The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form. A
nucleic acid is "isolated" or "rendered substantially pure" when
purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987).
[0266] When given an amino acid sequence, one versed in the art can
make conservative substitutions to the nucleotide sequence encoding
it without altering the amino acid sequence, given the redundancy
in the genetic code. The nucleic acid compositions, while often in
a native sequence (except for modified restriction sites and the
like), from either cDNA, genomic or mixtures thereof may be
mutated, in accordance with standard techniques to provide gene
sequences. For coding sequences, these mutations, may affect amino
acid sequence as desired. In particular, DNA sequences
substantially homologous to or derived from native V, D, J,
constant, switches and other such sequences described herein are
contemplated (where "derived" indicates that a sequence is
identical or modified from another sequence).
[0267] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid,"
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the disclosure
is intended to include such other forms of expression vectors, such
as viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0268] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
[0269] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, in need of such treatment, a human antibody of the present
disclosure, for example, a subject in need of an enhanced immune
response against a particular antigen (e.g., Zika virus) or a
subject who ultimately may acquire such a disorder, in order to
prevent, cure, delay, reduce the severity of, or ameliorate one or
more symptoms of the disorder or recurring disorder, or in order to
prolong the survival of a subject beyond that expected in the
absence of such treatment.
[0270] The term "effective dose" or "effective dosage" is defined
as an amount sufficient to achieve or at least partially achieve
the desired effect. The term "therapeutically effective dose" is
defined as an amount sufficient to cure or at least partially
arrest the disease and its complications in a patient already
suffering from the disease. Amounts effective for this use will
depend upon the severity of the disorder being treated and the
general state of the patient's own immune system.
[0271] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0272] As used herein, the term "subject" includes any human or
non-human animal. For example, the methods and compositions of the
present disclosure can be used to treat a subject with an immune
disorder. The term "non-human animal" includes all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc. In some embodiments,
the subject is pregnant.
[0273] As used herein, the term "vertical infection" refers to
mother-to-child transmission of a pathogen (e.g., Zika virus). In
some embodiments, the anti-Zika virus EP antibodies described
herein prevent vertical infection in a pregnant subject.
[0274] Various aspects of the disclosure are described in further
detail in the following subsections.
Production of Antibodies to Zika Virus Envelope Protein
[0275] The present disclosure encompasses antibodies that bind Zika
virus EP. In some embodiments, antibodies that bind Zika virus EP
are optimized monoclonal antibodies which include CDRs, or
optimized CDRs, based on an anti-TDRD3 (Tudor Domain Containing 3)
human monoclonal antibody. Provided herein are isolated monoclonal
antibodies or antigen binding portions thereof, comprising heavy
and light chain variable region sequences comprising (further
described in Tables 2 and 3):
[0276] (a) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 20, 26 and 31, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 38, 44 and 50, respectively;
[0277] (b) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 22, 28 and 33, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 39, 45 and 51, respectively;
[0278] (c) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 20, 26 and 31, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 39, 45 and 51, respectively;
[0279] (d) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 20, 26 and 31, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 40, 46 and 50, respectively;
[0280] (e) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 38, 44 and 50, respectively;
[0281] (f) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 27 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 40, 46 and 50, respectively;
[0282] (g) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 27 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 38, 44 and 50, respectively;
[0283] (h) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 27 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 37, 43 and 49, respectively;
[0284] (i) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 37, 43 and 49, respectively;
[0285] (j) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 22, 28 and 33, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 37, 43 and 49, respectively;
[0286] (k) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 23, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 37, 43 and 49, respectively;
[0287] (l) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 37, 43 and 49, respectively;
[0288] (m) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 27 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 39, 45 and 51, respectively;
[0289] (n) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 39, 45 and 51, respectively;
[0290] (o) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 32, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 40, 46 and 50, respectively;
[0291] (p) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 22, 28 and 33, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 38, 44 and 50, respectively;
[0292] (q) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 22, 28 and 33, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 40, 46 and 50, respectively;
[0293] (r) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 23, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 38, 44 and 50, respectively;
[0294] (s) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 23, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 39, 45 and 51, respectively;
[0295] (t) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 23, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 40, 46 and 50, respectively;
[0296] (u) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 38, 44 and 50, respectively;
[0297] (v) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 39, 45 and 51, respectively; and
[0298] (w) a heavy chain comprising CDR1, CDR2 and CDR3 sequences
set forth in SEQ ID NOs: 21, 28 and 34, respectively, and a light
chain comprising CDR1, CDR2 and CDR3 sequences set forth in SEQ ID
NOs: 40, 46 and 50, respectively.
[0299] In some embodiments, antibodies that bind Zika virus EP are
optimized monoclonal antibodies which include heavy and/or light
chain variable regions, or optimized heavy and/or light chain
variable regions, based on an anti-TDRD3 human monoclonal antibody.
Also provided herein, are isolated monoclonal antibodies or antigen
binding portions thereof, comprising heavy and light chain variable
sequences comprising:
[0300] (a) SEQ ID NOs: 4 and 14, respectively;
[0301] (b) SEQ ID NOs: 4 and 15, respectively;
[0302] (c) SEQ ID NOs: 9 and 16, respectively;
[0303] (d) SEQ ID NOs: 4 and 16, respectively;
[0304] (e) SEQ ID NOs: 4 and 17, respectively;
[0305] (f) SEQ ID NOs: 8 and 14, respectively;
[0306] (g) SEQ ID NOs: 7 and 17, respectively;
[0307] (h) SEQ ID NOs: 6 and 15, respectively;
[0308] (i) SEQ ID NOs: 6 and 5, respectively;
[0309] (j) SEQ ID NOs: 7 and 5, respectively;
[0310] (k) SEQ ID NOs: 8 and 5, respectively;
[0311] (l) SEQ ID NOs: 9 and 5, respectively;
[0312] (m) SEQ ID NOs: 10 and 5, respectively;
[0313] (n) SEQ ID NOs: 11 and 5, respectively;
[0314] (o) SEQ ID NOs: 6 and 14, respectively;
[0315] (p) SEQ ID NOs: 6 and 16, respectively;
[0316] (q) SEQ ID NOs: 6 and 17, respectively;
[0317] (r) SEQ ID NOs: 7 and 14, respectively;
[0318] (s) SEQ ID NOs: 7 and 15, respectively;
[0319] (t) SEQ ID NOs: 7 and 16, respectively;
[0320] (u) SEQ ID NOs: 8 and 15, respectively;
[0321] (v) SEQ ID NOs: 8 and 16, respectively;
[0322] (w) SEQ ID NOs: 8 and 17, respectively;
[0323] (x) SEQ ID NOs: 9 and 14, respectively;
[0324] (y) SEQ ID NOs: 9 and 15, respectively;
[0325] (z) SEQ ID NOs: 9 and 17, respectively;
[0326] (aa) SEQ ID NOs: 10 and 14, respectively;
[0327] (bb) SEQ ID NOs: 10 and 15, respectively;
[0328] (cc) SEQ ID NOs: 10 and 16, respectively;
[0329] (dd) SEQ ID NOs: 10 and 17, respectively;
[0330] (ee) SEQ ID NOs: 11 and 14, respectively;
[0331] (ff) SEQ ID NOs: 11 and 15, respectively;
[0332] (gg) SEQ ID NOs: 11 and 16, respectively; and
[0333] (hh) SEQ ID NOs: 11 and 17, respectively.
[0334] Monoclonal antibodies described herein can be produced using
a variety of known techniques, such as the standard somatic cell
hybridization technique described by Kohler and Milstein, Nature
256: 495 (1975). Although somatic cell hybridization procedures are
preferred, in principle, other techniques for producing monoclonal
antibodies also can be employed, e.g., viral or oncogenic
transformation of B lymphocytes, phage display technique using
libraries of human antibody genes.
[0335] Accordingly, in certain embodiments, a hybridoma method is
used for producing an antibody that binds Zika virus EP. In this
method, a mouse or other appropriate host animal can be immunized
with a suitable antigen in order to elicit lymphocytes that produce
or are capable of producing antibodies that will specifically bind
to the antigen used for immunization. Alternatively, lymphocytes
may be immunized in vitro. Lymphocytes can then be fused with
myeloma cells using a suitable fusing agent, such as polyethylene
glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:
Principles and Practice, pp. 59-103 (Academic Press, 1986)).
Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against the antigen.
After hybridoma cells are identified that produce antibodies of the
desired specificity, affinity, and/or activity, the clones may be
subcloned by limiting dilution procedures and grown by standard
methods (Goding, Monoclonal Antibodies:Principles and Practice, pp.
59-103 (Academic Press, 1986)). Suitable culture media for this
purpose include, for example, DMEM or RPMI-1640 medium. In
addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal. The monoclonal antibodies secreted by the
subclones can be separated from the culture medium, ascites fluid,
or serum by conventional immunoglobulin purification procedures
such as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[0336] In certain embodiments, antibodies and antibody portions
that bind Zika virus EP can be isolated from antibody phage
libraries generated using the techniques described in, for example,
McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,
Nature, 352:624-628 (1991), Marks et al., J. Mol. Biol.,
222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23,
344-348; U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.
5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717
to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to
McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;
6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.
Additionally, production of high affinity (nM range) human
antibodies by chain shuffling (Marks et al., Bio/Technology,
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266
(1993)) may also be used.
[0337] In certain embodiments, the antibody that binds Zika virus
EP is produced using the phage display technique described by Hoet
et al., supra. This technique involves the generation of a human
Fab library having a unique combination of immunoglobulin sequences
isolated from human donors and having synthetic diversity in the
heavy-chain CDRs is generated. The library is then screened for
Fabs that bind to Zika virus EP.
[0338] The preferred animal system for generating hybridomas which
produce antibodies of the disclosure is the murine system.
Hybridoma production in the mouse is well known in the art,
including immunization protocols and techniques for isolating and
fusing immunized splenocytes.
[0339] In certain embodiments, antibodies directed against Zika
virus EP are generated using transgenic or transchromosomal mice
carrying parts of the human immune system rather than the mouse
system. In some embodiments, antibodies described herein are
generated using transgenic mice, referred to herein as "HuMAb mice"
which contain a human immunoglobulin gene miniloci that encodes
unrearranged human heavy (.mu. and .gamma.) and .kappa. light chain
immunoglobulin sequences, together with targeted mutations that
inactivate the endogenous .mu. and .kappa. chain loci (Lonberg, N.
et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice
exhibit reduced expression of mouse IgM or .kappa., and in response
to immunization, the introduced human heavy and light chain
transgenes undergo class switching and somatic mutation to generate
high affinity human IgG.kappa. monoclonal antibodies (Lonberg, N.
et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of
Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D.
(1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and
Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). The
preparation of HuMAb mice is described in detail below and in
Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen,
J. et al. (1993) International Immunology 5: 647-656; Tuaillon et
al. (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi et al.
(1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J.
12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920;
Lonberg et al., (1994) Nature 368(6474): 856-859; Lonberg, N.
(1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L.
et al. (1994) International Immunology 6: 579-591; Lonberg, N. and
Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F.
and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild,
D. et al. (1996) Nature Biotechnology 14: 845-851. See further,
U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and
5,770,429; all to Lonberg and Kay, and GenPharm International; U.S.
Pat. No. 5,545,807 to Surani et al.; International Publication Nos.
WO 98/24884, published on Jun. 11, 1998; WO 94/25585, published
Nov. 10, 1994; WO 93/1227, published Jun. 24, 1993; WO 92/22645,
published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992).
[0340] In certain embodiments, antibodies described herein can be
raised using a mouse that carries human immunoglobulin sequences on
transgenes and transchomosomes, such as a mouse that carries a
human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to in the art as "KM mice",
are described in detail in PCT Publication WO 02/43478 to Ishida et
al.
[0341] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-Zika virus EP antibodies of the
disclosure. For example, an alternative transgenic system referred
to as the Xenomouse (Abgenix, Inc.) can be used; such mice are
described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181;
6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al.
[0342] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-Zika virus EP antibodies described
herein. For example, mice carrying both a human heavy chain
transchromosome and a human light chain tranchromosome, referred to
in the art as "TC mice" can be used; such mice are described in
Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.
Furthermore, cows carrying human heavy and light chain
transchromosomes have been described in the art (Kuroiwa et al.
(2002) Nature Biotechnology 20:889-894) and can be used to raise
anti-Zika virus EP antibodies of the disclosure.
[0343] Additional mouse systems described in the art for raising
human antibodies also can be applied to raising anti-Zika virus EP
antibodies of the disclosure, including but not limited to (i) the
VelocImmune.RTM. mouse (Regeneron Pharmaceuticals, Inc.), in which
the endogenous mouse heavy and light chain variable regions have
been replaced, via homologous recombination, with human heavy and
light chain variable regions, operatively linked to the endogenous
mouse constant regions, such that chimeric antibodies (human
V/mouse C) are raised in the mice, and then subsequently converted
to fully human antibodies using standard recombinant DNA
techniques; and (ii) the MeMo.RTM. mouse (Merus Biopharmaceuticals,
Inc.), in which the mouse contains unrearranged human heavy chain
variable regions but a single rearranged human common light chain
variable region. Such mice, and use thereof to raise antibodies,
are described in, for example, WO 2009/15777, US 2010/0069614, WO
2011/072204, WO 2011/097603, WO 2011/163311, WO 2011/163314, WO
2012/148873, US 2012/0070861 and US 2012/0073004.
[0344] Human monoclonal antibodies described herein can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
[0345] In certain embodiments, the mAbs described herein can be
produced in plants using deconstructed viral vectors, as described
in Olinger et al., PNAS 2012; 109, 18030-18035, herein incorporated
by reference. In certain embodiments, the mAbs are produced in
tobacco plants.
[0346] In certain embodiments, chimeric antibodies can be prepared
based on the sequence of a murine monoclonal antibodies described
herein. A chimeric antibody refers to an antibody whose light and
heavy chain genes have been constructed, typically by genetic
engineering, from immunoglobulin gene segments belonging to
different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as IgG1 and IgG4. Human isotype IgG1 is
preferred. A typical chimeric antibody is thus a hybrid protein
consisting of the V or antigen-binding domain from a mouse antibody
and the C or effector domain from a human antibody.
Production of Humanized Antibodies
[0347] The term "humanized antibody" refers to an antibody
comprising at least one chain comprising variable region framework
residues substantially from a human antibody chain (referred to as
the acceptor immunoglobulin or antibody) and at least one
complementarity determining region substantially from a mouse
antibody, (referred to as the donor immunoglobulin or antibody).
See, Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033
(1989), U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762,
Selick et al., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539
(incorporated by reference in their entirety for all purposes). The
constant region(s), if present, are also substantially or entirely
from a human immunoglobulin.
[0348] The substitution of mouse CDRs into a human variable domain
framework is most likely to result in retention of their correct
spatial orientation if the human variable domain framework adopts
the same or similar conformation to the mouse variable framework
from which the CDRs originated. This is achieved by obtaining the
human variable domains from human antibodies whose framework
sequences exhibit a high degree of sequence identity with the
murine variable framework domains from which the CDRs were derived.
The heavy and light chain variable framework regions can be derived
from the same or different human antibody sequences. The human
antibody sequences can be the sequences of naturally occurring
human antibodies or can be consensus sequences of several human
antibodies. See Kettleborough et al., Protein Engineering 4:773
(1991); Kolbinger et al., Protein Engineering 6:971 (1993) and
Carter et al., WO 92/22653.
[0349] Having identified the complementarity determining regions of
the murine donor immunoglobulin and appropriate human acceptor
immunoglobulins, the next step is to determine which, if any,
residues from these components should be substituted to optimize
the properties of the resulting humanized antibody. In general,
substitution of human amino acid residues with murine should be
minimized, because introduction of murine residues increases the
risk of the antibody eliciting a human-anti-mouse-antibody (HAMA)
response in humans. Art-recognized methods of determining an immune
response can be performed to monitor a HAMA response in a
particular patient or during clinical trials. Patients administered
humanized antibodies can be given an immunogenicity assessment at
the beginning and throughout the administration of said therapy.
The HAMA response is measured, for example, by detecting antibodies
to the humanized therapeutic reagent, in serum samples from the
patient using a method known to one in the art, including surface
plasmon resonance technology (BIACORE) and/or solid-phase ELISA
analysis.
[0350] Certain amino acids from the human variable region framework
residues are selected for substitution based on their possible
influence on CDR conformation and/or binding to antigen. The
unnatural juxtaposition of murine CDR regions with human variable
framework region can result in unnatural conformational restraints,
which, unless corrected by substitution of certain amino acid
residues, lead to loss of binding affinity.
[0351] The selection of amino acid residues for substitution is
determined, in part, by computer modeling. Computer hardware and
software are described herein for producing three-dimensional
images of immunoglobulin molecules. In general, molecular models
are produced starting from solved structures for immunoglobulin
chains or domains thereof. The chains to be modeled are compared
for amino acid sequence similarity with chains or domains of solved
three-dimensional structures, and the chains or domains showing the
greatest sequence similarity is/are selected as starting points for
construction of the molecular model. Chains or domains sharing at
least 50% sequence identity are selected for modeling, and
preferably those sharing at least 60%, 70%, 80%, 90% sequence
identity or more are selected for modeling. The solved starting
structures are modified to allow for differences between the actual
amino acids in the immunoglobulin chains or domains being modeled,
and those in the starting structure. The modified structures are
then assembled into a composite immunoglobulin. Finally, the model
is refined by energy minimization and by verifying that all atoms
are within appropriate distances from one another and that bond
lengths and angles are within chemically acceptable limits.
[0352] The selection of amino acid residues for substitution can
also be determined, in part, by examination of the characteristics
of the amino acids at particular locations, or empirical
observation of the effects of substitution or mutagenesis of
particular amino acids. For example, when an amino acid differs
between a murine variable region framework residue and a selected
human variable region framework residue, the human framework amino
acid should usually be substituted by the equivalent framework
amino acid from the mouse antibody when it is reasonably expected
that the amino acid: [0353] (1) noncovalently binds antigen
directly, [0354] (2) is adjacent to a CDR region, [0355] (3)
otherwise interacts with a CDR region (e.g., is within about 3-6
angstroms of a CDR region as determined by computer modeling), or
[0356] (4) participates in the VL-VH interface.
[0357] Residues which "noncovalently bind antigen directly" include
amino acids in positions in framework regions which have a good
probability of directly interacting with amino acids on the antigen
according to established chemical forces, for example, by hydrogen
bonding, Van der Waals forces, hydrophobic interactions, and the
like.
[0358] CDR and framework regions are as defined by Kabat et al. or
Chothia et al., supra. When framework residues, as defined by Kabat
et al., supra, constitute structural loop residues as defined by
Chothia et al., supra, the amino acids present in the mouse
antibody may be selected for substitution into the humanized
antibody. Residues which are "adjacent to a CDR region" include
amino acid residues in positions immediately adjacent to one or
more of the CDRs in the primary sequence of the humanized
immunoglobulin chain, for example, in positions immediately
adjacent to a CDR as defined by Kabat, or a CDR as defined by
Chothia (See e.g., Chothia and Lesk J M B 196:901 (1987)). These
amino acids are particularly likely to interact with the amino
acids in the CDRs and, if chosen from the acceptor, to distort the
donor CDRs and reduce affinity. Moreover, the adjacent amino acids
may interact directly with the antigen (Amit et al., Science,
233:747 (1986), which is incorporated herein by reference) and
selecting these amino acids from the donor may be desirable to keep
all the antigen contacts that provide affinity in the original
antibody.
[0359] Residues that "otherwise interact with a CDR region" include
those that are determined by secondary structural analysis to be in
a spatial orientation sufficient to affect a CDR region. In certain
embodiments, residues that "otherwise interact with a CDR region"
are identified by analyzing a three-dimensional model of the donor
immunoglobulin (e.g., a computer-generated model). A
three-dimensional model, typically of the original donor antibody,
shows that certain amino acids outside of the CDRs are close to the
CDRs and have a good probability of interacting with amino acids in
the CDRs by hydrogen bonding, Van der Waals forces, hydrophobic
interactions, etc. At those amino acid positions, the donor
immunoglobulin amino acid rather than the acceptor immunoglobulin
amino acid may be selected. Amino acids according to this criterion
will generally have a side chain atom within about 3 angstrom units
(A) of some atom in the CDRs and must contain an atom that could
interact with the CDR atoms according to established chemical
forces, such as those listed above.
[0360] In the case of atoms that may form a hydrogen bond, the 3
Angstroms is measured between their nuclei, but for atoms that do
not form a bond, the 3 Angstroms is measured between their Van der
Waals surfaces. Hence, in the latter case, the nuclei must be
within about 6 Angstroms (3 Angstroms plus the sum of the Van der
Waals radii) for the atoms to be considered capable of interacting.
In many cases the nuclei will be from 4 or 5 to 6 Angstroms apart.
In determining whether an amino acid can interact with the CDRs, it
is preferred not to consider the last 8 amino acids of heavy chain
CDR 2 as part of the CDRs, because from the viewpoint of structure,
these 8 amino acids behave more as part of the framework.
[0361] Amino acids that are capable of interacting with amino acids
in the CDRs, may be identified in yet another way. The solvent
accessible surface area of each framework amino acid is calculated
in two ways: (1) in the intact antibody, and (2) in a hypothetical
molecule consisting of the antibody with its CDRs removed. A
significant difference between these numbers of about 10 square
angstroms or more shows that access of the framework amino acid to
solvent is at least partly blocked by the CDRs, and therefore that
the amino acid is making contact with the CDRs. Solvent accessible
surface area of an amino acid may be calculated based on a
three-dimensional model of an antibody, using algorithms known in
the art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee and
Richards, J. Mol. Biol. 55:379 (1971), both of which are
incorporated herein by reference). Framework amino acids may also
occasionally interact with the CDRs indirectly, by affecting the
conformation of another framework amino acid that in turn contacts
the CDRs.
[0362] The amino acids at several positions in the framework are
known to be capable of interacting with the CDRs in many antibodies
(Chothia and Lesk, supra, Chothia et al., supra and Tramontano et
al., J. Mol. Biol. 215:175 (1990), all of which are incorporated
herein by reference). Notably, the amino acids at positions 2, 48,
64 and 71 of the light chain and 26-30, 71 and 94 of the heavy
chain (numbering according to Kabat) are known to be capable of
interacting with the CDRs in many antibodies. The amino acids at
positions 35 in the light chain and 93 and 103 in the heavy chain
are also likely to interact with the CDRs. At all these numbered
positions, choice of the donor amino acid rather than the acceptor
amino acid (when they differ) to be in the humanized immunoglobulin
is preferred. On the other hand, certain residues capable of
interacting with the CDR region, such as the first 5 amino acids of
the light chain, may sometimes be chosen from the acceptor
immunoglobulin without loss of affinity in the humanized
immunoglobulin.
[0363] Residues which "participate in the VL-VH interface" or
"packing residues" include those residues at the interface between
VL and VH as defined, for example, by Novotny and Haber, Proc.
Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia et al, supra.
Generally, unusual packing residues should be retained in the
humanized antibody if they differ from those in the human
frameworks.
[0364] In general, one or more of the amino acids fulfilling the
above criteria is substituted. In some embodiments, all or most of
the amino acids fulfilling the above criteria are substituted.
Occasionally, there is some ambiguity about whether a particular
amino acid meets the above criteria, and alternative variant
immunoglobulins are produced, one of which has that particular
substitution, the other of which does not. Alternative variant
immunoglobulins so produced can be tested in any of the assays
described herein for the desired activity, and the preferred
immunoglobulin selected.
[0365] Usually the CDR regions in humanized antibodies are
substantially identical, and more usually, identical to the
corresponding CDR regions of the donor antibody. Although not
usually desirable, it is sometimes possible to make one or more
conservative amino acid substitutions of CDR residues without
appreciably affecting the binding affinity of the resulting
humanized immunoglobulin. By conservative substitutions is intended
combinations such as gly, ala; val, ile, leu; asp, glu; asn, gln;
ser, thr; lys, arg; and phe, tyr.
[0366] Additional candidates for substitution are acceptor human
framework amino acids that are unusual or "rare" for a human
immunoglobulin at that position. These amino acids can be
substituted with amino acids from the equivalent position of the
mouse donor antibody or from the equivalent positions of more
typical human immunoglobulins. For example, substitution may be
desirable when the amino acid in a human framework region of the
acceptor immunoglobulin is rare for that position and the
corresponding amino acid in the donor immunoglobulin is common for
that position in human immunoglobulin sequences; or when the amino
acid in the acceptor immunoglobulin is rare for that position and
the corresponding amino acid in the donor immunoglobulin is also
rare, relative to other human sequences. These criteria help ensure
that an atypical amino acid in the human framework does not disrupt
the antibody structure. Moreover, by replacing an unusual human
acceptor amino acid with an amino acid from the donor antibody that
happens to be typical for human antibodies, the humanized antibody
may be made less immunogenic.
[0367] The term "rare", as used herein, indicates an amino acid
occurring at that position in less than about 20% but usually less
than about 10% of sequences in a representative sample of
sequences, and the term "common", as used herein, indicates an
amino acid occurring in more than about 25% but usually more than
about 50% of sequences in a representative sample. For example, all
human light and heavy chain variable region sequences are
respectively grouped into "subgroups" of sequences that are
especially homologous to each other and have the same amino acids
at certain critical positions (Kabat et al., supra). When deciding
whether an amino acid in a human acceptor sequence is "rare" or
"common" among human sequences, it will often be preferable to
consider only those human sequences in the same subgroup as the
acceptor sequence.
[0368] Additional candidates for substitution are acceptor human
framework amino acids that would be identified as part of a CDR
region under the alternative definition proposed by Chothia et al.,
supra. Additional candidates for substitution are acceptor human
framework amino acids that would be identified as part of a CDR
region under the AbM and/or contact definitions.
[0369] Additional candidates for substitution are acceptor
framework residues that correspond to a rare or unusual donor
framework residue. Rare or unusual donor framework residues are
those that are rare or unusual (as defined herein) for murine
antibodies at that position. For murine antibodies, the subgroup
can be determined according to Kabat and residue positions
identified which differ from the consensus. These donor specific
differences may point to somatic mutations in the murine sequence
which enhance activity. Unusual residues that are predicted to
affect binding are retained, whereas residues predicted to be
unimportant for binding can be substituted.
[0370] Additional candidates for substitution are non-germline
residues occurring in an acceptor framework region. For example,
when an acceptor antibody chain (i.e., a human antibody chain
sharing significant sequence identity with the donor antibody
chain) is aligned to a germline antibody chain (likewise sharing
significant sequence identity with the donor chain), residues not
matching between acceptor chain framework and the germline chain
framework can be substituted with corresponding residues from the
germline sequence.
[0371] Other than the specific amino acid substitutions discussed
above, the framework regions of humanized immunoglobulins are
usually substantially identical, and more usually, identical to the
framework regions of the human antibodies from which they were
derived. Of course, many of the amino acids in the framework region
make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
humanized immunoglobulin. Thus, in one embodiment the variable
framework region of the humanized immunoglobulin shares at least
85% sequence identity to a human variable framework region sequence
or consensus of such sequences. In another embodiment, the variable
framework region of the humanized immunoglobulin shares at least
90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequence
identity to a human variable framework region sequence or consensus
of such sequences. In general, however, such substitutions are
undesirable.
[0372] The humanized antibodies preferably exhibit a specific
binding affinity for antigen of at least 10.sup.7, 10.sup.8,
10.sup.9 or 10.sup.10 M.sup.-1. Usually the upper limit of binding
affinity of the humanized antibodies for antigen is within a factor
of three, four or five of that of the donor immunoglobulin. Often
the lower limit of binding affinity is also within a factor of
three, four or five of that of donor immunoglobulin. Alternatively,
the binding affinity can be compared to that of a humanized
antibody having no substitutions (e.g., an antibody having donor
CDRs and acceptor FRs, but no FR substitutions). In such instances,
the binding of the optimized antibody (with substitutions) is
preferably at least two- to three-fold greater, or three- to
four-fold greater, than that of the unsubstituted antibody. For
making comparisons, activity of the various antibodies can be
determined, for example, by BIACORE (i.e., surface plasmon
resonance using unlabeled reagents) or competitive binding
assays.
Generation of Antibodies Having Modified Sequences
[0373] In certain embodiments, provided herein are antibodies
comprising heavy and light chain variable region CDRs from an
anti-TDRD3 monoclonal antibody (SEQ ID NOs: 4 and 5, respectively),
wherein at least one CDR comprises an amino acid substitution, such
that the antibody binds to Zika virus EP. In certain embodiments,
the heavy chain variable region CDR has an amino acid substitution
at position 28, 29, 31, 32, 52, 52A, 53, 54, 55, 100, 100A, 100B,
100C, 100D, 100E, 100F, 100G, 100H, 102, or combination thereof,
numbering according to Chothia. In certain embodiments, the amino
acid substitution at position 28 is serine. In certain embodiments,
the amino acid substitution at position 28 is threonine. In certain
embodiments, the amino acid at position 28 is deleted. In certain
embodiments, the amino acid substitution at position 29 is
phenylalanine. In certain embodiments, the amino acid substitution
at position 31 is threonine. In certain embodiments, the amino acid
substitution at position 32 is tyrosine. In certain embodiments,
the amino acid substitution at position 52 is threonine. In certain
embodiments, the amino acid substitution at position 52A is
glycine. In certain embodiments, the amino acid substitution at
position 53 is glutamic acid. In certain embodiments, the amino
acid substitution at position 54 is glycine. In certain
embodiments, the amino acid substitution at position 55 is aspartic
acid. In certain embodiments, the amino acid at position 99 is
deleted. In certain embodiments, the amino acid substitution at
position 100 is serine tyrosine. In certain embodiments, the amino
acid substitution at position 100A is serine. In certain
embodiments, the amino acid substitution at position 100A is
threonine. In certain embodiments, the amino acid substitution at
position 100B is asparagine. In certain embodiments, the amino acid
substitution at position 100C is phenylalanine. In certain
embodiments, the amino acid substitution at position 100D is
tyrosine. In certain embodiments, the amino acid substitution at
position 100E is tyrosine. In certain embodiments, the amino acid
substitution at position 100F is tyrosine. In certain embodiments,
the amino acid substitution at position 100G is tyrosine. In
certain embodiments, the amino acid substitution at position 100H
is threonine. In certain embodiments, the amino acid substitution
at position 102 is alanine. In certain embodiments, the amino acid
substitution at position 102 is valine.
[0374] In certain embodiments, the light chain variable region CDR
has an amino acid substitution at positions 26, 29, 31, 32, 33, 50,
53, 54, 55, 56, 91, 93, 94, 95, 95B, 96, 97, or combinations
thereof, numbering according to Chothia. In certain embodiments,
the amino acid substitution at position 26 is threonine. In certain
embodiments, the amino acid substitution at position 29 is
isoleucine. In certain embodiments, the amino acid substitution at
position 31 is valine. In certain embodiments, the amino acid
substitution at position 32 is phenylalanine. In certain
embodiments, the amino acid substitution at position 33 is leucine.
In certain embodiments, the amino acid substitution at position 50
is aspartic acid. In certain embodiments, the amino acid
substitution at position 53 is threonine. In certain embodiments,
the amino acid substitution at position 54 is arginine. In certain
embodiments, the amino acid substitution at position 54 is
asparagine. In certain embodiments, the amino acid substitution at
position 55 is alanine. In certain embodiments, the amino acid
substitution at position 56 is threonine. In certain embodiments,
the amino acid substitution at position 91 is arginine. In certain
embodiments, the amino acid substitution at position 93 is
tyrosine. In certain embodiments, the amino acid substitution at
position 94 is asparagine. In certain embodiments, the amino acid
substitution at position 95 is tryptophan. In certain embodiments,
the amino acid substitution at position 95B is proline. In certain
embodiments, the amino acid substitution at position 96 is
tyrosine. In certain embodiments, the amino acid substitution at
position 97 is serine. In certain embodiments, a proline is added
between amino acids at positions 95B and 96.
[0375] In certain embodiments, the antibodies described herein
comprise heavy and light chain variable region CDRs, wherein heavy
chain variable region CDR1 has the amino acid sequence
GFX.sub.1FSTY (SEQ ID NO: 54), wherein X.sub.1 may or may not be
present and is selected from serine and threonine; wherein heavy
chain variable region CDR2 has the amino acid sequence X.sub.2GEGDS
(SEQ ID NO: 55), wherein X.sub.2 is selected from serine and
threonine; and wherein heavy chain variable region CDR3 has the
amino acid sequence GYX.sub.3NFYYYTMDX.sub.4 (SEQ ID NO: 56),
wherein X.sub.3 is selected from serine and threonine, and X.sub.4
is selected from alanine and valine.
[0376] In certain embodiments, heavy chain CDR1 has the amino acid
sequence GFSFSTY (SEQ ID NO: 21). In certain embodiments, heavy
chain CDR1 has the amino acid sequence GFTGSTY (SEQ ID NO: 22). In
certain embodiments, heavy chain CDR1 has the amino acid sequence
GFFSTY (SEQ ID NO: 23). In certain embodiments, heavy chain CDR2
has the amino acid sequence TGEGDS (SEQ ID NO: 28). In certain
embodiments, heavy chain CDR2 has the amino acid sequence SGEGDS
(SEQ ID NO: 27). In certain embodiments, heavy chain CDR3 has the
amino acid sequence GYSNFYYYYTMDA (SEQ ID NO: 32). In certain
embodiments, heavy chain CDR3 has the amino acid sequence
GYSNFYYYYTMDV (SEQ ID NO: 33). In certain embodiments, heavy chain
CDR3 has the amino acid sequence GYTNFYYYTMDA (SEQ ID NO: 34).
[0377] In certain embodiments, the antibodies described herein
comprise heavy and light chain variable region CDRs, wherein light
chain variable region CDR1 has the amino acid sequence
RAX.sub.5QSIX.sub.6TFLA (SEQ ID NO: 57), wherein X.sub.5 is
selected from serine and threonine, and wherein X.sub.6 is selected
from serine and valine; wherein light chain variable region CDR2
has the amino acid sequence DASTX.sub.7AX.sub.8 (SEQ ID NO: 58),
wherein X.sub.7 is selected from arginine and asparagine, and
X.sub.8 is selected from serine and threonine; and wherein light
chain variable region CDR3 has the amino acid sequence
QQRYNWPPYX.sub.9 (SEQ ID NO: 59), wherein X.sub.9 is selected from
serine and threonine.
[0378] In certain embodiments, light chain CDR1 has the amino acid
sequence RATQSISTFLA (SEQ ID NO: 38). In certain embodiments, light
chain CDR1 has the amino acid sequence RASQSISTFLA (SEQ ID NO: 39).
In certain embodiments, light chain CDR1 has the amino acid
sequence RATQSIVTFLA (SEQ ID NO: 40). In certain embodiments, light
chain CDR2 has the amino acid sequence DASTRAS (SEQ ID NO: 44). In
certain embodiments, light chain CDR2 has the amino acid sequence
DASTRAT (SEQ ID NO: 45). In certain embodiments, light chain CDR2
has the amino acid sequence DASTNAS (SEQ ID NO: 46). In certain
embodiments, light chain CDR3 has the amino acid sequence
QQRYNWPPYS (SEQ ID NO: 50). In certain embodiments, light chain
CDR3 has the amino acid sequence QQRYNWPPYT (SEQ ID NO: 51).
[0379] In certain embodiments, the anti-Zika virus EP antibodies
described herein contain framework mutations in the variable region
sequences. In some embodiments, the antibodies described herein
comprise heavy and light chain variable region sequences, wherein
the heavy chain variable region sequence comprises amino acid
substitutions at positions 1, 5, 23, 33, 38, 47, 49, 50, 57, 58,
68, 71, 73, 78, 80, 81, 82B, 82C, 84, 93, 108, or combinations
thereof, numbering according to Chothia. In certain embodiments,
the amino acid substitution at position 1 is glutamine. In some
embodiments, the amino acid substitution at position 5 is leucine.
In some embodiments, the amino acid substitution at position 23 is
serine. In some embodiments, the amino acid substitution at
position 33 is serine. In some embodiments, the amino acid
substitution at position 38 is lysine. In some embodiments, the
amino acid substitution at position 47 is tyrosine. In some
embodiments, the amino acid substitution at position 49 is serine.
In some embodiments, the amino acid substitution at position 50 is
alanine. In some embodiments, the amino acid substitution at
position 57 is alanine. In some embodiments, the amino acid
substitution at position 58 is phenylalanine. In some embodiments,
the amino acid substitution at position 68 is glutamic acid. In
some embodiments, the amino acid substitution at position 71 is
arginine. In some embodiments, the amino acid substitution at
position 73 is asparagine. In some embodiments, the amino acid
substitution at position 78 is leucine. In some embodiments, the
amino acid substitution at position 80 is phenylalanine. In some
embodiments, the amino acid substitution at position 81 is glutamic
acid. In some embodiments, the amino acid substitution at position
82(B) is lysine. In some embodiments, the amino acid substitution
at position 82(C) is valine. In some embodiments, the amino acid
substitution at position 84 is proline. In some embodiments, the
amino acid substitution at position 93 is valine. In some
embodiments, the amino acid substitution at position 108 is serine.
In some embodiments, the amino acid substitution at position 108 is
threonine. In some embodiments, the amino acid substitution at
position 108 is methionine.
[0380] In certain embodiments, the anti-Zika virus EP antibodies
described herein contain framework mutations in the variable region
sequences. In some embodiments, the antibodies described herein
comprise heavy and light chain variable region sequences, wherein
the light chain variable region sequence comprises amino acid
substitutions at positions 1, 3, 4, 9, 10, 13, 15, 17, 19, 21, 22,
38, 42, 45, 58, 60, 76, 77, 79, 85, 104, or combinations thereof,
numbering according to Chothia. In some embodiments, the amino acid
substitution at position 1 is glutamic acid. In some embodiments,
the amino acid substitution at position 3 is valine. In some
embodiments, the amino acid substitution at position 4 is leucine.
In some embodiments, the amino acid substitution at position 9 is
alanine. In some embodiments, the amino acid substitution at
position 10 is threonine. In some embodiments, the amino acid
substitution at position 13 is leucine. In some embodiments, the
amino acid substitution at position 15 is proline. In some
embodiments, the amino acid substitution at position 17 is glutamic
acid. In some embodiments, the amino acid substitution at position
19 is alanine. In some embodiments, the amino acid substitution at
position 21 is leucine. In some embodiments, the amino acid
substitution at position 22 is serine. In some embodiments, the
amino acid substitution at position 38 is histidine. In some
embodiments, the amino acid substitution at position 42 is
glutamine. In some embodiments, the amino acid substitution at
position 45 is arginine. In some embodiments, the amino acid
substitution at position 58 is isoleucine. In some embodiments, the
amino acid substitution at position 60 is alanine. In some
embodiments, the amino acid substitution at position 76 is
threonine. In some embodiments, the amino acid substitution at
position 77 is arginine. In some embodiments, the amino acid
substitution at position 77 is threonine. In some embodiments, the
amino acid substitution at position 79 is glutamic acid. In some
embodiments, the amino acid substitution at position 85 is valine.
In some embodiments, the amino acid substitution at position 104 is
leucine.
[0381] In another embodiment, the variable region sequences, or
portions thereof, of the anti-Zika virus EP antibodies described
herein are modified to create structurally related anti-Zika virus
EP antibodies that retain binding (i.e., to the same epitope as the
unmodified antibody).
[0382] Accordingly, in one aspect of the disclosure, the CDR1, 2,
and/or 3 regions of the engineered antibodies described above can
comprise the exact amino acid sequence(s) as those of antibodies
disclosed herein. However, in other aspects of the disclosure, the
antibodies comprise derivatives from the exact CDR sequences of the
antibodies disclosed herein, still retain the ability of to bind
Zika virus EP effectively. Such sequence modifications may include
one or more amino acid additions, deletions, or substitutions,
e.g., conservative sequence modifications as described above.
Sequence modifications may also be based on the consensus sequences
described above for the particular CDR1, CDR2, and CDR3 sequences
of antibodies disclosed herein.
[0383] Accordingly, in another embodiment, the engineered antibody
may be composed of one or more CDRs that are, for example, 90%,
95%, 98% or 99.5% identical to one or more CDRs of antibodies
disclosed herein. Ranges intermediate to the above-recited values,
e.g., CDRs that are 90-95%, 95-98%, or 98-100% identical identity
to one or more of the above sequences are also intended to be
encompassed by the present disclosure.
[0384] In another embodiment, one or more residues of a CDR may be
altered to modify binding to achieve a more favored on-rate of
binding, a more favored off-rate of binding, or both, such that an
idealized binding constant is achieved. Using this strategy, an
antibody having ultra-high binding affinity of, for example,
10.sup.10 M.sup.-1 or more, can be achieved. Affinity maturation
techniques, well known in the art and those described herein, can
be used to alter the CDR region(s) followed by screening of the
resultant binding molecules for the desired change in binding.
Accordingly, as CDR(s) are altered, changes in binding affinity as
well as immunogenicity can be monitored and scored such that an
antibody optimized for the best combined binding and low
immunogenicity are achieved.
[0385] Thus, for variable region modification within the VH and/or
VL CDR1, CDR2 and/or CDR3 regions, site-directed mutagenesis or
PCR-mediated mutagenesis can be performed to introduce the
mutation(s) and the effect on antibody binding, or other functional
property of interest, can be evaluated in in vitro or in vivo
assays. Preferably conservative modifications (as discussed herein)
are introduced. The mutations can be amino acid substitutions,
additions or deletions, but are preferably substitutions. Moreover,
typically no more than one, two, three, four or five residues
within a CDR region are altered.
[0386] In general, the framework regions of antibodies are usually
substantially identical, and more usually, identical to the
framework regions of the human germline sequences from which they
were derived. Of course, many of the amino acids in the framework
region make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
immunoglobulin. Thus, in one embodiment the variable framework
region of the antibody shares at least 85% sequence identity to a
human germline variable framework region sequence or consensus of
such sequences. In another embodiment, the variable framework
region of the antibody shares at least 90%, 95%, 96%, 97%, 98% or
99% sequence identity to a human germline variable framework region
sequence or consensus of such sequences.
[0387] Framework modifications can also be made to reduce
immunogenicity of the antibody or to reduce or remove T cell
epitopes that reside therein, as described for instance by Carr et
al in US2003/0153043.
[0388] Engineered antibodies of the disclosure include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "back-mutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation can contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived.
[0389] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043.
Use of Partial Antibody Sequences to Express Intact Antibodies
[0390] Antibodies interact with target antigens predominantly
through amino acid residues that are located in the six heavy and
light chain complementarity determining regions (CDRs). For this
reason, the amino acid sequences within CDRs are more diverse
between individual antibodies than sequences outside of CDRs.
Because CDR sequences are responsible for most antibody-antigen
interactions, it is possible to express recombinant antibodies that
mimic the properties of specific naturally occurring antibodies by
constructing expression vectors that include CDR sequences from the
specific naturally occurring antibody grafted onto framework
sequences from a different antibody with different properties (see,
e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et
al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc.
Natl. Acad. See. U.S.A. 86:10029-10033). Such framework sequences
can be obtained from public DNA databases that include germline
antibody gene sequences. These germline sequences will differ from
mature antibody gene sequences because they will not include
completely assembled variable genes, which are formed by V(D)J
joining during B cell maturation. Germline gene sequences will also
differ from the sequences of a high affinity secondary repertoire
antibody at individual evenly across the variable region. For
example, somatic mutations are relatively infrequent in the
amino-terminal portion of framework region. For example, somatic
mutations are relatively infrequent in the amino terminal portion
of framework region 1 and in the carboxy-terminal portion of
framework region 4. Furthermore, many somatic mutations do not
significantly alter the binding properties of the antibody. For
this reason, it is not necessary to obtain the entire DNA sequence
of a particular antibody in order to recreate an intact recombinant
antibody having binding properties similar to those of the original
antibody (see PCT/US99/05535 filed on Mar. 12, 1999). Partial heavy
and light chain sequence spanning the CDR regions is typically
sufficient for this purpose. The partial sequence is used to
determine which germline variable and joining gene segments
contributed to the recombined antibody variable genes. The germline
sequence is then used to fill in missing portions of the variable
regions. Heavy and light chain leader sequences are cleaved during
protein maturation and do not contribute to the properties of the
final antibody. To add missing sequences, cloned cDNA sequences can
be combined with synthetic oligonucleotides by ligation or PCR
amplification. Alternatively, the entire variable region can be
synthesized as a set of short, overlapping, oligonucleotides and
combined by PCR amplification to create an entirely synthetic
variable region clone. This process has certain advantages such as
elimination or inclusion or particular restriction sites, or
optimization of particular codons.
[0391] The nucleotide sequences of heavy and light chain
transcripts from a hybridoma are used to design an overlapping set
of synthetic oligonucleotides to create synthetic V sequences with
identical amino acid coding capacities as the natural sequences.
The synthetic heavy and kappa chain sequences can differ from the
natural sequences in three ways: strings of repeated nucleotide
bases are interrupted to facilitate oligonucleotide synthesis and
PCR amplification; optimal translation initiation sites are
incorporated according to Kozak's rules (Kozak, 1991, J. Biol.
Chem. 266:19867-19870); and, HindIII sites are engineered upstream
of the translation initiation sites.
[0392] For both the heavy and light chain variable regions, the
optimized coding, and corresponding non-coding, strand sequences
are broken down into 30-50 nucleotide approximately the midpoint of
the corresponding non-coding oligonucleotide. Thus, for each chain,
the oligonucleotides can be assembled into overlapping double
stranded sets that span segments of 150-400 nucleotides. The pools
are then used as templates to produce PCR amplification products of
150-400 nucleotides. Typically, a single variable region
oligonucleotide set will be broken down into two pools which are
separately amplified to generate two overlapping PCR products.
These overlapping products are then combined by PCR amplification
to form the complete variable region. It may also be desirable to
include an overlapping fragment of the heavy or light chain
constant region (including the BbsI site of the kappa light chain,
or the AgeI site if the gamma heavy chain) in the PCR amplification
to generate fragments that can easily be cloned into the expression
vector constructs.
[0393] The reconstructed heavy and light chain variable regions are
then combined with cloned promoter, leader sequence, translation
initiation, leader sequence, constant region, 3' untranslated,
polyadenylation, and transcription termination, sequences to form
expression vector constructs. The heavy and light chain expression
constructs can be combined into a single vector, co-transfected,
serially transfected, or separately transfected into host cells
which are then fused to form a host cell expressing both
chains.
[0394] Plasmids for use in construction of expression vectors were
constructed so that PCR amplified V heavy and V kappa light chain
cDNA sequences could be used to reconstruct complete heavy and
light chain minigenes. These plasmids can be used to express
completely human IgG.sub.1.kappa. or IgG.sub.4.kappa.
antibodies.
[0395] Fully human, humanized, and chimeric antibodies described
herein also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies.
Similar plasmids can be constructed for expression of other heavy
chain isotypes, or for expression of antibodies comprising lambda
light chains.
[0396] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 20, 26 and 31; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 38, 44 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0397] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 22, 28 and 33; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 39, 45 and 51; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0398] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 20, 26 and 31; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 39, 45 and 51; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0399] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 20, 26 and 31; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 40, 46 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0400] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 38, 44 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0401] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 27 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 40, 46 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0402] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 27 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 38, 44 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0403] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 27 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 37, 43 and 49; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0404] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 37, 43 and 49; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0405] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 22, 28 and 33; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 37, 43 and 49; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0406] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 23, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 37, 43 and 49; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0407] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 37, 43 and 49; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0408] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 27 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 39, 45 and 51; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0409] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 39, 45 and 51; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0410] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 32; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 40, 46 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0411] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 22, 28 and 33; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 38, 44 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0412] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 22, 28 and 33; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 40, 46 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0413] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 23, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 38, 44 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0414] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 23, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 40, 46 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0415] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 38, 44 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0416] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 39, 45 and 51; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
[0417] In certain embodiments, methods for preparing an anti-Zika
virus EP antibody are provided, including: preparing an antibody
including (1) heavy chain framework regions and heavy chain CDRs,
where at least one of the heavy chain CDRs includes an amino acid
sequence selected from the amino acid sequences of CDRs shown in
SEQ ID NOs: 21, 28 and 34; and (2) light chain framework regions
and light chain CDRs, where at least one of the light chain CDRs
includes an amino acid sequence selected from the amino acid
sequences of CDRs shown in SEQ ID NOs: 40, 46 and 50; where the
antibody binds to Zika virus EP. The ability of the antibody to
bind Zika virus EP can be determined using standard binding assays
(e.g., an ELISA or a FLISA).
Additional Antibody Modifications
[0418] Antibodies of the present disclosure can contain one or more
glycosylation sites in either the light or heavy chain variable
region. Such glycosylation sites may result in increased
immunogenicity of the antibody or an alteration of the pK of the
antibody due to altered antigen binding (Marshall et al (1972) Annu
Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol
172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro
(2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N-X-S/T sequence. In some instances, it is preferred to have an
anti-Zika virus antibody that does not contain variable region
glycosylation. This can be achieved either by selecting antibodies
that do not contain the glycosylation motif in the variable region
or by mutating residues within the glycosylation region.
[0419] For example, in certain embodiments, the glycosylation of an
antibody is modified, e.g., the variable region is altered to
eliminate one or more glycosylation sites resident in the variable
region. More particularly, it is desirable in the sequence of the
present antibodies to eliminate sites prone to glycosylation. This
is achieved by altering the occurrence of one or more N-X-(S/T)
sequences that occur in the parent variable region (where X is any
amino acid residue), particularly by substituting the N residue
and/or the S or T residue. In one embodiment, T95 is mutated to
K95. In another embodiment, N47 is mutated to R47.
[0420] For example, aglycoslated antibodies can be made (i.e.,
which lack glycosylation). Glycosylation can be altered to, for
example, increase the affinity of the antibody for antigen. Such
carbohydrate modifications can be accomplished by, for example,
altering one or more sites of glycosylation within the antibody
sequence. For example, one or more amino acid substitutions can be
made that result in elimination of one or more variable region
framework glycosylation sites to thereby eliminate glycosylation at
that site. Such aglycosylation may increase the affinity of the
antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and
6,350,861.
[0421] Additionally or alternatively, the antibody can have an
altered type of glycosylation, such as a hypofucosylated antibody
having reduced amounts of fucosyl residues or an antibody having
increased bisecting GlcNac structures. Such altered glycosylation
patterns have been demonstrated to increase the ADCC ability of
antibodies. Such carbohydrate modifications can be accomplished by,
for example, expressing the antibody in a host cell with altered
glycosylation machinery. Cells with altered glycosylation machinery
have been described in the art and can be used as host cells in
which to express recombinant antibodies described herein to thereby
produce an antibody with altered glycosylation. For example, the
cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase
gene, FUT8 (.alpha.(1,6)-fucosyltransferase), such that antibodies
expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on
their carbohydrates. The Ms704, Ms705, and Ms709 FUT8.sup.-/- cell
lines were created by the targeted disruption of the FUT8 gene in
CHO/DG44 cells using two replacement vectors (see U.S. Patent
Publication No. 20040110704 and Yamane-Ohnuki et al. (2004)
Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195
describes a cell line with a functionally disrupted FUT8 gene,
which encodes a fucosyl transferase, such that antibodies expressed
in such a cell line exhibit hypofucosylation by reducing or
eliminating the .alpha.-1,6 bond-related enzyme. EP 1,176,195 also
describes cell lines which have a low enzyme activity for adding
fucose to the N-acetylglucosamine that binds to the Fc region of
the antibody or does not have the enzyme activity, for example the
rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT Publication WO
03/035835 describes a variant CHO cell line, Lec13 cells, with
reduced ability to attach fucose to Asn(297)-linked carbohydrates,
also resulting in hypofucosylation of antibodies expressed in that
host cell (see also Shields et al. (2002) J. Biol. Chem.
277:26733-26740). Antibodies with a modified glycosylation profile
can also be produced in chicken eggs, as described in PCT
Publication WO 06/089231. Alternatively, antibodies with a modified
glycosylation profile can be produced in plant cells, such as
Lemna. Methods for production of antibodies in a plant system are
disclosed in the U.S. patent application corresponding to Alston
& Bird LLP attorney docket No. 040989/314911, filed on Aug. 11,
2006. PCT Publication WO 99/54342 describes cell lines engineered
to express glycoprotein-modifying glycosyl transferases (e.g.,
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed in the engineered cell lines exhibit increased
bisecting GlcNac structures which results in increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180). Alternatively, the fucose residues of the
antibody can be cleaved off using a fucosidase enzyme; e.g., the
fucosidase .alpha.-L-fucosidase removes fucosyl residues from
antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).
[0422] The variable segments of antibodies produced as described
supra (e.g., the heavy and light chain variable regions of chimeric
or humanized antibodies) are typically linked to at least a portion
of an immunoglobulin constant region (Fc region), typically that of
a human immunoglobulin. Human constant region DNA sequences can be
isolated in accordance with well-known procedures from a variety of
human cells, but preferably immortalized B cells (see Kabat et al.,
supra, and Liu et al., WO87/02671) (each of which is incorporated
by reference in its entirety for all purposes). Ordinarily, the
antibody will contain both light chain and heavy chain constant
regions. The heavy chain constant region usually includes CH1,
hinge, CH2, CH3, and CH4 regions. The antibodies described herein
include antibodies having all types of constant regions, including
IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2,
IgG3 and IgG4. When it is desired that the antibody (e.g.,
humanized antibody) exhibit cytotoxic activity, the constant domain
is usually a complement fixing constant domain and the class is
typically IgG1. Human isotype IgG1 is preferred. Light chain
constant regions can be lambda or kappa. The humanized antibody may
comprise sequences from more than one class or isotype. Antibodies
can be expressed as tetramers containing two light and two heavy
chains, as separate heavy chains, light chains, as Fab,
Fab'F(ab')2, and Fv, or as single chain antibodies in which heavy
and light chain variable domains are linked through a spacer.
[0423] In certain embodiments, the antibody comprises a variable
region that is mutated to improve the physical stability of the
antibody. In one embodiment, the antibody is an IgG4 isotype
antibody comprising a serine to proline mutation at a position
corresponding to position 228 (S228P; EU index) in the hinge region
of the heavy chain constant region. This mutation has been reported
to abolish the heterogeneity of inter-heavy chain disulfide bridges
in the hinge region (Angal et al. supra; position 241 is based on
the Kabat numbering system). For example, in certain embodiments,
an anti-Zika virus EP antibody described herein can comprise the
heavy chain variable region of any of the antibodies described
herein linked to a human IgG4 constant region in which the Serine
at a position corresponding to position 241 as described in Angal
et al., supra, has been mutated to Proline. Thus, for the heavy
chain variable regions linked to a human IgG4 constant region, this
mutation corresponds to an S228P mutation by the EU index.
[0424] In certain embodiments, the hinge region of CH1 is modified
such that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425. The number of cysteine residues
in the hinge region of CH1 is altered to, for example, facilitate
assembly of the light and heavy chains or to increase or decrease
the stability of the antibody.
[0425] In addition, the antibody can be pegylated, for example, to
increase the biological (e.g., serum) half-life of the antibody. To
pegylate an antibody, the antibody, or fragment thereof, typically
is reacted with polyethylene glycol (PEG), such as a reactive ester
or aldehyde derivative of PEG, under conditions in which one or
more PEG groups become attached to the antibody or antibody
fragment. Preferably, the pegylation is carried out via an
acylation reaction or an alkylation reaction with a reactive PEG
molecule (or an analogous reactive water-soluble polymer). As used
herein, the term "polyethylene glycol" is intended to encompass any
of the forms of PEG that have been used to derivatize other
proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene
glycol or polyethylene glycol-maleimide. In certain embodiments,
the antibody to be pegylated is an aglycosylated antibody. Methods
for pegylating proteins are known in the art and can be applied to
the antibodies described herein. See, e.g., EP 0 154 316 and EP 0
401 384.
Immunizations
[0426] To generate fully human antibodies to Zika virus EP,
transgenic or transchromosomal mice containing human immunoglobulin
genes (e.g., HCol2, HCo7 or KM mice) can be immunized with a
purified or enriched preparation of the Zika virus EP antigen
and/or cells expressing Zika virus EP, as described, for example,
by Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al.
(1996) Nature Biotechnology 14: 845-851 and WO 98/24884. As
described herein, HuMAb mice are immunized either with recombinant
Zika virus EP proteins or cell lines expressing Zika virus EP as
immunogens. Alternatively, mice can be immunized with DNA encoding
Zika virus EP. Preferably, the mice will be 6-16 weeks of age upon
the first infusion. For example, a purified or enriched preparation
(5-50 .mu.g) of the recombinant Zika virus EP antigen can be used
to immunize the HuMAb mice intraperitoneally.
[0427] Cumulative experience with various antigens has shown that
the HuMAb transgenic mice respond best when initially immunized
intraperitoneally (IP) or subcutaneously (SC) with antigen in
complete Freund's adjuvant, followed by every other week IP/SC
immunizations (up to a total of 10) with antigen in incomplete
Freund's adjuvant. The immune response can be monitored over the
course of the immunization protocol with plasma samples being
obtained by retroorbital bleeds. The plasma can be screened by
ELISA (as described below), and mice with sufficient titers of
anti-Zika virus EP human immunoglobulin can be used for fusions.
Mice can be boosted intravenously with antigen 3 days before
sacrifice and removal of the spleen.
Generation of Hybridomas Producing Monoclonal Antibodies to Zika
Virus EP
[0428] To generate hybridomas producing monoclonal antibodies to
Zika virus EP, splenocytes and lymph node cells from immunized mice
can be isolated and fused to an appropriate immortalized cell line,
such as a mouse myeloma cell line. The resulting hybridomas can
then be screened for the production of antigen-specific antibodies.
For example, single cell suspensions of splenic lymphocytes from
immunized mice can be fused to SP2/0-Ag8.653 nonsecreting mouse
myeloma cells (ATCC, CRL 1580) with 50% PEG (w/v). Cells can be
plated at approximately 1.times.10.sup.5 in flat bottom microtiter
plate, followed by a two week incubation in selective medium
containing besides usual reagents 10% fetal Clone Serum, 5-10%
origin hybridoma cloning factor (IGEN) and 1.times.HAT (Sigma).
After approximately two weeks, cells can be cultured in medium in
which the HAT is replaced with HT. Individual wells can then be
screened by ELISA for human anti-Zika virus EP monoclonal IgM and
IgG antibodies. Once extensive hybridoma growth occurs, medium can
be observed usually after 10-14 days. The antibody secreting
hybridomas can be replated, screened again, and if still positive
for IgG, anti-Zika virus EP monoclonal antibodies can be subcloned
at least twice by limiting dilution. The stable subclones can then
be cultured in vitro to generate antibody in tissue culture medium
for characterization.
Generation of Transfectomas Producing Monoclonal Antibodies to Zika
Virus EP
[0429] Antibodies described herein also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (Morrison, S. (1985) Science 229:1202).
[0430] For example, in certain embodiments, the gene(s) of
interest, e.g., human antibody genes, can be ligated into an
expression vector such as a eukaryotic expression plasmid such as
used by GS gene expression system disclosed in WO 87/04462, WO
89/01036 and EP 338 841 or other expression systems well known in
the art. The purified plasmid with the cloned antibody genes can be
introduced in eukaryotic host cells such as CHO-cells or NSO-cells
or alternatively other eukaryotic cells like a plant derived cells,
fungi or yeast cells. The method used to introduce these genes
could be methods described in the art such as electroporation,
lipofectine, lipofectamine or other. After introducing these
antibody genes in the host cells, cells expressing the antibody can
be identified and selected. These cells represent the transfectomas
which can then be amplified for their expression level and upscaled
to produce antibodies. Recombinant antibodies can be isolated and
purified from these culture supernatants and/or cells.
[0431] Alternatively these cloned antibody genes can be expressed
in other expression systems such as E. coli or in complete
organisms or can be synthetically expressed.
Expression of Recombinant Antibodies
[0432] Chimeric and humanized antibodies are typically produced by
recombinant expression. Nucleic acids encoding light and heavy
chain variable regions, optionally linked to constant regions, are
inserted into expression vectors. The light and heavy chains can be
cloned in the same or different expression vectors. The DNA
segments encoding immunoglobulin chains are operably linked to
control sequences in the expression vector(s) that ensure the
expression of immunoglobulin polypeptides. Expression control
sequences include, but are not limited to, promoters (e.g.,
naturally-associated or heterologous promoters), signal sequences,
enhancer elements, and transcription termination sequences.
Preferably, the expression control sequences are eukaryotic
promoter systems in vectors capable of transforming or transfecting
eukaryotic host cells. Once the vector has been incorporated into
the appropriate host, the host is maintained under conditions
suitable for high level expression of the nucleotide sequences, and
the collection and purification of the crossreacting
antibodies.
[0433] These expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers (e.g., ampicillin-resistance,
hygromycin-resistance, tetracycline resistance, kanamycin
resistance or neomycin resistance) to permit detection of those
cells transformed with the desired DNA sequences (see, e.g.,
Itakura et al., U.S. Pat. No. 4,704,362).
[0434] E. coli is one prokaryotic host particularly useful for
cloning the polynucleotides (e.g., DNA sequences) described herein.
Other microbial hosts suitable for use include bacilli, such as
Bacillus subtilis, and other enterobacteriaceae, such as
Salmonella, Serratia, and various Pseudomonas species. In these
prokaryotic hosts, one can also make expression vectors, which will
typically contain expression control sequences compatible with the
host cell (e.g., an origin of replication). In addition, any number
of a variety of well-known promoters will be present, such as the
lactose promoter system, a tryptophan (trp) promoter system, a
beta-lactamase promoter system, or a promoter system from phage
lambda. The promoters will typically control expression, optionally
with an operator sequence, and have ribosome binding site sequences
and the like, for initiating and completing transcription and
translation. Other microbes, such as yeast, are also useful for
expression.
[0435] Saccharomyces is a preferred yeast host, with suitable
vectors having expression control sequences (e.g., promoters), an
origin of replication, termination sequences and the like as
desired. Typical promoters include 3-phosphoglycerate kinase and
other glycolytic enzymes. Inducible yeast promoters include, among
others, promoters from alcohol dehydrogenase, isocytochrome C, and
enzymes responsible for maltose and galactose utilization.
[0436] In addition to microorganisms, mammalian tissue cell culture
may also be used to express and produce the polypeptides of
described herein (e.g., polynucleotides encoding immunoglobulins or
fragments thereof). See Winnacker, From Genes to Clones, VCH
Publishers, N.Y., N.Y. (1987). Eukaryotic cells are actually
preferred, because a number of suitable host cell lines capable of
secreting heterologous proteins (e.g., intact immunoglobulins) have
been developed in the art, and include CHO cell lines, various Cos
cell lines, HeLa cells, preferably, myeloma cell lines, or
transformed B-cells or hybridomas. Preferably, the cells are
nonhuman. Expression vectors for these cells can include expression
control sequences, such as an origin of replication, a promoter,
and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and
necessary processing information sites, such as ribosome binding
sites, RNA splice sites, polyadenylation sites, and transcriptional
terminator sequences. Preferred expression control sequences are
promoters derived from immunoglobulin genes, SV40, adenovirus,
bovine papilloma virus, cytomegalovirus and the like. See Co et
al., J. Immunol. 148:1149 (1992).
[0437] Alternatively, antibody-coding sequences can be incorporated
in transgenes for introduction into the genome of a transgenic
animal and subsequent expression in the milk of the transgenic
animal (see, e.g., Deboer et al., U.S. Pat. No. 5,741,957, Rosen,
U.S. Pat. No. 5,304,489, and Meade et al., U.S. Pat. No.
5,849,992). Suitable transgenes include coding sequences for light
and/or heavy chains in operable linkage with a promoter and
enhancer from a mammary gland specific gene, such as casein or beta
lactoglobulin.
[0438] The vectors containing the polynucleotide sequences of
interest (e.g., the heavy and light chain encoding sequences and
expression control sequences) can be transferred into the host cell
by well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transfection is commonly
utilized for prokaryotic cells, whereas calcium phosphate
treatment, electroporation, lipofection, biolistics or viral-based
transfection may be used for other cellular hosts. (See generally
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Press, 2nd ed., 1989) (incorporated by reference in
its entirety for all purposes). Other methods used to transform
mammalian cells include the use of polybrene, protoplast fusion,
liposomes, electroporation, and microinjection (see generally,
Sambrook et al., supra). For production of transgenic animals,
transgenes can be microinjected into fertilized oocytes, or can be
incorporated into the genome of embryonic stem cells, and the
nuclei of such cells transferred into enucleated oocytes.
[0439] When heavy and light chains are cloned on separate
expression vectors, the vectors are co-transfected to obtain
expression and assembly of intact immunoglobulins. Once expressed,
the whole antibodies, their dimers, individual light and heavy
chains, or other immunoglobulin forms of the present disclosure can
be purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, HPLC purification, gel electrophoresis and the like
(see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,
(1982)). Substantially pure immunoglobulins of at least about 90 to
95% homogeneity are preferred, and 98 to 99% or more homogeneity
most preferred, for pharmaceutical uses.
Antibody Fragments
[0440] Also contemplated within the scope of the instant disclosure
are antibody fragments. In one embodiment, fragments of non-human,
and/or chimeric antibodies are provided. In another embodiment,
fragments of humanized antibodies are provided. Typically, these
fragments exhibit specific binding to antigen with an affinity of
at least 10.sup.7, and more typically 10.sup.8 or 10.sup.9
M.sup.-1. Humanized antibody fragments include separate heavy
chains, light chains, Fab, Fab', F(ab')2, Fabc, and Fv. Fragments
are produced by recombinant DNA techniques, or by enzymatic or
chemical separation of intact immunoglobulins.
Assays for Characterization of Antibodies
[0441] Antibodies described herein can be tested for binding to
Zika virus envelope protein (EP) by, for example, standard ELISA.
Briefly, serial dilutions of Zika virus envelope protein, or Zika
virus itself, is mixed with antibodies described herein. These
mixtures are incubated overnight at room temperature to allow
equilibrium to be reached. An indirect ELISA is used to measure the
concentration of unbound and singly bound antibody. Alternatively,
microtiter plates are coated with purified Zika virus and then
blocked with 5% nonfat dry milk in PBS. After washing, purified
antibodies described herein are added to wells containing antigen
and incubated for 2 hours at room temperature. Bound antibodies are
detected using horseradish peroxidase conjugated anti-mouse IgG
secondary antibodies and ABTS substrate (Kirkegaard and Perry
Laboratories).
[0442] In some embodiments, the antibodies described herein bind
Zika virus EP with a Kd value between 3 and 25 .mu.g/mL as
determined by ELISA. In some embodiments, the antibodies described
herein bind Zika virus EP with a Kd value of at least 3 g/mL as
determined by ELISA. In some embodiments, the antibodies described
herein bind Zika virus EP with a Kd value of at least 5 g/mL as
determined by ELISA. In some embodiments, the antibodies described
herein bind Zika virus EP with a Kd value of at least g/mL as
determined by ELISA. In some embodiments, the antibodies described
herein bind Zika virus EP with a Kd value of at least 15 .mu.g/mL
as determined by ELISA. In some embodiments, the antibodies
described herein bind Zika virus EP with a Kd value of at least
g/mL as determined by ELISA. In some embodiments, the antibodies
described herein bind Zika virus EP with a Kd value of at least 25
.mu.g/mL as determined by ELISA.
[0443] An ELISA assay as described above can be used to screen for
antibodies and, thus, hybridomas that produce antibodies that show
positive reactivity with the Zika virus EP. Hybridomas that produce
antibodies that bind, preferably with high affinity, to Zika virus
EP can then be subcloned and further characterized. One clone from
each hybridoma, which retains the reactivity of the parent cells
(by ELISA), can then be chosen for making a cell bank, and for
antibody purification.
[0444] The ELISA assay described above can also be used to confirm
that framework mutation(s) do not affect the ability of the anti-EP
antibodies disclosed herein to bind to EP.
[0445] To purify anti-Zika virus EP antibodies, selected hybridomas
can be grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis
and high performance liquid chromatography to ensure purity. The
buffer solution can be exchanged into PBS, and the concentration
can be determined by OD.sub.280 using 1.43 extinction coefficient.
The monoclonal antibodies can be aliquoted and stored at
-80.degree. C.
[0446] To determine if the selected anti-Zika virus EP monoclonal
antibodies bind to unique epitopes, each antibody can be
biotinylated using commercially available reagents (Pierce,
Rockford, Ill.). Biotinylated MAb binding can be detected with a
streptavidin labeled probe. Competition studies using unlabeled
monoclonal antibodies and biotinylated monoclonal antibodies can be
performed using Zika virus EP coated-ELISA plates as described
above.
[0447] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for antibodies of a
particular isotype. For example, plates are coated with anti-IgG,
IgA, or IgM heavy-chain specific antibodies (100 ng/well) and
incubated with hybridoma culture supernatants. The subtype of the
mAb is detected by using anti-IgG1, IgG2a, IgG2b, IgG3, IgM, or IgA
heavy-chain specific antibodies conjugated to alkaline
phosphatase.
[0448] Anti-Zika virus EP antibodies can be further tested for
reactivity with the Zika virus EP antigen by Western blotting.
Briefly, unlabeled Zika virus EP is resolved on a 10%
SDS-polyacrylaminde gel and transferred to PVDF membranes. After
nonspecific bindings sites are blocked using nonfat dry milk in PBS
containing 0.02% Tween-20, purified mAb (10 ug/ml) are added to the
membranes for 1 hour at room temperature. Membranes are then
incubated with horseradish peroxidase-conjugated goat anti-mouse
IgA+IgG+IgM secondary antibodies for 1 hour and developed using ECL
chemiluminescence kit (Amersham).
[0449] Methods for analyzing binding affinity, cross-reactivity,
and binding kinetics of various anti-Zika virus EP antibodies
include standard assays known in the art, for example, Biacore.TM.
surface plasmon resonance (SPR) analysis using a Biacore.TM. 2000
SPR instrument (Biacore AB, Uppsala, Sweden).
[0450] To determine the neutralization of antibodies described
herein, in vitro plaque reduction neutralization (PRNT) assays can
be done. Briefly, plaque assays are done using confluent BHK-21
cells. Two-fold serial dilutions of antibodies described herein are
mixed with 50 PFU of Zika virus at 37.degree. C. for 1 hour. The
mixture is applied to cell monoloayers and incubated for 4-7 days.
Infection is quantified by 4G2 immunostaining followed by TMB
peroxidase substrate (KPL) and absorbance is measured in a plate
reader. 100% infection corresponds to the average OD600 in wells
not exposed to antibody. In some embodiments, the output of the
PRNT assay is PRNT50 (concentration of antibody that reduces plaque
formation by 50%). In some embodiments, the antibodies described
herein have a PRNT50 value of at least 0.03 .mu.g/mL.
[0451] In some embodiments, anti-Zika virus EP antibodies reduce
antibody-dependent enhancement (ADE) of infection. ADE is a
phenomenon that has been proposed to mediate increased disease
severity when infection occurs in a background of preexisting
enhancing antibodies. Mechanistically, this occurs when enhancing
antibodies bind the mature as well as immature virus particles and
mediate virus entry via antibody engagement of Fc.gamma. receptors
present on host cells. Several FLE-directed antibodies have been
described in literature including 4G2, E53, and the E-dimer epitope
(EDE) directed mAbs (Dejnirattisai W, et al., 2015). Several
studies have shown that when DENV is opsonized with antibody levels
that are phagocytosed by Fc.gamma. receptors, only antibodies that
are able to inhibit virus fusion with phagosomal membranes will
prevent infection and thus ADE (Chan K R et al., 2011, Wu R et al,
2012). Accordingly, in some embodiments, the ability of the
anti-Zika virus EP antibodies described herein to engage the Zika
virus FLE epitope at the E-dimer interface, results in reduction of
ADE activity by fusion inhibition.
[0452] In some embodiments, reduction of ADE is tested by using
cells that express leukocyte immunoglobulin like receptor B1
(LILRB1) and are thus highly susceptible to ADE. A non-limiting
example of such cells is THP1.2S monocytes. Cells are incubated
with an anti-Zika virus antibody described herein prior to
infection. Virus replication is then measured by plaque assay using
cells susceptible to viral infection. A non-limiting example of
such cells is BHK21 cells. Plaque titers are then measured, and a
reduction in plaque titers compared to a control indicates the
anti-Zika virus EP antibody tested is effective in reducing ADE.
Other methods of measuring ADE are known in the art and can be used
to determine the effect of an anti-Zika virus EP antibody described
herein on ADE.
Competitive Binding Antibodies
[0453] In certain embodiments, antibodies described herein compete
(e.g., cross-compete) for binding to Zika virus EP with the
particular anti-Zika virus EP antibodies described herein. Such
competing antibodies can be identified based on their ability to
competitively inhibit binding to Zika virus EP of one or more of
mAbs described herein in standard Zika virus EP binding assays. For
example, standard ELISA assays can be used in which a recombinant
Zika virus EP is immobilized on the plate, one of the antibodies is
fluorescently labeled and the ability of non-labeled antibodies to
compete off the binding of the labeled antibody is evaluated.
Additionally or alternatively, BIAcore analysis can be used to
assess the ability of the antibodies to cross-compete. The ability
of a test antibody to inhibit the binding of an anti-EP antibody
described herein to Zika virus EP demonstrates that the test
antibody can compete with the antibody for binding to Zika virus
EP.
[0454] In certain embodiments, the competing antibody is an
antibody that binds to the same epitope on Zika virus EP as the
particular anti-EP monoclonal antibodies described herein. Standard
epitope mapping techniques, such as x-ray crystallography and
2-dimensional nuclear magnetic resonance, can be used to determine
whether an antibody binds to the same epitope as a reference
antibody (see, e.g., Epitope Mapping Protocols in Methods in
Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
[0455] In certain embodiments, the antibody that competes for
binding to Zika virus EP and/or binds to the same epitope on Zika
virus EP is a humanized antibody.
[0456] In some embodiments, the anti-Zika virus antibodies
described herein bind an epitope on the fusion loop comprising
residues D98, R99 and W101 (SEQ ID NO: 3). In some embodiments, the
antibody that competes for binding to Zika virus EP binds the
epitope on the fusion loop comprising residues D98, R99 and W101
(SEQ ID NO: 3).
[0457] Once a single, archtypal anti-EP mAb has been isolated that
has the desired properties described herein, it is straightforward
to generate other mAbs with similar properties, e.g., having the
same epitope, by using art-known methods. For example, mice may be
immunized with Zika virus as described herein, hybridomas produced,
and the resulting mAbs screened for the ability to compete with the
archtypal mAb for binding to Zika virus EP. Mice can also be
immunized with a smaller fragment of Zika virus EP containing the
epitope to which the archtypal mAb binds. The epitope can be
localized by, e.g., screening for binding to a series of
overlapping peptides spanning Zika virus EP. Alternatively, the
method of Jespers et al., Biotechnology 12:899, 1994 may be used to
guide the selection of mAbs having the same epitope and therefore
similar properties to the archtypal mAb. Using phage display, first
the heavy chain of the archtypal antibody is paired with a
repertoire of (preferably human) light chains to select an Zika
virus EP-binding mAb, and then the new light chain is paired with a
repertoire of (preferably human) heavy chains to select an
(preferably human) Zika virus EP-binding mAb having the same
epitope as the archtypal mAb. Alternatively variants of the
archetypal mAb can be obtained by mutagenesis of cDNA encoding the
heavy and light chains of the antibody.
[0458] Epitope mapping, e.g., as described in Champe et al. (1995)
J. Biol. Chem. 270:1388-1394, can be performed to determine whether
the antibody binds an epitope of interest. "Alanine scanning
mutagenesis," as described by Cunningham and Wells (1989) Science
244: 1081-1085, or some other form of point mutagenesis of amino
acid residues in human Zika virus EP may also be used to determine
the functional epitope for an anti-EP antibody described herein.
Mutagenesis studies, however, may also reveal amino acid residues
that are crucial to the overall three-dimensional structure of Zika
virus EP but that are not directly involved in antibody-antigen
contacts, and thus other methods may be necessary to confirm a
functional epitope determined using this method.
[0459] The epitope bound by a specific antibody may also be
determined by assessing binding of the antibody to peptides
comprising fragments of Zika virus EP. A series of overlapping
peptides encompassing the sequence of Zika virus EP may be
synthesized and screened for binding, e.g. in a direct ELISA, a
competitive ELISA (where the peptide is assessed for its ability to
prevent binding of an antibody to Zika virus EP bound to a well of
a microtiter plate), or on a chip. Such peptide screening methods
may not be capable of detecting some discontinuous functional
epitopes, i.e. functional epitopes that involve amino acid residues
that are not contiguous along the primary sequence of the Zika
virus EP polypeptide chain.
[0460] The epitope bound by antibodies described herein may also be
determined by structural methods, such as X-ray crystal structure
determination (e.g., WO2005/044853), molecular modeling and nuclear
magnetic resonance (NMR) spectroscopy, including NMR determination
of the H-D exchange rates of labile amide hydrogens in Zika virus
EP when free and when bound in a complex with an antibody of
interest (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347;
Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).
[0461] With regard to X-ray crystallography, crystallization may be
accomplished using any of the known methods in the art (e.g. Giege
et al. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur.
J. Biochem. 189:1-23), including microbatch (e.g. Chayen (1997)
Structure 5:1269-1274), hanging-drop vapor diffusion (e.g.
McPherson (1976) J. Biol. Chem. 251:6300-6303), seeding and
dialysis. It is desirable to use a protein preparation having a
concentration of at least about 1 mg/mL and preferably about 10
mg/mL to about 20 mg/mL. Crystallization may be best achieved in a
precipitant solution containing polyethylene glycol 1000-20,000
(PEG; average molecular weight ranging from about 1000 to about
20,000 Da), preferably about 5000 to about 7000 Da, more preferably
about 6000 Da, with concentrations ranging from about 10% to about
30% (w/v). It may also be desirable to include a protein
stabilizing agent, e.g. glycerol at a concentration ranging from
about 0.5% to about 20%. A suitable salt, such as sodium chloride,
lithium chloride or sodium citrate may also be desirable in the
precipitant solution, preferably in a concentration ranging from
about 1 mM to about 1000 mM. The precipitant is preferably buffered
to a pH of from about 3.0 to about 5.0, preferably about 4.0.
Specific buffers useful in the precipitant solution may vary and
are well-known in the art (Scopes, Protein Purification: Principles
and Practice, Third ed., (1994) Springer-Verlag, New York).
Examples of useful buffers include, but are not limited to, HEPES,
Tris, MES and acetate. Crystals may be grow at a wide range of
temperatures, including 2.degree. C., 4.degree. C., 8.degree. C.
and 26.degree. C.
[0462] Antibody:antigen crystals may be studied using well-known
X-ray diffraction techniques and may be refined using computer
software such as X-PLOR (Yale University, 1992, distributed by
Molecular Simulations, Inc.; see e.g. Blundell & Johnson (1985)
Meth. Enzymol. 114 & 115, H. W. Wyckoff et al., eds., Academic
Press; U.S. Patent Application Publication No. 2004/0014194), and
BUSTER (Bricogne (1993) Acta Cryst. D49:37-60; Bricogne (1997)
Meth. Enzymol. 276A:361-423, Carter & Sweet, eds.; Roversi et
al. (2000) Acta Cryst. D56:1313-1323), the disclosures of which are
hereby incorporated by reference in their entireties.
[0463] Antibody competition assays, as described herein, can be
used to determine whether an antibody "binds to the same epitope"
as another antibody. Typically, competition of 50% or more, 60% or
more, 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%,
90%, 95% or more, of an antibody known to interact with the epitope
by a second antibody under conditions in which the second antibody
is in excess and the first saturates all sites, is indicative that
the antibodies "bind to the same epitope." To assess the level of
competition between two antibodies, for example, radioimmunoassays
or assays using other labels for the antibodies, can be used. For
example, a Zika virus EP antigen can be incubated with a saturating
amount of a first anti-EP antibody or antigen-binding fragment
thereof conjugated to a labeled compound (e.g., .sup.3H, .sup.125I,
biotin, or rubidium) in the presence the same amount of a second
unlabeled anti-EP antibody. The amount of labeled antibody that is
bound to the antigen in the presence of the unlabeled blocking
antibody is then assessed and compared to binding in the absence of
the unlabeled blocking antibody. Competition is determined by the
percentage change in binding signals in the presence of the
unlabeled blocking antibody compared to the absence of the blocking
antibody. Thus, if there is a 50% inhibition of binding of the
labeled antibody in the presence of the blocking antibody compared
to binding in the absence of the blocking antibody, then there is
competition between the two antibodies of 50%. Thus, reference to
competition between a first and second antibody of 50% or more, 60%
or more, 70% or more, such as 70%, 71%, 72%, 73%, 74%, 75%, 80%,
85%, 90%, 95% or more, means that the first antibody inhibits
binding of the second antibody (or vice versa) to the antigen by
50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more
(compared to binding of the antigen by the second antibody in the
absence of the first antibody). Thus, inhibition of binding of a
first antibody to an antigen by a second antibody of 50%, 60%, 70%,
71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95% or more indicates that
the two antibodies bind to the same epitope.
Testing Antibodies for Therapeutic Efficacy in Animal Models
[0464] Animal models are effective for testing the therapeutic
efficacy of antibodies against Zika virus EP. In certain
embodiments, rodents (i.e., mice) can be used for Zika infection.
Briefly, mice are challenged with a strain of mouse adapted Zika
virus (e.g., H/PF/2013 (French Polynesia 2013)) by intraperitoneal
inoculation, approximately 300 times the dose lethal for 50% of
adult mice. In certain embodiments, guinea pigs can be used for
Zika infection. Guinea pigs are challenged with guinea pig-adapted
virus. Additionally, in certain embodiments, non-human primates are
used as an animal model of infection. In all animal models,
antibodies can be administered 24 or 48 hours after infection to
test for therapeutic efficacy. In some embodiments, antibodies are
administered 24 or 48 hours before infection to test for
prophylactic efficacy.
[0465] Efficacy of the antibodies to prevent or treat Zika virus is
determined by analyzing the mortality rate, viral load, and/or body
weight of the animals over time. In some embodiments, untreated
animals infected with Zika virus die within 10-11 days post
infection. In some embodiments, animals treated with the anti-Zika
antibodies described herein before or after Zika virus infection,
live significantly longer compared to untreated animals. In some
embodiments, animals treated with the anti-Zika antibodies
described herein before or after Zika virus infection, live up to
22 days. In some embodiments, animals treated with the anti-Zika
antibodies described herein before or after Zika virus infection,
maintain body weight over time compared to untreated animals. In
some embodiments, animals treated with the anti-Zika antibodies
described herein before or after Zika virus infection, have a
reduced viral load compared to untreated animals.
[0466] In some embodiments, animal models (e.g., mice) are used to
determine the efficacy of an anti-Zika virus EP antibody in
treating or preventing vertical infection and fetal mortality in
pregnancy. In some embodiments, animal models are used to determine
the efficacy of an anti-Zika virus EP antibody in reducing or
reducing the risk of vertical infection and fetal mortality in
pregnancy. In some embodiments, pregnant animals are infected with
an adapted Zika virus (e.g., H/PF/2013 for mice) intravenously.
Anti-Zika virus EP antibodies described herein are administered
before or after infection to test for therapeutic or prophylactic
efficacy, respectively. In some embodiments, the efficacy of the
antibodies is determined by measuring the viral load in the mother,
fetus and placenta, along with determining the fetus survival rate.
In some embodiments, mothers treated with the anti-Zika antibodies
described herein have a reduced viral load. In some embodiments,
fetuses from mothers treated with the anti-Zika antibodies
described herein have a higher survival rate and reduced viral
load. In some embodiments, the higher survival rate of fetuses is
measured as reduction in percent lethality. In some embodiments,
the placenta from mothers treated with the anti-Zika antibodies
described herein has a reduced viral load. In some embodiments,
fetuses from mothers treated with the anti-Zika virus antibodies
described herein have normal embryo development. In some
embodiments, fetuses from mothers treated with the anti-Zika virus
antibodies described herein have normal embryo development,
compared to untreated mothers. In some embodiments, the fetuses
from untreated mothers are dead (i.e., 100% lethality).
Immunotoxins, Immunoconjugates and Antibody Derivatives
[0467] In another embodiment, the antibodies described herein are
linked to a therapeutic moiety, such as a cytotoxin, a drug or a
radioisotope. When conjugated to a cytotoxin, these antibody
conjugates are referred to as "immunotoxins." A cytotoxin or
cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells.
[0468] Techniques for conjugating such therapeutic moiety to
antibodies are well known in the art.
[0469] The toxin component of the immunotoxin can be, for example,
a chemotherapeutic agent, a toxin such as an enzymatically active
toxin of bacterial, fungal, plant or animal origin, or fragments
thereof, or a small molecule toxin.
[0470] Additional toxins and fragments thereof which can be used
include diphtheria A chain, nonbonding active fragments of
diphtheria toxin, cholera toxin, botulinus toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, phytolaca Americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, sapaonaria,
officinalis inhibitor, gelonin, saporin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothcenes. Small molecule toxins
include, for example, calicheamicins, maytansinoids, palytoxin and
CC1065.
[0471] Antibodies described herein also can be used for diagnostic
purposes, including sample testing and in vivo imaging, and for
this purpose the antibody (or binding fragment thereof) can be
conjugated to an appropriate detectable agent, to form an
immunoconjugate. For diagnostic purposes, appropriate agents are
detectable labels that include radioisotopes, for whole body
imaging, and radioisotopes, enzymes, fluorescent labels and other
suitable antibody tags for sample testing.
[0472] For Zika virus EP detection, the detectable labels can be
any of the various types used currently in the field of in vitro
diagnostics, including particulate labels including metal sols such
as colloidal gold, isotopes such as I.sup.125 or Tc.sup.99
presented for instance with a peptidic chelating agent of the
N.sub.2S.sub.2, N.sub.3S or N.sub.4 type, chromophores including
fluorescent markers, luminescent markers, phosphorescent markers
and the like, as well as enzyme labels that convert a given
substrate to a detectable marker, and polynucleotide tags that are
revealed following amplification such as by polymerase chain
reaction. Suitable enzyme labels include horseradish peroxidase,
alkaline phosphatase and the like. For instance, the label can be
the enzyme alkaline phosphatase, detected by measuring the presence
or formation of chemiluminescence following conversion of 1,2
dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyl
dioxetane (AMPPD), disodium
3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1
3,7}decan}-4-yl) phenyl phosphate (CSPD), as well as CDP and
CDP-Star.RTM. or other luminescent substrates well-known to those
in the art, for example the chelates of suitable lanthanides such
as Terbium(III) and Europium(III). The detection means is
determined by the chosen label. Appearance of the label or its
reaction products can be achieved using the naked eye, in the case
where the label is particulate and accumulates at appropriate
levels, or using instruments such as a spectrophotometer, a
luminometer, a fluorimeter, and the like, all in accordance with
standard practice.
[0473] In certain embodiments, an antibody provided herein may be
further modified to contain additional non-proteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
Compositions
[0474] In certain embodiments, a composition, e.g., a composition,
containing one or more monoclonal antibodies described herein,
formulated together with a carrier (e.g., a pharmaceutically
acceptable carrier), is provided. In some embodiments, the
compositions include a combination of multiple (e.g., two or more)
isolated antibodies described herein. Preferably, each of the
antibodies of the composition binds to a distinct, pre-selected
epitope of Zika virus EP.
[0475] Pharmaceutical compositions described herein also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include a
composition described herein with at least one or more additional
therapeutic agents. Co-administration with other antibodies is also
encompassed by the disclosure.
[0476] As used herein, the terms "carrier" and "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. Preferably, the carrier is suitable for intravenous,
intramuscular, subcutaneous, parenteral, spinal or epidermal
administration (e.g., by injection or infusion). Depending on the
route of administration, the active compound, e.g., antibody, may
be coated in a material to protect the compound from the action of
acids and other natural conditions that may inactivate the
compound.
[0477] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0478] A composition described herein can be administered by a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. The active compounds can be
prepared with carriers that will protect the compound against rapid
release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0479] To administer a compound described herein by certain routes
of administration, it may be necessary to coat the compound with,
or co-administer the compound with, a material to prevent its
inactivation. For example, the compound may be administered to a
subject in an appropriate carrier, for example, liposomes, or a
diluent. Acceptable diluents include saline and aqueous buffer
solutions. Liposomes include water-in-oil-in-water CGF emulsions as
well as conventional liposomes (Strejan et al. (1984) J.
Neuroimmunol. 7:27).
[0480] Carriers include sterile aqueous solutions or dispersions
and sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. The use of such media and
agents for pharmaceutically active substances is known in the art.
Except insofar as any conventional media or agent is incompatible
with the active compound, use thereof in the pharmaceutical
compositions described herein is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0481] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0482] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0483] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. For example, the antibodies described herein may be
administered once or twice weekly by subcutaneous or intramuscular
injection or once or twice monthly by subcutaneous or intramuscular
injection.
[0484] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms described herein are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0485] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0486] For the therapeutic compositions, formulations described
herein include those suitable for oral, nasal, topical (including
buccal and sublingual), rectal, vaginal and/or parenteral
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods known in the
art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the subject being treated, and the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the composition which
produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 0.001 percent to about
ninety percent of active ingredient, preferably from about 0.005
percent to about 70 percent, most preferably from about 0.01
percent to about 30 percent.
[0487] Formulations described herein which are suitable for vaginal
administration also include pessaries, tampons, creams, gels,
pastes, foams or spray formulations containing such carriers as are
known in the art to be appropriate. Dosage forms for the topical or
transdermal administration of compositions described herein include
powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants which
may be required.
[0488] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0489] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions described herein
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0490] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0491] When the compounds described herein are administered as
pharmaceuticals, to humans and animals, they can be given alone or
as a pharmaceutical composition containing, for example, 0.001 to
90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of active
ingredient in combination with a pharmaceutically acceptable
carrier.
[0492] Regardless of the route of administration selected, the
compounds described herein, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions described
herein, are formulated into pharmaceutically acceptable dosage
forms by conventional methods known to those of skill in the
art.
[0493] Actual dosage levels of the active ingredients in the
pharmaceutical compositions described herein may be varied so as to
obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. The selected dosage level will depend upon a variety of
pharmacokinetic factors including the activity of the particular
compositions described herein employed, or the ester, salt or amide
thereof, the route of administration, the time of administration,
the rate of excretion of the particular compound being employed,
the duration of the treatment, other drugs, compounds and/or
materials used in combination with the particular compositions
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts. A physician or veterinarian having
ordinary skill in the art can readily determine and prescribe the
effective amount of the pharmaceutical composition required. For
example, the physician or veterinarian could start doses of the
compounds described herein employed in the pharmaceutical
composition at levels lower than that required in order to achieve
the desired therapeutic effect and gradually increase the dosage
until the desired effect is achieved. In general, a suitable daily
dose of a composition described herein will be that amount of the
compound which is the lowest dose effective to produce a
therapeutic effect. Such an effective dose will generally depend
upon the factors described above. It is preferred that
administration be intravenous, intramuscular, intraperitoneal, or
subcutaneous, preferably administered proximal to the site of the
target. If desired, the effective daily dose of a therapeutic
composition may be administered as two, three, four, five, six or
more sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. While it is
possible for a compound described herein to be administered alone,
it is preferable to administer the compound as a pharmaceutical
formulation (composition).
[0494] Therapeutic compositions can be administered with medical
devices known in the art. For example, in certain embodiments, a
therapeutic composition described herein can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. No. 5,399,163, 5,383,851, 5,312,335,
5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of
well-known implants and modules useful in the present disclosure
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. Many other such implants, delivery systems, and
modules are known to those skilled in the art.
[0495] In certain embodiments, the antibodies described herein can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds described
herein cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134), different species of which may comprise the
formulations described herein, as well as components of the
invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In
one embodiment, the therapeutic compounds described herein are
formulated in liposomes; in certain embodiments, the liposomes
include a targeting moiety. In certain embodiments, the therapeutic
compounds in the liposomes are delivered by bolus injection to a
site proximal to the tumor or infection. The composition must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi.
[0496] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier can be an isotonic buffered saline solution, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures thereof.
Proper fluidity can be maintained, for example, by use of coating
such as lecithin, by maintenance of required particle size in the
case of dispersion and by use of surfactants. In many cases, it is
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition. Long-term absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0497] When the active compound is suitably protected, as described
above, the compound may be orally administered, for example, with
an inert diluent or an assimilable edible carrier.
Uses and Methods
[0498] In certain embodiments, the antibodies, bispecific
molecules, and compositions described herein can be used to treat
and/or prevent (e.g., immunize against) Zika virus infection. The
ability of the antibodies described herein to bind the fusion loop
within domain II of the envelope protein, suggests cross reactivity
within the flavivirus family. Therefore, in some embodiments, the
antibodies, bispecific molecules and compositions described herein
can be used to treat and/or prevent (e.g., immunize against) any
member of the flavivirus family.
[0499] For use in therapy, the antibodies described herein can be
administered to a subject directly (i.e., in vivo), either alone or
with other therapies such as an immunostimulatory agent. In all
cases, the antibodies, compositions, and immunostimulatory agents
and other therapies are administered in an effective amount to
exert their desired therapeutic effect. The term "effective amount"
refers to that amount necessary or sufficient to realize a desired
biologic effect. One of ordinary skill in the art can empirically
determine the effective amount of a particular molecule without
necessitating undue experimentation.
[0500] Preferred routes of administration for vaccines include, for
example, injection (e.g., subcutaneous, intravenous, parenteral,
intraperitoneal, intrathecal). The injection can be in a bolus or a
continuous infusion. Other routes of administration include oral
administration.
[0501] Antibodies described herein also can be coadministered with
adjuvants and other therapeutic agents. It will be appreciated that
the term "coadministered" as used herein includes any or all of
simultaneous, separate, or sequential administration of the
antibodies and conjugates described herein with adjuvants and other
agents, including administration as part of a dosing regimen. The
antibodies are typically formulated in a carrier alone or in
combination with such agents. Examples of such carriers include
solutions, solvents, dispersion media, delay agents, emulsions and
the like. The use of such media for pharmaceutically active
substances is well known in the art. Any other conventional carrier
suitable for use with the molecules falls within the scope of the
instant disclosure.
[0502] In certain embodiments, the antibodies described herein can
be utilized for prophylactic applications. In certain embodiments,
prophylactic applications involve systems and methods for
preventing, inhibiting progression of, and/or delaying the onset of
Zika virus infection, and/or any other Zika virus-associated
condition in individuals susceptible to and/or displaying symptoms
of Zika virus infection. In certain embodiments, prophylactic
applications involve systems and methods for preventing, inhibiting
progression of, and/or delaying the development of microcephaly in
newborn babies of mothers with Zika virus infection.
[0503] In some embodiments, the antibodies described herein are
utilized for treating or preventing vertical transmission of Zika
virus infection in a pregnant subject. In some embodiments, the
antibodies described herein are utilized for reducing or reducing
the risk of vertical transmission of Zika virus infection in a
pregnant subject. In some embodiments, the antibodies are
administered to a pregnant subject infected with Zika virus. In
some embodiments, the antibodies are administered to a pregnant
subject at risk of being infected with Zika virus. In some
embodiments, the antibodies described herein are utilized for
treating or preventing fetal mortality in a pregnant subject
infected with Zika virus or at risk of being infected with Zika
virus. In some embodiments, the antibodies described herein are
utilized for reducing or reducing the risk of fetal mortality in a
pregnant subject infected with Zika virus or at risk of being
infected with Zika virus. In some embodiments, the antibodies
described herein are utilized to reduce viral load in a pregnant
subject, the fetus, and/or the placenta, wherein the pregnant
subject is infected with Zika virus. Viral load can be measured as
described herein.
Peptides and Compositions Based on Zika Virus Envelope Protein (EP)
Epitopes
[0504] The antibodies described above are formulated into vaccine
compositions. These vaccine compositions may be employed to
immunize a subject in order to elicit a highly anti-Zika antibody
immune response. Vaccine compositions are also useful to administer
to subjects in need thereof to induce a protective immune response.
Such vaccine compositions are well known in the art and include,
for example, physiologically compatible buffers, preservatives, and
saline and the like, as well adjuvants.
[0505] "Adjuvants" are agents that nonspecifically increase an
immune response to a particular antigen, thus reducing the quantity
of antigen necessary in any given vaccine, and/or the frequency of
injection necessary in order to generate an adequate immune
response to the antigen of interest. Suitable adjuvants for the
vaccination of animals include, but are not limited to, Adjuvant 65
(containing peanut oil, mannide monooleate and aluminum
monostearate); Freund's complete or incomplete adjuvant; mineral
gels, such as aluminum hydroxide, aluminum phosphate and alum;
surfactants, such as hexadecylamine, octadecylamine, lysolecithin,
dimethyldioctadecylammonium bromide, N,N-dioctadecylN',
N'-bis(2-hydroxymethyl) propanediamine, methoxyhexadecylglycerol
and pluronic polyols; polyanions, such as pyran, dextran sulfate,
poly IC, polyacrylic acid and carbopol; peptides, such as muramyl
dipeptide, dimethylglycine and tuftsin; and oil emulsions. The
protein or peptides could also be administered following
incorporation into liposomes or other microcarriers. Information
concerning adjuvants and various aspects of immunoassays are
disclosed, e.g., in the series by P. Tijssen, Practice and Theory
of Enzyme Immunoassays, 3rd Edition, 1987, Elsevier, N.Y.,
incorporated by reference herein.
[0506] The vaccine composition includes a sufficient amount of the
desired immunogen, such as the peptides of the disclosure, to
elicit an immune response. The amount administered can range from
about 0.0001 g/kg to about 1.0 g/kg, relative to the mass of the
animal. Any suitable vertebrate animal is readily employed to
obtain polyclonal antiserum. Preferably, the animal is a mammal,
and includes, but is not limited to, rodents, such as a mice, rats,
rabbits, horses, canines, felines, bovines, ovines, e.g., goats and
sheep, primates, e.g., monkeys, great apes and humans, and the
like.
[0507] The vaccine composition is readily administered by any
standard route, including intravenously, intramuscularly,
subcutaneously, intraperitoneally, and/or orally. The artisan will
appreciate that the vaccine composition is preferably formulated
appropriately for each type of recipient animal and route of
administration.
[0508] Other aspects of the disclosure relate to methods of
treating or preventing of Zika virus infection by administering to
a subject in need thereof an effective amount of a vaccine
according to the disclosure.
[0509] The present disclosure is further illustrated by the
following examples which should not be construed as further
limiting. The contents of Sequence Listing, figures and all
references, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
EXAMPLES
Example 1
Generation of Anti-Zika Virus EP Antibodies
[0510] To generate anti-Zika virus envelope protein (EP)
antibodies, the FLEP-region located in the ectodomain II (E-DII) of
the envelope protein was investigated. To determine which scaffold
to employ, the RCSB Protein Data Bank (PDB) was utilized and over
500 antigen-antibody structural complexes were analyzed for
interface formation. An anti-TDRD3 (Tudor Domain Containing 3)
human antibody was identified as a scaffold with promising
potential to interact with the FLEP-region of Zika virus.
Therefore, this antibody was chosen for optimization to generate
antibodies that target the Zika virus EP. The heavy and light chain
variable regions of the anti-TDRD3 are shown in SEQ ID NOs: 4 and
5, respectively.
[0511] Antibodies targeting Zika virus EP were designed by
computing the epitope-paratope connectivity network, as described
in Robinson, L. et al., Cell, Vol. 162: 493-504 (2015). The
antibodies were designed to target residues D98, R99 and W101
within the fusion loop. Briefly, the crystal structure of the
anti-TDRD3 antibody in complex with the FLEP was used to determine
the various inter-residue inter-atomic contacts across the
antigen-antibody interface. The interactions between a CDR residue
and its neighboring epitope residues were rendered in a 2D network
graph to analyze the connectivity network. Mutations in the CDRs
and/or framework regions that contributed to more favorable
contacts, as evaluated by the structural analysis and connectivity
network, were identified and various amino acid residues which
potentially mediate new or improved contacts were analyzed. The
variable regions and CDRs generated are shown in Tables 2 and 3 as
well as in FIGS. 1A and 1B.
[0512] The antibodies were then expressed in Freestyle 293 cells by
transient transfection with polyethyleneimine (PEI) and purified by
protein A chromatography. The purified antibodies were quantified
by IgG ELISA. Briefly, 96-well plates were coated overnight at
4.degree. C. with appropriate antigen. The plates were washed and
blocked with 1% blott (Santa Cruz Biotechnologies). Serial dilution
of antibodies were added to the plate and incubated for 2 hours at
room temperature. Antigen bound IgG was detected using Rb.alpha.Hu
IgG HRP conjugated secondary antibody (Jackson ImmunoResearch)
followed by TMB substrate (KPL) addition.
Example 2
Binding Affinity of Zika Virus EP Antibodies
[0513] To determine whether the antibodies generated were capable
of binding to the Zika virus, a sandwich ELISA was used.
Specifically, mAbs 3, 6, 7, and 8, as shown in Table 2, were
tested. In addition, a fusion loop targeting pan-flavivirus
antibody (4G2) and HIV-1 neutralizing antibody (PGT124) were
utilized. The heavy and light variable region sequences for 4G2 are
set forth in SEQ ID NOs: 12 and 18, respectively. The heavy and
light variable region sequences for PGT124 are set forth in SEQ ID
NOs: 13 and 19, respectively. The PGT124 antibody targets N-glycan
which is present near the fusion loop epitope region of the Zika
virus.
[0514] Microtiter plates were coated with 0.05 .mu.g of purified
mouse 4G2 in carbonate buffer (pH 9.6) overnight at 4.degree. C.
and blocked with 10% BSA for 2 hours at room temperature.
Thereafter, 5.times.10.sup.4 pfu of Zika virus were added.
Specifically, the strains H/PF/2013 (French Polynesia 2013), ILM
(Brazil Paraiba 2015), or MR766-NIID (Uganda 1947) were utilized.
After 1 hour incubation at room temperature, serial two-fold
dilutions of antibodies were added for 1 hour, followed by goat
anti-human IgG Fc cross-adsorbed HRP-conjugated anti-human IgG for
45 minutes. Antibody binding was visualized by adding
3,3',5,5'-tetramethylbenzidene substrate and reaction was stopped
after 10 minutes with sulphuric acid. In between the different
steps, plates were washed twice with PBST (PBS+0.05% Tween).
Absorbance was read at 450 nm using a plate reader. The results are
shown in FIG. 2 and in Table 1 below.
TABLE-US-00001 TABLE 1 Kd .mu.g/mL Zika strain Zika strain Zika
strain Antibody H/PF/2013 ILM MR766 4G2 10.29 6.405 0.0422 PGT124
No/weak 388.2 No/weak binding binding mAb 3 7.976 0.8088 0.02479
mAb 6 6.542 3.847 0.02852 mAb 7 6.341 5.208 0.02651 mAb 8 22.99
19.32 0.02984
[0515] These results indicated the antibodies generated in Example
1 were capable of binding to various Zika virus strains.
Example 3
Neutralization of Zika Virus Envelope Protein Antibodies
[0516] To determine whether the anti-Zika virus antibodies could
neutralize Zika virus in vitro, purified mAbs were evaluated for
their ability to inhibit plaque formation by Zika virus. The plaque
neutralization test (PRNT) was performed as previously described
(Robinson, L. et al., Cell, Vol. 162: 493-504, 2015). Briefly, the
PRNT was performed on BHK-21 cells. Serial two-fold dilution of
sera containing mAbs 6 and 8 in RPMI maintenance media (MM) was
incubated with 50 pfu of Zika virus (ILM strain or H/PF/2013
strain) in equal volumes for 1 hour before adding to BHK-21. After
1 hour incubation at 37.degree. C., media was aspirated and cells
were overlaid with 1% methyl cellulose in MM. After 5 days at
37.degree. C., cells were fixed with 20% formaldehyde and stained
with 1% crystal violet. PRNT50 values were determined using a
sigmoid dose-response curve fit and reported as reciprocal
values.
[0517] As shown in FIGS. 3A and 3B, mAbs 6 and 8 were capable of
neutralizing Zika virus. The PRNT50 of mAb 8 was 5.122 .mu.g/mL
whereas the PRNT50 of mAb 6 was 0.0597 .mu.g/mL for the ILM strain.
The PRNT50 of mAb 8 was 5.092 .mu.g/mL for the H/PF/2013 strain.
These results indicated that antibodies generated against Zika
virus could bind and neutralize the virus.
Example 4
In Vivo Prophylactic and Therapeutic Study
[0518] To determine whether antibodies capable of binding and
neutralizing Zika virus in vitro had protective and therapeutic
efficacy in vivo, mAbs 6 and 8 were tested in adult A129 mice (8-11
weeks old). A129 mice were infected intraperitoneally (ip) with
H/PF/2013 strain (French Polynesia) at 10.sup.3 pfu. To assess
protective or prophylactic efficacy of mAbs, mice were injected ip
with mAbs (50 .mu.g) one day prior to infection. To assess
therapeutic efficacy, mice were injected ip with mAbs (50 .mu.g)
one day after infection. Efficacy of mAb was monitored by assessing
mortality, weight loss and viremia reduction. In short, mouse blood
was collected from facial vein on days 1-8 post-infection to
measure serum viremia level by real-time PCR. Weight was monitored
daily until the mice succumbed to infection. Mouse survival was
monitored until 20 days post-infection.
[0519] As shown in FIGS. 4C and 4D, virus infection caused 100%
mortality by day 10 post-infection. In addition, the mice had
symptoms of neuro-related disease (paralysis; data not shown).
Treatment with mAb 6 or 8 significantly reduced the mortality rate
and the loss in body weight in both prophylactic and treatment
models (FIGS. 4A-4D). In addition, FIG. 5 shows mAbs 6 and 8 were
able to reduce viremia in mice administered the antibody either
prophylactically or therapeutically. Specifically, viral load was
reduced by about 2-3 logs and delayed peak viremia.
[0520] The results indicated the anti-Zika virus antibodies
generated had both prophylactic and therapeutic effects.
Example 5
In Vivo Dosage and Antibody-Dependent Enhancement Study
[0521] The effect of dosing of anti-Zika virus antibodies on weight
loss, viremia and survival was evaluated. A129 mice were
administered varying doses of mAb 8 (10 mg/kg, 2 mg/kg or 0.2
mg/kg) a day after infection with Zika virus (strain H/PF/2013).
FIGS. 6A-6C show that at 2 mg/kg (.about.60 g), mAb 8 reduced
viremia and provided complete protection against weight loss and
survival. Remarkably, there were no observable differences in
viremia or accelerated death even when significantly lower doses
(0.2 mg/kg 6 g) of mAb 8 were administered. These results indicated
that mAb 8 has the potential to provide partial protection, even at
significantly low concentrations without increasing viremia
associated with disease progression.
[0522] In addition, antibody-dependent enhancement (ADE) activity
of anti-Zika virus mAb 8 was analyzed. ADE is a phenomenon that has
been proposed to mediate increased disease severity when infection
occurs in a background of preexisting enhancing antibodies.
Mechanistically, this occurs when enhancing antibodies bind the
mature as well as immature virus particles and mediate virus entry
via antibody engagement of Fc.gamma. receptors present on host
cells. Several FLE-directed antibodies have been described in
literature including 4G2, E53, and the E-dimer epitope (EDE)
directed mAbs (Dejnirattisai W, et al., 2015). Several studies have
shown that when DENV is opsonized with antibody levels that are
phagocytosed by Fc.gamma. receptors, only antibodies that are able
to inhibit virus fusion with phagosomal membranes will prevent
infection and thus ADE (Chan K R et al., 2011, Wu R et al, 2012).
Accordingly, the ability of mAb8's engagement of the Zika virus FLE
epitope at the E-dimer interface to reduce its ADE activity by
fusion inhibition was tested.
[0523] Specifically, THP1.2S monocyte cells were utilized, which
are known to express LILRB1 and are thus highly susceptible to ADE.
An early design variant of mAb 8 which showed very weak binding and
neutralization of ZIKV H/PF/2013 strain (Kd=50.85 ug/ml;
PRNT50>500 ug/ml), was used as a control ("mAb control").
Different concentrations of antibody were incubated with ZIKV
(strain H/PF/2013) for 1 hour prior to infecting THP1.2S monocytes.
Seventy hours later the virus replication in culture supernatants
was measured by plaque assay on BHK21 cells. As shown in FIG. 7A,
all mAbs tested showed ADE in THP1.2S cells but mAbl3 exhibited
high ADE activity without any ZIKV neutralization activity despite
high antibody concentrations. In contrast, mAb8 showed ADE of at
least 3 fold lower viral titers at peak enhancement comparable with
4G2, which is another fusion loop antibody but which has no
neutralization activity against ZIKV (FIG. 7B). These results
indicated the anti-Zika virus EP antibodies generated in Example 1
were capable of reducing ADE.
Example 6
Efficacy on Maternal Transfer
[0524] Placental and fetal infection, along with fetal mortality,
has been observed in pregnant A129 mice infected with ZIKV.
Accordingly, the potential of mAb 8 to prevent vertical infection
and fetal mortality in pregnant A129 mice was evaluated. The
overall study design is provided in FIG. 8A. Specifically, 18
pregnant A129 mice that were mated to male mice for 4 days
(starting on day 0 evening and separated on day 4 morning), were
infected with 10.sup.3 PFU of Zika virus (H/PF/2013 strain of Asian
lineage) intravenously on day 10 (corresponding to embryo day 7-10,
i.e., E7-E10). The mice were then treated with 50 g of mAb 8 (n=9)
or an isotype control IgG (n=9) for 24 hours (E8-E11) after
infection. Mice were sacrificed on day 17 (E14-E17) and viral RNA
levels were analyzed on day 12 (blood) and day 17 (blood, placenta
and fetal compartments).
[0525] The results of the study are provided in FIGS. 8B-8H. When
compared with isotype control IgG treated mice, the mAb 8 treated
mice has substantially lower levels of viral RNA in blood on day 12
(p-value <0.0001; two-sided t-test) and day 17 (p-value=0.018;
two-sided t-test) (FIGS. 8B and 8C). Significantly, mAb 8 treatment
reduced placental (p-value <0.0001; two sided t-test) and fetal
(p-value <0.0001; two-sided t-test) infection and provided
protection against fetal mortality. In contrast, fetuses harvested
from the control group had 100% lethality, with significant viremia
in fetal and placental compartments (FIGS. 8D-8F). Fetuses
harvested from mice treated with mAb 8 showed signs of normal
embryo development without any developmental impairment. In
contrast, fetuses from isotype control IgG treated mice were dead
before harvesting, revealing a stark morphological difference
compared with normal fetuses (data not shown). These results
indicated the anti-Zika virus antibodies described herein are
capable of preventing vertical infection and fetal mortality in
pregnant mice.
Example 7
In Vivo Non-Human Primate Study
[0526] Cynomolgus macaques are used to test whether and how
effectively an anti-EP mAb can improve survival when administered
after high-dose ZIKV infection.
[0527] Zika virus (ZIKV) is produced on Vero cells in complete
minimal essential medium (cMEM), 2% FBS, and 1%
penicillin/stretopmycin.
[0528] Macaques are randomized into groups on the basis of
treatment regimens, plus one receiving only PBS as a positive
control for infection. Each subject is infected with 1000 PFU (1 mL
each into two sites intramuscularly) of ZIKV in Dulbecco's modified
Eagle's medium (DMEM). Half of the groups begin treatment 24 hours
post infection and the other half of the groups begin treatment 48
hours post infection. The subjects are treated intravenously with a
mAb (25 mg/kg), one of the ZIKV-EP-specific neutralizing antibodies
disclosed herein, as a 5 mL slow bolus in the saphenous vein. The
subjects are monitored daily and scored for disease progression
with an internal scoring protocol. Scoring rates changes in the
subject's posture/activity, attitude, activity level, feces/urine
output, food/water intake, weight, temperature, respiration, and
scored disease manifestations such as a visible rash, hemorrhage,
cyanosis, or flushed skin. Tests for weight, temperature, blood,
and oropharyngeal, nasal, and rectal swabs are taken at days 1, 4,
7, 14, 21, and 28 post infections for the 24-hour group or at 2, 5,
8, 14, 21, and 28 days post infection for the 48-hour group, before
the animals receive the mAb.
Example 8
Human Study
[0529] Humans infected with Zika virus are given a single anti-EP
antibody disclosed herein. Ideally, the antibodies will be given 24
or 48 hours post infection. Subjects will be monitored for disease
manifestations such as visible rash, fevers, or joint pain. In
addition, newborns from pregnant women receiving an anti-EP
antibody disclosed herein, will be monitored for microcephaly.
Viral titers will also be monitored.
EQUIVALENTS
[0530] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the disclosure described
herein. Such equivalents are intended to be encompassed by the
following claims.
TABLE-US-00002 TABLE 2 Antibody Pairs by SEQ ID Number V.sub.H CDR
V.sub.L CDR Antibody V.sub.H V.sub.L CDR1 CDR2 CDR3 CDR1 CDR2 CDR3
mAb 1 4 14 20 26 31 38 44 50 mAb 2 4 15 20 26 31 38 44 50 mAb 3 9
16 22 28 33 39 45 51 mAb 4 4 16 20 26 31 39 45 51 mAb 5 4 17 20 26
31 40 46 50 mAb 6 8 14 21 28 32 38 44 50 mAb 7 7 17 21 27 32 40 46
50 mAb 8 6 15 21 27 32 38 44 50 mAb 9 6 5 21 27 32 37 43 49 mAb 10
7 5 21 27 32 37 43 49 mAb 11 8 5 21 28 32 37 43 49 mAb 12 9 5 22 28
33 37 43 49 mAb 13 10 5 23 28 34 37 43 49 mAb 14 11 5 21 28 34 37
43 49 mAb 15 6 14 21 27 32 38 44 50 mAb 16 6 16 21 27 32 39 45 51
mAb 17 6 17 21 27 32 40 46 50 mAb 18 7 14 21 27 32 38 44 50 mAb 19
7 15 21 27 32 38 44 50 mAb 20 7 16 21 27 32 39 45 51 mAb 21 8 15 21
28 32 38 44 50 mAb 22 8 16 21 28 32 39 45 51 mAb 23 8 17 21 28 32
40 46 50 mAb 24 9 14 22 28 33 38 44 50 mAb 25 9 15 22 28 33 38 44
50 mAb 26 9 17 22 28 33 40 46 50 mAb 27 10 14 23 28 34 38 44 50 mAb
28 10 15 23 28 34 38 44 50 mAb 29 10 16 23 28 34 39 45 51 mAb 30 10
17 23 28 34 40 46 50 mAb 31 11 14 21 28 34 38 44 50 mAb 32 11 15 21
28 34 38 44 50 mAb 33 11 16 21 28 34 39 45 51 mAb 34 11 17 21 28 34
40 46 50
TABLE-US-00003 TABLE 3 Summary Sequence Table SEQ ID NO Description
Sequence 1 Human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
Heavy Chain GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 Human IgG2
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV Light Chain
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA (kappa)
CEVTHQGLSSPVTKSFNRGEC 3 Zika virus DRGWGNGCGLFG fusion protein loop
(residues 98-109 of E- DII) 4 anti-TDRD3
EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSYMHWVRQAPG V.sub.H Wild Type
KGLEWVASISSSYGSTYYADSVKGRFTISADTSKNTAYLQMNSL
RAEDTAVYYCARTVRGSKKPYFSGWAMDYWGQGTLVTVSS 5 anti-TDRD3
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGK V.sub.L Wild Type
APKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC
QQHGPFYW-LFTFGQGTKVEIK 6 V.sub.H .1
EVQLLESGGGLVQPGGSLRLSCAASGFSFSTYSMHWVRQAPG
KGLEWVSAISGEGDSAYYADSVKGRFTISRDNSKNTLYLQMN
KVRAEDTAVYYCV----GGYSNFYYYYTMDAWGQGTMVTVSS (V5L, N285, L29F, S31T,
532Y, Y335, A495, S50A, S52(A)G, 553E, Y54G, G55D, T57A, A71R,
T73N, A78L, S82(B)K, L82(C)V, A93V, S99G, K100Y, K100(A)S,
P100(B)N, Y100(C)F, F100(D)Y, S100(E)Y, G100(F)Y, W100(G)Y,
A100(H)T, Y102(A), L108M) 7 V.sub.H .2
EVQLLESGGGLVQPGGSLRLSCAASGFSFSTYSMHWVRQAPG
KGLEWVSAISGEGDSAYYADSVKGRFEISRDNSKNTLYLQMN
KVRAEDTAVYYCV----GGYSNFYYYYTMDAWGQGTMVTVSS ##STR00001## 8 V.sub.H
.3 EVQLVESGGGLVQPGGSLRLSCSASGFSFSTYSMHWVRQAPG
KGLEYVSAITGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNS
LRPEDTAVYYCV----GGYSNFYYYYTMDAWGQGTSVTVSS ##STR00002## 9 V.sub.H .4
EVQLVESUGGLVQPGGSLRLSCSASGFTFSTYSMHWVRQAPG
KGLEYVSAITGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNS
LRPEDTAVYYCV----GGYSNFYYYYTMDVWGQGTTVTVSS ##STR00003## 10 V.sub.H
.5 QVQLVESGGGLVQPGGSLRLSCSASGFFSTYSMHWVKQAPGK
GLEYVSAITGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNSL
RPEDTAVYYCV----GGYTNFYYYYTMDAWGQGTSVTVSS ##STR00004## 11 V.sub.H .6
QVQLVESGGGLVQPGGSLRLSCSASGFSFSTYSMHWVKQAPG
KGLEYVSAITGEGDSAFYADSVKGRFTISRDNSKNTLYFEMNS
LRPEDTAVYYCV----GGYTNFYYYYTMDAWGQGTSVTVSS (EQ1, A23S, N28S, L29F,
S31T, S32Y, Y33S, R38K, W47Y, A49S, S50A, S52T, S52(A)G, S53E,
Y54G, G55D, T57A, Y58F, A71R, T73N, A78L, L80F, Q81E, A84P, A93V,
S99G, K100Y, K100(A)T, P100(B)N, Y100(C)F, F100(D)Y, S100(E)Y,
G100(F)Y, W100(G)Y, A100(H)T, Y102A, L108S) 12 V.sub.H of fusion
EVQLQQSGPELVKPGTSVKISCKTSGYTFTEYTIHWVKQSHGK loop targeting
SLAWIGGIDPNSGGTNYSPNFKGKATLTVDKSSSTAYMDLRSL pan-flavivirus
SSEDSAVYFCARIYHYDGYFDVWGAGTAVTVSS antibody 4g2 13 V.sub.H of anti-
QVQLQESGPGLVRPSETLSVTCIVSGGSISNYYWTWIRQSPGKG HIV
LEWIGYISDRETTTYNPSLNSRAVISRDTSKNQLSLQLRSVTTA neutralizing
DTAIYFCATARRGQRIYGVVSFGEFFYYYYMDVWG antibody KGTAVTVSS PGT124 14
V.sub.L .1 EIVLTQSPASLSLSPGERATLSCRATOSISTFLAWYQHKPGQA
PRLLIYDASTRASGVPARFSGSRSGTDFTLTISSLEPEDFAVYYC QQR-YNWPPYSFGQGTKVEIK
(D1E, Q3V, M4L, S9A, A13L, V15P, D17E, V19A, I21L, Y22S, S26T,
V29I, S31T, A32F, V33L, Q38H, K42Q, K45R, S50D, S53T, L54R, Y55A,
S60A, Q79E, T85V, H91R, F94Y, Y95N, L95(b)P, F96Y, T97S) 15 V.sub.L
.2 DIVMTQSPASLSLSPGERATLSCRATOSISTFLAWYQQKPGQ
APRLLIYDASTRASGIPARFSGSRSGTDFTLTITRLEPEDFAVYY
CQQR-YNWPPYSFGQGTKLEIK ##STR00005## 16 V.sub.L .3
EIVLTQSPATLSLSPGERATLSCRASOSISTFLAWYQHKPGQAP
RLLIYDASTRATGVPARFSGSRSGTDFTLTISTLEPEDFAVYYC QQR-YNWPPYTFGQGTKVEIK
##STR00006## 17 V.sub.L .4
DIVMTQSPASLSLSPGERATLSCRATQSIVTFLAWYQQKPGQ
APRLLIYDASTNASGIPARFSGSRSGTDFTLTITRLEPEDFAVYY
CQQR-YNWPPYSFGQGTKLEIK ##STR00007## 18 V.sub.L of fusion
DIKMTQSPSSMYASLGERVTITCKASQDINSYLTWFQQKPGKS loop targeting
PKTLIYRANRLIDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYY pan-flavivirus
CLQYDEFPPTFGGGTKLEIKR antibody 4g2 19 V.sub.L of anti-
SYVSPLSVALGETARISCGRQALGSRAVQWYQHKPGQAPILLI HIV
YNNQDRPSGIPERFSGTPUNFGTTATLTISGVEVGDEADYYCH neutralizing
MWDSRSGFSWSFGGATRLTVLSQP antibody PGT124 20 anti-TDRD3 GFNLSSS
V.sub.HCDR1 21 V.sub.H.1 CDR1 GFSFSTY 22 V.sub.H.4 CDR1 GFTFSTY 23
V.sub.H.5CDR1 GF-FSTY 24 4g2 V.sub.H CDR1 GYTFTEY 25 PGT124 V.sub.H
GGSISNY CDR1 26 anti-TDRD3 SSSYGS V.sub.H CDR2 27 V.sub.H.1 CDR2
SGEGDS 28 V.sub.H.3 CDR2 TGEGDS 29 4g2 V.sub.H CDR2 DPNSGG 30
PGT124 V.sub.H SDRET CDR2 31 anti-TDRD3 TVRGSKKPYFSGWAMDY V.sub.H
CDR3 32 V.sub.H.1 CDR3 ----GYSNFYYYYTMDA 33 V.sub.H.4 CDR3
----GYSNFYYYYTMDV 34 V.sub.H.5 CDR3 ----GYTNFYYYYTMDA 35 4g2
V.sub.H CDR3 IYHYDGYFDV 36 PGT124 V.sub.H ARRGQRIYGVVSFGEFFYYYYMDV
CDR3 37 anti-TDRD3 RASQSVSSAVA V.sub.L CDR1 38 V.sub.L.1 CDR1
RATQSISTFLA 39 V.sub.L.3 CDR1 RASQSISTFLA 40 V.sub.L.4 CDR1
RATQSIVTFLA 41 4g2 V.sub.L CDR1 KASQDINSYLT 42 PGT124 V.sub.L
GRQALGSRAVQ CDR1 43 anti-TDRD3 SASSLYS V.sub.L CDR2 44 V.sub.L.1
CDR2 DASTRAS 45 V.sub.L.3 CDR2 DASTRAT 46 V.sub.L.4 CDR2 DASTNAS 47
4g2 V.sub.L CDR2 RANRLID 48 PGT124 V.sub.L NNQDRPS CDR2 49
anti-TDRD3 QQHGPFYWLFT V.sub.L CDR3 50 V.sub.L.1 CDR3 QQR--YNWPPYS
51 V.sub.L.3 CDR3 QQR--YNWPPYT 52 4g2 V.sub.L CDR3 LQYDEFPPT 53
PGT124 V.sub.L HMWDSRSGFSWS CDR3 54 V.sub.H CDR1 GFX.sub.1FSTY 55
V.sub.H CDR2 X.sub.2GEGDS 56 V.sub.H CDR3 GYX.sub.3NFYYYYTMDX.sub.4
57 V.sub.L CDR1 RAX.sub.5QSIX.sub.6TFLA 58 V.sub.L CDR2
DASTX.sub.7AX.sub.8 59 V.sub.L CDR3 QQRYNWPPYX.sub.9
Sequence CWU 1
1
591330PRTHomo sapiensmisc_featureHuman IgG1 Heavy Chain 1Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
3302107PRTHomo sapiensmisc_featureHuman IgG2 Light Chain (kappa)
2Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5
10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 105312PRTArtificial SequenceSynthetic Zika virus fusion
protein loop (residues 98-109 of E-DII) 3Asp Arg Gly Trp Gly Asn
Gly Cys Gly Leu Phe Gly1 5 104126PRTArtificial SequenceSynthetic
anti-TDRD3 VH Wild Type 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asn Leu Ser Ser Ser 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Ser Ile Ser Ser Ser Tyr
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr
Val Arg Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala 100 105 110Met
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
1255109PRTArtificial SequenceSynthetic anti-TDRD3 VL Wild Type 5Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Gly Pro Phe Tyr Trp 85 90 95Leu Phe Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 1056122PRTArtificial SequenceSynthetic VH .1 6Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Thr Tyr
20 25 30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Glu Gly Asp Ser Ala Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Lys Val Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Val Gly Gly Tyr Ser Asn Phe Tyr Tyr Tyr
Tyr Thr Met Asp Ala Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val
Ser Ser 115 1207122PRTArtificial SequenceSynthetic VH .2 7Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Thr Tyr 20 25
30Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Glu Gly Asp Ser Ala Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Glu Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Lys Val Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Val Gly Gly Tyr Ser Asn Phe Tyr Tyr Tyr Tyr
Thr Met Asp Ala Trp 100 105 110Gly Gln Gly Thr Met Val Thr Val Ser
Ser 115 1208122PRTArtificial SequenceSynthetic VH .3 8Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ser Ala Ser Gly Phe Ser Phe Ser Thr Tyr 20 25 30Ser
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40
45Ser Ala Ile Thr Gly Glu Gly Asp Ser Ala Phe Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80Phe Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Val Gly Gly Tyr Ser Asn Phe Tyr Tyr Tyr Tyr Thr
Met Asp Ala Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 1209122PRTArtificial SequenceSynthetic VH .4 9Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ser Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser
Ala Ile Thr Gly Glu Gly Asp Ser Ala Phe Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Phe Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Val Gly Gly Tyr Ser Asn Phe Tyr Tyr Tyr Tyr Thr Met Asp
Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
12010121PRTArtificial SequenceSynthetic VH .5 10Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ser Ala Ser Gly Phe Phe Ser Thr Tyr Ser 20 25 30Met His Trp
Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ser 35 40 45Ala Ile
Thr Gly Glu Gly Asp Ser Ala Phe Tyr Ala Asp Ser Val Lys 50 55 60Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Phe65 70 75
80Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95Gly Gly Tyr Thr Asn Phe Tyr Tyr Tyr Tyr Thr Met Asp Ala Trp
Gly 100 105 110Gln Gly Thr Ser Val Thr Val Ser Ser 115
12011122PRTArtificial SequenceSynthetic VH .6 11Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ser Ala Ser Gly Phe Ser Phe Ser Thr Tyr 20 25 30Ser Met His
Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45Ser Ala
Ile Thr Gly Glu Gly Asp Ser Ala Phe Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Phe Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Val Gly Gly Tyr Thr Asn Phe Tyr Tyr Tyr Tyr Thr Met Asp Ala
Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115
12012119PRTArtificial SequenceSynthetic VH of fusion loop targeting
pan-flavivirus antibody 4g2 12Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser Val Lys Ile Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30Thr Ile His Trp Val Lys Gln
Ser His Gly Lys Ser Leu Ala Trp Ile 35 40 45Gly Gly Ile Asp Pro Asn
Ser Gly Gly Thr Asn Tyr Ser Pro Asn Phe 50 55 60Lys Gly Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Asp Leu
Arg Ser Leu Ser Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg
Ile Tyr His Tyr Asp Gly Tyr Phe Asp Val Trp Gly Ala Gly 100 105
110Thr Ala Val Thr Val Ser Ser 11513132PRTArtificial
SequenceSynthetic VH of anti-HIV neutralizing antibody PGT124 13Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Glu1 5 10
15Thr Leu Ser Val Thr Cys Ile Val Ser Gly Gly Ser Ile Ser Asn Tyr
20 25 30Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45Gly Tyr Ile Ser Asp Arg Glu Thr Thr Thr Tyr Asn Pro Ser
Leu Asn 50 55 60Ser Arg Ala Val Ile Ser Arg Asp Thr Ser Lys Asn Gln
Leu Ser Leu65 70 75 80Gln Leu Arg Ser Val Thr Thr Ala Asp Thr Ala
Ile Tyr Phe Cys Ala 85 90 95Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly
Val Val Ser Phe Gly Glu 100 105 110Phe Phe Tyr Tyr Tyr Tyr Met Asp
Val Trp Gly Lys Gly Thr Ala Val 115 120 125Thr Val Ser Ser
13014108PRTArtificial SequenceSynthetic VL .1 14Glu Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Thr Gln Ser Ile Ser Thr Phe 20 25 30Leu Ala Trp
Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Thr Arg Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Tyr Asn Trp Pro Pro
85 90 95Tyr Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10515108PRTArtificial SequenceSynthetic VL .2 15Asp Ile Val Met Thr
Gln Ser Pro Ala Ser Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Thr Gln Ser Ile Ser Thr Phe 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Thr Arg Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Tyr Asn Trp Pro Pro
85 90 95Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10516108PRTArtificial SequenceSynthetic VL .3 16Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Thr Phe 20 25 30Leu Ala Trp
Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Thr Arg Ala Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Tyr Asn Trp Pro Pro
85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10517108PRTArtificial SequenceSynthetic VL .4 17Asp Ile Val Met Thr
Gln Ser Pro Ala Ser Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Thr Gln Ser Ile Val Thr Phe 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Thr Asn Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Tyr Asn Trp Pro Pro
85 90 95Tyr Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
10518108PRTArtificial SequenceSynthetic VL of fusion loop targeting
pan- flavivirus antibody 4g2 18Asp Ile Lys Met Thr Gln Ser Pro Ser
Ser Met Tyr Ala Ser Leu Gly1 5 10 15Glu Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30Leu Thr Trp Phe Gln Gln Lys
Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu
Ile Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Gln
Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr65 70 75 80Glu Asp Met
Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Pro 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 10519111PRTArtificial
SequenceSynthetic VL of anti-HIV neutralizing antibody PGT124 19Ser
Tyr Val Ser Pro Leu Ser Val Ala Leu Gly Glu Thr Ala Arg Ile1 5 10
15Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln Trp Tyr Gln
20 25 30His Lys Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn Asn Gln
Asp 35 40 45Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro Asp
Ile Asn 50 55 60Phe Gly Thr Thr Ala Thr Leu Thr Ile Ser Gly Val Glu
Val Gly Asp65 70 75 80Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser
Arg Ser Gly Phe Ser 85 90 95Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr
Val Leu Ser Gln Pro 100 105 110207PRTArtificial
SequenceSynthetic
anti-TDRD3 VH CDR1 20Gly Phe Asn Leu Ser Ser Ser1 5217PRTArtificial
SequenceSynthetic VH.1 CDR1 21Gly Phe Ser Phe Ser Thr Tyr1
5227PRTArtificial SequenceSynthetic VH.4 CDR1 22Gly Phe Thr Phe Ser
Thr Tyr1 5236PRTArtificial SequenceSynthetic VH.5CDR1 23Gly Phe Phe
Ser Thr Tyr1 5247PRTArtificial SequenceSynthetic 4g2 VH CDR1 24Gly
Tyr Thr Phe Thr Glu Tyr1 5257PRTArtificial SequenceSynthetic PGT124
VH CDR1 25Gly Gly Ser Ile Ser Asn Tyr1 5266PRTArtificial
SequenceSynthetic anti-TDRD3 VH CDR2 26Ser Ser Ser Tyr Gly Ser1
5276PRTArtificial SequenceSynthetic VH.1 CDR2 27Ser Gly Glu Gly Asp
Ser1 5286PRTArtificial SequenceSynthetic VH.3 CDR2 28Thr Gly Glu
Gly Asp Ser1 5296PRTArtificial SequenceSynthetic 4g2 VH CDR2 29Asp
Pro Asn Ser Gly Gly1 5305PRTArtificial SequenceSynthetic PGT124 VH
CDR2 30Ser Asp Arg Glu Thr1 53117PRTArtificial SequenceSynthetic
anti-TDRD3 VH CDR3 31Thr Val Arg Gly Ser Lys Lys Pro Tyr Phe Ser
Gly Trp Ala Met Asp1 5 10 15Tyr3213PRTArtificial SequenceSynthetic
VH.1 CDR3 32Gly Tyr Ser Asn Phe Tyr Tyr Tyr Tyr Thr Met Asp Ala1 5
103313PRTArtificial SequenceSynthetic VH.4 CDR3 33Gly Tyr Ser Asn
Phe Tyr Tyr Tyr Tyr Thr Met Asp Val1 5 103413PRTArtificial
SequenceSynthetic VH.5 CDR3 34Gly Tyr Thr Asn Phe Tyr Tyr Tyr Tyr
Thr Met Asp Ala1 5 103510PRTArtificial SequenceSynthetic 4g2 VH
CDR3 35Ile Tyr His Tyr Asp Gly Tyr Phe Asp Val1 5
103624PRTArtificial SequenceSynthetic PGT124 VH CDR3 36Ala Arg Arg
Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu Phe1 5 10 15Phe Tyr
Tyr Tyr Tyr Met Asp Val 203711PRTArtificial SequenceSynthetic
anti-TDRD3 VL CDR1 37Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala1 5
103811PRTArtificial SequenceSynthetic VL.1 CDR1 38Arg Ala Thr Gln
Ser Ile Ser Thr Phe Leu Ala1 5 103911PRTArtificial
SequenceSynthetic VL.3 CDR1 39Arg Ala Ser Gln Ser Ile Ser Thr Phe
Leu Ala1 5 104011PRTArtificial SequenceSynthetic VL.4 CDR1 40Arg
Ala Thr Gln Ser Ile Val Thr Phe Leu Ala1 5 104111PRTArtificial
SequenceSynthetic 4g2 VL CDR1 41Lys Ala Ser Gln Asp Ile Asn Ser Tyr
Leu Thr1 5 104211PRTArtificial SequenceSynthetic PGT124 VL CDR1
42Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln1 5 10437PRTArtificial
SequenceSynthetic anti-TDRD3 VL CDR2 43Ser Ala Ser Ser Leu Tyr Ser1
5447PRTArtificial SequenceSynthetic VL.1 CDR2 44Asp Ala Ser Thr Arg
Ala Ser1 5457PRTArtificial SequenceSynthetic VL.3 CDR2 45Asp Ala
Ser Thr Arg Ala Thr1 5467PRTArtificial SequenceSynthetic VL.4 CDR2
46Asp Ala Ser Thr Asn Ala Ser1 5477PRTArtificial SequenceSynthetic
4g2 VL CDR2 47Arg Ala Asn Arg Leu Ile Asp1 5487PRTArtificial
SequenceSynthetic PGT124 VL CDR2 48Asn Asn Gln Asp Arg Pro Ser1
54911PRTArtificial SequenceSynthetic anti-TDRD3 VL CDR3 49Gln Gln
His Gly Pro Phe Tyr Trp Leu Phe Thr1 5 105010PRTArtificial
SequenceSynthetic VL.1 CDR3 50Gln Gln Arg Tyr Asn Trp Pro Pro Tyr
Ser1 5 105110PRTArtificial SequenceSynthetic VL.3 CDR3 51Gln Gln
Arg Tyr Asn Trp Pro Pro Tyr Thr1 5 10529PRTArtificial
SequenceSynthetic 4g2 VL CDR3 52Leu Gln Tyr Asp Glu Phe Pro Pro
Thr1 55312PRTArtificial SequenceSynthetic PGT124 VL CDR3 53His Met
Trp Asp Ser Arg Ser Gly Phe Ser Trp Ser1 5 10547PRTArtificial
SequenceSynthetic VH CDR1misc_feature(3)..(3)Xaa may or may not be
present, and if present is a polar amino acid residue 54Gly Phe Xaa
Phe Ser Thr Tyr1 5556PRTArtificial SequenceSynthetic VH
CDR2misc_feature(1)..(1)Xaa is a polar amino acid residue 55Xaa Gly
Glu Gly Asp Ser1 55613PRTArtificial SequenceSynthetic VH
CDR3misc_feature(3)..(3)Xaa is a polar amino acid
residuemisc_feature(13)..(13)Xaa is a nonpolar amino acid residue
56Gly Tyr Xaa Asn Phe Tyr Tyr Tyr Tyr Thr Met Asp Xaa1 5
105711PRTArtificial SequenceSynthetic VL
CDR1misc_feature(3)..(3)Xaa is a polar amino acid
residuemisc_feature(7)..(7)Xaa is a polar amino acid residue or a
hydrophobic amino acid residue 57Arg Ala Xaa Gln Ser Ile Xaa Thr
Phe Leu Ala1 5 10587PRTArtificial SequenceSynthetic VL
CDR2misc_feature(5)..(5)Xaa is a polar amino acid
residuemisc_feature(7)..(7)Xaa is a polar amino acid residue 58Asp
Ala Ser Thr Xaa Ala Xaa1 55910PRTArtificial SequenceSynthetic VL
CDR3misc_feature(10)..(10)Xaa is a polar amino acid residue 59Gln
Gln Arg Tyr Asn Trp Pro Pro Tyr Xaa1 5 10
* * * * *
References