U.S. patent application number 16/685087 was filed with the patent office on 2020-05-07 for non-immunogenic single domain antibodies.
This patent application is currently assigned to Inhibrx, Inc.. The applicant listed for this patent is Inhibrx, Inc.. Invention is credited to Quinn Deveraux, Brendan P. Eckelman, John C. Timmer.
Application Number | 20200140525 16/685087 |
Document ID | / |
Family ID | 56407322 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200140525 |
Kind Code |
A1 |
Eckelman; Brendan P. ; et
al. |
May 7, 2020 |
Non-Immunogenic Single Domain Antibodies
Abstract
The invention provides modifications within human or humanized
single domain antibody fragments (sdAbs) that prevent recognition
by pre-existing antibodies, to isolated polypeptides that include
these modifications, and to methods and uses thereof.
Inventors: |
Eckelman; Brendan P.; (La
Jolla, CA) ; Timmer; John C.; (La Jolla, CA) ;
Deveraux; Quinn; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inhibrx, Inc. |
La Jolla |
CA |
US |
|
|
Assignee: |
Inhibrx, Inc.
La Jolla
CA
|
Family ID: |
56407322 |
Appl. No.: |
16/685087 |
Filed: |
November 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15003234 |
Jan 21, 2016 |
10526397 |
|
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16685087 |
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62106035 |
Jan 21, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 2317/567 20130101; A61P 37/06 20180101; C07K 16/4283 20130101;
C07K 2317/24 20130101; C07K 2317/56 20130101; C07K 2317/569
20130101; C07K 16/00 20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 16/42 20060101 C07K016/42 |
Claims
1.-20. (canceled)
21. A single domain antibody (sdAb) comprising a mutation at
position Ala88 according to Kabat, wherein the mutation at position
Ala88 is Ala88Glu (A88E), Ala88Asp (A88D), Ala88Arg (A88R), or
Ala88Lys (A88K).
22. The single domain antibody of claim 21, wherein the carboxy
terminus comprises an amino acid sequence selected from TVE, TVK,
LVTVS (SEQ ID NO: 7), LVTVSS (SEQ ID NO: 113), TVSE (SEQ ID NO:
24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVSK (SEQ ID NO:
27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ ID
NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG (SEQ
ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49), TVRPG
(SEQ ID NO: 50), TVRPGG (SEQ ID NO: 51), TVEP (SEQ ID NO: 52),
TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ ID NO:
55), TVDPG (SEQ ID NO: 56), and TVDPGG (SEQ ID NO: 57).
23. The single domain antibody of claim 21, wherein the carboxy
terminus comprises an amino acid sequence selected from SEQ ID NOs:
2-10 and 30-43.
24. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Lys (A88K) and the carboxy terminus
comprises an amino acid sequence selected from TVE, TVSE (SEQ ID
NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ ID
NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ
ID NO: 55), TVDPG (SEQ ID NO: 56), and TVDPGG (SEQ ID NO: 57).
25. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Lys (A88K) and the carboxy terminus
comprises an amino acid sequence selected from TVK, TVSK (SEQ ID
NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ
ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG
(SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49),
TVRPG (SEQ ID NO: 50), and TVRPGG (SEQ ID NO: 51).
26. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Glu (A88E) and the carboxy terminus
comprises an amino acid sequence selected from TVE, TVSE (SEQ ID
NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ ID
NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ
ID NO: 55), TVDPG (SEQ ID NO: 56), and TVDPGG (SEQ ID NO: 57).
27. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Glu (A88E) and the carboxy terminus
comprises an amino acid sequence selected from TVK, TVSK (SEQ ID
NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ
ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG
(SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49),
TVRPG (SEQ ID NO: 50), and TVRPGG (SEQ ID NO: 51).
28. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Arg (A88R) and the carboxy terminus
comprises an amino acid sequence selected from TVE, TVSE (SEQ ID
NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ ID
NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ
ID NO: 55), TVDPG (SEQ ID NO: 56), and TVDPGG (SEQ ID NO: 57).
29. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Arg (A88R) and the carboxy terminus
comprises an amino acid sequence selected from TVK, TVSK (SEQ ID
NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ
ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG
(SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49),
TVRPG (SEQ ID NO: 50), and TVRPGG (SEQ ID NO: 51).
30. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Asp (A88D) and the carboxy terminus
comprises an amino acid sequence selected from TVE, TVSE (SEQ ID
NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ ID
NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ
ID NO: 55), TVDPG (SEQ ID NO: 56), and TVDPGG (SEQ ID NO: 57).
31. The single domain antibody of claim 21, wherein the mutation at
position Ala88 is Ala88Asp (A88D) and the carboxy terminus
comprises an amino acid sequence selected from TVK, TVSK (SEQ ID
NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ
ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG
(SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49),
TVRPG (SEQ ID NO: 50), and TVRPGG (SEQ ID NO: 51).
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/003,234, filed Jan. 21, 2016, which claims the benefit
of U.S. Provisional Application No. 62/106,035, filed Jan. 21,
2015, the contents of each of which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to modifications within
human or humanized single domain antibody fragments (sdAbs) that
prevent recognition by pre-existing antibodies, to isolated
polypeptides that include these modifications, and to methods and
uses thereof.
INCORPORATION OF SEQUENCE LISTING
[0003] The contents of the text file named
"2019-11-15_01202-0001-01US_ST25", which was created on Nov. 10,
2019, and is 10.0 KB in size, are hereby incorporated by reference
in their entirety.
BACKGROUND OF THE INVENTION
[0004] Clinical immunogenicity of biotherapeutics can limit
efficacy and contribute to adverse events. Both cellular and
humoral immunogenicity can be problematic in a clinical setting.
Most often observed in antibodies derived from non-human sources,
immunogenicity or the threat thereof has led to extensive protein
engineering and platform innovations to mitigate potential
immunogenicity. These include humanization, humaneering, veneering,
and development of fully human antibody platforms. However, even
fully human antibodies and antibody fragments have the risk of
being immunogenic. Immunogenicity is often directed toward the CDR
regions of an antibody. Conversely, framework region-directed
immunogenicity has also been observed, as in the case of single
domain antibodies. It has been noted that a significant proportion
of people have pre-existing antibodies to the cryptic epitopes
within single domain antibodies. Therefore, there exists a need for
the generation non-immunogenic single domain antibodies.
SUMMARY OF THE INVENTION
[0005] The modified single domain antibody (sdAb) fragments, of the
present invention can be derived from numerous sources, including
but not limited to V.sub.HH, V.sub.NAR, engineered V.sub.H or
V.sub.K domains. V.sub.HHs can be generated from camelid heavy
chain only antibodies and libraries thereof. V.sub.NARs can be
generated from cartilaginous fish heavy chain only antibodies and
libraries thereof. Various methods have been implemented to
generate monomeric sdAbs from conventionally heterodimeric V.sub.H
and V.sub.K domains, including interface engineering and selection
of specific germline families. In preferred embodiments the
modified sdAb of the present invention are human or humanized.
[0006] Pre-existing anti-human single domain antibody (sdAb)
antibodies (ASDA) are most prevalently observed in formats wherein
an sdAb is positioned such that is has a free carboxy-terminus. The
present invention provides mutations within the human or humanized
sdAb regions that disable ASDA recognition. In some embodiments of
the present invention, the sdAb is altered or modified within the
framework 1 region (FW1). In other embodiments, the sdAb is altered
or modified within the framework 2 region (FW2). In other
embodiments, the sdAb is altered or modified within the framework 3
region (FW3). In other embodiments, the sdAb is altered or modified
within the framework 4 region (FW4). These modifications include
mutations, truncation, extensions, or a combination thereof. In
some embodiments, the sdAb is modified within a single region. In
other embodiments, the sdAb is modified at more than one region.
For example, an sdAb may be modified within framework 1 and
framework 4. The sdAb modifications of the present invention
disable ASDA recognition without substantially decreasing binding
affinity, specificity, expressability or stability of the protein.
All numbering used herein refers to Kabat Numbering.
[0007] Exemplary modifications with the framework 4 of the sdAb are
shown below aligned to the human VH carboxy-terminal residues
(Leu/Thr108, Val109, Thr110, Val111, Ser112, Ser113; LVTVSS):
TABLE-US-00001 (natural human VH sequence, SEQ ID NO: 58) 108
LVTVSS 113 (SEQ ID NO: 1) 108 LVEIK 112 (SEQ ID NO: 2) 108 LVTVE
112 (SEQ ID NO: 3) 108 LVTVSE 113 (SEQ ID NO: 4) 108 LVTVEG 113
(SEQ ID NO: 5) 108 LVTVSEG 114 (SEQ ID NO: 6) 108 LVTVK 112 (SEQ ID
NO: 7) 108 LVTVS 112 (SEQ ID NO: 8) 108 LVTVSK 113 (SEQ ID NO: 9)
108 LVTVKG 113 (SEQ ID NO: 10) 108 LVTVSKG 114 (SEQ ID: NO 30) 108
LVTVSKPG 115 (SEQ ID: NO 31) 108 LVTVSKPGG 116 (SEQ ID: NO 32) 108
LVTVKP 113 (SEQ ID: NO 33) 108 LVTVKPG 114 (SEQ ID: NO 34) 108
LVTVKPGG 115 (SEQ ID: NO 35) 108 LVTVRP 113 (SEQ ID: NO 36) 108
LVTVRPG 114 (SEQ ID: NO 37) 108 LVTVRPGG 115 (SEQ ID: NO 38) 108
LVTVEP 113 (SEQ ID: NO 39) 108 LVTVEPG 114 (SEQ ID: NO 40) 108
LVTVEPGG 115 (SEQ ID: NO 41) 108 LVTVDP 113 (SEQ ID: NO 42) 108
LVTVDPG 114 (SEQ ID: NO 43) 108 LVTVDPGG 115 (SEQ ID NO: 11) 108
EVTVSS 113 (SEQ ID NO: 12) 108 KVTVSS 113 (SEQ ID NO: 13) 108
LVEVSS 113 (SEQ ID NO: 14) 108 LVKVSS 113 (SEQ ID NO: 15) 108
EVEVSS 113 (SEQ ID NO: 16) 108 EVKVSS 113 (SEQ ID NO: 17) 108
KVKVSS 113 (SEQ ID NO: 18) 108 KVEVSS 113
[0008] Exemplary carboxy-terminal extensions to the sdAb are shown
below aligned to the human VH carboxy-terminal residues
(Leu/Thr108, Val109, Thr110, Val111, Ser112, Ser113; LVTVSS):
TABLE-US-00002 (natural human VH sequence, SEQ ID NO: 58) 108
LVTVSS 113 (SEQ ID NO: 19) 108 LVTVSSA 114 (SEQ ID NO: 20) 108
LVTVSSG 114 (SEQ ID NO: 21) 108 LVTVSSGG 115 (SEQ ID NO: 22) 108
LVTVSSGGG 116 (SEQ ID NO: 23) 108 LVTVSSGGGG 117
[0009] In some embodiments the sdAb is modified at position Leu11
within framework 1. For example, the modification at position Leu11
is Leu11Lys (L11K), Leu11Arg (L11R), Leu11Glu (L11E) or Leu11Asp
(L11D). In some embodiments, the sdAb is modified position Leu11
and within its carboxy-terminal region. For example L11K, L11R,
L11E or L11D and comprising the terminal sequence of TVE, TVSE (SEQ
ID NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVK, TVSK
(SEQ ID NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29),
TVSKPG (SEQ ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO:
46), TVKPG (SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID
NO: 49), TVRPG (SEQ ID NO: 50), TVRPGG (SEQ ID NO: 51), TVEP (SEQ
ID NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP
(SEQ ID NO: 55), TVDPG (SEQ ID NO: 56), TVDPGG (SEQ ID NO: 57).
[0010] In some embodiments, the sdAb is modified at position 11
with a positively charged residue (e.g. L11K or L11R) and paired
with a carboxy-terminal modification that contains a negatively
charged residue at position 112 or 113. For example, L11K or L11R
is paired with carboxy-terminal modification of TVE, TVSE (SEQ ID
NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ ID
NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ
ID NO: 55), TVDPG (SEQ ID NO: 56), or TVDPGG (SEQ ID NO: 57).
[0011] In some embodiments, the sdAb is modified at position 11
with a negatively charged residue (e.g. L11E or L11D) and is paired
with a carboxy-terminal modification that contains a positively
charged residue at position 112 or 113. For example, L11E or L11D
is paired with carboxy-terminal modification of TVK, TVSK (SEQ ID
NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ
ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG
(SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49),
TVRPG (SEQ ID NO: 50), or TVRPGG (SEQ ID NO: 51).
[0012] In some embodiments, the sdAb is modified at position 11
with a positively charged residue (e.g. L11K or L11R) and paired
with a carboxy-terminal modification that contains a positively
charged residue at position 112 or 113. For example, L11K or L11R
is paired with carboxy-terminal modification of TVK, TVSK (SEQ ID
NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ
ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG
(SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49),
TVRPG (SEQ ID NO: 50), or TVRPGG (SEQ ID NO: 51).
[0013] In some embodiments, the sdAb is modified at position 11
with a negatively charged residue (e.g. L11E or L11D) and paired
with a carboxy-terminal modification that contains a negatively
charged residue at position 112 or 113. For example, L11E or L11D
is paired with carboxy-terminal modification of TVE, TVSE (SEQ ID
NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ ID
NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ
ID NO: 55), TVDPG (SEQ ID NO: 56), or TVDPGG (SEQ ID NO: 57).
[0014] Presumably, in embodiments wherein there are opposing
charged residues introduced into position 11 and 112 or position 11
and 113 of the sdAb, a new electrostatic interaction enabled by the
newly added negative and positive charge pair. Presumably, in
embodiments, wherein the residues introduced at position 11 and 112
or position 11 and 113, are of the same charge, the orientation of
these sides of these amino acids will be position away from each
other, however, are still capable of occluding the recognition site
be pre-existing ASDA recognition of the modified sdAb.
[0015] In some embodiments, the sdAb is modified at position Ala88
within framework 3. For example, the modification at position Ala88
is Ala88Glu (A88E), Ala88Asp (A88D), Ala88Lys (A88K) or Ala88Arg
(A88R). In some embodiments, the sdAb is modified position Ala88
and within its carboxy-terminal region. For example A88E, A88D,
A88K or A88R and comprising the terminal sequence of TVE, TVSE (SEQ
ID NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVK, TVSK
(SEQ ID NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID NO: 29),
TVSKPG (SEQ ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ ID NO:
46), TVKPG (SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID
NO: 49), TVRPG (SEQ ID NO: 50), TVRPGG (SEQ ID NO: 51), TVEP (SEQ
ID NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP
(SEQ ID NO: 55), TVDPG (SEQ ID NO: 56), or TVDPGG (SEQ ID NO:
57).
[0016] In some embodiments, the sdAb is modified position Leu11 and
Ala88. For example, L11E/A88E, L11E/A88D, L11E/A88K, L11E/A88R,
L11D/A88E, L11D/A88D, L11D/A88K, L11D/A88R, L11K/A88E, L11K/A88D,
L11K/A88K, L11K/A88R, L11R/A88E, L11R/A88D, L11R/A88K or L11R/A88R.
In some embodiments, the sdAb is modified position Leu11 and Ala88
and paired with a modified carboxy-terminal region. For example,
L11E/A88E, L11E/A88D, L11E/A88K, L11E/A88R, L11D/A88E, L11D/A88D,
L11D/A88K, L11D/A88R, L11K/A88E, L11K/A88D, L11K/A88K, L11K/A88R,
L11R/A88E, L11R/A88D, L11R/A88K or L11R/A88R and comprising the
terminal sequence of TVE, TVSE (SEQ ID NO: 24), TVEG (SEQ ID NO:
25), TVSEG (SEQ ID NO: 26), TVK, TVSK (SEQ ID NO: 27), TVKG (SEQ ID
NO: 28), TVSKG (SEQ ID NO: 29), TVSKPG (SEQ ID NO: 44), TVSKPGG
(SEQ ID NO: 45), TVKP (SEQ ID NO: 46), TVKPG (SEQ ID NO: 47),
TVKPGG (SEQ ID NO: 48), TVRP (SEQ ID NO: 49), TVRPG (SEQ ID NO:
50), TVRPGG (SEQ ID NO: 51), TVEP (SEQ ID NO: 52), TVEPG (SEQ ID
NO: 53), TVEPGG (SEQ ID NO: 54), TVDP (SEQ ID NO: 55), TVDPG (SEQ
ID NO: 56), or TVDPGG (SEQ ID NO: 57).
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a schematic of structural model of VH domain
highlighting exemplary positions that were modified to prevent ASDA
recognition.
[0018] FIG. 2 is a graph of an ASDA assay demonstrating the impact
of various exemplary modifications of an immunogenic sdAb in ASDA
recognition. Enbrel (TNFR2-Fc) was included as non-ASDA recognized
control. C-terminal extensions are indicated by a "." between the
natural terminal residue and start of the extension. C-terminal
truncations are indicated by a "_" at the site of excluded
residue.
[0019] FIGS. 3A and 3B are a series of graphs of an ASDA assay
demonstrating the impact of various modifications of an immunogenic
sdAb in ASDA recognition. Enbrel (TNFR2-Fc) was included as
non-ASDA recognized control.
[0020] FIG. 4 is a graph of an ASDA assay demonstrating the impact
of various modifications of an immunogenic sdAb in ASDA
recognition. Enbrel (TNFR2-Fc) was included as non-ASDA recognized
control. Herein framework 1 modifications were combined with
framework 4 modifications.
[0021] FIGS. 5A and 5B are a series of graphs of an indirect ASDA
assay demonstrating the impact the dual modifications of a
humanized VHH having of L11E paired with a carboxy-terminal TVKPGG
compared with a humanized VHH unmodified at L11 and having the
native carboxy-terminal sequence, TVSS.
DETAILED DESCRIPTION
[0022] A single-domain antibody (sdAb) is an antibody fragment
consisting of a single monomeric variable antibody domain that is
able to bind selectively to a specific antigen. See e.g.,
Hamers-Casterman, C., et al., "Naturally occurring antibodies
devoid of light chains," Nature vol. 363: 446-448 (1993); Nguyen,
V. K., et al., "Camel heavy-chain antibodies: diverse germline
V(H)H and specific mechanisms enlarge the antigen-binding
repertoire," EMBO, vol. 19: 921-30 (2000); Achour, I., et al.,
"Tetrameric and homodimeric camelid IgGs originate from the same
IgH locus," J. Immunol., vol. 181: 2001-2009 (2008); Harmsen, et
al., "Llama heavy-chain V regions consist of at least four distinct
subfamilies revealing novel sequence features," Mol. Immunol., vol.
37: 579-590 (2000); Arbabi Ghahroudi, M., et al., "Selection and
identification of single domain antibody fragments from camel
heavy-chain antibodies," FEBS Lett. vol. 414: 521-526 (1997); and
Vincke et al., "General Strategy to Humanize a Camelid
Single-domain Antibody and Identification of a Universal Humanized
Nanobody Scaffold," J. Biol. Chem., vol. 284(5): 3273-84 (2009),
the contents of each of which are hereby incorporated by reference
in their entireties).
[0023] With a molecular weight of only 12-15 kDa, single-domain
antibodies are much smaller than common antibodies (150-160 kDa)
which are composed of two heavy protein chains and two light
chains, and even smaller than Fab fragments (.about.50 kDa, one
light chain and half a heavy chain) and single-chain variable
fragments (.about.25 kDa, two variable domains, one from a light
and one from a heavy chain).
[0024] Single domain antibodies are antibodies whose complementary
determining regions are part of a single domain polypeptide.
Examples include, but are not limited to, heavy chain antibodies,
antibodies naturally devoid of light chains, single domain
antibodies derived from conventional 4-chain antibodies, engineered
antibodies and single domain scaffolds other than those derived
from antibodies. Single domain antibodies may be derived from any
species including, but not limited to mouse, human, camel, llama,
goat, rabbit, and/or bovine. In some embodiments, a single domain
antibody as used herein is a naturally occurring single domain
antibody known as heavy chain antibody devoid of light chains. For
clarity reasons, this variable domain derived from a heavy chain
antibody naturally devoid of light chain is known herein as a VHH
to distinguish it from the conventional VH of four chain
immunoglobulins. Such a VHH molecule can be derived from antibodies
raised in Camelidae species, for example in camel, llama,
dromedary, alpaca and guanaco. Other species besides Camelidae may
produce heavy chain antibodies naturally devoid of light chain;
such VHHs are within the scope of the invention.
[0025] A single-domain antibody can be obtained by immunization of
dromedaries, camels, llamas, alpacas or sharks with the desired
antigen and subsequent isolation of the mRNA coding for heavy-chain
antibodies. By reverse transcription and polymerase chain reaction,
a gene library of single-domain antibodies containing several
million clones is produced. Screening techniques like phage display
and ribosome display help to identify the clones binding the
antigen. (See e.g., Arbabi Ghahroudi, M.; Desmyter, A.; et al.
(1997). "Selection and identification of single domain antibody
fragments from camel heavy-chain antibodies". FEBS Letters 414 (3):
521-526.)
[0026] A different method uses gene libraries from animals that
have not been immunized beforehand. Such naive libraries usually
contain only antibodies with low affinity to the desired antigen,
making it necessary to apply affinity maturation by random
mutagenesis as an additional step. (Saerens, D.; et al. (2008).
"Single-domain antibodies as building blocks for novel
therapeutics". Current Opinion in Pharmacology 8 (5): 600-608.)
[0027] When the most potent clones have been identified, their DNA
sequence is optimized, for example to improve their stability
towards enzymes. Another goal is humanization to prevent
immunological reactions of the human organism against the antibody.
Humanization is unproblematic because of the homology between
camelid VHH and human VH fragments. (See e.g., Saerens, et al.,
(2008). "Single-domain antibodies as building blocks for novel
therapeutics". Current Opinion in Pharmacology 8 (5): 600-608.) The
final step is the translation of the optimized single-domain
antibody in E. coli, Saccharomyces cerevisiae or other suitable
organisms.
[0028] Single domain antibody fragments are also derived from
conventional antibodies. In some embodiments, single-domain
antibodies can be made from common murine or human IgG with four
chains. (Holt, L. J.; et al. (2003). "Domain antibodies: proteins
for therapy". Trends in Biotechnology 21 (11): 484-490.) The
process is similar, comprising gene libraries from immunized or
naive donors and display techniques for identification of the most
specific antigens. A problem with this approach is that the binding
region of common IgG consists of two domains (VH and VL), which
tend to dimerize or aggregate because of their lipophilicity.
Monomerization is usually accomplished by replacing lipophilic by
hydrophilic amino acids, but often results in a loss of affinity to
the antigen. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002).
"Antibody evolution beyond Nature". Nature Biotechnology 20 (12):
1189-90.) If affinity can be retained, the single-domain antibodies
can likewise be produced in E. coli, S. cerevisiae or other
organisms.
[0029] The modifications within human or humanized single domain
antibody fragments described herein are useful with any single
domain antibody fragment, regardless of method of production.
[0030] The invention provides a single domain antibody (sbAb) or
antigen-binding fragments thereof that comprise at least one
mutation that prevents recognition of the sbAb by an antibody that
specifically recognizes single domain antibodies. In some
embodiments, the mutation is at least one mutation in a framework
region.
[0031] In some embodiments, the mutation is at least one framework
4 mutation. In some embodiments, the framework 4 mutation comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-18 or 30-43.
[0032] In some embodiments, the mutation is a carboxy-terminal
amino acid extension. In some embodiments, the carboxy-terminal
amino acid extension comprises an amino acid sequence selected from
the group consisting of SEQ ID NO: 19-23.
[0033] In some embodiments, the mutation is at least one framework
1 mutation. In some embodiments, the framework 1 mutation comprises
a mutation at position Leu11. In some embodiments, the mutation at
position Leu11 is Leu11Lys (L11K), Leu11Arg (L11R), Leu11Asp
(L11D), or Leu11Glu (L11E). In some embodiments, the single domain
antibody further comprises a mutation in the carboxy terminus. In
some embodiments, the mutation in the carboxy terminus comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO: 24-29 or 30-43. In some embodiments, the framework 1 region
comprises Leu11Lys (L11K) and the mutation in the carboxy terminus
comprises an amino acid sequence selected from the group consisting
of TVE, TVSE (SEQ ID NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID
NO: 26), TVEP (SEQ ID NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ
ID NO: 54), TVDP (SEQ ID NO: 55), TVDPG (SEQ ID NO: 56), and TVDPGG
(SEQ ID NO: 57). In some embodiments, the framework 1 region
comprises Leu11Glu (L11E) and the mutation in the carboxy terminus
comprises an amino acid sequence selected from the group consisting
of TVK, TVSK (SEQ ID NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID
NO: 29), TVSKPG (SEQ ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ
ID NO: 46), TVKPG (SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP
(SEQ ID NO: 49), TVRPG (SEQ ID NO: 50), and TVRPGG (SEQ ID NO:
51).
[0034] In some embodiments, the mutation is at least one framework
3 mutation. In some embodiments, the framework 3 mutation comprises
a mutation at position Ala88. In some embodiments, the mutation at
position Ala88 is Ala88Glu (A88E), Ala88Asp (A88D), Ala88Arg
(A88R), or Ala88Lys (A88K). In some embodiments, the single domain
antibody further comprises a mutation in the carboxy terminus. In
some embodiments, the mutation in the carboxy terminus comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO: 24-29 and 44-57. In some embodiments, the framework 3 region
comprises Ala88Glu (A88E) and the mutation in the carboxy terminus
comprises an amino acid sequence selected from the group consisting
of TVK, TVSK (SEQ ID NO: 27), TVKG (SEQ ID NO: 28), TVSKG (SEQ ID
NO: 29), TVSKPG (SEQ ID NO: 44), TVSKPGG (SEQ ID NO: 45), TVKP (SEQ
ID NO: 46), TVKPG (SEQ ID NO: 47), TVKPGG (SEQ ID NO: 48), TVRP
(SEQ ID NO: 49), TVRPG (SEQ ID NO: 50), and TVRPGG (SEQ ID NO: 51).
In some embodiments, the framework 3 region comprises Ala88Lys
(A88K) and the mutation in the carboxy terminus comprises an amino
acid sequence selected from the group consisting of TVE, TVSE (SEQ
ID NO: 24), TVEG (SEQ ID NO: 25), TVSEG (SEQ ID NO: 26), TVEP (SEQ
ID NO: 52), TVEPG (SEQ ID NO: 53), TVEPGG (SEQ ID NO: 54), TVDP
(SEQ ID NO: 55), TVDPG (SEQ ID NO: 56), and TVDPGG (SEQ ID NO:
57).
[0035] All patents and publications mentioned in this specification
are herein incorporated by reference to the same extent as if each
independent patent and publication was specifically and
individually indicated to be incorporated by reference.
Definitions
[0036] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well-known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
foregoing techniques and procedures are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See e.g.,
Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989)). The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well-known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0037] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0038] As used herein, "specifically bind" or "immunoreacts with"
"or directed against" is meant that the antibody reacts with one or
more antigenic determinants of the desired antigen and does not
react with other polypeptides or binds at much lower affinity
(K.sub.d>10.sup.-6). Antibodies include, but are not limited to,
polyclonal, monoclonal, chimeric, dAb (domain antibody), single
chain, Fab, Fab' and F(ab').sub.2 fragments, Fv, scFvs, an Fab
expression library, and single domain antibody (sdAb) fragments,
for example V.sub.HH, V.sub.NAR, engineered V.sub.H or V.sub.K.
[0039] The term "antigen-binding site" or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences which are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in
three-dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs." The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature
342:878-883 (1989).
[0040] As used herein, the term "epitope" includes any protein
determinant capable of specific binding to/by an immunoglobulin or
fragment thereof, or a T-cell receptor. The term "epitope" includes
any protein determinant capable of specific binding to/by an
immunoglobulin or T-cell receptor. Epitopic determinants usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. An antibody is said to specifically bind an
antigen when the dissociation constant is .ltoreq.1 .mu.M; e.g.,
.ltoreq.100 nM, preferably .ltoreq.10 nM and more preferably
.ltoreq.1 nM.
[0041] As used herein, the terms "immunological binding," and
"immunological binding properties" refer to the non-covalent
interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific.
The strength, or affinity of immunological binding interactions can
be expressed in terms of the dissociation constant (K.sub.d) of the
interaction, wherein a smaller K.sub.d represents a greater
affinity. Immunological binding properties of selected polypeptides
can be quantified using methods well known in the art. One such
method entails measuring the rates of antigen-binding site/antigen
complex formation and dissociation, wherein those rates depend on
the concentrations of the complex partners, the affinity of the
interaction, and geometric parameters that equally influence the
rate in both directions. Thus, both the "on rate constant"
(k.sub.on) and the "off rate constant" (k.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of k.sub.off/k.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
invention is said to specifically bind to an antigen, when the
equilibrium binding constant (K.sub.d) is .ltoreq.1 .mu.M,
preferably .ltoreq.100 nM, more preferably .ltoreq.10 nM, and most
preferably .ltoreq.100 pM to about 1 pM, as measured by assays such
as radioligand binding assays, surface plasmon resonance (SPR),
flow cytometry binding assay, or similar assays known to those
skilled in the art.
[0042] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity, and most preferably at least 99 percent sequence
identity.
[0043] Preferably, residue positions which are not identical differ
by conservative amino acid substitutions.
[0044] Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine valine,
glutamic-aspartic, and asparagine-glutamine.
[0045] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75%, more preferably at least 80%, 90%, 95%, and most
preferably 99%. In particular, conservative amino acid replacements
are contemplated. Conservative replacements are those that take
place within a family of amino acids that are related in their side
chains. Genetically encoded amino acids are generally divided into
families: (1) acidic amino acids are aspartate, glutamate; (2)
basic amino acids are lysine, arginine, histidine; (3) non-polar
amino acids are alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan, and (4) uncharged polar
amino acids are glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. The hydrophilic amino acids include arginine,
asparagine, aspartate, glutamine, glutamate, histidine, lysine,
serine, and threonine. The hydrophobic amino acids include alanine,
cysteine, isoleucine, leucine, methionine, phenylalanine, proline,
tryptophan, tyrosine and valine. Other families of amino acids
include (i) serine and threonine, which are the aliphatic-hydroxy
family; (ii) asparagine and glutamine, which are the amide
containing family; (iii) alanine, valine, leucine and isoleucine,
which are the aliphatic family; and (iv) phenylalanine, tryptophan,
and tyrosine, which are the aromatic family. For example, it is
reasonable to expect that an isolated replacement of a leucine with
an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
binding or properties of the resulting molecule, especially if the
replacement does not involve an amino acid within a framework site.
Whether an amino acid change results in a functional peptide can
readily be determined by assaying the specific activity of the
polypeptide derivative. Assays are described in detail herein.
Fragments or analogs of antibodies or immunoglobulin molecules can
be readily prepared by those of ordinary skill in the art.
Preferred amino- and carboxy-termini of fragments or analogs occur
near boundaries of functional domains. Structural and functional
domains can be identified by comparison of the nucleotide and/or
amino acid sequence data to public or proprietary sequence
databases. Preferably, computerized comparison methods are used to
identify sequence motifs or predicted protein conformation domains
that occur in other proteins of known structure and/or function.
Methods to identify protein sequences that fold into a known
three-dimensional structure are known. Bowie et al. Science 253:164
(1991). Thus, the foregoing examples demonstrate that those of
skill in the art can recognize sequence motifs and structural
conformations that may be used to define structural and functional
domains in accordance with the invention.
[0046] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various muteins of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain(s)
forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).
[0047] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino terminal and/or carboxy-terminal
deletion, but where the remaining amino acid sequence is identical
to the corresponding positions in the naturally-occurring sequence
deduced, for example, from a full length cDNA sequence. Fragments
typically are at least 5, 6, 8 or 10 amino acids long, preferably
at least 14 amino acids long' more preferably at least 20 amino
acids long, usually at least 50 amino acids long, and even more
preferably at least 70 amino acids long.
[0048] In this disclosure, "comprises," "comprising," "containing,"
"having," and the like can have the meaning ascribed to them in
U.S. Patent law and can mean "includes," "including," and the like;
the terms "consisting essentially of" or "consists essentially"
likewise have the meaning ascribed in U.S. Patent law and these
terms are open-ended, allowing for the presence of more than that
which is recited so long as basic or novel characteristics of that
which is recited are not changed by the presence of more than that
which is recited, but excludes prior art embodiments.
[0049] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule. This portion contains, preferably, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of
the reference nucleic acid molecule or polypeptide. A fragment may
contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400,
500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
[0050] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, or 50.
[0051] Unless specifically stated or obvious from context, as used
herein, the terms "a," "an," and "the" are understood to be
singular or plural. Unless specifically stated or obvious from
context, as used herein, the term "or" is understood to be
inclusive.
[0052] Unless specifically stated or obvious from context, as used
herein, the term "about" is understood as within a range of normal
tolerance in the art, for example within 2 standard deviations of
the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%,
5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated
value. Unless otherwise clear from the context, all numerical
values provided herein are modified by the term "about."
Examples
[0053] A direct anti-single domain antibody (ASDA) detection assay
is useful in determining pre-existing ASDA in serum from human
donors. Generally, pre-existing antibodies within human serum can
be identified by an ELISA wherein the sdAb is immobilized on a
plate and blocked with BSA. Human serum (neat or diluted in
PBS+0.01% Tween-20) is then incubated with the plate-bound sdAb.
Unbound serum IgG is washed away and any remaining antibodies to
the sdAb are detected using both anti-human IgKappa and anti-human
IgLamda HRP-conjugated antibodies.
[0054] An indirect ASDA assay is useful for the analysis of
multiple variants of a given sdAb so as to measure the sdAb
specific serum antibody reactivity. The indirect ASDA involves
immobilization of an sdAb and blocked with BSA. Human serum (neat
or diluted in PBS+0.01% Tween-20) is then pre-incubated with
soluble sdAb (either the same sdAb or variants of the parental sdAb
with modifications of the present invention [mutations, truncation
or extensions]), and then added to the plate bound sdAb. The
ability of a soluble sdAb variant to block the ASDA recognition of
the immobilized sdAb is indicative of the soluble sdAb being
recognized by the ASDAs and is thus an indirect measurement of the
pre-existing ASDA response. Conversely, the inability of a soluble
sdAbs to prevent the ASDA recognition of the immobilized sdAb is
indicative of the soluble sdAb variant being non-immunogenic or
otherwise not recognized by the pre-existing ASDAs. Generally, 8-16
assays are conducted in parallel with serum from separate donors
previously determined to contain ASDAs.
[0055] In the studies provided herein, various exemplary
modifications of an immunogenic sdAb were evaluated using an ASDA
assay. Enbrel (TNFR2-Fc) and BSA were sometimes included as
non-ASDA recognized control. In some experiments purified serum IgG
was coated as a positive control for the secondary antibodies.
C-terminal extensions are indicated by a "." between the natural
terminal residue and start of the extension. C-terminal truncations
are indicated by a "_" at the site of excluded residue. The results
are shown in FIGS. 2-4.
[0056] An indirect ASDA assay demonstrated the impact the dual
modifications of a humanized VHH having of L11E paired with a
carboxy-terminal TVKPGG compared with a humanized VHH unmodified at
L11 and having the native carboxy-terminal sequence, TVSS (FIGS.
5A-5B).
Other Embodiments
[0057] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following.
Sequence CWU 1
1
5815PRTArtificial Sequencechemically synthesized 1Leu Val Glu Ile
Lys1 525PRTArtificial Sequencechemically synthesized 2Leu Val Thr
Val Glu1 536PRTArtificial Sequencechemically synthesized 3Leu Val
Thr Val Ser Glu1 546PRTArtificial Sequencechemically synthesized
4Leu Val Thr Val Glu Gly1 557PRTArtificial Sequencechemically
synthesized 5Leu Val Thr Val Ser Glu Gly1 565PRTArtificial
Sequencechemically synthesized 6Leu Val Thr Val Lys1
575PRTArtificial Sequencechemically synthesized 7Leu Val Thr Val
Ser1 586PRTArtificial Sequencechemically synthesized 8Leu Val Thr
Val Ser Lys1 596PRTArtificial Sequencechemically synthesized 9Leu
Val Thr Val Lys Gly1 5107PRTArtificial Sequencechemically
synthesized 10Leu Val Thr Val Ser Lys Gly1 5116PRTArtificial
Sequencechemically synthesized 11Glu Val Thr Val Ser Ser1
5126PRTArtificial Sequencechemically synthesized 12Lys Val Thr Val
Ser Ser1 5136PRTArtificial Sequencechemically synthesized 13Leu Val
Glu Val Ser Ser1 5146PRTArtificial Sequencechemically synthesized
14Leu Val Lys Val Ser Ser1 5156PRTArtificial Sequencechemically
synthesized 15Glu Val Glu Val Ser Ser1 5166PRTArtificial
Sequencechemically synthesized 16Glu Val Lys Val Ser Ser1
5176PRTArtificial Sequencechemically synthesized 17Lys Val Lys Val
Ser Ser1 5186PRTArtificial Sequencechemically synthesized 18Lys Val
Glu Val Ser Ser1 5197PRTArtificial Sequencechemically synthesized
19Leu Val Thr Val Ser Ser Ala1 5207PRTArtificial Sequencechemically
synthesized 20Leu Val Thr Val Ser Ser Gly1 5218PRTArtificial
Sequencechemically synthesized 21Leu Val Thr Val Ser Ser Gly Gly1
5229PRTArtificial Sequencechemically synthesized 22Leu Val Thr Val
Ser Ser Gly Gly Gly1 52310PRTArtificial Sequencechemically
synthesized 23Leu Val Thr Val Ser Ser Gly Gly Gly Gly1 5
10244PRTArtificial Sequencechemically synthesized 24Thr Val Ser
Glu1254PRTArtificial Sequencechemically synthesized 25Thr Val Glu
Gly1265PRTArtificial Sequencechemically synthesized 26Thr Val Ser
Glu Gly1 5274PRTArtificial Sequencechemically synthesized 27Thr Val
Ser Lys1284PRTArtificial Sequencechemically synthesized 28Thr Val
Lys Gly1295PRTArtificial Sequencechemically synthesized 29Thr Val
Ser Lys Gly1 5308PRTArtificial Sequencechemically synthesized 30Leu
Val Thr Val Ser Lys Pro Gly1 5319PRTArtificial Sequencechemically
synthesized 31Leu Val Thr Val Ser Lys Pro Gly Gly1
5326PRTArtificial Sequencechemically synthesized 32Leu Val Thr Val
Lys Pro1 5337PRTArtificial Sequencechemically synthesized 33Leu Val
Thr Val Lys Pro Gly1 5348PRTArtificial Sequencechemically
synthesized 34Leu Val Thr Val Lys Pro Gly Gly1 5356PRTArtificial
Sequencechemically synthesized 35Leu Val Thr Val Arg Pro1
5367PRTArtificial Sequencechemically synthesized 36Leu Val Thr Val
Arg Pro Gly1 5378PRTArtificial Sequencechemically synthesized 37Leu
Val Thr Val Arg Pro Gly Gly1 5386PRTArtificial Sequencechemically
synthesized 38Leu Val Thr Val Glu Pro1 5397PRTArtificial
Sequencechemically synthesized 39Leu Val Thr Val Glu Pro Gly1
5408PRTArtificial Sequencechemically synthesized 40Leu Val Thr Val
Glu Pro Gly Gly1 5416PRTArtificial Sequencechemically synthesized
41Leu Val Thr Val Asp Pro1 5427PRTArtificial Sequencechemically
synthesized 42Leu Val Thr Val Asp Pro Gly1 5438PRTArtificial
Sequencechemically synthesized 43Leu Val Thr Val Asp Pro Gly Gly1
5446PRTArtificial Sequencechemically synthesized 44Thr Val Ser Lys
Pro Gly1 5457PRTArtificial Sequencechemically synthesized 45Thr Val
Ser Lys Pro Gly Gly1 5464PRTArtificial Sequencechemically
synthesized 46Thr Val Lys Pro1475PRTArtificial Sequencechemically
synthesized 47Thr Val Lys Pro Gly1 5486PRTArtificial
Sequencechemically synthesized 48Thr Val Lys Pro Gly Gly1
5494PRTArtificial Sequencechemically synthesized 49Thr Val Arg
Pro1505PRTArtificial Sequencechemically synthesized 50Thr Val Arg
Pro Gly1 5516PRTArtificial Sequencechemically synthesized 51Thr Val
Arg Pro Gly Gly1 5524PRTArtificial Sequencechemically synthesized
52Thr Val Glu Pro1535PRTArtificial Sequencechemically synthesized
53Thr Val Glu Pro Gly1 5546PRTArtificial Sequencechemically
synthesized 54Thr Val Glu Pro Gly Gly1 5554PRTArtificial
Sequencechemically synthesized 55Thr Val Asp Pro1565PRTArtificial
Sequencechemically synthesized 56Thr Val Asp Pro Gly1
5576PRTArtificial Sequencechemically synthesized 57Thr Val Asp Pro
Gly Gly1 5586PRTArtificial Sequencechemically synthesized 58Leu Val
Thr Val Ser Ser1 5
* * * * *