U.S. patent application number 17/650792 was filed with the patent office on 2022-07-28 for novel ankyrin repeat binding proteins and their uses.
The applicant listed for this patent is Molecular Partners AG. Invention is credited to Patrick AMSTUTZ, Valerie Perrine CALABRO, Marcel WALSER.
Application Number | 20220235103 17/650792 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-28 |
United States Patent
Application |
20220235103 |
Kind Code |
A1 |
AMSTUTZ; Patrick ; et
al. |
July 28, 2022 |
NOVEL ANKYRIN REPEAT BINDING PROTEINS AND THEIR USES
Abstract
The present invention relates to recombinant binding proteins
comprising one or more designed ankyrin repeat domains with binding
specificity for coronavirus spike proteins, nucleic acids encoding
such proteins, pharmaceutical compositions comprising such proteins
or nucleic acids, and the use of such proteins, nucleic acids or
pharmaceutical compositions in the treatment of coronavirus
diseases, particularly diseases caused by SARS-CoV-2.
Inventors: |
AMSTUTZ; Patrick;
(Kilchberg, CH) ; CALABRO; Valerie Perrine;
(Bergdietikon, CH) ; WALSER; Marcel; (Winterthur,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Molecular Partners AG |
Zurich-Schlieren |
|
CH |
|
|
Appl. No.: |
17/650792 |
Filed: |
February 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17313543 |
May 6, 2021 |
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17650792 |
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63020882 |
May 6, 2020 |
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63021024 |
May 6, 2020 |
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63057477 |
Jul 28, 2020 |
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63069174 |
Aug 24, 2020 |
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63145192 |
Feb 3, 2021 |
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International
Class: |
C07K 14/47 20060101
C07K014/47; A61P 31/14 20060101 A61P031/14 |
Claims
1-25. (canceled)
26. A method of treating a coronavirus infection in a subject, the
method comprising the step of administering an effective amount of
at least one binding protein to a subject in need thereof; wherein
the binding protein comprises an amino acid sequence selected from
the group consisting of SEQ ID NO: 3, 5, and 6.
27. The method of claim 26, wherein the binding protein comprises
the amino acid sequence of SEQ ID NO: 3.
28. The method of claim 26, wherein the binding protein comprises
the amino acid sequence of SEQ ID NO: 5.
29. The method of claim 26, wherein the binding protein comprises
the amino acid sequence of SEQ ID NO: 6.
30. The method of claim 26, wherein the binding protein further
comprises the amino acid sequence of SEQ ID NO: 47.
31. The method of claim 26, wherein the binding protein comprises
the amino acid sequences of SEQ ID NO: 3, 5 and 6.
32. The method of claim 26, wherein the binding protein comprises
the amino acid sequences of SEQ ID NO: 3, 5, 6, and 47.
33. The method of claim 26, wherein the binding protein comprises a
sequence having at least 90% sequence identity to SEQ ID NO:
31.
34. The method of claim 26, wherein the binding protein comprises a
sequence having at least 95% sequence identity to SEQ ID NO:
31.
35. The method of claim 26, wherein the binding protein comprises a
sequence having at least 98% sequence identity to SEQ ID NO:
31.
36. The method of claim 26, wherein the binding protein comprises
the sequence of SEQ ID NO: 31.
37. The method of claim 26, wherein the coronavirus infection is
caused by SARS-CoV-2.
38. The method of claim 26, wherein the coronavirus infection is
caused by a variant of SARS-CoV-2.
39. The method of claim 26, wherein the step of administering the
binding protein to the subject is repeated.
40. A method of treating a coronavirus infection in a subject, the
method comprising the step of administering an effective amount of
at least one binding protein to a subject in need thereof; wherein
the binding protein comprises the amino acid sequence of SEQ ID NO:
31.
41. The method of claim 40, wherein the coronavirus infection is
caused by SARS-CoV-2.
42. The method of claim 40, wherein the coronavirus infection is
caused by a variant of SARS-CoV-2.
43. The method of claim 40, wherein the step of administering the
binding protein to the subject is repeated.
44. A binding protein comprising an amino acid sequence, wherein
the amino acid sequence is selected from the group consisting of
SEQ ID NO: 3, 5, and 6.
45. The binding protein of claim 44, wherein the binding protein
further comprises the amino acid sequence of SEQ ID NO: 47.
46. The binding protein of claim 44, wherein the binding protein
comprises a sequence having at least 90% sequence identity to SEQ
ID NO: 31.
47. The binding protein of claim 44, wherein the binding protein
comprises the sequence of SEQ ID NO: 31.
48. A nucleic acid encoding the binding protein of claim 44.
49. The nucleic acid of claim 48, wherein the nucleic acid
comprises the sequence of SEQ ID NO: 74.
50. A binding protein comprising an amino acid sequence, wherein
the amino acid sequence is selected from the group consisting of
SEQ ID NO: 3, 76, and 77.
51. The binding protein of claim 50, wherein the binding protein
further comprises the amino acid sequence of SEQ ID NO: 47.
52. The binding protein of claim 50, wherein the binding protein
comprises a sequence having at least 90% sequence identity to SEQ
ID NO: 75.
53. The binding protein of claim 50, wherein the binding protein
comprises the sequence of SEQ ID NO: 75.
54. A nucleic acid encoding the binding protein of claim 50.
55. The nucleic acid of claim 54, wherein the nucleic acid
comprises the sequence of SEQ ID NO: 78.
56. A method of treating a coronavirus infection in a subject, the
method comprising the step of administering an effective amount of
the binding protein of claim 50 to a subject in need thereof.
57. The method of claim 56, wherein the coronavirus infection is
caused by SARS-CoV-2.
58. The method of claim 56, wherein the coronavirus infection is
caused by a variant of SARS-CoV-2.
59. The method of claim 56, wherein the step of administering the
binding protein to the subject is repeated.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to recombinant binding
proteins comprising one or more designed ankyrin repeat domains
with binding specificity for coronavirus spike proteins, nucleic
acids encoding such proteins, pharmaceutical compositions
comprising such proteins or nucleic acids, and the use of such
proteins, nucleic acids or pharmaceutical compositions in the
treatment of coronavirus diseases, particularly diseases caused by
SARS-CoV-2.
BACKGROUND OF THE INVENTION
[0002] With a positive-stranded RNA genome of 28 to 32 kb, the
Coronaviridae are the largest enveloped RNA viruses. Coronaviruses
infect many different mammalian and avian species. They are
responsible for a variety of acute and chronic diseases of the
respiratory, hepatic, gastrointestinal, and neurological
systems.
[0003] The common cold is an example of a mild form of coronavirus
infection. The 2003 SARS outbreak and the 2012 MERS outbreaks were
both caused by coronaviruses. SARS-CoV-2 (also called 2019-nCoV) is
the virus strain that causes COVID-19.
[0004] Coronaviruses have four structural proteins, known as the
spike protein, envelope protein, membrane protein, and nucleocapsid
protein. The spike protein is the viral membrane protein
responsible for cell entry.
[0005] Coronaviruses make use of a densely glycosylated spike
protein to gain entry into host cells. The spike protein consists
of three subunits and is a trimeric class I fusion protein that
exists in a metastable prefusion conformation that undergoes a
substantial structural rearrangement to fuse the viral membrane
with the host cell membrane. This process is triggered when the S1
subunit binds to a host cell receptor. Receptor binding
destabilizes the prefusion trimer, resulting in shedding of the S1
subunit and transition of the S2 subunit to a stable post-fusion
conformation. To engage a host cell receptor, the receptor-binding
domain (RBD) of S1 undergoes hinge-like conformational movements
that transiently hide or expose the determinants of receptor
binding. These two states are referred to as the "down"
conformation and the "up" conformation, where down corresponds to
the receptor-inaccessible state and up corresponds to the receptor
accessible state, which is thought to be less stable. Once the
spike protein is in the "up" conformation, binding to the
angiotensin-converting enzyme 2 (ACE2) receptor in the host cell
can occur, allowing the virus into the cell. "Activation" of the
spike protein to the "up" conformation can be carried out by
enzymes such as furin or TMPRSS2 which act by opening the spike
protein, allowing the nucleocapsid protein out of the viral capsid
and into the cell, resulting in infection.
[0006] Once the cell is infected with the coronavirus, treatment
options become more difficult as the immune system (or therapeutic
agent) must only target virus-infected cells, without damaging
non-infected cells. Because of the indispensable function of the
spike protein, it represents a target for antibody-mediated
neutralization. Thus, one approach to coronavirus therapy is to
inhibit binding of the virus to the cell by neutralizing the spike
proteins, preventing infection of the cell.
[0007] DARPin.RTM. proteins are genetically engineered ankyrin
repeat proteins, which can function like antibody mimetic proteins,
typically exhibiting highly specific and high-affinity target
binding. DARPin.RTM. proteins comprise one or more designed ankyrin
repeat domains. Designed ankyrin repeat domains are derived from
natural ankyrin repeat proteins and each designed ankyrin repeat
domain typically binds a target protein with high specificity and
affinity. Due to their high specificity, stability, potency and
affinity and due to their flexibility in formatting to generate
mono-, bi- or multi-specific proteins, DARPin.RTM. proteins are
attractive therapeutic agents for a wide variety of clinical
applications. For example, WO 2011/135067 describes DARPin.RTM.
proteins for use in the treatment of cancer and other pathological
conditions including eye diseases such as age-related macular
degeneration. DARPin.RTM. is a registered trademark owned by
Molecular Partners AG.
[0008] The technical problem underlying the present invention is
identifying novel recombinant binding proteins comprising one or
more designed ankyrin repeat domains with binding specificity for
coronavirus, preferably SARS-CoV-2. Such recombinant binding
proteins may be useful for inhibiting binding of the coronavirus to
cells and for preventing viral infection of cells. Such recombinant
binding proteins and pharmaceutical compositions comprising such
proteins may further be useful for methods of preventing, treating
or diagnosing coronavirus diseases, such as coronavirus diseases
caused by SARS-CoV-2, and/or for methods of detecting coronavirus,
preferably SARS-CoV-2.
SUMMARY OF THE INVENTION
[0009] Based on the disclosure provided herein, those skilled in
the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific
embodiments of the invention described herein. Such equivalents are
intended to be encompassed by the following embodiments (E).
[0010] 1. In a first embodiment, the present invention relates to a
recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain comprises an amino
acid sequence that has at least about 90% sequence identity with an
ankyrin repeat domain selected from the group consisting of SEQ ID
NOs 1 to 11, 76, 77 and 85.
[0011] 1a. In embodiment 1a, the present invention relates to a
recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain comprises an amino
acid sequence that has at least about 90% sequence identity with an
ankyrin repeat domain selected from the group consisting of SEQ ID
NOs 1 to 11.
[0012] 1b. In embodiment 1b, the present invention relates to a
recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain comprises an amino
acid sequence that has at least about 90% sequence identity with an
ankyrin repeat domain selected from the group consisting of SEQ ID
NOs 1 to 11, 76 and 77.
[0013] 2. In a second embodiment, the present invention relates to
the recombinant binding protein according to embodiment 1, wherein
said first ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76, 77 and 85.
[0014] 2a. In embodiment 2a, the present invention relates to the
recombinant binding protein according to embodiment 1a, wherein
said first ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11.
[0015] 2b. In embodiment 2b, the present invention relates to the
recombinant binding protein according to embodiment 1b, wherein
said first ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76 and 77.
[0016] 3. In a third embodiment, the present invention relates to
the recombinant binding protein according to embodiment 1, wherein
said first ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11, 76, 77 and 85.
[0017] 3a. In embodiment 3a, the present invention relates to the
recombinant binding protein according to embodiment 1a, wherein
said first ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11.
[0018] 3b. In embodiment 3b, the present invention relates to the
recombinant binding protein according to embodiment 1b, wherein
said first ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11, 76 and 77.
[0019] 4. In a fourth embodiment, the present invention relates to
the recombinant binding protein according to any one of embodiments
1 to 3 further comprising a second ankyrin repeat domain, wherein
said second ankyrin repeat domain comprises an amino acid sequence
that has at least about 90% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76, 77 and 85.
[0020] 4a. In embodiment 4a, the present invention relates to the
recombinant binding protein according to any one of embodiments 1a,
2a or 3a further comprising a second ankyrin repeat domain, wherein
said second ankyrin repeat domain comprises an amino acid sequence
that has at least about 90% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11.
[0021] 4b. In embodiment 4b, the present invention relates to the
recombinant binding protein according to any one of embodiments 1b,
2b or 3b further comprising a second ankyrin repeat domain, wherein
said second ankyrin repeat domain comprises an amino acid sequence
that has at least about 90% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76 and 77.
[0022] 5. In a fifth embodiment, the present invention relates to
the recombinant binding protein according to embodiment 4, wherein
said second ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76, 77 and 85.
[0023] 5a. In embodiment 5a, the present invention relates to the
recombinant binding protein according to embodiment 4a, wherein
said second ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11.
[0024] 5b. In embodiment 5b, the present invention relates to the
recombinant binding protein according to embodiment 4b, wherein
said second ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76 and 77.
[0025] 6. In a sixth embodiment, the present invention relates to
the recombinant binding protein according to embodiment 4, wherein
said second ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11, 76, 77 and 85.
[0026] 6a. In embodiment 6a, the present invention relates to the
recombinant binding protein according to embodiment 4a, wherein
said second ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11.
[0027] 6b. In embodiment 6b, the present invention relates to the
recombinant binding protein according to embodiment 4b, wherein
said second ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11, 76 and 77.
[0028] 7. In a seventh embodiment, the present invention relates to
the recombinant binding protein according to any one of embodiments
4 to 6 further comprising a third ankyrin repeat domain, wherein
said third ankyrin repeat domain comprises an amino acid sequence
that has at least about 90% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76, 77 and 85.
[0029] 7a. In embodiment 7a, the present invention relates to the
recombinant binding protein according to any one of embodiments 4a,
5a or 6a further comprising a third ankyrin repeat domain, wherein
said third ankyrin repeat domain comprises an amino acid sequence
that has at least about 90% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11.
[0030] 7b. In embodiment 7b, the present invention relates to the
recombinant binding protein according to any one of embodiments 4b,
5b or 6b further comprising a third ankyrin repeat domain, wherein
said third ankyrin repeat domain comprises an amino acid sequence
that has at least about 90% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76 and 77.
[0031] 8. In an eighth embodiment, the present invention relates to
the recombinant binding protein according to embodiment 7, wherein
said third ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76, 77 and 85.
[0032] 8a. In embodiment 8a, the present invention relates to the
recombinant binding protein according to embodiment 7a, wherein
said third ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11.
[0033] 8b. In embodiment 8b, the present invention relates to the
recombinant binding protein according to embodiment 7b, wherein
said third ankyrin repeat domain comprises an amino acid sequence
that has at least about 95% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76 and 77.
[0034] 9. In a ninth embodiment, the present invention relates to
the recombinant binding protein according to embodiment 7, wherein
said third ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11, 76, 77 and 85.
[0035] 9a. In embodiment 9a, the present invention relates to the
recombinant binding protein according to embodiment 7a, wherein
said third ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11.
[0036] 9b. In embodiment 9b, the present invention relates to the
recombinant binding protein according to embodiment 7b, wherein
said third ankyrin repeat domain is selected from the group
consisting of SEQ ID NOs 1 to 11, 76 and 77.
[0037] 10. In a tenth embodiment, the present invention relates to
the recombinant binding protein according to embodiment 7, 7a or
7b, wherein said first, second and third ankyrin repeat domains
comprise amino acid sequences and are arranged, from the N-terminus
to C-terminus, as follows:
[0038] (i) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 6, 1 and 3;
[0039] (ii) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 4, 2 and 1;
[0040] (iii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 4, 6 and 3;
[0041] (iv) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 6, 3 and 6;
[0042] (v) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 7, 3 and 6;
[0043] (vi) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 8, 4 and 1;
[0044] (vii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 6 and 7;
[0045] (viii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 4, 1 and 8;
[0046] (ix) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 3, 6 and 9;
[0047] (x) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 9, 3 and 6;
[0048] (xi) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 1, 6 and 9;
[0049] (xii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 9, 6 and 1;
[0050] (xiii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 6, 9 and 10;
[0051] (xiv) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 9 and 11;
[0052] (xv) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 10, 9 and 6;
[0053] (xvi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 11, 9 and 3;
[0054] (xvii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 5, 1 and 3;
[0055] (xviii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 1, 2 and 5;
[0056] (xix) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 5 and 6;
[0057] (xx) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 6, 3 and 5;
[0058] (xxi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 7, 3 and 5;
[0059] (xxii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 8, 5 and 6;
[0060] (xxiii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 6, 10 and 11;
[0061] (xxiv) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 10 and 10;
[0062] (xxv) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 5, 6 and 9;
[0063] (xxvi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 9, 3 and 5;
[0064] (xxvii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 9, 6 and 5;
[0065] (xxviii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 5, 9 and 10;
[0066] (xxix) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 6, 9 and 11;
[0067] (xxx) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 10, 9 and 5;
[0068] (xxxi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 11, 9 and 6;
[0069] (xxxii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 76 and 77; or (xxxiii) amino
acid sequences having at least about 90% sequence identity with SEQ
ID NOs 3, 85 and 77.
[0070] 10a. In embodiment 10a, the present invention relates to the
recombinant binding protein according to embodiment 10 (xx).
[0071] 10b. In embodiment 10b, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 68.
[0072] 10c. In embodiment 10c, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 68.
[0073] 10d. In embodiment 10d, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 68.
[0074] 10e. In embodiment 10e, the present invention relates to the
recombinant binding protein according to embodiment 10
(xxviii).
[0075] 10f. In embodiment 10f, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 69.
[0076] 10g. In embodiment 10g, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 69.
[0077] 10h. In embodiment 10h, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 69.
[0078] 10i. In embodiment 10i, the present invention relates to the
recombinant binding protein according to embodiment 10 (xxxii).
[0079] 10j. In embodiment 10j, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 79.
[0080] 10k. In embodiment 10k, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 79.
[0081] 10l. In embodiment 10l, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 79.
[0082] 10m. In embodiment 10m, the present invention relates to the
recombinant binding protein according to embodiment 10
(xxxiii).
[0083] 10n. In embodiment 10n, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 89 to 91.
[0084] 10o. In embodiment 10o, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 89 to 91.
[0085] 10p. In embodiment 10p, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence selected from the group
consisting of SEQ ID NOs: 89 to 91.
[0086] 11. In an eleventh embodiment, the present invention relates
to the recombinant binding protein according to any one of
embodiments 1 to 10p, wherein said binding protein binds to a
coronavirus spike protein.
[0087] 12. In a twelfth embodiment, the present invention relates
to the recombinant binding protein according to embodiment 11,
wherein said spike protein is SARS-CoV-2 spike protein.
[0088] 13. In a thirteenth embodiment, the present invention
relates to the recombinant binding protein according to any one of
embodiments 11 and 12, wherein said first, second and/or third
ankyrin repeat domain binds said coronavirus spike protein with a
dissociation constant (K.sub.D) of or below about 100 nM.
[0089] 14. In a fourteenth embodiment, the present invention
relates to a recombinant binding protein comprising at least one
ankyrin repeat domain, wherein said ankyrin repeat domain binds a
coronavirus spike protein with a dissociation constant (K.sub.D) of
or below about 100 nM.
[0090] 15. In a fifteenth embodiment, the present invention relates
to the recombinant binding protein according to any preceding
embodiment further comprising at least one serum albumin binding
domain.
[0091] 16. In a sixteenth embodiment, the present invention relates
to the recombinant binding protein according to embodiment 15,
wherein said serum albumin binding domain comprises an amino acid
sequence that has at least about 90% sequence identity with a
sequence selected from the group consisting of SEQ ID NOs:
47-49.
[0092] 16a. In embodiment 16a, the present invention relates to a
recombinant binding protein according to any one of embodiments 15
and 16, wherein said recombinant binding protein has a terminal
half-life in mice of at least about 30 hours, preferably at least
about 35 hours, at least about 40 hours, or at least about 45
hours.
[0093] 17. In a seventeenth embodiment, the present invention
relates to a recombinant binding protein comprising a polypeptide,
wherein said polypeptide has an amino acid sequence that has at
least about 90% sequence identity with a sequence selected from the
group consisting of SEQ ID NOs: 12-42, 75, 84, 87 and 88.
[0094] 17a. In embodiment 17a, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 31.
[0095] 17b. In embodiment 17b, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 39.
[0096] 17c. In embodiment 17c, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 75.
[0097] 17d. In embodiment 17d, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 84.
[0098] 17e. In embodiment 17e, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 87.
[0099] 17f. In embodiment 17f, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 88.
[0100] 17g. In embodiment 17g, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 12-42.
[0101] 17h. In embodiment 17h, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 12-42 and 75.
[0102] 18. In an eighteenth embodiment, the present invention
relates to the recombinant binding protein according to embodiment
17, wherein said polypeptide has an amino acid sequence that has at
least about 95% sequence identity with a sequence selected from the
group consisting of SEQ ID NOs: 12-42, 75, 84, 87 and 88.
[0103] 18a. In embodiment 18a, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 31.
[0104] 18b. In embodiment 18b, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 39.
[0105] 18c. In embodiment 18c, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 75.
[0106] 18d. In embodiment 18d, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 84.
[0107] 18e. In embodiment 18e, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 87.
[0108] 18f. In embodiment 18f, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 95%
sequence identity with SEQ ID NO: 88.
[0109] 18g. In embodiment 18g, the present invention relates to the
recombinant binding protein according to embodiment 17g, wherein
said polypeptide has an amino acid sequence that has at least about
95% sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 12-42.
[0110] 18h. In embodiment 18h, the present invention relates to the
recombinant binding protein according to embodiment 17h, wherein
said polypeptide has an amino acid sequence that has at least about
95% sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 12-42 and 75.
[0111] 19. In a nineteenth embodiment, the present invention
relates to the recombinant binding protein according to embodiment
17, wherein said polypeptide has an amino acid sequence that is
selected from the group consisting of SEQ ID NOs: 12-42, 75, 84, 87
and 88.
[0112] 19a. In embodiment 19a, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 31.
[0113] 19b. In embodiment 19b, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 39.
[0114] 19c. In embodiment 19c, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 75.
[0115] 19d. In embodiment 19d, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 84.
[0116] 19e. In embodiment 19e, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 87.
[0117] 19f. In embodiment 19f, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has the amino acid sequence of SEQ ID NO: 88.
[0118] 19g. In embodiment 19g, the present invention relates to the
recombinant binding protein according to embodiment 17g, wherein
said polypeptide has an amino acid sequence that is selected from
the group consisting of SEQ ID NOs: 12-42.
[0119] 19h. In embodiment 19h, the present invention relates to the
recombinant binding protein according to embodiment 17h, wherein
said polypeptide has an amino acid sequence that is selected from
the group consisting of SEQ ID NOs: 12-42 and 75.
[0120] 20. In a twentieth embodiment, the present invention relates
to the recombinant binding protein according to any one of
embodiments 17 to 19h, wherein said binding protein binds to a
coronavirus spike protein.
[0121] 21. In a twenty-first embodiment, the present invention
relates to the recombinant binding protein according to embodiment
20, wherein said spike protein is SARS-CoV-2 spike protein.
[0122] 22. In a twenty-second embodiment, the present invention
relates to the recombinant binding protein according to any one of
embodiments 20 and 21, wherein said binding protein binds said
coronavirus spike protein with a dissociation constant (K.sub.D) of
or below about 100 nM.
[0123] 23. In a twenty-third embodiment, the present invention
relates to the recombinant binding protein according to any one of
embodiments 1 to 22, wherein said binding protein is capable of
inhibiting infection of cells by a coronavirus.
[0124] 24. In a twenty-fourth embodiment, the present invention
relates to the recombinant binding protein according to any one of
embodiments 1 to 22, wherein said binding protein is capable of
inhibiting infection of cells by SARS-CoV-2.
[0125] 25. In a twenty-fifth embodiment, the present invention
relates to a nucleic acid encoding a recombinant binding protein
according to any one of embodiments 1 to 24.
[0126] 25a. In embodiment 25a, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 70 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 70.
[0127] 25b. In embodiment 25b, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 71 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 71.
[0128] 25c. In embodiment 25c, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 72 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 72.
[0129] 25d. In embodiment 25d, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 73 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 73.
[0130] 25e. In embodiment 25e, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 74 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 74.
[0131] 25f. In embodiment 25f, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 80 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 80.
[0132] 25g. In embodiment 25g, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 81 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 81.
[0133] 25h. In embodiment 25h, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 82 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 82.
[0134] 25i. In embodiment 25i, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 83 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 83.
[0135] 25j. In embodiment 25j, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 78 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 78.
[0136] 25k. In embodiment 25k, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 86 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 86.
[0137] 25l. In embodiment 25l, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 92 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 92.
[0138] 25m. In embodiment 25m, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 93 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 93.
[0139] 25n. In embodiment 25n, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 94 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 94.
[0140] 25o. In embodiment 250, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 95 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 95.
[0141] 26. In a twenty-sixth embodiment, the present invention
relates to a host cell comprising the nucleic acid molecule of any
one of embodiments 25 to 250.
[0142] 27. In a twenty-seventh embodiment, the present invention
relates to a method of making the recombinant binding protein
according to any one of embodiments 1 to 24, comprising culturing
the host cell of embodiment 26 under conditions wherein said
recombinant binding protein is expressed.
[0143] 28. In a twenty-eighth embodiment, the present invention
relates to a pharmaceutical composition comprising the binding
protein of any one of embodiments 1 to 24 or the nucleic acid of
any one of embodiments 25 to 250, and a pharmaceutically acceptable
carrier or excipient.
[0144] 29. In a twenty-ninth embodiment, the present invention
relates to a method of treating a coronavirus infection in a
subject, the method comprising the step of administering an
effective amount of at least one binding protein according to any
one of embodiments 1 to 24, or of the nucleic acid of any one of
embodiments 25 to 250, or of the pharmaceutical composition
according to embodiment 28, to a subject in need thereof.
[0145] 29a. In embodiment 29a, the present invention relates to a
method of treating according to embodiment 29, wherein said method
is a therapeutic treatment method.
[0146] 29b. In embodiment 29b, the present invention relates to a
method of treating according to embodiment 29, wherein said method
is a prophylactic treatment method.
[0147] 29c. In embodiment 29c, the present invention relates to a
method of preventing a coronavirus infection in a subject, the
method comprising the step of administering an effective amount of
at least one binding protein according to any one of embodiments 1
to 24, or of the nucleic acid of any one of embodiments 25 to 250,
or of the pharmaceutical composition according to embodiment 28, to
a subject in need thereof.
[0148] 29d. In embodiment 29d, the present invention relates to at
least one binding protein according to any one of embodiments 1 to
24, or the nucleic acid of any one of embodiments 25 to 250, or the
pharmaceutical composition according to embodiment 28 for use in a
method of diagnosing a coronavirus infection in a subject.
[0149] 29e. In embodiment 29e, the present invention relates to a
method of diagnosing a coronavirus infection in a subject
comprising the steps of contacting a sample from the subject in
vitro or ex vivo with at least one binding protein according to any
one of embodiments 1 to 24.
[0150] 29f. In embodiment 29f, the present invention relates to a
method of detecting a coronavirus infection in a subject, said
method comprising:
[0151] a) obtaining a sample from a subject;
[0152] b) contacting said sample with at least one binding protein
according to any one of embodiments 1 to 24; and
[0153] c) detecting the presence of a coronavirus infection.
[0154] 29g. In embodiment 29g, the present invention relates to at
least one binding protein according to any one of embodiments 1 to
24, or of the nucleic acid of any one of embodiments 25 to 250, or
of the pharmaceutical composition according to embodiment 28 for
use in treating or preventing a coronavirus infection in a
subject.
[0155] 30. In a thirtieth embodiment, the present invention relates
to the method according to any one of embodiments 29 to 29g,
wherein the coronavirus infection is caused by SARS-CoV-2.
[0156] 31. In a thirty-first embodiment, the present invention
relates to the method according to any one of embodiments 29, 29a,
29b, 29c, 29e, 29f, 29g and 30, or the use according to embodiment
29d wherein said subject is a human.
BRIEF DESCRIPTION OF THE FIGURES
[0157] FIG. 1: 2019-nCoV spike protein protomer showing the
proposed binding sites for different ankyrin repeat proteins
(DARPin.RTM. proteins).
[0158] FIG. 2: 2019-nCoV spike protein protomer in down
conformation.
[0159] FIG. 3: 2019-nCoV spike protein protomer in up conformation,
showing the hACE2 binding site elevated. hACE2 is thought to bind
to the up conformation of the spike protein, but not to the down
conformation.
[0160] FIG. 4: 2019-nCoV spike protein protomer, indicating the
location of the hACE2 binding site, S1/S2 cleavage site and S2'
cleavage site. During molecular maturation, the spike protein
trimerizes and is cleaved at the S1/S2 site. It is displayed at the
membrane as a non-covalent complex. A concerted action of
receptor-binding and proteolytic processing of the spike protein is
required for membrane fusion. An initial energy barrier for
conformational transition is necessary. Without wishing to be bound
by theory, this energy barrier is overcome by (i) binding to the
hACE2 receptor; and (ii) proteolytic priming at the S2' site. The
interaction with ACE2 at the host cell surface is believed to
trigger the cleavage of the S2' site. This cleavage has been
proposed to activate the protein for membrane fusion via extensive
irreversible conformational changes.
[0161] FIG. 5: SARS-CoV-2 VSV pseudotype virus inhibition at 100 nM
of various recombinant binding proteins comprising a single ankyrin
repeat domain that binds to the spike protein (mono-domain and
mono-paratopic DARPin.RTM. binding proteins). Shorter bars are
indicative of stronger virus inhibition.
[0162] FIG. 6: Representative SPR (surface plasmon resonance) trace
of a recombinant binding protein comprising a single ankyrin repeat
domain that binds to the spike protein (mono-domain and
mono-paratopic DARPin.RTM. binding protein).
[0163] FIG. 7: SARS-CoV-2 VSV pseudotype virus inhibition at 100 nM
of various recombinant binding proteins comprising three ankyrin
repeat domains that bind to the spike protein (multi-domain and
multi-paratopic DARPin.RTM. binding proteins). Shorter bars are
indicative of stronger virus inhibition.
[0164] FIG. 8: SARS-CoV-2 VSV pseudotype virus inhibition at 1 nM
of various recombinant binding proteins comprising three ankyrin
repeat domains that bind to the spike protein (multi-domain and
multi-paratopic DARPin.RTM. binding proteins). Shorter bars are
indicative of stronger virus inhibition.
[0165] FIGS. 9a-c: SPR (surface plasmon resonance) trace of
recombinant binding proteins comprising a single ankyrin repeat
domain that binds to the spike protein. Four or five concentration
SPR fitted curves confirm the high binding affinity of these
mono-domain, mono-paratopic DARPin.RTM. binding proteins (e.g. in
the double-digit pM range). In FIG. 9c, the upper panel represents
SEQ ID NO: 9 and the lower panel represents SEQ ID NO: 10.
[0166] FIG. 10: Fluorescence microscopy image showing GFP positive
Vero E06 cells which were infected with the GFP-labeled VSV
pseudotype SARS-CoV-2 virus. DARPin.RTM. constructs ALE043 (SEQ ID
NO: 25) and vS07_M101 E04 do not show any infected cells in well 1
(at 100 nM concentration) and well 6 (at 3.125 nM) while there is
infection of the Vero E06 cells with the GFP-labeled VSV pseudotype
SARS-CoV-2 virus visible in wells 1 and 6 for the isotype negative
control (his-tagged MP0250). At lower DARPin.RTM. protein
concentrations in well 12 (0.049 nM) infected Vero E06 cells (GFP
positive) are visible for all constructs.
[0167] FIG. 11: Neutralization of VSV pseudotype SARS-CoV-2 virus
by multi-domain DARPin.RTM. binding proteins. The names of the
tested constructs (ALE030, ALE031, etc.) are indicated in the
Figure.
[0168] FIG. 12: Neutralization of VSV pseudotype SARS-CoV-2 virus
by multi-domain DARPin.RTM. binding proteins. The names of the
tested constructs (ALE030, ALE033, etc.) are indicated in the
Figure.
[0169] FIG. 13: A map of the test plates used in Example 4 with
border zones around the edge and triplicate wells for each dilution
value from 0.0064 to 100 nm, and control wells.
[0170] FIGS. 14a-f: Photographs of the test plates obtained from
Example 4.
[0171] FIGS. 15a-b: Characterization of ALE033 (see Table 5, sample
3). FIG. 15a is an SPR (surface plasmon resonance) trace showing
high affinity binding to the coronavirus spike protein. No loss of
target binding was observed over time. FIG. 15b shows a size
exclusion chromatography (SEC) profile (molar mass vs time). No
aggregates or oligomers were observed. No unfolding was detectable
up to 85.degree. C. on CD (circular dichroism) spectra (not
provided).
[0172] FIG. 16: SPR (surface plasmon resonance) trace for ALE030
(see Table 5, sample 1).
[0173] FIG. 17: SPR (surface plasmon resonance) trace for ALE038
(see Table 5, sample 7).
[0174] FIGS. 18a-d: Photographs of the test plates obtained from
Example 5.
[0175] FIG. 19: Cell protection as measured with CellTiter-Glo.RTM.
luminescent cell viability assay (Promega), see Example 7.
[0176] FIG. 20: Photographs of the test plates obtained following
violet crystal staining, see Example 8.
[0177] FIG. 21a-c: Cell protection as measured with
CellTiter-Glo.RTM. luminescent cell viability assay (Promega), see
Example 8.
[0178] FIGS. 22a-b: (A) Molecular model of ALE049 (yellow:
HSA-binding domains; cyan, blue and magenta: RBD-binding domains)
bound to the spike ectodomain (gray) of SARS-CoV-2. (B) Molecular
model of ALE058 (yellow: HSA-binding domains; blue: RBD-binding
domain; green: S1-NTD-binding domain; red: S2-binding domain) bound
to the spike ectodomain (grey) of SARS-CoV-2.
[0179] FIG. 23: Neutralization of SARS-CoV-2 VSV pseudotype virus
with multi-specific binding proteins ALE049 and ALE058, see Example
8.
[0180] FIG. 24: ELISA method as used in Example 9.
[0181] FIG. 25: Mean serum concentration data for ALE033, ALE048
and ALE049, see Example 9.
[0182] FIGS. 26a-e: Efficacy of ALE049 in treating SARS-CoV-2
infection in a preventative Syrian gold hamster model, see Example
10.
[0183] FIG. 27: Representative histopathology microscopic pictures
of hamster lung tissue taken at day 4. Left panel: healthy hamster
lung tissue of an animal treated with 1600 .mu.g ALE049 (group 1);
right panel: diseased lung tissue of an animal which received the
placebo injection (group 4).
[0184] FIG. 28: Structural visualization of mutations of the
SARS-CoV-2 spike protein evaluated in Examples 11 and 12. A)
Representation of the full trimeric SARS-CoV-2 spike protein with
all residues analyzed in the Pseudovirus neutralization assay
visualized as blue spheres. Binding regions for the individual
DARPin.RTM. domains incorporated in ALE049 and ALE109 are colored
in blue (RBD), green (NTD) and red (S2); B) monomeric spike protein
structure representing the variant first identified in the UK
B.1.1.7 (del69-70, del145, N501Y, A570D, D614G, P681H, T7161,
S982A, D1118H); C) monomeric spike protein structure representing
the variant first identified in South Africa B.1.351 (D80A, D215G,
E484K, N501Y, A701V). The PDB file 6xcn was used for generating the
figures with PyMol version 2.1.1 (Schrodinger, LLC). In order to
visualize all mutations, the loops 518-520, 676-689, 811-813 and
the regions of the NTD domain missing in the cryo-EM structure,
were modelled with MODELLER included in the BIOVIA Discovery Studio
software using the PDB file 6zge as template for the NTD domain
(BIOVIA, Dassault Systemes, BIOVIA Discovery Studio 2021).
[0185] FIG. 29: (A) A visual representation of the ALE109
constructs generated for knock out experiments. For each knock out
(k.o.) construct, the indicated SARS-CoV-2-binding DARPin.RTM.
domain was replaced with a non-binding DARPin.RTM. domain. HSA:
HSA-binding DARPin.RTM. domain, RBD: RBD-binding DARPin.RTM.
domain, NTD: NTD-binding DARPin.RTM. domain, S2: S2-binding
DARPin.RTM. domain, see Example 11. (B) Neutralization profiles of
ALE109 and k.o. constructs against VSV-SARS-CoV-2 pseudoviruses
expressing the wild-type spike protein. (C) Upper panel: protective
effect of DARPin.RTM. molecules against SARS-CoV-2 (100
pfu)-mediated cytopathic effect. Depicted are the percentage of
cell protection conferred by ALE109 or the k.o. constructs. Cell
protection was determined after 3 days of incubation by measuring
intracellular ATP levels in a cell viability assay using Cell
Titer-Glo. Lower panel: inhibition of SARS-CoV-2 viral replication
quantified by real-time RT-PCR and expressed as percentage of viral
genome equivalents present in the supernatant of Vero E6 cells
exposed to 100 pfu SARS-CoV-2 with increasing amounts of ALE109 or
k.o. constructs. (D) IC.sub.50/E.sub.50 values and potency ranking
of the constructs analyzed.
[0186] FIG. 30: Schematic representation of the procedure of
Example 12.
[0187] FIG. 31: Tables showing the cytopathic effects observed in
Example 12. The DARPin.RTM. binding protein R1b is called RBD-2 in
this Figure.
[0188] FIG. 32: Neutralization of VSV pseudotype SARS-CoV-2 virus
by multi-domain DARPin.RTM. binding proteins. The names of the
tested constructs (ALE049, ALE058, etc.) are indicated in the
Figure.
[0189] FIG. 33: Mean serum concentration-time profile of ALE058 in
BALB/c mice following administration of 1 mg/kg.
[0190] FIG. 34: Mean serum concentration-time profile of ALE109,
ALE126, ALE129, and ALE133 in BALB/c mice following administration
of 1 mg/kg.
[0191] FIG. 35. Schematic study outline. Body weight and
temperature were measured daily and swabs, blood and tissues were
collected from 3 animals for each group, which were euthanized at
day 3 and day 5, respectively.
[0192] FIG. 36. Average and SEM of body weight measurements of all
five study groups over the time course from day 0 to day 5.
[0193] FIG. 37a to 37d: Virus quantification by live virus
titration of lung homogenate at day 3 (A) and at day 5 (B) and by
qPCR measurement of genome copies in the lung at day 3 (C) and at
day 5 (D), of three animals for each of the time points.
[0194] FIG. 38a to 38d: Sum of the averaged histopathological
scores grouped into four categories for signs of inflammation (A),
affected blood vessels (B), alveoli (C) or bronchi (D).
DETAILED DESCRIPTION OF THE INVENTION
[0195] Overview Disclosed herein are recombinant binding proteins
comprising one or more designed ankyrin repeat domains with binding
specificity for coronavirus spike proteins, particularly SARS-CoV-2
spike proteins. Also disclosed are nucleic acids encoding the
binding proteins, pharmaceutical compositions comprising the
binding proteins or nucleic acids, and methods of using the binding
proteins, nucleic acids, or pharmaceutical compositions.
[0196] The recombinant binding proteins according to the present
invention bind to the coronavirus spike protein at one or more
binding sites, thereby neutralizing the virus. These binding sites
are illustrated in FIG. 1. In one embodiment, the recombinant
binding proteins bind to three sites on the spike protein.
[0197] Without wishing to be bound by theory, the designed ankyrin
repeat proteins of the present invention are believed to act by (i)
inhibiting receptor binding; (ii) providing allosteric inhibition
of spike protein conformational change; and/or (iii) blocking
protease sites needed for spike protein activation. As shown in
FIG. 1, designed ankyrin repeat domain 1 (DARPin.RTM. 1) is
understood to act by blocking angiotensin-converting enzyme 2
(ACE2) receptor binding. Designed ankyrin repeat domains 1 and 2
(DARPin.RTM.1 and 2) are further understood to act by preventing
conformational change in the spike protein, effectively locking the
spike protein in the closed configuration. Designed ankyrin repeat
domain 3 (DARPin.RTM.3) is understood to further inhibit
conformational change and to block protease binding. These designed
ankyrin repeat domains can bind and/or inhibit the spike protein as
individual proteins. Multi-epitope targeting by multi-domain,
multi-specific proteins is believed to provide even more potent
neutralization of the spike proteins, and to minimise the
likelihood of escape mutations.
[0198] Further advantages to the described designed ankyrin repeat
proteins are that they may reduce the incidence of Acute Lung
Inflammation (ALI) due to lack of Fc-mediated macrophage or
complement activation (as described by Liu et al., JCI Insight,
2019 4(4):e123158). Designed ankyrin repeat proteins may also
address epitopes which are not accessible with monoclonal
antibodies.
[0199] Further advantages to the described designed ankyrin repeat
proteins are that they have low immunogenic potential and no
off-target effects. DARPin.RTM. candidates also display favorable
development properties including rapid, low-cost and high-yield
manufacturing and up to several years of shelf-life at 4.degree.
C.
Definitions
[0200] Unless otherwise defined herein, 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 used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well-known
and commonly used in the art.
[0201] The terms "comprising", "having", "including" and
"containing" are to be construed as open-ended terms unless
otherwise noted. If aspects of the invention are described as
"comprising" a feature, embodiments also are contemplated
"consisting of" or "consisting essentially of" the feature. The use
of any and all examples, or exemplary language (e.g., "such as")
provided herein, is intended merely to better illustrate the
disclosure and does not pose a limitation on the scope of the
disclosure unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the disclosure. Other than
in the operating examples, or where otherwise indicated, all
numbers expressing quantities of ingredients or reaction conditions
used herein should be understood as modified in all instances by
the term "about" as that term would be interpreted by the person
skilled in the relevant art. The term "about" as used herein is
equivalent to .+-.10% of a given numerical value, unless otherwise
stated.
[0202] Recitation of ranges of values herein are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range and each endpoint, unless
otherwise indicated herein, and each separate value and endpoint is
incorporated into the specification as if it were individually
recited herein.
[0203] The term "nucleic acid" or "nucleic acid molecule" refers to
a polynucleotide molecule, which may be a ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) molecule, either single stranded or
double stranded, and includes modified and artificial forms of DNA
or RNA. A nucleic acid molecule may either be present in isolated
form or be comprised in recombinant nucleic acid molecules or
vectors.
[0204] In the context of the present invention the term "protein"
refers to a molecule comprising a polypeptide, wherein at least
part of the polypeptide has, or is able to acquire, a defined
three-dimensional arrangement by forming secondary, tertiary,
and/or quaternary structures within a single polypeptide chain
and/or between multiple polypeptide chains. If a protein comprises
two or more polypeptide chains, the individual polypeptide chains
may be linked non-covalently or covalently, e.g. by a disulfide
bond between two polypeptides. A part of a protein, which
individually has, or is able to acquire, a defined
three-dimensional arrangement by forming secondary and/or tertiary
structure, is termed "protein domain". Such protein domains are
well known to the practitioner skilled in the art.
[0205] The term "recombinant" as used in recombinant protein,
recombinant polypeptide and the like, means that said protein or
polypeptide is produced by the use of recombinant DNA technologies
well known to the practitioner skilled in the art. For example, a
recombinant DNA molecule (e.g. produced by gene synthesis) encoding
a polypeptide can be cloned into a bacterial expression plasmid
(e.g. pQE30, QIAgen), yeast expression plasmid, mammalian
expression plasmid, or plant expression plasmid, or a DNA enabling
in vitro expression. If, for example, such a recombinant bacterial
expression plasmid is inserted into appropriate bacteria (e.g.
Escherichia coli), these bacteria can produce the polypeptide(s)
encoded by this recombinant DNA. The correspondingly produced
polypeptide or protein is called a recombinant polypeptide or
recombinant protein.
[0206] In the context of the present invention, the term "binding
protein" refers to a protein comprising a binding domain. A binding
protein may also comprise two, three, four, five or more binding
domains. Preferably, said binding protein is a recombinant binding
protein. More preferably, the binding proteins of the instant
invention comprise an ankyrin repeat domain with binding
specificity for a coronavirus spike protein.
[0207] The term "target" refers to an individual molecule such as a
nucleic acid molecule, a peptide, polypeptide or protein, a
carbohydrate, or any other naturally occurring molecule, including
any part of such individual molecule, or to complexes of two or
more of such molecules, or to a whole cell or a tissue sample, or
to any non-natural compound. Preferably, a target is a naturally
occurring or non-natural polypeptide or protein, or a polypeptide
or protein containing chemical modifications, for example,
naturally occurring or non-natural phosphorylation, acetylation, or
methylation.
[0208] In the context of the present invention, the term
"polypeptide" relates to a molecule consisting of a chain of
multiple, i.e. two or more, amino acids linked via peptide bonds.
Preferably, a polypeptide consists of more than eight amino acids
linked via peptide bonds. The term "polypeptide" also includes
multiple chains of amino acids, linked together by S-S bridges of
cysteines. Polypeptides are well-known to the person skilled in the
art.
[0209] Patent application WO2002/020565 and Forrer et al., 2003
(Forrer, P., Stumpp, M. T., Binz, H. K., Pluckthun, A., 2003. FEBS
Letters 539, 2-6), contain a general description of repeat protein
features and repeat domain features, techniques and applications.
The term "repeat protein" refers to a protein comprising one or
more repeat domains. Preferably, a repeat protein comprises one,
two, three, four, five or six repeat domains. Furthermore, said
repeat protein may comprise additional non-repeat protein domains,
polypeptide tags and/or peptide linkers. The repeat domains can be
binding domains.
[0210] The term "repeat domain" refers to a protein domain
comprising two or more consecutive repeat modules as structural
units, wherein said repeat modules have structural and sequence
homology. Preferably, a repeat domain also comprises an N-terminal
and/or a C-terminal capping module. For clarity, a capping module
can be a repeat module. Such repeat domains, repeat modules, and
capping modules, sequence motives, as well as structural homology
and sequence homology are well known to the practitioner in the art
from examples of ankyrin repeat domains (Binz et al., J. Mol. Biol.
332, 489-503, 2003; Binz et al., Nature Biotech. 22(5): 575-582
(2004); WO2002/020565; WO2012/069655), leucine-rich repeat domains
(WO2002/020565), tetratricopeptide repeat domains (Main, E. R.,
Xiong, Y., Cocco, M. J., D'Andrea, L., Regan, L., Structure 11(5),
497-508, 2003), and armadillo repeat domains (WO2009/040338). It is
further well known to the practitioner in the art, that such repeat
domains are different from proteins comprising repeated amino acid
sequences, where every repeated amino acid sequence is able to form
an individual domain (for example FN3 domains of Fibronectin).
[0211] The term "ankyrin repeat domain" refers to a repeat domain
comprising two or more consecutive ankyrin repeat modules as
structural units, wherein said ankyrin repeat modules have
structural and sequence homology.
[0212] The term "designed" as used in designed repeat protein,
designed repeat domain and the like refers to the property that
such repeat proteins and repeat domains, respectively, are man-made
and do not occur in nature. The binding proteins of the instant
invention are designed repeat proteins and they comprise at least
one designed repeat domain. Preferably, the designed repeat domain
is a designed ankyrin repeat domain.
[0213] The term "target interaction residues" refers to amino acid
residues of a repeat module, which contribute to the direct
interaction with a target.
[0214] The terms "framework residues" or "framework positions"
refer to amino acid residues of a repeat module, which contribute
to the folding topology, i.e. which contribute to the fold of said
repeat module or which contribute to the interaction with a
neighboring module. Such contribution may be the interaction with
other residues in the repeat module, or the influence on the
polypeptide backbone conformation as found in .alpha.-helices or
.beta.-sheets, or the participation in amino acid stretches forming
linear polypeptides or loops. Such framework and target interaction
residues may be identified by analysis of the structural data
obtained by physicochemical methods, such as X-ray crystallography,
NMR and/or CD spectroscopy, or by comparison with known and related
structural information well known to practitioners in structural
biology and/or bioinformatics.
[0215] The term "repeat modules" refers to the repeated amino acid
sequence and structural units of the designed repeat domains, which
are originally derived from the repeat units of naturally occurring
repeat proteins. Each repeat module comprised in a repeat domain is
derived from one or more repeat units of a family or subfamily of
naturally occurring repeat proteins, preferably the family of
ankyrin repeat proteins. Furthermore, each repeat module comprised
in a repeat domain may comprise a "repeat sequence motif" deduced
from homologous repeat modules obtained from repeat domains
selected on a target, e.g. as described in Example 1, and having
the same target specificity.
[0216] Accordingly, the term "ankyrin repeat module" refers to a
repeat module, which is originally derived from the repeat units of
naturally occurring ankyrin repeat proteins. Ankyrin repeat
proteins are well known to the person skilled in the art.
[0217] Repeat modules may comprise positions with amino acid
residues which have not been randomized in a library for the
purpose of selecting target-specific repeat domains
("non-randomized positions" or "fixed positions" used
interchangeably herein) and positions with amino acid residues
which have been randomized in the library for the purpose of
selecting target-specific repeat domains ("randomized positions").
The non-randomized positions comprise framework residues. The
randomized positions comprise target interaction residues. "Have
been randomized" means that two or more amino acids were allowed at
an amino acid position of a repeat module, for example, wherein any
of the usual twenty naturally occurring amino acids were allowed,
or wherein most of the twenty naturally occurring amino acids were
allowed, such as amino acids other than cysteine, or amino acids
other than glycine, cysteine and proline.
[0218] The term "repeat sequence motif" refers to an amino acid
sequence, which is deduced from one or more repeat modules.
Preferably, said repeat modules are from repeat domains having
binding specificity for the same target. Such repeat sequence
motifs comprise framework residue positions and target interaction
residue positions. Said framework residue positions correspond to
the positions of framework residues of the repeat modules.
Likewise, said target interaction residue positions correspond to
the positions of target interaction residues of the repeat modules.
Repeat sequence motifs comprise non-randomized positions and
randomized positions.
[0219] The term "repeat unit" refers to amino acid sequences
comprising sequence motifs of one or more naturally occurring
proteins, wherein said "repeat units" are found in multiple copies,
and exhibit a defined folding topology common to all said motifs
determining the fold of the protein. Examples of such repeat units
include leucine-rich repeat units, ankyrin repeat units, armadillo
repeat units, tetratricopeptide repeat units, HEAT repeat units,
and leucine-rich variant repeat units.
[0220] The term "ankyrin repeat domain" refers to a domain that
comprises at least one ankyrin repeat motif, which is originally
derived from the repeat units of naturally occurring ankyrin repeat
proteins. In general, the ankyrin repeat motif comprises about 33
residues that form two alpha helices, separated by loops. Ankyrin
repeat proteins are known in the art. See, for example,
International Patent Publication Nos. WO 2002/020565, WO
2010/060748, WO 2011/135067, WO 2012/069654, WO 2012/069655, WO
2014/001442, WO 2014/191574, WO 2014/083208, WO 2016/156596, and WO
2018/054971, all of which are incorporated by reference in their
entireties. Ankyrin repeat domains optionally further comprise
appropriate capping modules.
[0221] Ankyrin repeat domains may be modularly assembled into
larger ankyrin repeat proteins according to the present disclosure,
optionally with half-life extension domains, using standard
recombinant DNA technologies (see, e.g., Forrer, P., et al., FEBS
letters 539, 2-6, 2003, WO 2012/069655, WO 2002/020565).
[0222] An ankyrin repeat domain "specifically binds" or
"preferentially binds" (used interchangeably herein) to a target if
it reacts or associates more frequently, more rapidly, with greater
duration and/or with greater affinity with a particular target
(e.g., cell or substance) than it does with alternative targets
(e.g., cells or substances). For example, an ankyrin repeat domain
that specifically binds to coronavirus spike protein is an ankyrin
repeat domain that binds coronavirus spike protein with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other non-coronavirus spike proteins. It is also
understood by reading this definition that, for example, an ankyrin
repeat domain which specifically or preferentially binds to a first
target may or may not specifically or preferentially bind to a
second target. As such, "specific binding" does not necessarily
require (although it can include) exclusive binding. In general,
under designated assay conditions, an ankyrin repeat domain binds
preferentially to a particular target molecule and does not bind in
a significant amount to other components present in a test
sample.
[0223] A variety of assay formats may be used to select or
characterize an ankyrin repeat domain that specifically binds a
molecule of interest. For example, solid-phase ELISA immunoassay,
immunoprecipitation, BIAcore.TM. (GE Healthcare, Piscataway, N.J.),
fluorescence-activated cell sorting (FACS), Octet.TM. (ForteBio,
Inc., Menlo Park, Calif.) and Western blot analysis are among many
assays that may be used to identify an ankyrin repeat domain that
specifically reacts with a target. Typically, a specific or
selective reaction will be at least twice background signal or
noise and more typically more than 10 times background. Even more
specifically, an ankyrin repeat domain is said to "specifically
bind" a target when the equilibrium dissociation constant (K.sub.D)
value is <1 .mu.M, such as <100 nM, <10 nM, <1 nM,
<100 .mu.M, <10 .mu.M, or <1 .mu.M.
[0224] The K.sub.D value is often referred to as binding affinity.
Binding affinity measures the strength of the sum total of
non-covalent interactions between contact residue(s) of one binding
partner and contact residue(s) of its binding partner. Unless
indicated otherwise, as used herein, binding affinity refers to
binding affinity that reflects a 1:1 interaction between members of
a binding pair or binding partners. In case of a binding protein
comprising two binding domains for one binding partner, binding
affinity may refer to binding affinity that reflects a 1:2
interaction between the binding protein and the binding
partner.
[0225] A variety of methods of measuring binding affinity are known
in the art, any of which can be used for purposes of the present
invention. For example, as exemplified herein, the binding affinity
can be expressed as K.sub.D value, which refers to the dissociation
rate of a particular ankyrin repeat domain and its binding target.
K.sub.D is the ratio of the rate of dissociation, also called the
"off-rate (K.sub.off)", to the association rate, or "on-rate
(K.sub.on)". Thus, K.sub.D equals K.sub.off/K.sub.on and is
expressed as a molar concentration (M), and the smaller the
K.sub.D, the stronger the affinity of binding.
[0226] K.sub.D values can be determined using any suitable method.
One exemplary method for measuring K.sub.D is surface plasmon
resonance (SPR) (see, e.g., Nguyen et al. Sensors (Basel). 2015 May
5; 15(5):10481-510). K.sub.D value may be measured by SPR using a
biosensor system such as a BIACORE.RTM. system. BIAcore kinetic
analysis comprises analyzing the binding and dissociation of an
antigen from chips with immobilized molecules (e.g., molecules
comprising epitope binding domains), on their surface. Another
method for determining the K.sub.D of a protein is by using
Bio-Layer Interferometry (see, e.g., Shah et al. J Vis Exp. 2014;
(84): 51383). K.sub.D value may be measured using OCTET.RTM.
technology (Octet QKe system, ForteBio). Alternatively, or in
addition, a KinExA.RTM. (Kinetic Exclusion Assay) assay, available
from Sapidyne Instruments (Boise, Id.) can also be used. Any method
suitable for assessing the binding affinity between two binding
partners is encompassed herein. Surface plasmon resonance (SPR) is
particularly preferred. Most preferably, the K.sub.D values are
determined in PBS and by SPR.
[0227] The term "PBS" means a phosphate buffered water solution
containing 137 mM NaCl, 10 mM phosphate and 2.7 mM KCl and having a
pH of 7.4.
[0228] The term "treat," as well as words related thereto, does not
necessarily imply 100% or complete cure. Rather, there are varying
degrees of treatment of which one of ordinary skill in the art
recognizes as having a potential benefit or therapeutic effect. In
this respect, the methods of treating coronavirus infections
described herein can provide any amount or any level of treatment.
Furthermore, the treatment provided by the method of the present
disclosure can include treatment of (i.e., relief from) one or more
conditions or symptoms. In exemplary aspects, the methods treat by
way increasing the survival of the subject. The term "treatment"
also includes prophylactic (preventive) treatment.
[0229] Therapeutic responses in any given disease or condition can
be determined by standardized response criteria specific to that
disease or condition. The subject undergoing therapy may experience
the beneficial effect of an improvement in the symptoms associated
with the disease.
[0230] Recombinant Binding Proteins that Target Coronavirus Spike
Proteins
[0231] Described herein are recombinant binding proteins comprising
one, two, three or more designed ankyrin repeat domains with
binding specificity for coronavirus spike proteins. In a preferred
embodiment, such recombinant binding proteins comprising two, three
or more designed ankyrin repeat domains with binding specificity
for coronavirus spike proteins target two, three or more different
epitopes on coronavirus spike proteins.
[0232] The described recombinant binding proteins, or binding
domains thereof, comprising designed ankyrin repeat motifs or
modules are also referred herein as DARPin.RTM. proteins. See
Stumpp et al., Curr Opin Drug Discov Devel. 10(2): 153-9 (2007);
and Binz et al., Nature Biotech. 22(5): 575-582 (2004). DARPin.RTM.
proteins can be considered as antibody mimetics with high
specificity and high binding affinity to a target protein. In
general, a DARPin.RTM. protein comprises at least one ankyrin
repeat domain, for example, at least 1, 2, 3, 4, 5, or more ankyrin
repeat domains.
[0233] The ankyrin repeat domains described herein generally
comprise a core scaffold that provides structure, and target
binding residues that bind to a target. The structural core
includes conserved amino acid residues, and the target binding
surface includes amino acid residues that differ depending on the
target. International Patent Publication No. WO 2002/020565 and
Binz et al., Nature Biotech. 22(5): 575-582 (2004) describe
libraries of ankyrin repeat proteins that can be used for the
selection/screening of a protein that binds specifically to a
target. Methods of making such libraries are also provided.
[0234] Multiple ankyrin repeat domains can be linked (either
through a covalent bond or non-covalent association) to form
bispecific or multi-specific molecules. One such molecule is shown
in FIG. 1, where three separate coronavirus spike protein binding
domains are linked to form a multi-specific molecule. The linkers
are illustrated by dashed lines joining the three binding
domains.
[0235] Coronavirus Spike Protein
[0236] As set out above, the coronavirus spike protein is an
attractive therapeutic target. Neutralizing the coronavirus spike
protein can prevent infection of mammalian cells, stopping the
coronavirus disease from taking hold in a subject. The recombinant
binding proteins according to the present invention are specific
for a mammalian coronavirus. Preferably, the designed ankyrin
repeat proteins are specific for a coronavirus of mice, rat, dog,
rabbit, monkey or human origin. More preferably, the designed
ankyrin repeat proteins are specific for a coronavirus of human
origin. The coronavirus SARS-CoV-2 is most preferred. As used
herein, the term "SARS-CoV-2" includes both wild-type virus (such
as SARS-CoV-2 found in infected humans at the beginning of the
COVID-19 pandemic) and mutated forms or variants thereof. In one
embodiment, the term "SARS-CoV-2" includes wild type and the
specific variants B.1.1.7 (the so-called "UK variant") and B.1.351
(the so-called "South African variant").
[0237] The recombinant binding protein described herein comprises
an ankyrin repeat domain that specifically binds to coronavirus
spike protein. In one embodiment, the recombinant binding protein
described herein comprises two, three or more ankyrin repeat
domains that specifically bind to coronavirus spike protein. In one
embodiment, the recombinant binding protein described herein
comprises one, two, three or more ankyrin repeat domains that
specifically bind to SARS-CoV-2 spike protein.
[0238] The target domains of interest in this disclosure on the
coronavirus spike protein include, but are not limited to, the
receptor binding domain (RBD domain); the S1 NTD domain; and the S2
domain. These domains are known in the art (see, e.g. Wrapp et al.,
Science 367, 1260-1263 (2020).
[0239] Ankyrin repeat domains according to the present invention
that bind coronavirus spike protein are provided in Table 1:
TABLE-US-00001 TABLE 1 DARPin .RTM. protein Spike Protein SEQ ID NO
name Abbreviation Target Domain SEQ ID NO 1 vS07_19G10 R2a RBD SEQ
ID NO 2 vS07_06F12 R1a RBD SEQ ID NO 3 vS07_12C06 R1b RBD SEQ ID NO
4 vS07_22E12 R3a RBD SEQ ID NO 5 vS07_23E04 R3c RBD SEQ ID NO 6
vS07_29B10 R3b RBD SEQ ID NO 7 vS07_07F02 RN1 RBD SEQ ID NO 8
vS07_26C03 RN2 RBD SEQ ID NO 9 vS07_08F10 S1a S1-NTD SEQ ID NO 10
vS07_14G03 S2a S2 SEQ ID NO 11 vS07_18A05 S2b S2 SEQ ID NO 76
vS07_08F10v27 S1-NTD SEQ ID NO 77 vS07_14G03v19 S2 SEQ ID NO 85
vS07_08F10v47 S1-NTD
[0240] Thus, in one embodiment, the present invention relates to a
recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain comprises an amino
acid sequence that has at least about 90% sequence identity with an
ankyrin repeat domain selected from the group consisting of SEQ ID
NOs 1 to 11, 76, 77 and 85, as illustrated in Table 1 above.
[0241] In one embodiment, the present invention relates to a
recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain comprises an amino
acid sequence that has at least about 80%, at least about 81%, at
least about 82%, at least about 83%, at least about 84%, at least
about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98% or about 99% sequence identity with an ankyrin repeat
domain selected from the group consisting of SEQ ID NOs 1 to 11,
76, 77 and 85, as illustrated in Table 1 above. In one embodiment,
the present invention relates to a recombinant binding protein
comprising a first ankyrin repeat domain, wherein said first
ankyrin repeat domain comprises an amino acid sequence that has at
least about 80%, at least about 81%, at least about 82%, at least
about 83%, at least about 84%, at least about 85%, at least about
86%, at least about 87%, at least about 88%, at least about 89%, at
least about 90%, at least about 91%, about 92%, about 93%, about
94%, about 95%, about 96%, about 97%, about 98% or about 99%
sequence identity with an ankyrin repeat domain selected from the
group consisting of SEQ ID NOs 1 to 11, 76 and 77, as illustrated
in Table 1 above. In one embodiment, the present invention relates
to a recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain comprises an amino
acid sequence that has at least about 80%, at least about 81%, at
least about 82%, at least about 83%, at least about 84%, at least
about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98% or about 99% sequence identity with an ankyrin repeat
domain selected from the group consisting of SEQ ID NOs 1 to 11, as
illustrated in Table 1 above.
[0242] In one embodiment, the present invention relates to a
recombinant binding protein comprising a first ankyrin repeat
domain, wherein said first ankyrin repeat domain is selected from
the group consisting of SEQ ID NOs 1 to 11, 76, 77 and 85, as
illustrated in Table 1 above.
[0243] The ankyrin repeat domains listed in Table 1 may be combined
in any manner to provide a bi-specific or multi-specific molecule.
The first, second and third ankyrin repeat domains may have
identical sequences. The first, second and third ankyrin repeat
domains may have different sequences.
[0244] Thus, in one embodiment, the present invention relates to a
recombinant binding protein further comprising a second ankyrin
repeat domain, wherein said second ankyrin repeat domain comprises
an amino acid sequence that has at least about 90% sequence
identity with an ankyrin repeat domain selected from the group
consisting of SEQ ID NOs 1 to 11, 76, 77 and 85, as illustrated in
Table 1 above.
[0245] In one embodiment, the present invention relates to a
recombinant binding protein comprising a second ankyrin repeat
domain, wherein said second ankyrin repeat domain comprises an
amino acid sequence that has at least about 80%, at least about
81%, at least about 82%, at least about 83%, at least about 84%, at
least about 85%, at least about 86%, at least about 87%, at least
about 88%, at least about 89%, at least about 90%, at least about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
97%, about 98% or about 99% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76, 77 and 85, as illustrated in Table 1 above. In one
embodiment, the present invention relates to a recombinant binding
protein comprising a second ankyrin repeat domain, wherein said
second ankyrin repeat domain comprises an amino acid sequence that
has at least about 80%, at least about 81%, at least about 82%, at
least about 83%, at least about 84%, at least about 85%, at least
about 86%, at least about 87%, at least about 88%, at least about
89%, at least about 90%, at least about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98% or about 99%
sequence identity with an ankyrin repeat domain selected from the
group consisting of SEQ ID NOs 1 to 11, 76 and 77, as illustrated
in Table 1 above. In one embodiment, the present invention relates
to a recombinant binding protein comprising a second ankyrin repeat
domain, wherein said second ankyrin repeat domain comprises an
amino acid sequence that has at least about 80%, at least about
81%, at least about 82%, at least about 83%, at least about 84%, at
least about 85%, at least about 86%, at least about 87%, at least
about 88%, at least about 89%, at least about 90%, at least about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
97%, about 98% or about 99% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, as illustrated in Table 1 above.
[0246] In one embodiment, the present invention relates to a
recombinant binding protein comprising a second ankyrin repeat
domain, wherein said second ankyrin repeat domain is selected from
the group consisting of SEQ ID NOs 1 to 11, 76, 77 and 85, as
illustrated in Table 1 above.
[0247] In one embodiment, the present invention relates to a
recombinant binding protein as defined above further comprising a
third ankyrin repeat domain, wherein said third ankyrin repeat
domain comprises an amino acid sequence that has at least about 90%
sequence identity with an ankyrin repeat domain selected from the
group consisting of SEQ ID NOs 1 to 11, 76, 77 and 85, as
illustrated in Table 1 above.
[0248] In one embodiment, the present invention relates a
recombinant binding protein as defined above further comprising a
third ankyrin repeat domain, wherein said third ankyrin repeat
domain comprises an amino acid sequence that has at least about
80%, at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98% or about 99% sequence identity
with an ankyrin repeat domain selected from the group consisting of
SEQ ID NOs 1 to 11, 76, 77 and 85, as illustrated in Table 1 above.
In one embodiment, the present invention relates a recombinant
binding protein as defined above further comprising a third ankyrin
repeat domain, wherein said third ankyrin repeat domain comprises
an amino acid sequence that has at least about 80%, at least about
81%, at least about 82%, at least about 83%, at least about 84%, at
least about 85%, at least about 86%, at least about 87%, at least
about 88%, at least about 89%, at least about 90%, at least about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about
97%, about 98% or about 99% sequence identity with an ankyrin
repeat domain selected from the group consisting of SEQ ID NOs 1 to
11, 76 and 77, as illustrated in Table 1 above. In one embodiment,
the present invention relates a recombinant binding protein as
defined above further comprising a third ankyrin repeat domain,
wherein said third ankyrin repeat domain comprises an amino acid
sequence that has at least about 80%, at least about 81%, at least
about 82%, at least about 83%, at least about 84%, at least about
85%, at least about 86%, at least about 87%, at least about 88%, at
least about 89%, at least about 90%, at least about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or
about 99% sequence identity with an ankyrin repeat domain selected
from the group consisting of SEQ ID NOs 1 to 11, as illustrated in
Table 1 above.
[0249] In one embodiment, the present invention relates to a
recombinant binding protein as defined above further comprising a
third ankyrin repeat domain, wherein said third ankyrin repeat
domain is selected from the group consisting of SEQ ID NOs 1 to 11,
76, 77 and 85, as illustrated in Table 1 above.
[0250] The present invention further relates to specific
combinations of first, second and third ankyrin repeat domains
having amino acid sequences and being arranged from the N-terminus
to the C-terminus as follows:
[0251] (i) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 6, 1 and 3;
[0252] (ii) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 4, 2 and 1;
[0253] (iii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 4, 6 and 3;
[0254] (iv) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 6, 3 and 6;
[0255] (v) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 7, 3 and 6;
[0256] (vi) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 8, 4 and 1;
[0257] (vii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 6 and 7;
[0258] (viii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 4, 1 and 8;
[0259] (ix) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 3, 6 and 9;
[0260] (x) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 9, 3 and 6;
[0261] (xi) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 1, 6 and 9;
[0262] (xii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 9, 6 and 1;
[0263] (xiii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 6, 9 and 10;
[0264] (xiv) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 9 and 11;
[0265] (xv) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 10, 9 and 6;
[0266] (xvi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 11, 9 and 3;
[0267] (xvii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 5, 1 and 3;
[0268] (xviii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 1, 2 and 5;
[0269] (xix) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 5 and 6;
[0270] (xx) amino acid sequences having at least about 90% sequence
identity with SEQ ID NOs 6, 3 and 5;
[0271] (xxi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 7, 3 and 5;
[0272] (xxii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 8, 5 and 6;
[0273] (xxiii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 6, 10 and 11;
[0274] (xxiv) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 10 and 10;
[0275] (xxv) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 5, 6 and 9;
[0276] (xxvi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 9, 3 and 5;
[0277] (xxvii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 9, 6 and 5;
[0278] (xxviii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 5, 9 and 10;
[0279] (xxix) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 6, 9 and 11;
[0280] (xxx) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 10, 9 and 5;
[0281] (xxxi) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 11, 9 and 6;
[0282] (xxxii) amino acid sequences having at least about 90%
sequence identity with SEQ ID NOs 3, 76 and 77; or (xxxiii) amino
acid sequences having at least about 90% sequence identity with SEQ
ID NOs 3, 85 and 77.
[0283] In one embodiment, the present invention relates to the
recombinant binding protein according to embodiment (xx) as listed
above. In a further embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98% or about 99% sequence identity with SEQ ID NO:
68. In another embodiment, the recombinant binding protein
comprises a polypeptide, wherein said polypeptide has the amino
acid sequence of SEQ ID NO: 68.
[0284] In one embodiment, the present invention relates to the
recombinant binding protein according to embodiment (xxviii), as
listed above. In a further embodiment, the present invention
relates to a recombinant binding protein comprising a polypeptide,
wherein said polypeptide has an amino acid sequence that has at
least about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98% or about 99% sequence identity
with SEQ ID NO: 69. In another embodiment, the recombinant binding
protein comprises a polypeptide, wherein said polypeptide has the
amino acid sequence of SEQ ID NO: 69.
[0285] In one embodiment, the present invention relates to the
recombinant binding protein according to (xxxii), as listed above.
In a further embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98% or about 99% sequence identity with SEQ ID NO:
79. In another embodiment, the recombinant binding protein
comprises a polypeptide, wherein said polypeptide has the amino
acid sequence of SEQ ID NO: 79.
[0286] In one embodiment, the present invention relates to the
recombinant binding protein according to (xxxiii), as listed above.
In a further embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98% or about 99% sequence identity with a sequence
selected from the group consisting of SEQ ID NOs: 89 to 91. In
another embodiment, the recombinant binding protein comprises a
polypeptide, wherein said polypeptide has an amino acid sequence
selected from the group consisting of SEQ ID NOs: 89 to 91.
[0287] In another embodiment, the recombinant binding protein of
the present invention binds to a coronavirus spike protein. In
another embodiment, the spike protein is SARS-CoV-2 spike
protein.
[0288] In another embodiment, the recombinant binding protein of
the invention comprising at least one ankyrin repeat domain binds
to a coronavirus spike protein with a binding affinity (K.sub.D) of
or below about 100 nM. In another embodiment, the spike protein is
SARS-CoV-2 spike protein.
[0289] In another embodiment, the recombinant binding protein of
the invention comprises first, second and/or third ankyrin repeat
domains and said first, second and/or third ankyrin repeat domains
bind to a coronavirus spike protein with a binding affinity
(K.sub.D) of or below about 100 nM. In another embodiment, the
spike protein is SARS-CoV-2 spike protein.
[0290] In exemplary embodiments, the recombinant binding protein of
the invention binds coronavirus spike protein, preferably
SARS-CoV-2 spike protein, with an K.sub.D value of, or less than:
about 100 nM; about 50 nM, about 40 nM, about 30 nM, about 20 nM,
about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM,
about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400
pM, about 300 pM, about 250 pM, about 200 pM, about 150 pM, about
100 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about
20 pM, about 15 pM, about 10 pM, about 5 pM, or about 1 pM. In one
exemplary embodiment, the recombinant binding protein binds
coronavirus spike protein, preferably SARS-CoV-2 spike protein,
with a K.sub.D value of less than or equal to about 10 nM. In
another exemplary embodiment, the recombinant binding protein binds
coronavirus spike protein, preferably SARS-CoV-2 spike protein,
with a K.sub.D value of less than or equal to about 1 nM.
[0291] In certain embodiments, the coronavirus spike protein is
human coronavirus spike protein. In certain embodiments, the
coronavirus spike protein is human SARS-CoV-2 spike protein.
[0292] In certain embodiments, the recombinant binding protein may
further comprise at least one human serum albumin binding domain.
In embodiments, the at least one human serum albumin domain may be
located at the N-terminus, the C-terminus, or both.
[0293] In certain embodiments, the serum albumin binding domain
comprises an amino acid sequence that has at least 90% sequence
identity with a sequence selected from the group consisting of SEQ
ID NOs: 47-49. In one embodiment, the serum albumin binding domain
comprises an amino acid sequence that has at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% sequence identity with SEQ ID NO: 47.
[0294] In further embodiments, the recombinant binding protein of
the invention has a terminal half-life in mice of at least about 30
hours, preferably at least about 35 hours, more preferably at least
about 40 hours, and more preferably at least about 45 hours. Said
terminal half-life is preferably determined in Balb/c mice, as
described in Example 9.
[0295] Particularly preferred combinations of ankyrin repeat
domains are listed in Table 2, wherein H denotes human serum
albumin and R31b, R2a etc are as defined in Table 1 above:
TABLE-US-00002 TABLE 2 5 Domain DARPin .RTM. Designs # 1 2 3 4 5 1
SEQ ID NO: 12 H H R3b R2a R1b 2 SEQ ID NO: 13 H H R3a R1a R2a 3 SEQ
ID NO: 14 H H R3a R3b R1b 4 SEQ ID NO: 15 H H R3b R1b R3b 5 SEQ ID
NO: 16 H H RN1 R1b R3b 6 SEQ ID NO: 17 H H RN2 R3a R2a 7 SEQ ID NO:
18 H H R1b R3b RN1 8 SEQ ID NO: 19 H H R3a R2a RN2 9 SEQ ID NO: 20
H H R1b R3b S1a 10 SEQ ID NO: 21 H H S1a R1b R3b 11 SEQ ID NO: 22 H
H R2a R3b S1a 12 SEQ ID NO: 23 H H S1a R3b R2a 13 SEQ ID NO: 24 H H
R3b S1a S2a 14 SEQ ID NO: 25 H H R1b S1a S2b 15 SEQ ID NO: 26 H H
S2a S1a R3b 16 SEQ ID NO: 27 H H S2b S1a R1b 17 SEQ ID NO: 28 H H
R3c R2a R1b 18 SEQ ID NO: 29 H H R2a R1a R3c 19 SEQ ID NO: 30 H H
R1b R3c R3b 20 SEQ ID NO: 31 H H R3b R1b R3c 21 SEQ ID NO: 32 H H
RN1 R1b R3c 22 SEQ ID NO: 33 H H RN2 R3c R3b 23 SEQ ID NO: 34 H H
R3b S2a S2b 24 SEQ ID NO: 35 H H R1b S2a S2a 25 SEQ ID NO: 36 H H
R3c R3b S1a 26 SEQ ID NO: 37 H H S1a R1b R3c 27 SEQ ID NO: 38 H H
S1a R3b R3c 28 SEQ ID NO: 39 H H R3c S1a S2a 29 SEQ ID NO: 40 H H
R3b S1a S2b 30 SEQ ID NO: 41 H H S2a S1a R3c 31 SEQ ID NO: 42 H H
S2b S1a R3b 32 SEQ ID NO: 75 H H R1b SEQ ID SEQ ID NO 76 NO 77 33
SEQ ID NOs: H H R1b SEQ ID SEQ ID 84, 87 and 88 NO 85 NO 77
[0296] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with a sequence selected from the group
consisting of SEQ ID NOs: 12-42, 75, 84, 87 and 88. In one
embodiment, said binding protein binds to a coronavirus spike
protein. In one embodiment, said spike protein is SARS-CoV-2 spike
protein. In one embodiment, said binding protein binds said
coronavirus spike protein with a binding affinity (K.sub.D) of or
below about 100 nM.
[0297] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with a sequence selected from the group consisting of SEQ
ID NOs: 12-42, 75, 84, 87 and 88. In another embodiment, the
present invention relates to a recombinant binding protein
comprising a polypeptide, wherein said polypeptide has an amino
acid sequence that has at least about 80%, at least about 81%, at
least about 82%, at least about 83%, at least about 84%, at least
about 85%, at least about 86%, at least about 87%, at least about
88%, at least about 89%, at least about 90%, at least about 91%, at
least about 92%, at least about 93%, at least about 94%, at least
about 95%, at least about 96%, at least about 97%, at least about
98% or at least about 99% sequence identity with a sequence
selected from the group consisting of SEQ ID NOs: 12-42 and 75. In
another embodiment, the present invention relates to a recombinant
binding protein comprising a polypeptide, wherein said polypeptide
has an amino acid sequence that has at least about 80%, at least
about 81%, at least about 82%, at least about 83%, at least about
84%, at least about 85%, at least about 86%, at least about 87%, at
least about 88%, at least about 89%, at least about 90%, at least
about 91%, at least about 92%, at least about 93%, at least about
94%, at least about 95%, at least about 96%, at least about 97%, at
least about 98% or at least about 99% sequence identity with a
sequence selected from the group consisting of SEQ ID NOs: 12-42.
In one embodiment, said binding protein binds to a coronavirus
spike protein. In one embodiment, said spike protein is SARS-CoV-2
spike protein. In one embodiment, said binding protein binds said
coronavirus spike protein with a binding affinity (K.sub.D) of or
below about 100 nM.
[0298] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that is selected from the
group consisting of SEQ ID NOs: 12-42, 75, 84, 87 and 88. In one
embodiment, said binding protein binds to a coronavirus spike
protein. In one embodiment, said spike protein is SARS-CoV-2 spike
protein. In one embodiment, said binding protein binds said
coronavirus spike protein with a binding affinity (K.sub.D) of or
below about 100 nM.
[0299] In another embodiment, the present invention relates to the
recombinant binding protein as described herein, wherein said
binding protein is capable of inhibiting infection of cells by a
coronavirus. In another embodiment, the present invention relates
to the recombinant binding protein as described herein, wherein
said binding protein is capable of inhibiting infection of cells by
SARS-CoV-2.
[0300] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with SEQ ID NO: 31. In another embodiment, the recombinant
binding protein comprises a polypeptide, wherein said polypeptide
has the amino acid sequence of SEQ ID NO: 31. In one embodiment,
said binding protein binds to a coronavirus spike protein. In one
embodiment, said spike protein is SARS-CoV-2 spike protein. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 100 nM,
of or below about 10 nM, of or below about 1 nM, of or below about
100 pM, of or below about 10 pM, or of or below about 1 pM. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 1 nM.
In one embodiment, said binding protein has a terminal half-life in
mice of at least about 20 hours, at least about 25 hours, at least
about 30 hours, at least about 35 hours, at least about 40 hours,
or at least about 45 hours. In one embodiment, said binding protein
has a terminal half-life in mice of at least about 40 hours. In one
embodiment, said binding protein exhibits a high thermal stability
with a Tm above 50.degree. C., above 60.degree. C., above
70.degree. C., or above 80.degree. C. In one embodiment, said
binding protein exhibits a high thermal stability with a Tm above
60.degree. C. In one embodiment, said binding protein inhibits
viral entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 100 nM, of or below 10 nM, of or below
1 nM, or of or below 0.5 nM. In one embodiment, said binding
protein inhibits viral entry of SARS-CoV-2 VSV pseudovirus in
VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 100 nM, of or
below 10 nM, of or below 1 nM, or of or below 0.1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein has a combination of two, three,
four, five or six properties selected from the properties listed in
this paragraph relating to amino acid sequence, binding affinity,
terminal half-life, thermal stability, IC.sub.50 of SARS-CoV-2 VSV
pseudovirus inhibition and IC.sub.50 of SARS-CoV-2 inhibition. In
one exemplary embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 31, and wherein said binding
protein binds to SARS-CoV-2 spike protein with a binding affinity
(K.sub.D) of or below about 1 nM, wherein said binding protein has
a terminal half-life in mice of at least about 40 hours, wherein
said binding protein exhibits a high thermal stability with a Tm
above 60.degree. C., wherein said binding protein inhibits viral
entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 1 nM, and/or wherein said binding
protein inhibits viral entry of SARS-CoV-2 in VeroE6 cells with an
IC.sub.50 value of or below 1 nM.
[0301] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with SEQ ID NO: 39. In another embodiment, the recombinant
binding protein comprises a polypeptide, wherein said polypeptide
has the amino acid sequence of SEQ ID NO: 39. In one embodiment,
said binding protein binds to a coronavirus spike protein. In one
embodiment, said spike protein is SARS-CoV-2 spike protein. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 100 nM,
of or below about 10 nM, of or below about 1 nM, of or below about
100 pM, of or below about 10 pM, or of or below about 1 pM. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 1 nM.
In one embodiment, said binding protein has a terminal half-life in
mice of at least about 20 hours, at least about 25 hours, at least
about 30 hours, at least about 35 hours, at least about 40 hours,
or at least about 45 hours. In one embodiment, said binding protein
has a terminal half-life in mice of at least about 20 hours. In one
embodiment, said binding protein exhibits a high thermal stability
with a Tm above 50.degree. C., above 60.degree. C., above
70.degree. C., or above 80.degree. C. In one embodiment, said
binding protein exhibits a high thermal stability with a Tm above
60.degree. C. In one embodiment, said binding protein inhibits
viral entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 100 nM, of or below 10 nM, of or below
1 nM, or of or below 0.5 nM. In one embodiment, said binding
protein inhibits viral entry of SARS-CoV-2 VSV pseudovirus in
VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 100 nM, of or
below 10 nM, of or below 1 nM, or of or below 0.4 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein has a combination of two, three,
four, five or six properties selected from the properties listed in
this paragraph relating to amino acid sequence, binding affinity,
terminal half-life, thermal stability, IC.sub.50 of SARS-CoV-2 VSV
pseudovirus inhibition and IC.sub.50 of SARS-CoV-2 inhibition. In
one exemplary embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 39, and wherein said binding
protein binds to SARS-CoV-2 spike protein with a binding affinity
(K.sub.D) of or below about 1 nM, wherein said binding protein has
a terminal half-life in mice of at least about 20 hours, wherein
said binding protein exhibits a high thermal stability with a Tm
above 60.degree. C., wherein said binding protein inhibits viral
entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 1 nM, and/or wherein said binding
protein inhibits viral entry of SARS-CoV-2 in VeroE6 cells with an
IC.sub.50 value of or below 1 nM.
[0302] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with SEQ ID NO: 75. In another embodiment, the recombinant
binding protein comprises a polypeptide, wherein said polypeptide
has the amino acid sequence of SEQ ID NO: 75. In one embodiment,
said binding protein binds to a coronavirus spike protein. In one
embodiment, said spike protein is SARS-CoV-2 spike protein. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 100 nM,
of or below about 10 nM, of or below about 1 nM, of or below about
100 pM, of or below about 10 pM, or of or below about 1 pM. In one
embodiment, said binding protein has a terminal half-life in mice
of at least about 20 hours, at least about 25 hours, at least about
30 hours, at least about 35 hours, at least about 40 hours, or at
least about 45 hours. In one embodiment, said binding protein has a
terminal half-life in mice of at least about 30 hours. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 1 nM.
In one embodiment, said binding protein exhibits a high thermal
stability with a Tm above 50.degree. C., above 60.degree. C., above
70.degree. C., or above 80.degree. C. In one embodiment, said
binding protein exhibits a high thermal stability with a Tm above
60.degree. C. In one embodiment, said binding protein inhibits
viral entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 100 nM, of or below 10 nM, of or below
1 nM, or of or below 0.5 nM. In one embodiment, said binding
protein inhibits viral entry of SARS-CoV-2 VSV pseudovirus in
VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 100 nM, of or
below 10 nM, of or below 1 nM, or of or below 0.4 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein has a combination of two, three,
four, five or six properties selected from the properties listed in
this paragraph relating to amino acid sequence, binding affinity,
terminal half-life, thermal stability, IC.sub.50 of SARS-CoV-2 VSV
pseudovirus inhibition and IC.sub.50 of SARS-CoV-2 inhibition. In
one exemplary embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 75, and wherein said binding
protein binds to SARS-CoV-2 spike protein with a binding affinity
(K.sub.D) of or below about 1 nM, wherein said binding protein has
a terminal half-life in mice of at least about 30 hours, wherein
said binding protein exhibits a high thermal stability with a Tm
above 60.degree. C., wherein said binding protein inhibits viral
entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 1 nM, and/or wherein said binding
protein inhibits viral entry of SARS-CoV-2 in VeroE6 cells with an
IC.sub.50 value of or below 1 nM.
[0303] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with SEQ ID NO: 84. In another embodiment, the recombinant
binding protein comprises a polypeptide, wherein said polypeptide
has the amino acid sequence of SEQ ID NO: 84. In one embodiment,
said binding protein binds to a coronavirus spike protein. In one
embodiment, said spike protein is SARS-CoV-2 spike protein. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 100 nM,
of or below about 10 nM, of or below about 1 nM, of or below about
100 pM, of or below about 10 pM, or of or below about 1 pM. In one
embodiment, said binding protein has a terminal half-life in mice
of at least about 20 hours, at least about 25 hours, at least about
30 hours, at least about 35 hours, at least about 40 hours, or at
least about 45 hours. In one embodiment, said binding protein has a
terminal half-life in mice of at least about 40 hours. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 1 nM.
In one embodiment, said binding protein exhibits a high thermal
stability with a Tm above 50.degree. C., above 60.degree. C., above
70.degree. C., or above 80.degree. C. In one embodiment, said
binding protein exhibits a high thermal stability with a Tm above
60.degree. C. In one embodiment, said binding protein inhibits
viral entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 100 nM, of or below 10 nM, of or below
1 nM, or of or below 0.5 nM. In one embodiment, said binding
protein inhibits viral entry of SARS-CoV-2 VSV pseudovirus in
VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 100 nM, of or
below 10 nM, of or below 1 nM, or of or below 0.4 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein has a combination of two, three,
four, five or six properties selected from the properties listed in
this paragraph relating to amino acid sequence, binding affinity,
terminal half-life, thermal stability, IC.sub.50 of SARS-CoV-2 VSV
pseudovirus inhibition and IC.sub.50 of SARS-CoV-2 inhibition. In
one exemplary embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 84, and wherein said binding
protein binds to SARS-CoV-2 spike protein with a binding affinity
(K.sub.D) of or below about 1 nM, wherein said binding protein has
a terminal half-life in mice of at least about 40 hours, wherein
said binding protein exhibits a high thermal stability with a Tm
above 60.degree. C., wherein said binding protein inhibits viral
entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 1 nM, and/or wherein said binding
protein inhibits viral entry of SARS-CoV-2 in VeroE6 cells with an
IC.sub.50 value of or below 1 nM.
[0304] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with SEQ ID NO: 87. In another embodiment, the recombinant
binding protein comprises a polypeptide, wherein said polypeptide
has the amino acid sequence of SEQ ID NO: 87. In one embodiment,
said binding protein binds to a coronavirus spike protein. In one
embodiment, said spike protein is SARS-CoV-2 spike protein. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 100 nM,
of or below about 10 nM, of or below about 1 nM, of or below about
100 pM, of or below about 10 pM, or of or below about 1 pM. In one
embodiment, said binding protein has a terminal half-life in mice
of at least about 20 hours, at least about 25 hours, at least about
30 hours, at least about 35 hours, at least about 40 hours, or at
least about 45 hours. In one embodiment, said binding protein has a
terminal half-life in mice of at least about 35 hours. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 1 nM.
In one embodiment, said binding protein exhibits a high thermal
stability with a Tm above 50.degree. C., above 60.degree. C., above
70.degree. C., or above 80.degree. C. In one embodiment, said
binding protein exhibits a high thermal stability with a Tm above
60.degree. C. In one embodiment, said binding protein inhibits
viral entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 100 nM, of or below 10 nM, of or below
1 nM, or of or below 0.5 nM. In one embodiment, said binding
protein inhibits viral entry of SARS-CoV-2 VSV pseudovirus in
VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 100 nM, of or
below 10 nM, of or below 1 nM, or of or below 0.4 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein has a combination of two, three,
four, five or six properties selected from the properties listed in
this paragraph relating to amino acid sequence, binding affinity,
terminal half-life, thermal stability, IC.sub.50 of SARS-CoV-2 VSV
pseudovirus inhibition and IC.sub.50 of SARS-CoV-2 inhibition. In
one exemplary embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 87, and wherein said binding
protein binds to SARS-CoV-2 spike protein with a binding affinity
(K.sub.D) of or below about 1 nM, wherein said binding protein has
a terminal half-life in mice of at least about 35 hours, wherein
said binding protein exhibits a high thermal stability with a Tm
above 60.degree. C., wherein said binding protein inhibits viral
entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 1 nM, and/or wherein said binding
protein inhibits viral entry of SARS-CoV-2 in VeroE6 cells with an
IC.sub.50 value of or below 1 nM.
[0305] In another embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 80%,
at least about 81%, at least about 82%, at least about 83%, at
least about 84%, at least about 85%, at least about 86%, at least
about 87%, at least about 88%, at least about 89%, at least about
90%, at least about 91%, at least about 92%, at least about 93%, at
least about 94%, at least about 95%, at least about 96%, at least
about 97%, at least about 98% or at least about 99% sequence
identity with SEQ ID NO: 88. In another embodiment, the recombinant
binding protein comprises a polypeptide, wherein said polypeptide
has the amino acid sequence of SEQ ID NO: 88. In one embodiment,
said binding protein binds to a coronavirus spike protein. In one
embodiment, said spike protein is SARS-CoV-2 spike protein. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 100 nM,
of or below about 10 nM, of or below about 1 nM, of or below about
100 pM, of or below about 10 pM, or of or below about 1 pM. In one
embodiment, said binding protein has a terminal half-life in mice
of at least about 20 hours, at least about 25 hours, at least about
30 hours, at least about 35 hours, at least about 40 hours, or at
least about 45 hours. In one embodiment, said binding protein has a
terminal half-life in mice of at least about 40 hours. In one
embodiment, said binding protein binds said coronavirus spike
protein with a binding affinity (K.sub.D) of or below about 1 nM.
In one embodiment, said binding protein exhibits a high thermal
stability with a Tm above 50.degree. C., above 60.degree. C., above
70.degree. C., or above 80.degree. C. In one embodiment, said
binding protein exhibits a high thermal stability with a Tm above
60.degree. C. In one embodiment, said binding protein inhibits
viral entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 100 nM, of or below 10 nM, of or below
1 nM, or of or below 0.5 nM. In one embodiment, said binding
protein inhibits viral entry of SARS-CoV-2 VSV pseudovirus in
VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 100 nM, of or
below 10 nM, of or below 1 nM, or of or below 0.4 nM. In one
embodiment, said binding protein inhibits viral entry of SARS-CoV-2
in VeroE6 cells with an IC.sub.50 value of or below 1 nM. In one
embodiment, said binding protein has a combination of two, three,
four, five or six properties selected from the properties listed in
this paragraph relating to amino acid sequence, binding affinity,
terminal half-life, thermal stability, IC.sub.50 of SARS-CoV-2 VSV
pseudovirus inhibition and IC.sub.50 of SARS-CoV-2 inhibition. In
one exemplary embodiment, the present invention relates to a
recombinant binding protein comprising a polypeptide, wherein said
polypeptide has an amino acid sequence that has at least about 90%
sequence identity with SEQ ID NO: 88, and wherein said binding
protein binds to SARS-CoV-2 spike protein with a binding affinity
(K.sub.D) of or below about 1 nM, wherein said binding protein has
a terminal half-life in mice of at least about 40 hours, wherein
said binding protein exhibits a high thermal stability with a Tm
above 60.degree. C., wherein said binding protein inhibits viral
entry of SARS-CoV-2 VSV pseudovirus in VeroE6 cells with an
IC.sub.50 value of or below 1 nM, and/or wherein said binding
protein inhibits viral entry of SARS-CoV-2 in VeroE6 cells with an
IC.sub.50 value of or below 1 nM.
[0306] Half-Life Extending Moieties
[0307] The "half-life extending moiety" extends the serum half-life
in vivo of the recombinant binding proteins described herein,
compared to the same protein without the half-life extending
moiety. Examples of half-life extending moieties include, but are
not limited to, polyhistidine, Glu-Glu, glutathione S transferase
(GST), thioredoxin, protein A, protein G, an immunoglobulin domain,
maltose binding protein (MBP), human serum albumin (HSA) binding
domain, or polyethylene glycol (PEG). In some embodiments, the
half-life extending moieties are glutathione S transferase (GST),
protein A, protein G, an immunoglobulin domain, human serum albumin
(HSA) binding domain, or polyethylene glycol (PEG).
[0308] In some embodiments, the recombinant binding protein
described herein comprises an ankyrin repeat domain that
specifically binds serum albumin (such as preferably human serum
albumin), also referred herein as "serum albumin binding domain".
The recombinant binding protein described herein may also comprise
more than one serum albumin binding domain, for example, two or
three or more serum albumin binding domains. Thus, the recombinant
binding protein described herein may comprise a first and a second
serum albumin binding domain, or a first, a second and a third
serum albumin binding domain. The embodiments provided below
describe such a first serum albumin binding domain, second serum
albumin binding domain, and/or third serum albumin binding
domain.
[0309] In some embodiments, the half-life extending moiety
described herein comprises a serum albumin binding domain
comprising an amino acid sequence that is at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical to any one of SEQ ID NOs: 47 to 49. In an exemplary
embodiment, the half-life extending moiety described herein
comprises an amino acid sequence that is at least 90% identical to
any one of SEQ ID NOs: 47 to 49. In some embodiments, the half-life
extending moiety described herein comprises an amino acid sequence
that is at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, or 100% identical to SEQ ID NO: 47. In an
exemplary embodiment, the half-life extending moiety described
herein comprises an amino acid sequence that is at least 90%
identical to SEQ ID NO: 47.
[0310] In some embodiments, two or more serum albumin binding
domains are preferred. In some embodiments, two serum albumin
binding domains are located at the N-terminus. In exemplary
embodiments, the recombinant binding protein comprises, from the
N-terminus to C-terminus: (i) an ankyrin repeat domain that
specifically binds serum albumin; (ii) an ankyrin repeat domain
that specifically binds serum albumin; and (iii) one or more
ankyrin repeat domains that specifically bind coronavirus spike
protein. In certain embodiments, the N-terminal serum albumin
binding domain (also referred to herein as serum albumin binding
domain 1) comprises an amino acid sequence that is at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical to SEQ ID NO: 47. In certain embodiments, the second
serum albumin binding domain (also referred to herein as serum
albumin binding domain 2) comprises an amino acid sequence that is
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% identical to SEQ ID NO: 47.
[0311] In some embodiments, the half-life extending moiety
comprises an immunoglobulin domain. In some embodiments, the
immunoglobulin domain comprises an Fc domain. In some embodiments,
the Fc domain is derived from any one of the known heavy chain
isotypes: IgG (.gamma.), IgM (.mu.), IgD (.delta.), IgE
(.epsilon.), or IgA (.alpha.). In some embodiments, the Fc domain
is derived from any one of the known heavy chain isotypes or
subtypes: IgG1 (.gamma.1), IgG2 (.gamma.2), IgG3 (.gamma.3), IgG4
(.gamma.4), IgA1 (.alpha.1), IgA2 (.alpha.2). In some embodiments,
the Fc domain is the Fc domain of human IgG1.
[0312] In some embodiments, the Fc domain comprises an
uninterrupted native sequence (i.e., wild type sequence) of an Fc
domain. In some embodiments, the immunoglobulin Fc domain comprises
a variant Fc domain resulting in altered biological activity. For
example, at least one point mutation or deletion may be introduced
into the Fc domain so as to reduce or eliminate the effector
activity (e.g., International Patent Publication No. WO
2005/063815), and/or to increase the homogeneity during the
production of the recombinant binding protein. In some embodiments,
the Fc domain is the Fc domain of human IgG1 and comprises one or
more of the following effector-null substitutions: L234A, L235A,
and G237A (Eu numbering). In some embodiments, the Fc domain does
not comprise the lysine located at the C-terminal position of human
IgG1 (i.e., K447 by Eu numbering). The absence of the lysine may
increase homogeneity during the production of the recombinant
binding protein. In some embodiments, the Fc domain comprises the
lysine located at the C-terminal position (K447, Eu numbering).
[0313] Ankyrin Repeat Domains
[0314] In some embodiments, no more than 10, no more than 9, no
more than 8, no more than 7, no more than 6, no more than 5, no
more than 4, no more than 3, no more than 2, or no more than 1
substitution is made in any ankyrin repeat domain of a recombinant
binding protein of the invention relative to the sequences of SEQ
ID NOs: 1 to 11, 47, 76, 77 and 85. In some embodiments, no more
than 5 substitutions are made relative to the sequences of SEQ ID
NOs: 1 to 11, 47, 76, 77 and 85. In some embodiments, no more than
4 substitutions are made relative to the sequences of SEQ ID NOs: 1
to 11, 47, 76, 77 and 85. In some embodiments, no more than 3
substitutions are made relative to the sequences of SEQ ID NOs: 1
to 11, 47, 76, 77 and 85. In some embodiments, no more than 2
substitutions are made relative to the sequences of SEQ ID NOs: 1
to 11, 47, 76, 77 and 85. In some embodiments, no more than 1
substitution is made relative to the sequences of SEQ ID NOs: 1 to
11, 47, 76, 77 and 85. In some embodiments, the substitution(s) do
not change the K.sub.D value by more than 1000-fold, more than
100-fold, or more than 10-fold, compared to the K.sub.D value of
the protein comprising the sequences of SEQ ID NOs: 1 to 11, 47,
76, 77 and 85. In certain embodiments, the substitution is a
conservative substitution according to Table 3. In certain
embodiments, the substitution is made outside the structural core
residues of the ankyrin repeat domain, e.g. in the beta loops that
connect the alpha-helices.
TABLE-US-00003 TABLE 3 Amino Acid Substitutions Original
Conservative Residue Substitutions Exemplary Substitutions Ala (A)
Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His;
Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn
Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln;
Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L)
Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn
Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro
(P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe
Tyr(Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0315] In certain embodiments, the substitution is made within the
structural core residues of the ankyrin repeat domain. For example,
the ankyrin domain may comprise the consensus sequence:
DxxGxTPLHLAxxxGxxxlVxVLLxxGADVNAx (SEQ ID NO: 50), wherein "x"
denotes any amino acid (preferably not cysteine, glycine, or
proline); or DxxGxTPLHLAAxxGHLEIVEVLLKzGADVNAx (SEQ ID NO: 51),
wherein "x" denotes any amino acid (preferably not cysteine,
glycine, or proline), and "z" is selected from the group consisting
of asparagine, histidine, or tyrosine. In one embodiment, the
substitution is made to residues designated as "x". In another
embodiment, the substitution is made outside the residues
designated as "x".
[0316] In addition, the second last position of any ankyrin repeat
domain of a recombinant binding protein of the invention can be "A"
or "L", and/or the last position can be "A" or "N". Accordingly, in
some embodiments, each ankyrin repeat domain comprises an amino
acid sequence that is at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical to any one
of SEQ ID NOs: 1 to 11, 47, 76, 77 and 85, and wherein optionally A
at the second last position is substituted with L and/or A at the
last position is substituted with N. In an exemplary embodiment,
each spike protein binding domain comprises an amino acid sequence
that is at least 90% identical to any one of SEQ ID NOs: 1 to 11,
47, 76, 77 and 85, and wherein optionally A at the second last
position is substituted with L and/or A at the last position is
substituted with N. Furthermore, the sequence of any ankyrin repeat
domain comprised in a binding protein of the invention may
optionally comprise at its N-terminus, a G, an S, or a GS (see
below).
[0317] In addition, each ankyrin repeat domain comprised in a
recombinant binding protein of the invention may optionally
comprise a "G," an "S," or a "GS" sequence at its N-terminus.
Accordingly, in some embodiments, each ankyrin repeat domain
comprises an amino acid sequence that is at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical to any one of SEQ ID NOs: 1 to 11, 47, 76, 77 and
85, and further comprises at its N-terminus a GS (as e.g. in SEQ ID
NOs: 1 to 11, 47, 76, 77 and 85) or only a G or an S instead of the
GS.
[0318] In certain embodiments, the affinity between the recombinant
binding protein and its target (spike protein or serum albumin) is
described in terms of K.sub.D. In exemplary embodiments, the
K.sub.D is about 10.sup.-1 M or less, about 10.sup.-2 M or less,
about 10.sup.-3 M or less, about 10.sup.-4 M or less, about
10.sup.-5 M or less, about 10.sup.-6 M or less, about 10.sup.-7 M
or less, about 10.sup.-8 M or less, about 10.sup.-9 M or less,
about 10.sup.-10 M or less, about 10.sup.-11 M or less, about
10.sup.-12 M or less, about 10.sup.-13 M or less, about 10.sup.-14
M or less, from about 10.sup.-5 M to about 10.sup.-15 M, from about
10.sup.-6 M to about 10.sup.-15 M, from about 10.sup.-7 M to about
10.sup.-15 M, from about 10.sup.-8 M to about 10.sup.-15 M, from
about 10.sup.-9 M to about 10.sup.-15 M, from about 10.sup.-10 M to
about 10.sup.-15 M, from about 10.sup.-5 M to about 10.sup.-14 M,
from about 10.sup.-6 M to about 10.sup.-14 M, from about 10.sup.-M
to about 10.sup.-14 M, from about 10.sup.-8 M to about 10.sup.-14
M, from about 10.sup.-9 M to about 10.sup.-14 M, from about
10.sup.-10 M to about 10.sup.-14 M, from about 10.sup.-5 M to about
10.sup.-13 M, from about 10.sup.-6 M to about 10.sup.-13 M, from
about 10.sup.-M to about 10.sup.-13 M, from about 10.sup.-8 M to
about 10.sup.-13 M, from about 10.sup.-9 M to about 10.sup.-13 M,
or from about 10.sup.-10 M to about 10.sup.-13 M.
[0319] In exemplary embodiments, the recombinant binding protein
binds spike protein or serum albumin with an K.sub.D value of, or
less than: about 900 nM, about 800 nM, about 700 nM, about 600 nM,
about 500 nM, about 400 nM, about 300 nM, about 250 nM, about 200
nM, about 150 nM, about 100 nM, about 50 nM, about 40 nM, about 30
nM, about 20 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM,
about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500
pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about
10 pM, or about 1 pM. In one exemplary embodiment, the recombinant
binding protein binds spike protein or serum albumin with a K.sub.D
value of less than or equal to 100 nM. In another exemplary
embodiment, the recombinant binding protein binds spike protein or
serum albumin with a K.sub.D value of less than or equal to 10
nM.
[0320] Linkers
[0321] The recombinant binding proteins described herein may
comprise a linker. A "linker" is a molecule or group of molecules
that binds two separate entities (for example DARPin.RTM.1 and
DARPin 2.RTM. as shown in FIG. 1) to one another and can provide
spacing and flexibility between the two entities such that they are
able to achieve a conformation in which they can bind their
respective targets. Protein linkers are particularly preferred, and
they may be expressed as a component of the recombinant binding
protein using standard recombinant DNA techniques well-known in the
art.
[0322] The ankyrin repeat domains can be linked either covalently,
for example, by a disulfide bond, a polypeptide bond or a
crosslinking agent; or non-covalently, to produce a heterodimeric
protein. The recombinant binding protein can comprise linkers
between the coronavirus spike binding domains, and the optional
half-life extending moiety.
[0323] In some embodiments, the linker is a peptidyl linker. In
some embodiments, the peptidyl linker comprises about 1 to 50 amino
acid residues. Exemplary linkers includes, e.g., a glycine rich
peptide; a peptide comprising glycine and serine; a peptide having
a sequence [Gly-Gly-Ser].sub.n, wherein n is 1, 2, 3, 4, 5, or 6;
or a peptide having a sequence [Gly-Gly-Gly-Gly-Ser].sub.n (SEQ ID
NO: 54), wherein n is 1, 2, 3, 4, 5, or 6. A glycine rich peptide
linker comprises a peptide linker, wherein at least 25% of the
residues are glycine. Glycine rich peptide linkers are well known
in the art (e.g., Chichili et al. Protein Sci. 2013 February;
22(2): 153-167).
[0324] In some embodiments, the peptidyl linker is a
proline-threonine rich peptide linker. In an exemplary embodiment,
the linker is the proline-threonine rich peptide linker of SEQ ID
NO: 52. In another exemplary embodiment, the linker is the
proline-threonine rich peptide linker of SEQ ID NO: 53.
[0325] In some embodiments, the linker comprises the amino acid
sequence of SEQ ID NO: 53. Examples of longer proline-threonine
rich peptide linkers are found in SEQ ID NOs: 84 and 88.
[0326] N-Terminal and C-Terminal Capping Sequences
[0327] The ankyrin repeat domains of the recombinant binding
protein disclosed herein may comprise N-terminal or C-terminal
capping sequences. Capping sequences refers to additional
polypeptide sequences fused to the N- or C-terminal end of the
ankyrin repeat sequence motif(s), wherein said capping sequences
form tight tertiary interactions (i.e. tertiary structure
interactions) with the ankyrin repeat sequence motif(s), thereby
providing a cap that shields the hydrophobic core of the ankyrin
repeat domain at the side from exposing to the solvent.
[0328] The N- and/or C-terminal capping sequences may be derived
from, a capping unit or other structural unit found in a naturally
occurring repeat protein adjacent to a repeat unit. Examples of
capping sequences are described in International Patent Publication
Nos. WO 2002/020565 and WO 2012/069655, in U.S. Patent Publication
No. US 2013/0296221, and by Interlandi et al., J Mol Biol. 2008
Jan. 18;375(3):837-54.
[0329] Examples of N-terminal ankyrin capping modules (i.e.
N-terminal capping repeats) are SEQ ID NOs: 55 to 57 and examples
of ankyrin C-terminal capping modules (i.e. C-terminal capping
repeats) includes SEQ ID NO: 58.
[0330] Nucleic Acids & Methods
[0331] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein as defined
herein.
[0332] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 70 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 70.
[0333] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 71 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 71.
[0334] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 72 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 72.
[0335] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 73 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 73.
[0336] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 74 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 74.
[0337] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 80 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 80.
[0338] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 81 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 81.
[0339] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 82 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 82.
[0340] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 83 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 83.
[0341] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 78 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 78.
[0342] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 86 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 86.
[0343] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 92 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 92.
[0344] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 93 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 93.
[0345] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 94 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 94.
[0346] In one embodiment, the present invention relates to a
nucleic acid encoding a recombinant binding protein according to
one of the preceding embodiments, wherein said nucleic acid
comprises or consists of SEQ ID NO 95 or a variant thereof encoding
the same amino acid sequence as SEQ ID NO 95.
[0347] The present invention further relates to a vector comprising
said nucleic acid molecule. In one embodiment, said vector is an
expression vector.
[0348] The present invention further relates to a host cell
comprising said nucleic acid molecule or said vector.
[0349] In one embodiment, the present invention relates to a method
of making the recombinant binding protein as defined herein,
comprising culturing the host cell defined herein under conditions
wherein said recombinant binding protein is expressed.
[0350] Compositions, Uses and Methods of Treatment
[0351] The recombinant binding proteins described herein can be
used to treat a subject infected with the coronavirus. In one
embodiment, the subject is infected with coronavirus
SARS-CoV-2.
[0352] Thus, in one embodiment, the present invention relates to a
pharmaceutical composition comprising the binding protein or
nucleic acid as defined herein and a pharmaceutically acceptable
carrier or excipient.
[0353] The pharmaceutical compositions may comprise a
pharmaceutically acceptable carrier, diluent, or excipient.
Standard pharmaceutical carriers include a phosphate buffered
saline solution, water, emulsions such as an oil/water or water/oil
emulsion, and various types of wetting agents.
[0354] The pharmaceutical compositions can comprise any
pharmaceutically acceptable ingredients, including, for example,
acidifying agents, additives, adsorbents, aerosol propellants, air
displacement agents, alkalizing agents, anticaking agents,
anticoagulants, antimicrobial preservatives, antioxidants,
antiseptics, bases, binders, buffering agents, chelating agents,
coating agents, colouring agents, desiccants, detergents, diluents,
disinfectants, disintegrants, dispersing agents, dissolution
enhancing agents, dyes, emollients, emulsifying agents, emulsion
stabilizers, fillers, film forming agents, flavour enhancers,
flavouring agents, flow enhancers, gelling agents, granulating
agents, humectants, lubricants, mucoadhesives, ointment bases,
ointments, oleaginous vehicles, organic bases, pastille bases,
pigments, plasticizers, polishing agents, preservatives,
sequestering agents, skin penetrants, solubilizing agents,
solvents, stabilizing agents, suppository bases, surface active
agents, surfactants, suspending agents, sweetening agents,
therapeutic agents, thickening agents, tonicity agents, toxicity
agents, viscosity-increasing agents, water-absorbing agents,
water-miscible cosolvents, water softeners, or wetting agents. See,
e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A.
H. Kibbe (Pharmaceutical Press, London, U K, 2000), which is
incorporated by reference in its entirety. Remington's
Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack
Publishing Co., Easton, Pa., 1980), which is incorporated by
reference in its entirety.
[0355] The pharmaceutical compositions can be formulated to achieve
a physiologically compatible pH. In some embodiments, the pH of the
pharmaceutical composition can be, for example, between about 4 or
about 5 and about 8.0, or between about 4.5 and about 7.5, or
between about 5.0 and about 7.5. In exemplary embodiments, the pH
of the pharmaceutical composition is between 5.5 and 7.5.
[0356] In another embodiment, the present invention relates to a
method of treating a coronavirus infection in a subject, the method
comprising the step of administering an effective amount of at
least one binding protein as defined herein, or the nucleic acid as
defined herein, or of the pharmaceutical composition as defined
herein, to a subject in need thereof. The subject may be exhibiting
any of the symptoms associated with a coronavirus infection, with
differing degrees of severity, when the method of treating is
administered.
[0357] In some embodiments, a single administration of the method
of treating may be sufficient. In other embodiments, repeated
administration may be necessary. Various factors will impact on the
number and frequency of administrations, such as the age and
general health of the subject, as well as the state of the
subject's coronavirus infection and the severity of the symptoms
associated with coronavirus infection.
[0358] In some embodiments, the method is a prophylactic method,
i.e. a method of preventing a coronavirus infection in a subject.
In such methods, an effective amount of at least one binding
protein as defined herein, or the nucleic acid as defined herein,
or of the pharmaceutical composition as defined herein is
administered to a subject. Typically, the subject will not be
exhibiting any of the symptoms associated with a coronavirus
infection when the prophylactic method is administered.
[0359] In some embodiments, a single administration of the
prophylactic method may be sufficient. In other embodiments,
repeated administration may be necessary. Various factors will
impact on the number and frequency of administrations, such as the
age and general health of the subject, as well as the subject's
risk of exposure to a coronavirus.
[0360] In certain embodiments, the coronavirus infection is caused
by SARS-CoV-2. In certain embodiments, the subject is a human.
[0361] The binding proteins described herein can be administered to
the subject via any suitable route of administration, such as
parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal
administration. Formulations suitable for parenteral administration
include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain anti-oxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. For additional
details, see Pharmaceutics and Pharmacy Practice, J. B. Lippincott
Company, Philadelphia, Pa., Banker and Chalmers, eds., pages
238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th
ed., pages 622-630 (1986)).
[0362] The binding proteins described herein may be used in
combination with another therapeutic agent, such as an analgesic.
Each therapeutic agent may be administered simultaneously (e.g., in
the same medicament or at the same time), concurrently (i.e., in
separate medicaments administered one right after the other in any
order) or sequentially in any order. Sequential administration may
be useful when the therapeutic agents in the combination therapy
are in different dosage forms (e.g., one agent is a tablet or
capsule and another agent is a sterile liquid) and/or are
administered on different dosing schedules, e.g., an analgesic that
is administered at least daily and a biotherapeutic that is
administered less frequently, such as once weekly or once every two
weeks.
[0363] Methods of Detection or Diagnosis
[0364] In one embodiment, the present invention relates to at least
one binding protein described herein for use in a method of
diagnosing a coronavirus infection in a subject.
[0365] In one embodiment, the present invention relates to a method
of diagnosing a coronavirus infection in a subject comprising the
steps of contacting a sample from the subject in vitro or ex vivo
with at least one binding protein as described herein.
[0366] In one embodiment, the present invention relates to a method
of detecting a coronavirus in a subject, said method
comprising:
[0367] a) obtaining a sample from a subject;
[0368] b) contacting said sample with at least one binding protein
as described herein; and
[0369] c) detecting the presence of a coronavirus.
[0370] In said methods and uses, the sample may be obtained from a
bodily fluid such as blood, cerebrospinal fluid, plasma or urine.
Samples may also be obtained from mucus (such as via nasal,
oropharyngeal or vaginal swabs) or may be solid tissue samples
(e.g. from biopsy).
[0371] Samples may be stored before use in any of these methods.
For example, samples may be subject to cryogenic freezing for a
suitable period of time before use in said methods.
[0372] In said methods and uses, the subject may be exhibiting
symptoms associated with a coronavirus infection, with differing
degrees of severity. Alternatively, the subject may be
asymptomatic. The methods and uses may also be carried out on
samples obtained from non-living subjects to investigate cause of
death.
EXAMPLES
[0373] Starting materials and reagents disclosed below are known to
those skilled in the art, are commercially available and/or can be
prepared using well-known techniques.
[0374] Materials
[0375] Chemicals were purchased from Sigma-Aldrich (USA).
Oligonucleotides were from Microsynth (Switzerland). Unless stated
otherwise, DNA polymerases, restriction enzymes and buffers were
from New England Biolabs (USA) or Fermentas/Thermo Fisher
Scientific (USA). Inducible E. coliexpression strains were used for
cloning and protein production, e.g. E. coliXL1-blue (Stratagene,
USA) or BL21 (Novagen, USA).
[0376] Molecular Biology
[0377] Unless stated otherwise, methods are performed according to
known protocols (see, e.g., Sambrook J., Fritsch E. F. and Maniatis
T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory 1989, New York).
[0378] Cells and Viruses
[0379] Vero E6 cells (African green monkey kidney cells, ATCC.RTM.
CRL1586.TM.) purchased from ATCC (Manassas, Va. 20110 USA) were
passaged in cell culture medium DMEM (FG0445) containing 10% FBS
and supplements (2 mM L-Glutamine, Non-essential amino acids and
100 U/ml Penicillin 100 .mu.g/ml Streptomycin and HEPES, all from
Biochrom, Berlin, Germany) at 37.degree. C. without CO.sub.2.
SARS-CoV-2 (2019-nCoV/IDF0372/2020) was propagated in Vero E6 cells
in MEM containing 2% FBS and supplements (2%-FBS-MEM) at 37.degree.
C. Viruses were cultured without CO.sub.2 in non-vented flasks, 24
well-, or 96 well-plates covered with sealing foil (Biorad,
microseal B-film, MSB 1001) for the duration of experiments.
[0380] Designed Ankyrin Repeat Protein Libraries
[0381] Methods to generate designed ankyrin repeat protein
libraries have been described, e.g. in U.S. Pat. No. 7,417,130;
Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.. By such
methods designed ankyrin repeat protein libraries having randomized
ankyrin repeat modules and/or randomized capping modules can be
constructed. For example, such libraries could accordingly be
assembled based on a fixed N-terminal capping module or a
randomized N-terminal capping module, one or more randomized repeat
modules, and a fixed C-terminal capping module or a randomized
C-terminal capping module. Preferably, such libraries are assembled
to not have any of the amino acids C, G, M, N (in front of a G
residue) and P at randomized positions of repeat or capping
modules.
[0382] Furthermore, such randomized modules in such libraries may
comprise additional polypeptide loop insertions with randomized
amino acid positions. Examples of such polypeptide loop insertions
are complement determining region (CDR) loop libraries of
antibodies or de novo generated peptide libraries. For example,
such a loop insertion could be designed using the structure of the
N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka,
N., Nakanishi, M, Kusakabe, Y, Goto, Y., Kitade, Y, Nakamura, K.
T., EMBO J. 23(30), 3929-3938, 2004) as guidance. In analogy to
this ankyrin repeat domain where ten amino acids are inserted in
the beta-turn present close to the border of two ankyrin repeats,
ankyrin repeat protein libraries may contain randomized loops (with
fixed and randomized positions) of variable length (e.g. 1 to 20
amino acids) inserted in one or more beta-turns of an ankyrin
repeat domain.
[0383] Any such N-terminal capping module of an ankyrin repeat
protein library preferably possesses the RILLAA, RILLKA or RELLKA
motif (e.g. present from position 21 to 26 in SEQ ID NO: 55) and
any such C-terminal capping module of an ankyrin repeat protein
library preferably possesses the KLN, KLA or KAA motif (e.g.
present at the last three amino acids in SEQ ID NO: 58).
[0384] The design of such an ankyrin repeat protein library may be
guided by known structures of an ankyrin repeat domain interacting
with a target. Examples of such structures, identified by their
Protein Data Bank (PDB) unique accession or identification codes
(PDB-IDs), are 1WDY, 3V31, 3V30, 3V2X, 3V20, 3UXG, 3TWQ-3TWX, 1N11,
1S70 and 2ZGD.
[0385] Examples of designed ankyrin repeat protein libraries, such
as N2C and N3C designed ankyrin repeat protein libraries, have been
described (U.S. Pat. No. 7,417,130; Binz et al. 2003, loc. cit.;
Binz et al. 2004, loc. cit.). The digit in N2C and N3C describes
the number of randomized repeat modules present between the
N-terminal and C-terminal capping modules.
[0386] The nomenclature used to define the positions inside the
repeat units and modules is based on Binz et al. 2004, loc. cit.
with the modification that borders of the ankyrin repeat modules
and ankyrin repeat units are shifted by one amino acid position.
For example, position 1 of an ankyrin repeat module of Binz et al.
2004 (loc. cit.) corresponds to position 2 of an ankyrin repeat
module of the current disclosure and consequently position 33 of an
ankyrin repeat module of Binz et al. 2004, loc. cit. corresponds to
position 1 of a following ankyrin repeat module of the current
disclosure.
Example 1: Selection of Binding Proteins Comprising an Ankyrin
Repeat Domain with Binding Specificity for SARS-CoV-2 Spike
Protein
[0387] Summary
[0388] Using ribosome display (Hanes, J. and Pluckthun, A., PNAS
94, 4937-42, 1997), multiple ankyrin repeat domains with binding
specificity for different domains of the SARS-CoV-2 spike protein
(RBD domain; S1 NTD domain; S2 domain) were selected from
DARPin.RTM. libraries in a way similar to the one described by Binz
et al. 2004 (loc. cit.), with specific conditions and additional
de-selection steps. The binding and specificity of the selected
clones towards recombinant SARS-CoV-2 spike protein target domains
were assessed by E. coli crude extract Homogeneous Time Resolved
Fluorescence (HTRF), indicating that multiple SARS-CoV-2 spike
protein specific binding proteins were successfully selected. For
example, the ankyrin repeat domains of SEQ ID NOs: 1 to 11
constitute amino acid sequences of selected binding proteins
comprising an ankyrin repeat domain with binding specificity for
SARS-CoV-2 spike protein.
[0389] Spike Protein Domains as Target and Selection Material
[0390] Spike protein domains were used as target and selection
material. Proteins used for selections comprised SARS-CoV-2 S
protein ectodomain (SARS2-Secto-d72-GCN4-Streptag), SARS-Cov-2 S
protein (S1+S2 ECT, His-tag; Sinobiological 40589-V08B1),
Bio-COVID-19_S1 protein_His_Avitag (Acro Biosystems),
SARS2-S1-Flag-3Streptag, COVID-19_S_protein_RBD_Fc (Acro
Biosystems), and SARS2-S1B-2Streptag. Such target proteins were
selected from the polypeptides of SEQ ID NOs: 43 to 45 and 59 to
67. Proteins were biotinylated using standard methods.
[0391] Selection of SARS-CoV-2 Spike Protein-Specific Ankyrin
Repeat Proteins by Ribosome Display
[0392] Designed ankyrin repeat protein libraries (N2C and N3C) were
used in ribosome display selections against the SARS-CoV-2 spike
protein fragments (see Binz et al., Nat Biotechnol 22, 575-582
(2004); Zahnd et al., Nat Methods 4, 269-279 (2007); Hanes et al.,
Proc Natl Acad Sci USA 95, 14130-14135 (1998)).
[0393] Four selection rounds were performed per target and library.
The four rounds of selection employed standard ribosome display
selection, using decreasing target concentrations and increasing
washing stringency to increase selection pressure from round 1 to
round 4 (Binz et al. 2004, loc. cit.). The number of reverse
transcription (RT)-PCR cycles after each selection round was
continuously reduced, adjusting to the yield due to enrichment of
binders. The 12 resulting pools were then subjected to a binder
screening.
[0394] Selected Clones Bind Specifically to the RBD, S2 and S1-NTD
Domains of the Spike Protein of SARS-CoV-2 as Shown by Crude
Extract HTRF
[0395] Individually selected ankyrin repeat proteins specifically
binding to the RBD, S2 and S1-NTD domains of the spike protein of
SARS-CoV-2 in solution were identified by a Homogeneous Time
Resolved Fluorescence (HTRF) assay using crude extracts of ankyrin
repeat protein-expressing Escherichia coli cells using standard
protocols. Ankyrin repeat protein clones selected by ribosome
display were cloned into a derivative of the pQE30 (Qiagen)
expression vector, transformed into E. coliXL1-Blue (Stratagene),
plated on LB-agar (containing 1% glucose and 50 .mu.g/ml
ampicillin) and then incubated overnight at 37.degree. C. Single
colonies were picked into a 96 well plate (each clone in a single
well) containing 165 .mu.l growth medium (LB containing 1% glucose
and 50 .mu.g/ml ampicillin) and incubated overnight at 37.degree.
C., shaking at 800 rpm. 150 .mu.l of fresh LB medium containing 50
.mu.g/ml ampicillin was inoculated with 8.5 .mu.l of the overnight
culture in a fresh 96-deep-well plate. After incubation for 120
minutes at 37.degree. C. and 850 rpm, expression was induced with
IPTG (0.5 mM final concentration) and continued for 6 hours. Cells
were harvested by centrifugation of the plates, supernatant was
discarded and the pellets were frozen at -20.degree. C. overnight
before resuspension in 8.5 .mu.l pl B-PERII (Thermo Scientific) and
incubation for one hour at room temperature with shaking (600 rpm).
Then, 160 .mu.l PBS was added and cell debris was removed by
centrifugation (3220 g for 15 min).
[0396] The extract of each lysed clone was applied as a 1:200
dilution (final concentration) in PBSTB (PBS supplemented with 0.1%
Tween 20.RTM. and 0.2% (w/v) BSA, pH 7.4) together with 20 nM
(final concentration) biotinylated spike protein domain, 1:400
(final concentration) of anti-6His-D2 HTRF antibody--FRET acceptor
conjugate (Cisbio) and 1:400 (final concentration) of anti-strep-Tb
antibody FRET donor conjugate (Cisbio, France) to a well of a
384-well plate and incubated for 120 minutes at 4.degree. C. The
HTRF was read-out on a Tecan M1000 using a 340 nm excitation
wavelength and a 620.+-.10 nm emission filter for background
fluorescence detection and a 665.+-.10 nm emission filter to detect
the fluorescence signal for specific binding.
[0397] The extract of each lysed clone was tested for binding to
the biotinylated spike protein domains, in order to assess specific
binding to the spike protein.
[0398] Further Analysis and Selection of Binding Proteins
[0399] A total of 909 binders and inhibitors were identified. Based
on binding profiles, 360 candidates were selected to be expressed
in 96-well format and purified to homogeneity in parallel to DNA
sequencing. Candidates were characterized biophysically by size
exclusion chromatography, Sypro-Orange thermal stability assessment
(see Niesen et al., Nat Protoc 2, 2212-2221, (2007)), ProteOn
surface plasmon resonance (SPR) target affinity assessment, ELISA,
hACE2-competition HTRF experiments, SDS-PAGE, and/or SARS-CoV-2
pseudotype virus inhibition assay. Based on these data, 11
candidates (SEQ ID NOs: 1 to 11), binding to the RBD, S1-NTD or the
S2 domain, were chosen for further analysis. This analysis also
included 31 combinations in multi-domain formats (SEQ ID NOs: 12 to
42), exploring novel modes of action, determining inhibition
potency, epitope and target diversity, sequence diversity, and/or
biophysical properties. Multi-domain constructs were prepared using
Gibson assembly as described previously (see Binz, H. K. et al.
MAbs 9, 1262-1269, (2017)). Binding proteins of the invention were
expressed with a His-tag (SEQ ID NO: 46) at their N-terminus for
ease of purification or detection and tested in this His-tagged
form in the experiments described below.
[0400] Engineering of Additional Binding Proteins
[0401] In further development of the initially identified binding
proteins, binding domains with improved properties, such as
increased affinity to and/or reduced off-rate from target protein
or improved pharmacokinetic characteristics in mouse, were
generated using various methods. In one approach, an initially
identified binding protein (the "parental" binding protein) was
selected as a suitable starting point for affinity maturation. The
affinity maturation procedure entailed saturation mutagenesis of
each randomized position of the ankyrin repeat domain used as a
starting point. Sequences generated by the affinity maturation
procedure were screened for lower off-rates by competition HTRF.
Beneficial mutations identified thereby were combined in binding
proteins by protein engineering. The binding properties of affinity
matured and engineered binding proteins were validated by surface
plasmon resonance (SPR). In another approach, certain amino acid
residues in the N-terminal and/or C-terminal capping modules of the
ankyrin repeat domain were altered in order to achieve improved
pharmacokinetic properties, including a prolonged terminal
half-life, of the ankyrin repeat domain and of proteins comprising
the ankyrin repeat domain. Such altered amino acid residues were
mostly surface exposed residues (see, e.g., PCT/EP2020/085855). In
one example, ankyrin repeat domains with binding specificity for
the S1-NTD domain of the SARS-CoV-2 spike protein, namely
vS07_08F10v27 (SEQ ID NO: 76) and vS07_08F10v47 (SEQ ID NO: 85),
were generated by introducing a number of mutations in ankyrin
repeat domain vS07_08F10 (SEQ ID NO: 9), in order to reduce
hydrophobicity and/or increase binding affinity to and/or reduce
off-rate from its target. Reduction of hydrophobicity (e.g. by
altering residues in the N-terminal and C-terminal capping modules)
reduced the amount of any multimerization detected by SEC, reduced
viscosity and/or improved the pharmacokinetic properties in mouse.
Several mutated residues were identified in an affinity maturation
process using a single site mutagenesis approach on "parental"
binding protein, whereby potential binding residues were randomized
to all 20 amino acids by PCR, using degenerated primers. Individual
variants were tested for an improved off-rate by using a
competitive HTRF screening. Some individual mutations increased the
HTRF signal at least up to 2 to 3-fold. As examples, mutations
found in vS07_08F10v47 (SEQ ID NO: 85) include the following:
[0402] IR1_V11T: In the first internal repeat module, Valine at
position 11 was mutated to Threonine based on a 2 to 3-fold higher
signal in a HTRF competition assay indicating an improved
off-rate;
[0403] IR2_S3K: In the second internal repeat module, Serine at
position 3 was mutated to Lysine based on a 1.5 to 2-fold higher
signal in a HTRF competition assay indicating an improved
off-rate;
[0404] IR2_I4V: In the second internal repeat module, Isoleucine at
position 4 was mutated to Valine based on a 1.5 to 2-fold higher
signal in a HTRF competition assay indicating an improved off-rate,
and reduced multimerization of the protein compared to parental
protein;
[0405] IR2_R14Q: In the second internal repeat module, Arginine at
position 14 was mutated to Glutamine based on a >3-fold higher
signal in a HTRF competition assay indicating an improved
off-rate;
[0406] IR2_V15S: In the second internal repeat module, Valine at
position 15 was mutated to Serine based on a 1.5 to 2-fold higher
signal in a HTRF competition assay indicating an improved
off-rate;
[0407] C_W3V: In the C-terminal capping module, Tryptophan at
position 3 was mutated to Valine based on a 1.2 to 1.5-fold higher
signal in a HTRF competition assay indicating an improved off-rate,
and reduced multimerization of the protein compared to parental
protein;
[0408] C_I4V: In the C-terminal capping module, Isoleucine at
position 4 was mutated to Valine based on a 2 to 3-fold higher
signal in a HTRF competition assay indicating an improved off-rate;
and
[0409] C_I6V: In the C-terminal capping module, Isoleucine at
position 6 was mutated to Valine based on a 2 to 3-fold higher
signal in a HTRF competition assay indicating an improved
off-rate.
[0410] In another example, an ankyrin repeat domain with binding
specificity for the S2 domain of the SARS-CoV-2 spike protein
having improved properties, namely the ankyrin repeat domain of SEQ
ID NO: 77, was generated by introducing a number of mutations in
ankyrin repeat domain vS07_14G03 (SEQ ID NO: 10).
[0411] Engineered binding proteins, such as SEQ ID NOs: 76, 77 and
85, were characterized biophysically similarly as described above
for SEQ ID NOs: 1 to 11. Furthermore, combinations in multi-domain
formats comprising one or more of such engineered binding domains
were generated (e.g. SEQ ID NOs: 75, 84, 87 and 88), exploring
novel modes of action, determining inhibition potency, epitope and
target diversity, sequence diversity, and/or biophysical
properties, similarly as described above for SEQ ID NOs: 12 to
42.
Example 2: SPR Binding Assays
[0412] Surface plasmon resonance (SPR) assays were used to
determine the binding affinity of the binding proteins of the
invention to the spike protein of SARS-CoV-2.
[0413] All SPR data were generated using a Bio-Rad ProteOn XPR36
instrument with PBS-T (0.005% Tween20) as running buffer. A new
neutravidin sensor chip (NLC) was air-initialized and conditioned
according to Bio-Rad manual.
[0414] Mono-domain DARPin proteins: In-house chemically
biotinylated (via lysines) SARS-CoV-2 Spike Protein (Sino
Biologics, cat. 40589-V08B1, Lot MF14MA0701) was captured to
.about.3400 RUs (30 ug/ml, 30 ul/min, 300s). Two buffer injections
(100 ul/min, 60s) followed by two 12.5 mM NaOH regeneration steps
(100 ul/min, 18s) were applied before the first injections.
Mono-domain DARPin proteins were injected (at 50/16.7/5.6/1.9/0.6
nM (or at 16.7/5.6/1.9/0.6 nM for SEQ ID NO: 9 and 10)) for 180s at
100 ul/min for association and dissociation was recorded for 3600s
(at 100 ul/min). The ligand was regenerated with a 12.5 mM NaOH
pulse (100 ul/min, 18s). The data was double referenced against the
empty surface and a buffer injection and fitted according to the
1:1 Langmuir model.
[0415] Multi-domain DARPin proteins: In-house chemically
biotinylated (via lysines) SARS-CoV-2 (COVID-19) S protein RBD
(cat. SPD-C5255, lot. BV3539b-203FF1-203K) was captured to
.about.1000 RUs (775 ng/ml, 30 ul/min, 300s). Two buffer injections
(100 ul/min, 60s) followed by two 12.5 mM NaOH regeneration steps
(100 ul/min, 18s) were applied before the first injections. One
single concentration of 25 nM of each multi-domain DARPin construct
(including, e.g. ALE033, ALE030, ALE038, ALE049, ALE058) was
injected for 180s at 100 ul/min for association and dissociation
was recorded for 36000s (at 100 ul/min). The data was double
referenced against the empty surface and a buffer injection. Due to
avidity gain, no significant dissociation can be recorded during
the measured time.
[0416] Exemplary results of SPR assays are shown in FIGS. 6, 9a-c,
15a, 16 and 17 and in Table 4. See also Example 4.
[0417] Ankyrin repeat domains according to SEQ ID Nos 1-11 were
tested for their binding affinity to specific coronavirus spike
protein domains using SPR (multi trace, unless indicated). In
addition, other biophysical and functional properties were also
tested, using methods described herein in the Examples, such as
size exclusion chromatography (SEC), thermal stability measurements
(Tm), and SARS-CoV-2 VSV pseudovirus neutralization assays.
[0418] Results are provided in FIGS. 9a-c and in Tables 4a and 4b
below:
TABLE-US-00004 TABLE 4a bio-S ecto Sino (SEQ ID NO: 44) SEQ ID NO
K.sub.D [M] 1 2.6E-10 2 2.5E-10 3 2.1E-11 4 2.4E-10 5 9.0E-11 6
8.1E-11 7 .sup. 1.4E-08 * 8 .sup. 2.3E-08 * * single trace
TABLE-US-00005 TABLE 4b VSV-SARS-CoV-2 IC.sub.50 SEQ ID NO SEC Tm
[.degree. C.] [10.sup.-9 M] 3 Monomer >85.degree. C. <2 5
Monomer >85.degree. C. <2 6 Monomer >85.degree. C. <2 9
Monomer >85.degree. C. 10 Monomer <100
[0419] SEQ ID NOs: 1 to 8 were shown by SPR (single trace) to bind
to the RBD domain or the S1 domain of the spike protein with
similar affinities as indicated in Table 4a, using the bio-RBD Fc
Acro (SEQ ID NO: 45) and the bio-S1 Acro (SEQ ID NO: 43) as target
materials. SEQ ID NO: 9 was shown to bind to the S1 domain of the
spike protein with a K.sub.D of 2.0E-08 M (single trace), using the
bio-S1 Acro (SEQ ID NO: 43) as target material. SEQ ID NO: 9 and
SEQ ID NO: 10 were shown to bind to the ecto-domain of the spike
protein with a K.sub.D of 1.2E-09 M and 7.9E-10 M, respectively,
using the S ecto U (SEQ ID NO: 61) as target material (see FIG.
9c). SEQ ID NO: 76 was shown to bind to the S1 domain or the
ecto-domain of the spike protein with about the same K.sub.D as
observed for SEQ ID NO: 9, while SEQ ID NO: 85 was found to bind to
the S1 domain or the ecto-domain of the spike protein with an even
higher binding affinity (i.e. a lower K.sub.D) than SEQ ID NO: 9 or
SEQ ID NO: 76. SEQ ID NOs: 10, 11 and 77 were shown to bind to the
S2 domain of the spike protein, e.g. by HTRF assay. Table 4b shows
that each of SEQ ID NOs: 3, 5, 6, 9 and 10 was monomeric in size
exclusion chromatography. Furthermore, high thermal stability
(>85.degree. C.) and IC.sub.50 values in the nanomolar range
(e.g. <2 nM) when tested against SARS-CoV-2 VSV pseudovirus are
indicated for several of the SEQ ID Nos.
[0420] For the multi-domain DARPin proteins, no significant
dissociation could be recorded during the measured time due to
avidity gain (see, e.g., FIGS. 15a, 16 and 17). The apparent
affinity of the multi-domain proteins (including, e.g., of ALE049
and ALE058) was beyond the limit of SPR, indicating sub-pM target
affinity (data not shown).
Example 3: Functional Screening
[0421] This Example describes functional screening of mono-domain
and multi domain proteins using the SARS-CoV-2 VSV pseudotype virus
assay. The results of this assay are provided in FIGS. 5 to 8.
[0422] Infection inhibition was assessed using a vesicular
stomatitis virus (VSV) pseudovirus assay (psVSV), where the
glycoprotein of VSV was replaced by the Wuhan variant of the
SARS-CoV-2 spike glycoprotein tagged with an enhanced green
fluorescent protein (EGFP) and firefly luciferase (LUC). Inhibition
of infection following addition of 1 nM, 10 nM, or 100 nM of
candidate was measured by simple quantification of EGFP and LUC
activity (see Torriani, G. et al., Virology 531, 57-68 (2019)).
[0423] FIGS. 5 and 7 show pseudotype SARS-CoV-2 virus inhibition at
100 nM of various recombinant binding proteins that bind to a
single site on the spike protein (mono-domain DARPin.RTM. proteins)
and three sites on the spike protein (multi-domain DARPin.RTM.
proteins), respectively. Shorter bars are indicative of stronger
virus inhibition. FIG. 8 repeats FIG. 7 but at 1 nM. FIG. 6 shows a
representative SPR trace of a mono-domain recombinant binding
protein. This data shows that the Applicant was able to rapidly
establish the structures of multi-domain DARPin.RTM. proteins
having sub-nanomolar antiviral activity. Further rational design of
the recombinant binding proteins further increased potency.
Example 4: Neutralization Assay Using SARS-CoV-2 VSV Pseudovirus
(PsV nCoV) Cells
[0424] Vero E6, plated in 9 Costar 3610, clear bottom, white
plate
[0425] Pseudo SARS-CoV-2 (PsV nCoV)
[0426] 2000 IU/well (25 .mu.L)
[0427] 80'000 IU/mL=8*1041 U/mL
[0428] 4000 IU/well made 1.6*10.sup.5 IU/mL
[0429] Per plate 100*35 .mu.L. Prepared 4 mL of virus.times.8
plates=32 mL.
[0430] Took C15 at about 1*10.sup.6 IU/mL
[0431] 6 ml stock into 26 mL medium 2% FCS (fetal calf serum).
Total 32 mL
[0432] Recombinant Binding Proteins
TABLE-US-00006 TABLE 5 Samples 5 Domain Multi-Specific DARPin .RTM.
Designs Stock Vol Sample no. Sample name 1 2 3 4 5 (.mu.M) (.mu.L)
1 ALE030 H H R3b R2a R1b 20 100 2 ALE031 H H R3a R1a R2a 20 100 3
ALE033 H H R3b R1b R3b 20 100 4 ALE034 H H RN1 R1b R3b 20 100 5
ALE035 H H RN2 R3a R2a 20 100 6 ALE037 H H R3a R2a RN2 20 100 7
ALE038 H H R1b R3b S1a 20 100 8 ALE039 H H S1a R1b R3b 20 100 9
ALE040 H H R2a R3b S1a 20 100 10 ALE041 H H S1a R3b R2a 20 100 11
ALE042 H H R3b S1a S2a 20 100 12 ALE043 H H R1b S1a S2b 20 100 13
ALE044 H H S2a S1a R3b 20 100 14 ALE045 H H S2b S1a R1b 20 100 15*
ACO268 167 16** vS07_M101E04 10 50 *negative control; **positive
control
[0433] Human Serum Albumin
[0434] A 3.0 mM stock solution of human serum albumin (HSA) was
used to prepare a 10 .mu.M solution of HSA. The medium for this
solution comprised DMEM (Dulbecco's Modified Eagle Medium) 2% FCS
(fetal calf serum) and 20 .mu.M HEPES buffer solution
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
[0435] Sample Dilutions
[0436] 2-fold dilutions were prepared. Each dilution was mixed with
one volume (PsV nCoV).
[0437] Samples 1-14: Stock at 20 .mu.M
[0438] Prepared 300 .mu.L (quadruplicates, 4.times.70 .mu.L) at 100
nM Dilution 1: /1/10:
[0439] Took 15 .mu.L of the stock 20 .mu.M+135 .mu.L PBS Final
conc. 2 .mu.M
[0440] Dilution 2: 1/20:
[0441] Took 15 .mu.L of the dilution 1+285 .mu.L de milieu DMEM 2%
FCS, with 20 .mu.M HSA.
[0442] Final conc. 100 nM
[0443] Negative Control:
[0444] Dilution 1 neg control.
[0445] Dilution 1: /1/16.5:
[0446] Took 10 .mu.L of the stock 167 .mu.M+157 .mu.L PBS Final
conc. 10 .mu.M
[0447] Dilution 2: 1/10:
[0448] Took 15 .mu.L of the stock 10 .mu.M+135 .mu.L PBS Final
conc. 1 .mu.M
[0449] Dilution 3: /1/10:
[0450] Took 30 .mu.L of the dilution 2+270 .mu.L de milieu DMEM 2%
FCS, with 20 .mu.M HSA.
[0451] Positive Control:
[0452] Prepared 300 .mu.L (quadruplicates, 4.times.70 .mu.L) at 100
nM
[0453] Dilution 1: /1/10:
[0454] Took 15 .mu.L of the stock 10 .mu.M+135 .mu.L PBS Final
conc. 1 .mu.M
[0455] Dilution 2: 1/10:
[0456] Took 30 .mu.L of the dilution 1+270 .mu.L de milieu DMEM 2%
FCS, with 20 .mu.M HSA.
[0457] Final conc. 100 nM
[0458] Prepared [0459] In a V-bottom plate [0460] Prepared an
initial 1/10 dilution of the samples. Volume needed 4.times.70
.mu.l=280 .mu.l. Prepared 300 .mu.l media containing 2% FCS and 10
mM HEPES and [0461] Distributed 70 .mu.l in the quadruplicate
samples [0462] Two-fold dilutions carried out in the V-bottom
plate
[0463] Method & Results
[0464] One volume (35 .mu.l) of PsV nCOV was added to each well
before incubation for one hour at 37.degree. C. The cells were then
infected with 50 .mu.l/well and incubated again 37.degree. C. for
90 minutes. The inoculum was then removed, and 150 .mu.l medium 2%
FCS was added before a final incubation at 37.degree. C. for 16
hours. After the final incubation period, the assay was stopped and
infected cells (EGFP+) were counted at the appropriate dilution
using an inverted fluorescence microscope. Fixation of the cells
was not required. A luciferase assay was then carried out. Part of
the cell media was removed (100 .mu.l out of the 150 .mu.l) and 50
.mu.l of Glow (PROMEGA) was added to each well. The results were
read using a Berthold.RTM. TriStar LB941 luminometer for
approximately 1 sec. The data was analysed using the software Graph
Pad Prism 7, and the results are provided in Table 6:
TABLE-US-00007 TABLE 6 Sample no. Sample name Stock (.mu.M)
IC.sub.50 (nM) 1 ALE030 20 0.11950 2 ALE031 20 0.07529 3 ALE033 20
0.12470 4 ALE034 20 0.24070 5 ALE035 20 0.23770 6 ALE037 20 0.26320
7 ALE038 20 0.26380 8 ALE039 20 0.27920 9 ALE040 20 0.41750 10
ALE041 20 0.47560 11 ALE042 20 0.09803 12 ALE043 20 1.26700 13
ALE044 20 0.14710 14 ALE045 20 0.69270 15 ACO268 167
>>250.0000 16 vS07_M101E04 10 0.35780
[0465] Samples 1-14 have been found to be potent inhibitors of
pseudo-SARS-CoV-2, showing an IC.sub.50 of less 1.5 nM, and in most
cases of less than 0.7 nM. Samples 2 and 11 were particularly
potent, with an IC.sub.50 of less than 0.1 nM. FIG. 5 shows
fluorescence microscopy images showing GFP positive Vero E06 cells
which were infected with the GFP-labeled VSV pseudotype SARS-CoV-2
virus. ALE043 in FIG. 5 corresponds to sample no. 12 in Table 6
above. AC0268 and vS07_M101E04 are the negative and positive
controls respectively.
[0466] FIG. 11 shows neutralization of SARS-CoV-2 VSV pseudotype
virus.
[0467] FIG. 12 shows neutralization of SARS-CoV-2 VSV pseudotype
virus for samples 1 (ALE030), 4 (ALE033), 9 (ALE038), 13 (ALE042)
and 14 (ALE043). The positive control is also included
(vS07_M101E04).
[0468] In FIGS. 11 and 12, titration of candidates was from 50
nM-50 .mu.M (2-fold dilutions). The presence of 10 .mu.M of HSA did
not seem to influence the assay (see the control M101E04 without
HSA-binders). The results demonstrate that half-life extended
multi-domain constructs are potent inhibitors of PsV nCoV, with
IC.sub.50 values around 100 .mu.M.
Example 5: Virus Neutralization Activity; Microtitration Assay of
DARPin.RTM. Proteins (Open Cell System)
[0469] In this example, samples were tested against SARS-CoV-2
virus samples (i.e. not pseudovirus). Samples of the compounds set
out in Table 8 below were prepared in dilutions of 100 nM, 20 nM, 4
nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM.
TABLE-US-00008 TABLE 8 Sample no. Sample name Stock (.mu.M)
IC.sub.50 (nM) 1 ALE030 20 0.11950 3 ALE033 20 0.12470 4 ALE034 20
0.24070 5 ALE035 20 0.23770 6 ALE037 20 0.26320 7 ALE038 20 0.26380
8 ALE039 20 0.27920 9 ALE040 20 0.41750 10 ALE041 20 0.47560 11
ALE042 20 0.09803 12 ALE043 20 1.26700 13 ALE044 20 0.14710 14
ALE045 20 0.69270
[0470] The following control samples were also prepared: [0471]
Antibody positive serum (from a patient): 1:100, 1:500, 1: 2500,
1:625, . . . [0472] Antibody negative serum: 1:100, 1:500, 1:2500,
1:625, . . . [0473] Negative control DARPin protein: AC0268, a
HSA-binding DARPin protein [0474] ACE2 [0475] Virus back
titration
[0476] Medium: MEM, 2% FCS, L-Glut, NEAA, Neo, Pen Add: 10 .mu.M
HSA (Human Serum Albumin) dilute stock 1:300
[0477] The day before the assay was carried out, a 96-well plate
was prepared with confluent VeroE6 cells (open system) per two
compounds to be tested. All tests were carried out in triplicate.
FIG. 13 shows a map of the test plates, with border zones around
the edge and triplicate wells for each dilution value from 0.0064
to 100 nm, and control wells.
[0478] The samples were diluted to 100 nM in 1 ml medium containing
2% FCS (fetal calf serum) and 10 .mu.M HSA (human serum albumin).
100 .mu.l medium containing 2% FCS and 10 .mu.M HSA was added to
all wells in lines 4-11. 100 .mu.l of diluted test compound or
control (100 nM) was added to line 2, and 125 .mu.l was added to
line 3. Starting from line 3, the serum was serially diluted 1:5,
by mixing 25 .mu.l of the upper row with the lower row (each time,
the wells were thoroughly mixed by transferring the liquid up and
down the pipette 5 times) until line 10.
[0479] 6 ml of virus suspension was prepared per plate with 1000
TCID50/ml in MEM, 2% FCS, 10 .mu.M HSA (TCID50 is the 50% tissue
culture infective dose). 100 .mu.l virus suspension (100 TCID50)
was added to each well of line 3-10. The plates were incubated for
1 h at 37.degree. C. The medium was then removed from the 96 well
plate containing VeroE6 cells. 200 .mu.l of the test compound/virus
mixture was transferred to the 96 well plate with cells, and the
plates were incubated for 3 days at 37.degree. C. CPE was then
determined by microscope and crystal violet staining.
[0480] The results of Example 4 are shown in FIGS. 14a to 14f. Blue
colored cells indicate 100% activity; colorless cells indicate no
activity. As clearly demonstrated, there was almost complete
protection of the cells down to 32 .mu.M showing that the
recombinant binding proteins of the present invention are very
potent inhibitors of coronavirus spike protein, and specifically
SARS-CoV-2 spike protein.
Example 6: Virus Neutralization Activity; Microtitration Assay of
DARPin.RTM. Proteins (Open Cell System)
[0481] In this example, ankyrin repeat binding domains were tested
against SARS-CoV-2 virus. Samples of the ankyrin repeat binding
domains set out in Table 9 below were prepared in dilutions of 200
nM, 100 nM, 20 nM, 2 nM and 0.2 nM.
TABLE-US-00009 TABLE 9 SEQ ID NO Sample name 3 vS07_12C06 1
vS07_19G10 6 vS07_29B10 5 vS07_23E04 10 vS07_14G03
[0482] The following control samples were also prepared: [0483]
ACE2 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM [0484] Virus back
titration
[0485] Material used: Biotinylated human ACE-2 Fc, Acro Biosystems
(cat #AC2-H82F9)
[0486] Medium: MEM, 2% FCS, L-Glut, NEAA, Neo, Pen Add: 10 .mu.M
HSA (Human Serum Albumin) dilute stock 1:300
[0487] The day before the assay was carried out, a 96-well plate
was prepared with confluent VeroE6 cells (open system) per two
compounds to be tested. All tests were carried out in
quintuplicate. As per FIG. 13 each 96-well plate included a border
zone, and then rows of differing concentration wells, from 200 nm
through 0.2 nM.
[0488] The samples were diluted to 200 nM in 1 ml medium containing
2% FCS (fetal calf serum) and 10 .mu.M HSA (human serum albumin).
100 .mu.l medium containing 2% FCS and 10 .mu.M HSA was added to
all wells. The ACE2 control wells were prepared in an analogous
fashion, using the indicated concentrations of ACE2.
[0489] 10 ml of virus suspension was prepared per plate with 1000
TCID50/ml in MEM, 2% FCS, 10 .mu.M HSA. 100 .mu.l virus suspension
(100 TCID50) was added to each well, except for the wells at the
edges of the plates (i.e. the border wells). The plates were sealed
and incubated for 1 h at 37.degree. C. The medium was then removed
from the 96 well plate containing VeroE6 cells. 200 .mu.l of the
test compound/virus mixture was transferred to the 96 well plate
with cells, and the plates were sealed and incubated for 2-3 days
at 37.degree. C. CPE was then determined by microscope or methyl
blue staining.
[0490] The results of Example 5 are shown in FIGS. 18a to 18d. Blue
colored cells indicate 100% activity; colorless cells indicate no
activity. As clearly demonstrated, there was complete or almost
complete protection of the cells down to 20 nM or even below,
showing that the recombinant binding proteins of the present
invention are very potent inhibitors of coronavirus spike protein,
and specifically SARS-CoV-2 spike protein. Specifically, full
protection was observed for vS07_12C06 down to 2 nM, for vS07_29B10
and vS07_23E04 down to 20 nM, and for vS07_19G10 down to 100 nM
(with almost full protection at 20 nM). For vS07_14G03, partial
protection was observed between 2 and 200 nM.
Example 7: Virus Neutralization Activity; Titration of DARPin.RTM.
Proteins in a Low Concentration Range
[0491] In order to further investigate the ability of recombinant
binding proteins of the invention to inhibit the infection of cells
with live SARS-CoV-2, two distinct assays were performed measuring
cell viability of Vero E06 cells with i) CellTiter-Glo.RTM. from
Promega and ii) crystal violet staining. The samples tested are
listed in Table 10, and the results are shown in FIG. 19.
TABLE-US-00010 TABLE 10 SEQ ID NO Sample name 15 ALE033 24 ALE042
30 ALE048 31 ALE049 35 ALE053 39 ALE058
[0492] All samples were provided in 20 .mu.M stock and were
initially diluted to 800 .mu.M, 400 .mu.M, 200 .mu.M, 100 .mu.M, 50
.mu.M, 25 .mu.M, 12.5 .mu.M, 6.25 .mu.M, and 3.125 .mu.M in 2% FCS
medium containing 10 .mu.M HSA, then further diluted 1:2 with the
virus suspension.
[0493] Preparation [0494] 96-well plates with 80% confluent VeroE6
cells (open system) per two compounds were prepared the day before
testing (compounds are tested in triplicates) [0495] Compounds were
diluted to 800 .mu.M in 1 ml MEM medium containing 2% FCS and 10
.mu.M HSA. [0496] All compounds were serially diluted 1:2 by mixing
100 .mu.l diluted compound in 100 .mu.l MEM medium containing 2%
FCS and 10 .mu.M HSA. [0497] Border wells were kept free (only
cells and medium) to avoid border effects in test [0498] To
identify unspecific effects of compounds on cells a control line
with only compound and cells was foreseen (control; line 2) [0499]
From line 3 to 9 the compounds were serially diluted 1:2 from 200
.mu.M to 3.125 .mu.M [0500] In line 10 and 11 MEM medium containing
2% FCS and 10 .mu.M HSA was added for the virus control and cell
control
[0501] Test Procedure
[0502] The plate layout was similar to the layout as shown in FIG.
13, but with the compound concentrations indicated above [0503]
Virus suspension (10 ml per plate) with 1000 TCID50/ml of
SARS-CoV-2 (2019-nCoV/IDF0372/2020) in MEM medium containing 2% FCS
and 10 .mu.M HSA [0504] 100 .mu.l of virus suspension (100 TCID50)
was added to each well of line 3-10, medium was added to all other
wells [0505] The plates were incubated for 1 hour at 37.degree. C.
[0506] From the 96 well plates containing the 80% confluent VeroE6
cells medium was removed and 200 .mu.l of the test compound/virus
mixture added to the 96 well plate with cells [0507] Cultures were
incubated 3 days at 37.degree. C. [0508] For analysis of virus
genome copies by qPCR: 100 .mu.l supernatant was inactivated in 400
.mu.l AVL buffer+400 .mu.l 100% EtOH->Inactivated supernatant
was sluiced out of the BSL3 lab [0509] To determine cell viability:
100 ul CellTiter-Glo.RTM. (Promega) substrate was prepared and
added according to the manufacturers protocol, plates were shaken
for 2 min and fluorescence red analysed in a GloMax.RTM.
(Promega).
[0510] Results
[0511] The results of testing by CellTiter-Glo.RTM. luminescent
viability assay are provided in FIG. 19. VK: viral control; ZK:
cell control. Full protection of Vero E06 cells was observed at
approximately 25 .mu.M of test compound. Complete protection of
cells was observed down to 25 .mu.M for ALE033, ALE042 and ALE048.
Protection was somewhat less efficient for ALE049, ALE053 and
ALE058, but at least partial protection of cells was observed at 25
.mu.M also for these compounds. In conclusion, multi-domain binding
proteins of the invention are capable of inhibiting infection of
cells by SARS-CoV-2 at picomolar concentrations.
Example 8: Further Characterization of Multi-Specific Binding
Proteins Comprising SEQ ID NO: 31 (ALE049) or SEQ ID NO: 39
(ALE058)
[0512] Further characterization of multi-specific binding proteins
comprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO:
39 included SDS-PAGE (result: fully intact size without
degradation; data not shown), mass spectrometry (result: expected
molecular weight; data not shown), size exclusion chromatography
coupled to static light scattering, circular dichroism, storage
stability (result: stable at 60.degree. C. for 1 week; data not
shown), serum stability (result: stable at 37.degree. C. in serum
for one week; data not shown), surface plasmon resonance,
SARS-CoV-2 pseudotype virus inhibition assay, live virus inhibition
assay, mouse pharmacokinetic analysis (see Example 9), and hamster
efficacy model (see Example 10).
[0513] Experimental Methods and Results
[0514] Circular Dichroism
[0515] Circular dichroism measurement was performed with a Jasco
J-815 using a 1 cm pathlength cuvette (Hellma) with the monitor
sensor inserted in the cuvette. The MRE at 222 nm was followed over
a temperature ramp from 20.degree. C. to 90.degree. C. (heating and
cooling). Spectra from 190-250 nm were taken before and after the
variable temperature measurement at 20.degree. C. The protein was
measured at 0.25 .mu.M in PBS.
[0516] Surface Plasmon Resonance Affinity Determination
[0517] SPR assays were used to determine the binding affinity of
the multi-specific binding proteins to the spike protein of
SARS-CoV-2. SPR experiments were performed as described in Example
2.
[0518] SARS-CoV-2 VSV Pseudotype Virus Assay
[0519] The binding proteins were assessed for inhibition potency in
a SARS-CoV-2 VSV pseudotype virus assay. This assay was performed
as described in detail, e.g., in Examples 3 and 4.
[0520] SARS-CoV-2 Live Virus Assay
[0521] The binding proteins were assessed for inhibition potency in
a SARS-CoV-2 virus assay, similar as described in Example 5. In
brief, the binding proteins were prepared in dilutions of 100 nM,
20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM in 96-well
plates as described below, using the following medium: MEM, 2% FCS,
L-Glut, NEAA, Neo, Pen; with addition of 10 .mu.M HSA (Human Serum
Albumin) (dilute stock 1:300). All tests were carried out in
triplicates. The day before the assay was carried out, a 96-well
plate was prepared with confluent VeroE6 cells (open system) per
two compounds to be tested. The test plate was designed similar as
shown in FIG. 13, with border zones around the edge and triplicate
wells for each final dilution value from 0.0032 nM to 50 nM, and
control wells. Samples were diluted to 100 nM in 1 ml medium
containing 2% FCS (fetal calf serum) and 10 .mu.M HSA (human serum
albumin) (see above). 100 .mu.l medium containing 2% FCS and 10
.mu.M HSA was added to all wells in lines 4-11. 100 .mu.l of
diluted test compound or control (100 nM) was added to line 2, and
125 .mu.l was added to line 3. Starting from line 3, the serum was
serially diluted 1:5, by mixing 25 .mu.l of the upper row with the
lower row (each time, the wells were thoroughly mixed by
transferring the liquid up and down the pipette 5 times) until line
10. 6 ml of virus suspension was prepared per plate with 1'000
TCID50/ml in MEM, 2% FCS, 10 .mu.M HSA. 100 .mu.l virus suspension
(100 TCID50) was added to each well of lanes 3-10. The plates were
incubated for 1 h at 37.degree. C. The medium was then removed from
the 96 well plate containing VeroE6 cells. 200 .mu.l of the test
compound/virus mixture was transferred to the 96 well plate with
cells, and the plates were incubated for 3 days at 37.degree. C.
Cytopathic effect was then determined either by microscope and
crystal violet staining, where blue colored cells indicate 100%
activity and colorless cells indicate no activity (see FIG. 20),
or, alternatively, using a CellTiter-Glo.RTM. luminescent cell
viability assay (Promega; see FIGS. 21a-c). For the latter, 1'000,
10'000, or 100'000 TCID50 were used.
[0522] As clearly demonstrated in FIG. 20, there was complete or
almost complete protection of the cells down to 0.08 nM for both
ALE049 and ALE058, showing that the recombinant binding proteins of
the present invention are very potent inhibitors of coronavirus
spike protein, and specifically SARS-CoV-2 spike protein, and of
infection of cells by a coronavirus, and specifically by
SARS-CoV-2. Corresponding results are shown in FIGS. 21a-c, which
also demonstrate potent inhibition for both ALE049 and ALE058. The
exact concentration of the recombinant binding proteins required to
achieve efficient inhibition in these assays was dependent on the
viral load used. For both ALE049 and ALE058, potent inhibition of
SARS-CoV-2 was observed in the picomolar range, for ALE049 down to
50 .mu.M. IC.sub.50 values for ALE049 and ALE058 are shown in Table
11 below. These values of virus inhibition represent the strongest
SARS-CoV-2 inhibition reported to date.
[0523] Molecular Model of Drug Candidates
[0524] A molecular model for ALE049 (FIG. 22A) was built based on
cryogenic electron microscopy data (data not shown). In the first
step, a model structure of binding domain #2 was generated. The
consensus designed ankyrin repeat domain PDB:2xee was used as
template. Mutations were introduced with RosettaRemodel with fixed
backbone, and the structure was refined with RosettaRelax. Forty
refined structures were clustered using RosettaCluster with 0.3
.ANG. radius, and the lowest-energy model from the largest cluster
served as the final model. This model was then used for fitting
domain #2 into the observed electron density generated from the
complex structure of the spike protein and domain #2, resulting in
a PDB file with the coordinates of the trimer of domain #2:RBD.
This trimeric model was used as an input structure for the
conceptual modeling of ALE049 bound to the spike ectodomain as
shown in FIG. 22A. Similarly, a molecular model was also built for
ALE058 (FIG. 22B). This model for ALE058 is based on the cryogenic
electron microscopy data as well as a schematic structural
prediction for the S2 binding domain.
[0525] Multi-specific binding proteins comprising the amino acid
sequence of SEQ ID NO: 31 (ALE049) or SEQ ID NO: 39 (ALE058) each
comprise combinations of 3 SARS-CoV-2 spike protein binders fused
C-terminally to 2 clinically validated serum albumin-binding
domains for systemic half-life extension. The resulting 5-domain
proteins were expressed, purified and characterized in detail
regarding biophysical properties, target affinity, and virus
inhibition. The multi-specific binding proteins were expressed in
soluble form at high levels in the cytoplasm of E. coli. Purified
proteins are monomeric and exhibit high thermal stability
(Tm>88.degree. C.) and reversible unfolding as assessed by
circular dichroism, and high stability in accelerated storage
stability assays at 60.degree. C. (data not shown). Their apparent
affinity is beyond the limit of SPR, indicating sub-pM target
affinity (data not shown). In psVSV assays, the multi-specific
binding proteins inhibited viral entry with IC.sub.50 values
ranging from 3 pM to 138 pM or 0.24 ng/ml to 11.04 ng/ml (see Table
11, FIG. 23). The psVSV assay results correlated well to live virus
assay results, where infection inhibition was observed with
concentrations of 25 pM to 100 pM or 2 ng/ml to 8 ng/ml (see Table
11, FIGS. 21a-c).
TABLE-US-00011 TABLE 11 Tm [.degree. C.] IC.sub.50 psVSV IC.sub.50
LV* Name SEC (CD) [10.sup.-12 M] [10.sup.-12 M] ALE049 Monomer
>88.degree. C. 46-138 25 ALE058 Monomer >88.degree. C. 3-99
100 *LV: Live virus cytopathic effect assay
[0526] FIG. 23 further shows neutralization of the SARS-CoV-2 VSV
pseudovirus by the recombinant binding proteins of the invention
tested in the presence of the RBD domain of the spike protein. FIG.
23 shows that the RBD domain competes strongly with ALE049, which
contains three RBD binding domains, but not with ALE058, which
contains one RBD binding domain, one S1-NTD binding domain and one
S2 binding domain. ALE049 lost potency when competing with the
isolated RBD-domain, while competition of the single RBD-binder in
ALE058 had no significant impact on the potency of ALE058. Without
wishing to be bound by theory, this data appears to confirm that
ALE049 and ALE058 inhibit SARS-CoV-2 by different modes of action.
While ALE049 seems to rely strongly on the neutralization of the
RBD/ACE-2 interaction, ALE058 seems to show multi-mode binding and
a diversified mode of action, which beyond the neutralization of
the RBD/ACE-2 interaction also utilizes an independent
neutralization potency of the S1-NTD-S2 arm of the molecule. Thus,
based on the data shown in FIG. 23, ALE049 and ALE058 appear to
have different modes of action, consistent with the molecular
models of the two molecules shown in FIG. 22.
[0527] Such high potency as observed for the binding proteins of
the invention is key for the use in SARS-CoV-2 treatment and
prophylaxis where very low virus titers at infection initiation are
envisioned. Importantly, several spike protein variants of the most
abundant SARS-CoV-2 serotypes were blocked with high potency by the
multi-specific binding proteins (see Table 12), indicating
robustness against viral escape and potential of use in
prophylactic treatment in the current pandemic and potentially also
future pandemics. In mouse experiments, no adverse events were
observed up to the highest dose (50 mg/kg, i.v.) tested.
TABLE-US-00012 TABLE 12 Potency of inhibition of SARS-CoV-2 spike
protein variants (IC.sub.50, [10.sup.-12 M]) wt G476S V483A D614G
D614G .times. Q675H ALE049 16.53 27.08 27.48 11.77 12.11 ALE058
5.48 14.46 32.40 4.64 22.44
Example 9: Pharmacokinetic Analysis of Multi-Specific Binding
Proteins of the Invention in Mice
[0528] A pharmacokinetic (PK) study was conducted to assess the PK
characteristics of multi-specific recombinant binding proteins of
the invention in mice. Such PK characteristics are useful for dose
predictions of multi-specific binding proteins of the invention in
animal pharmacodynamic studies, in toxicology studies or in human
clinical trials.
[0529] The investigated multi-specific binding proteins of the
invention comprise--from N-terminus to C-terminus--two HSA-specific
binding domains followed by three spike protein-specific binding
domains (see Table 2). The HSA-specific binding domains are
cross-reactive to serum albumin of the mouse.
[0530] For this PK study, naive female BALB/c mice received a
single intravenous bolus injection at a target dose level of 1
mg/kg of the compounds. Blood samples were collected at several
time points between 5 min and 165 h after compound administration.
Serum concentrations were determined with ELISA-based analytical
methods.
[0531] From 6 h onwards the concentration-time profiles indicate a
slow and steady decrease of the serum concentrations resembling
roughly mono-exponential declines until 165 h, the last sampling
time point.
[0532] From the concentration time profiles pharmacokinetic
parameters were determined using non-compartmental analysis.
[0533] The following multi-specific binding proteins were tested in
this example:
TABLE-US-00013 TABLE 13 SEQ ID NO Sample name 15 ALE033 30 ALE048
31 ALE049
[0534] In Vivo Animal Experiments
[0535] The test items were administered to healthy female BALB/c
mice (6 mice per test item) as a single intravenous bolus injection
into the tail vein. The target dose level was 1 mg/kg. For the
study of each compound, the 6 mice were split into 2 groups with
equal numbers of animals. For pharmacokinetic investigations, serum
samples, 4 from each mouse, were collected from the saphenous vein
at time points 5 min, 6 h, 24 h, 48 h, 72 h, 96 h and 165 h. The
assignment of the individual animals to the respective sampling
time points was according to a predetermined scheme. Blood was kept
at room temperature for approx. 30 min to allow clotting followed
by centrifugation (5 min/12000 g/4.degree. C.). Afterwards serum
was frozen and stored at -20.degree. C. pending analyses. No major
issues and no drug-related adverse effects were reported for the in
vivo experiment.
[0536] ELISA Method
[0537] An ELISA method (see FIG. 24) was used for measuring serum
concentrations of the multi-specific binding proteins making use of
a common epitope of DARPin.RTM. moieties recognized by the
anti-DARPin.RTM. antibody 1-1-1 for capturing and of the N-terminal
His-tag, which is present in the tested binding proteins, to
facilitate detection. The ELISA setup scheme illustrated in FIG. 24
(showing ALE049 as a binding protein example) uses monoclonal goat
anti-rabbit-IgG immobilized on the ELISA plate, which binds rabbit
anti-DARPin.RTM. antibody 1-1-1, capturing the multi-specific
binding proteins via DARPin.RTM. scaffold epitopes in serum sample.
The captured DARPin.RTM. molecule is detected using mouse
anti-RGS-His-IgG-HRP conjugate. aSA: anti serum albumin, aRBD: anti
receptor binding domain (RBD)
[0538] Test Procedure
[0539] One hundred .mu.L per well of 10 nmol/L polyclonal goat
anti-rabbit IgG antibody (Ab18) in PBS was coated onto a NUNC
Maxisorb ELISA plate overnight at 4.degree. C. After washing with
300 .mu.L PBST (PBS supplemented with 0.1% Tween20) per well five
times, the wells were blocked with 300 .mu.L PBST supplemented with
0.25% Casein (PBST-C) for 1 h at room temperature (RT) on a
Heidolph Titramax 1000 shaker (450 rpm). Plates were washed as
described above. One hundred .mu.L per well of 5 nmol/L rabbit
anti-DARPin.RTM.1-1-1 antibody in PBST-C was added and the plates
were incubated at RT (22.degree. C.) with orbital shaking (450 rpm)
for 1 h. Plates were washed as described above.
[0540] One hundred .mu.L per well of diluted serum samples
(1:20-1:312500, in 1:5 dilution steps), multi-specific binding
protein quality control samples (100, 10 and 1 nmol/L) or
multi-specific binding protein standard curve samples (0 and
50-0.001 nmol/L in 1:3 dilution steps) diluted in PBST-C
(supplemented with naive mouse serum to result in a final serum
concentration of 1% (initial 1:20 dilution final serum
concentration of 5%)) were applied for 2 h, at RT, shaking at 450
rpm. Plates were washed as described above.
[0541] Wells were then incubated with 100 .mu.L murine
anti-RGS-His-HRP IgG (Ab06) 1:2000 in PBST-C and incubated for 1 h,
at RT, 450 rpm. Plates were washed as described above. The ELISA
was developed using 100 .mu.L per well TMB substrate solution for 5
minutes and stopped by the addition of 100 .mu.L per well 1 mol/L
H.sub.2SO.sub.4. The difference between the absorbance at 450 nm
and the absorbance at 620 nm was calculated. Samples were measured
in duplicate on two different plates.
[0542] Quality control samples of known concentrations were
included in the measurements in order to monitor the performance of
the assay.
[0543] Pharmacokinetic data analysis was performed using Phoenix
WinNonlin.TM. 8.0 program from Certara.
[0544] Calculation of the pharmacokinetic parameters of the study
based on the mean concentration-time data of the animals dosed via
intravenous bolus injection was performed with non-compartmental
analysis (NCA model 200-202, IV bolus, linear trapezoidal linear
interpolation).
[0545] The calculated pharmacokinetic parameters included at least
the following: AUCinf_pred, AUClast, AUC_extrapol,
AUC_Back_Ext_pred, Cmax, Tmax, CI_pred, Vss_pred, t1/2
(HL_Lambda_z) The results are shown in Table 14 and FIG. 25:
TABLE-US-00014 TABLE 14 Parameter Unit ALE033 ALE048 ALE049
AUCINF_pred h*(nmol/L) 12428 12768 14329 AUClast h*(nmol/L) 11439
11461 12949 Cmax nmol/L 244 230 291 Tmax h 0.083 0.083 0.083
Cl_pred L/(h*kg) 0.00094 0.00091 0.00081 Vss_pred L/kg 0.058 0.063
0.055 HL_Lambda_z h 45.8 50.8 49.6 AUC_% Extrap_pred (%) 8 10 10
AUC_% Back_Ext_pred (%) 0 0 0
[0546] Results and Conclusions
[0547] In the mono-exponential elimination phases, serum
concentrations of ALE033, ALE048 and ALE049 declined with half-life
values of 45.8 h, 50.8 h and 49.6 h, respectively. Clearance of
ALE033, ALE048 and ALE049 was determined to be 0.00094, 0.00091 and
0.00081 L/(h*kg), respectively, and volume of distribution (Vss) of
ALE033, ALE048 and ALE049 was calculated to be 0.058, 0.063 and
0.055 L/kg, respectively. The values determined for Vss indicate
that ALE033, ALE048 and ALE049 are largely confined to the systemic
circulation of the animals, similarly to monoclonal antibodies. In
conclusion, following intravenous administration at a dose level of
1 mg/kg the three tested multi-specific binding proteins of the
invention display a systemic half-life in the range of the
half-life of albumin in mice. Considering the half-life of albumin
in mouse and human as well as previous data (Binz et al., MAbs 9,
1262-1269 (2017)), the terminal half-life of ALE049 in humans is
expected to extrapolate to around 3 weeks. The terminal half-lives
of ALE033 and ALE048 in humans are expected to extrapolate
similarly.
Example 10: SARS-CoV-2 Inhibition Efficacy Experiments in Syrian
Hamster
[0548] The efficacy of ALE049 was further assessed in a Syrian
hamster model of preventive treatment of SARS-CoV-2 infection.
[0549] Syrian hamsters were divided into 4 groups of 6 female
animals each. The groups were treated with of 16 .mu.g, 160 .mu.g,
or 1600 .mu.g of multi-specific binding protein having the amino
acid sequence of SEQ ID NO: 31 or with placebo in a blinded manner.
Treatment injection (i.p., intraperitoneal) was done 24 h prior
(Day -1) to intranasal infection (Day 0) of the animals with
5.times.10.sup.4 TCID50 (in 100 .mu.l) of SARS-CoV-2
(BetaCoV/Munich/BavPat1/2020). At Day -2, body weight was measured,
blood was taken, and the first throat swab performed. Animals were
euthanized on Day 4 and tissue was taken and gross pathology was
performed. Throat swabs were collected daily in virus transport
medium, aliquoted and stored. At the time of necropsy, gross
pathology was performed. Lung lobes were inspected and an
estimation of the percentage of affected lung tissue from the
dorsal view was performed. Left lung lobes and nasal turbinates
were preserved in 10% neutral buffered formalin for histopathology.
The right side of these tissues was homogenised and subjected to
Taqman PCR and virus titration. Additionally, other organs were
collected. Tissue samples were frozen for virological analysis,
weighed, homogenized in infection medium and centrifuged briefly
before titration. Histopathology was performed on lung and nasal
turbinates for all animals. After fixation with 10% formalin,
sections from left lung and left nasal turbinate were embedded in
paraffin and the tissue sections were stained by H&E for
histological examination. For virological analyses, quadruplicate
10-fold serial dilutions were used to determine the virus titers in
confluent layers of Vero E6. To this end, serial dilutions of the
samples (throat swabs and tissue homogenates) were made and
incubated on Vero E6 monolayers for 1 h at 37.degree. C. Vero E6
monolayers were washed and incubated for 4-6 days at 37.degree. C.
after which plates were scored WST8. Viral titers (TCID50) were
calculated using the method of Spearman-Karber. Readout included
observation of body weight, lung lesions, virus titers, and
histopathology.
[0550] Histopathology
[0551] After fixation with 10% neutral-buffered formalin, sections
of the left lung, left nasal turbinate and trachea were embedded in
paraffin. The tissue sections were stained with hematoxylin and
eosin (H&E) for histopathological evaluation. Semi-quantitative
scores of 0, 1, 2 or 3 were given when the extent of alveolitis and
alveolar damage were estimated at 0%; 1-25%; 26-50% or >50%,
respectively. The cumulative score for the extent and severity of
inflammation of the lung provided the total score of alveolitis per
animal (see Table 15, column "SUM of extent+severity"). For the
severity of alveolitis, bronchiolitis, and bronchitis,
semi-quantitative scores of 0, 1, 2 or 3 were given when no, few,
moderate numbers or many inflammatory cells were present,
respectively. For the presence of alveolar edema, alveolar
hemorrhage, and type II pneumocyte hyperplasia, scores of 0 or 1
were given upon their absence or presence, respectively. In Table
16, the presence of alveolar edema, alveolar hemorrhage, and type
II pneumocyte hyperplasia is indicated by "yes" and "no" instead of
the numerical score.
[0552] Readout included observation of body weight, lung lesions,
virus titers, and histopathology. At the 1600 .mu.g dose, ALE049
exhibited significant reduction of the viral titers in the lung
(FIG. 26a). While the model exhibited high inter-animal
variability, trends to a dose-dependent reduction of virus titers
(FIG. 26a), dose-dependent reduction of macroscopically determined
lung lesions (FIG. 26b), and dose-dependent reduction of body
weight loss (FIG. 26c) were observed, indicating both the 160 .mu.g
as well as the 1600 .mu.g dose exhibited anti-viral activity. Virus
titers in the throat swabs further showed that the 1600 .mu.g dose,
and to a lesser extent the 160 .mu.g dose, inhibited the virus
titers and/or accelerated the reduction of virus titers in the
throat during the four day post-infection time period (FIG. 26d).
Virus titers in nasal turbinates (FIG. 26e) and histopathology data
(Tables 15 and 16, FIG. 27) confirmed that the 1600 .mu.g dose had
the strongest anti-viral protective effects. Based on these
encouraging initial findings further animal experiments are
ongoing.
TABLE-US-00015 TABLE 15 Histopathology results (1) Extent of SUM of
Animal Group alveolitis/alveolar Severity of extent + Severity of
Severity of no. no. Compound Dose damage alveolitis severity
bronchitis bronchiolitis 1 1 ALE049 1600 .mu.g 1 1 2 2 1 2 0 0 0 1
1 3 1 2 3 3 1 4 1 1 2 3 1 5 1 1 2 2 1 6 0 0 0 1 1 7 2 ALE049 160
.mu.g 1 3 4 3 2 8 2 3 5 3 3 9 2 3 5 3 3 10 2 3 5 3 3 11 2 3 5 2 3
12 2 3 5 3 3 13 3 ALE049 16 .mu.g 2 3 5 3 3 14 2 3 5 3 3 15 3 3 6 3
3 16 2 3 5 3 3 17 1 3 4 3 2 18 3 3 6 3 3 19 4 Placebo N/A 2 3 5 3 3
20 2 3 5 3 3 21 2 3 5 3 3 22 2 3 5 3 3 23 2 3 5 3 3 24 2 3 5 3
3
TABLE-US-00016 TABLE 16 Histopathology results (2) Type II Alveolar
Alveolar pneumocyte edema hemorrhage hyperplasia Animal no. Group
no. Compound Dose presence presence presence 1 1 ALE049 1600 .mu.g
no no no 2 no no no 3 no no yes 4 no no yes 5 no no yes 6 no no no
7 2 ALE049 160 .mu.g yes yes yes 8 yes yes yes 9 yes yes yes 10 yes
yes yes 11 yes yes yes 12 yes yes yes 13 3 ALE049 16 .mu.g yes yes
yes 14 yes yes yes 15 yes yes yes 16 yes yes yes 17 yes yes yes 18
yes yes yes 19 4 Placebo N/A yes yes yes 20 yes yes yes 21 yes yes
yes 22 yes yes yes 23 yes yes yes 24 yes yes yes
Example 11: SARS-CoV-2 Variant Inhibition Efficacy Experiments
[0553] The efficacy of ALE049 (SEQ ID NO: 31) and ALE109 (SEQ ID
NO: 75) was assessed against SARS-CoV-2 variants B.1.1.7 (the "UK
variant") and B.1.351 (the "South African variant"), as well as
against SARS-CoV-2 variants having single mutations in the spike
protein.
[0554] The spike protein of SARS-CoV-2 mediates cell entry through
binding to the human ACE2 receptor. SARS-CoV-2 is also capable of
infecting non-primate hosts, such as felines and minks (Oude
Munnink et al., 2021, Science 371, 172-177). The promiscuity of a
multi-host lifestyle is often an indicator of early, still
sub-optimal adaptation of the virus to its new host. This suggests
inherent dynamic plasticity and potential for further human
adaptation. The receptor-binding domain (RBD) in the spike protein
forms the interface with ACE2. Site mutagenesis scanning and
structure analysis revealed amino acid residues important for this
interaction, such as L455, F456, A475, F486, F490 and Q493 (Yan et
al., 2020, Science 367, 1444-1448; Yi et al., 2020, Cell Mol
Immunol 17, 621-630). Notably, single amino acid substitutions
N439R, L452K, N470T, E484P, Q498Y and N501T have been shown to
increase the affinity for human ACE2 (Yi et al., 2020, loc. cit.).
Consistent with these experimental findings, mutation N439K and
mutation N501Y appeared in rapidly spreading SARS-CoV2 spike
variants in association with facilitated receptor binding and
increased transmissibility (Thomson et al., 2021, Cell,
https://doi.org/10.1016/j.cell.2021.01.037). The RBD domain is also
immunogenic, and among other residues, K444, E484, and F486 have
been shown to be important for the binding of neutralizing
antibodies (Ku et al., 2021, Nat Commun 12, 469).
[0555] In this example, we analyzed the impact of selected
mutations of the spike protein on the neutralization capacity of
ALE049 and ALE109 (FIG. 28).
[0556] Generation of His-Tagged Mono-Valent RBD Binders, ALE049,
ALE109 and the Domain Knockout Variants of ALE109
[0557] Ankyrin repeat protein constructs selected and cloned as
described in Example 1 and in Walser et al., 2020 (bioRxiv preprint
doi: https://doi.org/10,1101/2020,0f25.256339) were transformed in
E. coli BL21 cells, plated on LB-agar (containing 1% glucose and 50
.mu.g/ml ampicillin) and then incubated overnight at 37.degree. C.
For each construct, a single colony was picked into TB medium
(containing 1% glucose and 50 .mu.g/ml ampicillin) and incubated
overnight at 37.degree. C., shaking at 230 rpm. Fresh TB medium
(containing 50 .mu.g/ml ampicillin) was inoculated with 1:20 of
overnight culture and incubated at 37.degree. C. at 230 rpm. At
OD600=1.1 the culture was induced by addition of IPTG (0.5 mM final
concentration) and incubated for further 5 h at 37.degree. C. 230
rpm. Harvest was done by centrifugation (10 min 5000.times.g).
After cell disruption by sonication primary recovery was done by
heat treatment for 30 min at 62.5.degree. C. and subsequent
centrifugation (15 min, 12000.times.g). 20 mM Imidazole and 1%
Triton X-100 was added to the supernatant and the 0.22 .mu.m
centrifuged supernatant was further purified by immobilized metal
affinity chromatography (HisTrap FF crude, Cytiva, Sweden) using
the N-terminal His-tag including a wash step with 1% Triton X-100
and a step elution with 250 mM Imidazole. In a subsequent step, the
elution fraction of the IMAC step was applied on a size exclusion
chromatography (Superdex 200, Cytiva, Sweden) and fractions of
interest were pooled and concentrated. Finally, the concentrated
sample was filtered through a 0.22 .mu.m Mustang E filter for
Endotoxin removal and sterile filtration and quality
controlled.
[0558] Generation of Monoclonal Reference Antibodies, RA1 and
RA2
[0559] Publicly available sequences of variable domains from
monoclonal antibodies RA1 and RA2 (the U.S. Food and Drug
Administration issued an emergency use authorization for RA1 and
RA2 to be administered as a cocktail for the treatment of COVID-19)
were used to synthetize the corresponding cDNA fragments and cloned
into a proprietary expression vector at Evitria AG (Switzerland).
Generated vectors containing the constant immunoglobulin hIgG1
chain or kappa light chain were used for transfection in Chinese
hamster ovary cells by Evitria. Sterile filtered cell supernatants
were purified via affinity purification with HiTrap MabSelect
column followed by a size exclusion chromatography using HiLoad
26/600 Superdex 200 column in PBS pH7.4. Selected fractions were
pooled and quality controlled (by SDS-PAGE, size exclusion
chromatography and endotoxin measurement) before use in assays.
[0560] VSV-SARS-CoV-2 Pseudotype Mutation-Vector Generation
[0561] Plasmid pCAGGS encoding the spike protein of SARS-CoV-2
(Walser et al., 2020, oc. cit.) was used as template for generation
of single and multiple spike protein mutants. Forward and reverse
complementary primers encoding the mutation were synthesized by
Microsynth (Balgach, Switzerland). High-fidelity Phusion polymerase
(New England Biolabs, USA) was used for all DNA amplification.
[0562] Single mutations of the spike protein were generated via two
PCR fragments of the spike ORF using high-fidelity Phusion
polymerase (New England Biolabs, USA). The first fragment was
generated via a generic forward primer (pCAGGS-5) annealing
upstream of the spike ORF and the specific reverse primer encoding
the mutation. The second fragment was generated using the specific
forward primer encoding the mutation and a reverse primer
(rbglobpA-R). The two fragments were gel-purified and used as input
for an assembly PCR without addition of flanking primers.
[0563] For multi-mutation spike proteins, a complementary pair of
primers (forward and reverse) encoding each mutation was designed.
Fragment 1 was generated with forward primer pCAGGS-5 and reverse
primer encoding mutation 1. Fragment 2 was generated using forward
primer encoding mutation 1 and reverse primer encoding mutation 2.
All subsequent fragments were generated analogously. DNA fragments
were gel-purified and mixed in equimolar amounts. This mix was used
for re-assembly of the full spike ORF using outer primers pCAGGS-5
and rbglobpA-R.
[0564] For both single as well as multi-mutation spike protein, the
full-length spike ORF was isolated from an agarose gel, digested by
restriction enzymes NheI/EcoRI and inserted into the pCAGGS vector
backbone. The correct sequence was verified via sequencing the
whole ORF of the spike protein by Microsynth (Balgach,
Switzerland).
[0565] VSV-SARS-CoV-2 Pseudotype Neutralization Assay for
Mutational Analyses and ALE109 Domain Knock Outs
[0566] The pseudotype viral system was based on the recombinant
VSV*.DELTA.G-Luc vector in which the glycoprotein gene (G) had been
deleted and replaced with genes encoding green fluorescent protein
and luciferase (Berger Rentsch and Zimmer, PLoS One. 2011;
6(10):e25858). Pseudoviruses were generated as reported previously
(Torriani et al., Virology. 2019 May; 531:57-68; Torriani et al., J
Virol. 2019 Mar. 5; 93(6):e01744-18). For the neutralization assay,
an initial dilution of the compounds was followed by three-fold
dilutions in quadruplicates in DMEM-2% [vol/vol] FCS supplemented
with 20 .mu.M human serum albumin (CSL Behring). The mixture was
mixed with an equal volume of DMEM-2% FCS containing 250 IU per
well of SARS-CoV-2 pseudoviruses and incubated for 90 min at
37.degree. C. The mix was inoculated onto Vero E6 cells in a clear
bottom white walled 96-well plate during 90 min at 37.degree. C.
The inoculum was removed and fresh medium added, and cells further
incubated at 37.degree. C. for 16 h. Cells were lysed according to
the ONE-Glo.TM. luciferase assay system (Promega, Madison, US) and
light emission was recorded using a Berthold.RTM. TriStar LB941
luminometer. The raw data (relative light unit values) were
exported to GraphPad Prism v8.01, and the % neutralization values
were normalized to the untreated PsV signal. IC.sub.50 with 95%
confidence interval were estimated by model of nonlinear regression
fit with settings for log (inhibitor) vs normalized response
curves.
[0567] Cells and Viruses
[0568] Vero E6 cells were passaged in Minimum Essential Medium
(MEM) (Cat No M3303) containing 10% fetal bovine serum (FBS) and
supplements (2 mM L-Glutamine, 1% Non-essential amino acids, 100
U/ml Penicillin, 100 .mu.g/ml Streptomycin, 0.06% Sodium
bicarbonate, all from Bioswisstec, Schaffhausen, Switzerland) at
37.degree. C., >85% humidity and 5% CO.sub.2. SARS-CoV-2
(2019-nCoV/IDF0372/2020) was propagated in Vero E6 cells in MEM
containing 2% FBS and supplements (2%-FBS-MEM) at 37.degree. C.,
>85% humidity and 5% CO.sub.2. Viral titer was determined by
standard plaque assay as described elsewhere.
[0569] Virus Neutralization of Authentic SARS-CoV-2 Determined by
CellTiter-Glo and Real-Time RT-PCR
[0570] Virus neutralization capacity of mono-domain and
multi-domain ankyrin repeat binding proteins was determined for 100
TCID50 SARS-CoV-2 by measuring ATP levels of protected cells in a
cell viability assay. DARPin.RTM. proteins were serially diluted
1:4 from 40 nM to 2.4 .mu.M (in triplicates) in 100 .mu.l cell
culture medium (2%-FBS-MEM) enriched with 10 .mu.M HSA in 96 well
plates. The diluted DARPin.RTM. proteins were mixed with 100 TCID50
SARS-CoV-2 in 100 .mu.l 2%-FBS-MEM+HSA and incubated for 1 h at
37.degree. C. DARPin.RTM. protein/virus mixtures (200 .mu.l) were
transferred onto 80% confluent Vero E6 cells. The controls
consisted of Vero E6 cells exposed to virus suspension only, to
determine maximal cytopathic effect and of cells incubated with
medium only, to determine baseline state of cells. The plates were
incubated for 3 days at 37.degree. C., >85% humidity and 5%
CO.sub.2. Cell viability was determined by removing 100 .mu.l
supernatant from all wells and adding 100 .mu.l CellTiter-Glo
reagent as described in the manufacturers protocol
(CellTiter-Glo.RTM. Luminescent Cell Viability Assay, Promega,
Madison, USA). Luminescence was read after 2 minutes shaking on an
orbital shaker, transferring the mixture to an opaque-walled plate
and 10 mi incubation at room temperature using the GloMax
instrument (Promega). To determine inhibition of virus replication,
the previously removed supernatant (100 .mu.l) was inactivated in
400 .mu.l AVL-buffer (Qiagen, Hilden, Germany) and 400 .mu.l 100%
Ethanol and extracted and eluted in 100 .mu.l using the MagNAPure
96 system (Roche, Basel, Switzerland). Viral RNA was quantified by
real-time RT-PCR targeting the E gene (Ref. Eurosurveillance I
Detection of 2019 novel coronavirus (2019-nCoV) by real-time
RT-PCR) using 5 .mu.l RNA and 45 .mu.l TaqMan Fast Virus 1-Step
Master Mix (Life Technologies, Zug, Switzerland). Viral genome
equivalents (ge) were calculated using a regression analysis and an
internal standard.
[0571] The results of the neutralization tests with multi-specific
DARPin.RTM. molecules ALE049 and ALE109 or reference antibodies 1
or 2 (RA1 and RA2 respectively) are shown in Table 17. Table 18
shows the activity of the three spike protein-binding domains of
ALE049 (SEQ ID NO: 31) as individual binders against spike protein
variants.
TABLE-US-00017 TABLE 17 Efficacy Results VSV Pseudotype
Neutralization Assay IC.sub.50 [ng/mL] Variants Rational ALE049
ALE109 RA1 RA2 wild type (Wuhan) 1.0 3.1 3.9 6.1 B.1.351 (SA,
.DELTA.5)* 3.0 2.4 19 6.2 B.1.1.7 (UK, .DELTA.9)** 1.7 70 2.4 3.5
Individual Residues in variants Mutations N501Y in UK, SA, BRA
variants; 0.5 1.4 4.3 5.8 increases RBD/ACE2 interaction.sup.1
E484K in SA, BRA variants; 2.7 1.8 17 5.8 increases RBD/ACE2
interaction.sup.1 K417E residue mutated to N/T in SA, BRA 0.5 1.2
>100 1.5 variants Y453F key residue evolved in Danish mink 3.2
2.0 >100 12 farms variants Individual Highly frequent mutations
Mutations D614G Wide global spread 2.4 2.8 n.d. n.d. S477N Wide
global spread 1.9 0.8 n.d. n.d. N439K Widespread in Northern
America, 1.3 2.5 2.8 30 UK; increases RBD/ACE2 interaction.sup.1
A222V Wide European spread 2.2 3.1 7.0 2.9 Individual Within RBD
epitope of DARPin .RTM. Mutations binder or reported resistance
mutation for other therapeutics G446V 1.7 1.0 1.5 >100 G476S 1.5
3.1 n.d. n.d. T478I 2.7 2.8 4.0 7.0 P479S 2.1 1.5 3.7 9.8 V483A 2.3
1.9 n.d. n.d. F486V key residue for DARPin .RTM. RBD >100 7.7
>100 4.4 binder.sup.2; reduces RBD/ACE2 interaction.sup.1 Q493K
7.9 2.4 >100 10 F490S Reduces RBD/ACE2 interaction.sup.1 3.8 1.6
3.1 9.2 n.d.: not determined *Mutations (SA): D80A, D215G, E484K,
N501Y, A701V **Mutations (UK): del69-70, del145, N501Y, A570D,
D614G, P681H, T716I, S982A, D1118H .sup.1Influence of residue
mutations on spike protein binding to human ACE2 (Yi et al., 2020,
loc. cit.) .sup.2Predicted interaction residue for DARPin .RTM. RBD
binder (Walser et al., 2020, https://doi.
org/10.1101/2020.08.25.256339)
TABLE-US-00018 TABLE 18 Efficacy of RBD domains of ALE049 VSV
Pseudotype Neutralization Assay IC.sub.50 [ng/mL] Mono-valent RBD
Binders in ALE049 Variants Rational ALE049 R3b R1b R3c wild type
(Wuhan) 1.0 7.2 2.1 13.3 B.1.351 (SA, .DELTA.5)* 3.0 76 26 >100
B.1.1.7 (UK, .DELTA.9)** 1.7 4.6 5.4 11.7 Individual Residues in
variants Mutations N501Y in UK, SA, BRA variants; 0.5 9.1 4.8 27.8
increases RBD/ACE2 interaction.sup.1 E484K in SA, BRA variants; 2.7
64.2 10.2 >100 increases RBD/ACE2 interaction.sup.1 K417E
residue mutated to N/T in SA, 0.5 1.8 1.0 3.6 BRA variants Y453F
key residue evolved in Danish 3.2 10.9 5.9 3.3 mink farms variants
Individual Highly frequent mutations Mutations D614G Wide global
spread 2.4 11.9 6.2 23 S477N Wide global spread 1.9 3.0 2.0 9.0
N439K Widespread in Northern America, 1.3 7.3 5.3 12.9 UK;
increases RBD/ACE2 interaction.sup.1 A222V Wide European spread 2.2
3.3 4.6 19.5 Individual Within RBD epitope of DARPin .RTM.
Mutations binder or reported resistance mutation for other
therapeutics G446V 1.7 0.7 1.8 2.3 G476S 1.5 2.3 3.7 29 T478I 2.7
11.2 3.1 16.7 P479S 2.1 7.2 2.3 27.6 V483A 2.3 21.8 8.4 21.3 F486V
key residue for DARPin .RTM. RBD >100 >100 >100 >100
binder.sup.2; reduces RBD/ACE2 interaction.sup.1 Q493K 7.9 30 28.2
45.8 F490S Reduces RBD/ACE2 interaction.sup.1 3.8 2.3 1.7 8.1 n.d.:
not determined *Mutations (SA): D80A, D215G, E484K, N501Y, A701V
**Mutations (UK): del69-70, del145, N501Y, A570D, D614G, P681H,
T716I, S982A, D1118H .sup.1Influence of residue mutations on spike
protein binding to human ACE2 (Yi et al. 2020, loc. cit.)
.sup.2Predicted interaction residue for DARPin .RTM. RBD binder
(Walser et al. 2020)
[0572] These results show that ALE049 can neutralize variants
B.1.1.7 and B.1.351 as efficiently as the wild-type form with
IC.sub.50 values in the low single-digit ng/mL range. ALE109
neutralized the B.1.351 variant equally efficiently as the
wild-type form, with IC.sub.50 values in the low single-digit ng/mL
range. A slight potency loss was observed for ALE109 against the UK
variant B.1.1.7 (IC50 value of 70 ng/ml). Nevertheless, the potency
of ALE109 against the UK variant B.1.1.7 was within the therapeutic
range. It is interesting to note that the RBD binder of ALE109
(i.e. R1b) retained the same neutralization ability for B.1.1.7 as
for the wild-type. The observed slight potency drop observed for
ALE109 may be caused by the exposed mutations in the S2 domain
(potentially P681H and T7161) alone or in combination with the NTD
mutations. The structural determinants responsible for this slight
potency drop are currently under investigation. Taken together, the
results showed that both tested multi-specific binding proteins,
ALE049 and ALE109, potently neutralized the wild-type form with
IC.sub.50 values in the low single-digit ng/mL range and
neutralized the variants B.1.1.7 (UK) and B.1.351 (SA) with
IC.sub.50 values in the therapeutic range (i.e., low single-digit
to double-digit ng/mL range).
[0573] Both multi-specific DARPin.RTM. molecules ALE049 and ALE109
also protected well against all individual mutations tested, with
the notable exception of F486V for ALE049 and all three mono-valent
DARPin.RTM. RBD binders. As F486 is a critical residue for ACE2
binding, the selective pressure on the virus favors its
conservation, thus maintaining an important anchoring element for
the binding of ALE049. The major impact of this mutation on ALE049
is not surprising, as previous structural analysis identified F486
as a core interacting residue for the three related but different
RBD binders in ALE049 (Walser et al. 2020, loc. cit.).
Consequently, the mutation F486V destabilizes the binding of the
ALE049 molecule to the spike protein. Taken together, our analysis
confirms that multi-specific DARPin.RTM. molecules of the invention
remain highly potent against spike proteins carrying the most
frequently observed mutations, and mutations known to impact the
binding of neutralizing antibodies, as expected from the
multi-specific design of the DARPin.RTM. molecules.
[0574] FIG. 29 shows the neutralization potency of single domain
knock-out (k.o.) constructs of ALE109 against the wild type form of
SARS-CoV-2. These experiments determined the contribution of each
of the three spike protein-binding DARPin.RTM. domains of ALE109 to
the neutralization activity against SARS-CoV-2. No potency loss
compared to ALE109 was observed for the NTD knock out construct
while some potency loss was observed for the RBD and S2 knock-out
constructs. Without wishing to be bound by theory, the NTD binding
domain of ALE109 is believed to play a significant role in the
neutralization activity of ALE109 against mutated forms or variants
of SARS-CoV-2, e.g., by providing increased binding avidity to
mutated spike protein.
Example 12: Viral Passaging of SARS-CoV-2
[0575] Previous studies have shown that viral escape mutants may
rapidly appear under selective pressure of a therapy (Ku et al.,
2021, loc. cit.; Andreano et al., 2020, DOI:
10.1101/2020.12.28.424451). We used a viral passaging model adapted
from Baum et al., Science 369, 1014-1018 (2020), to estimate the
risk of viral escape from therapeutic pressure of multi-specific
DARPin.RTM. proteins ALE049 and ALE109 and of a cocktail of
reference antibodies RA1 and RA2, in comparison to the mono-valent
DARPin.RTM. binder R1b (SEQ ID NO: 3) and to the monoclonal
antibodies S309, RA1 and RA2 applied as single molecules. S309 is
an antibody that was isolated from a patient who recovered from
severe acute respiratory syndrome (SARS) in 2003 and has been shown
to be effective against SARS-CoV-2 infection in cells and in animal
models (Pinto et al., Nature, Vol 583, p. 290-295, 9 Jul. 2020).
S309 was prepared in the same manner as RA1 and RA2 (see Example 11
above).
[0576] Experimental Protocol:
[0577] 1:5 serial dilutions of DARPin.RTM. proteins and monoclonal
antibodies from 100 .mu.g/ml to 0.032 .mu.g/ml were prepared in
Minimum Essential Medium (MEM) containing 2% FBS, supplements and
10 .mu.M human serum albumin (HSA; CSL Behring, Switzerland;
2%-FBS-MEM+HSA). 500 ul of virus suspension containing
1.5.times.10.sup.6 plaque forming units (pfu) SARS-CoV-2 (a French
isolate with the following differences compared to wild-type:
V367F; E990A) in 2%-FBS-MEM+HSA were mixed with 500 .mu.l of
serially diluted DARPin.RTM. proteins or monoclonal antibodies and
subsequently incubated for 1 hour at 37.degree. C. The mixtures
were then transferred to 80% confluent Vero E6 cells in 12 well
plates and incubated for 4 days at 37.degree. C., >85% humidity
and 5% CO.sub.2. Each culture well was assessed for cytopathic
effect (CPE) by microscopy. Supernatant was removed from wells with
the highest DARPin.RTM. protein or antibody concentrations showing
significant CPE (>20%) and used for total RNA extraction and
further passaging. For subsequent rounds of passaging, remaining
900 .mu.l supernatant of selected wells was diluted to 4 ml in
2%-FCS-MEM+HSA and thereof 500 .mu.l mixed with serial dilutions of
DARPin.RTM. proteins or antibodies, incubated and the mixture
transferred to 12 well plate with fresh Vero E6 cells as described
above. Cell culture wells were assessed for CPE again after 4 days
and the supernatant of wells with highest DARPin.RTM. protein or
antibody concentrations with evident viral replication (CPE)
harvested and used for additional passages (see FIG. 30). A total
of 4 passages were performed this way.
[0578] Results:
[0579] Resistant escape variants were selected by passaging the
supernatant of cultures showing significant virus-induced
cytopathic effect under the greatest selective pressure onto fresh
cells while maintaining the selective pressure of increasing
concentrations of antiviral proteins. FIG. 31 shows the results
obtained after the first to fourth incubation cycles (passages #1
to #4). After the first incubation cycle of four days (passage #1)
the mono-valent DARPin.RTM. binder Rb1 and the multi-specific
DARPin.RTM. proteins ALE049 and ALE109, as well as the monoclonal
antibody RA1 and the cocktail of the two monoclonal antibodies RA1
and RA2 conferred protection at the same concentrations of 0.4
.mu.g/mL. The monoclonal antibody S309 was less efficient,
requiring higher concentration (10 .mu.g/mL) for protection and the
monoclonal antibody RA2 as a single molecule was not protective up
the highest concentration tested of 50 .mu.g/mL. Under continuous
selective pressure through passage 2 to 4, the monovalent
DARPin.RTM. binder Rb1, and the individual monoclonal antibodies
RA2 and RA1 lost the capacity to protect cells from virus-induced
cytopathic effect, which manifested in complete CPE up to the
highest selective pressure tested. In contrast, the two
multi-specific DARPin.RTM. proteins ALE049 and ALE109, as single
molecules or as a mixture, and the cocktail of two monoclonal
antibodies (RA1 and RA2) remained effective and protected cells
from CPE throughout the 4 passages.
[0580] The multi-specific DARPin.RTM. proteins ALE049 and ALE109 as
single agents prevented the selection of escape mutants at
concentrations of 2 .mu.g/mL and 10 .mu.g/mL, respectively, after 4
passages, while the combination of the two multi-specific
DARPin.RTM. proteins ALE049 and ALE109 retained effectiveness even
at a low concentration of 0.08 .mu.g/mL. The antibody cocktail RA1
& RA2 prevented the selection of escape mutants at a
concentration of 0.4 .mu.g/mL after passage 4.
Example 13: Comparison of Several Multi-Specific Binding Proteins
in a Neutralization Assay Using SARS-CoV-2 VSV Pseudovirus (PsV
nCoV)
[0581] Several multi-specific binding proteins of the invention
were compared in a neutralization assay using SARS-CoV-2 VSV
pseudovirus (PsV nCoV). The neutralization assay was performed
similar as described in Example 4 above. The tested multi-specific
binding proteins included ALE049, ALE058, ALE109, ALE126, ALE129
and ALE133. ALE049, ALE058 and ALE109 have been described above.
ALE126, ALE129 and ALE133 comprise a further engineered S1-NTD
binding domain (vS07_08F10v47; SEQ ID NO: 85) as compared to
ALE109, which comprises vS07_08F10v27 (SEQ ID NO: 76). ALE126,
ALE129 and ALE133 differ from each other only in the length of the
linker that connects the S1-NTD binding domain and the S2 binding
domain (SEQ ID NO: 77).
[0582] The results of the PsV nCoV assay are shown in FIG. 32, with
EC50 values provided in nM. The experiment demonstrated that all
the tested multi-specific binding proteins have overall comparable
neutralization potencies in this SARS-CoV-2 VSV pseudovirus
neutralization assay. The EC50 values of all tested constructs were
in the range of 20 to 50 .mu.M.
Example 14: Pharmacokinetic Analysis of Multi-Specific Binding
Proteins of the Invention in Mice
[0583] Another pharmacokinetic (PK) study was conducted to assess
the PK characteristics of several multi-specific recombinant
binding proteins of the invention in mice. Such PK characteristics
are useful for dose predictions of multi-specific binding proteins
of the invention in animal pharmacodynamic studies, in toxicology
studies or in human clinical trials.
[0584] The PK study was performed essentially as described in
Example 9.
[0585] The following multi-specific binding proteins were tested in
this study:
TABLE-US-00019 TABLE 19 SEQ ID NO Sample name 39 ALE058 75 ALE109
87 ALE126 88 ALE129 84 ALE133
[0586] Pharmacokinetic data analysis was performed, as also
described in Example 9, using Phoenix WinNonlin.TM. 8.0 program
from Certara. Calculation of the pharmacokinetic parameters of the
study based on the mean concentration-time data of the animals
dosed via intravenous bolus injection was performed with
non-compartmental analysis (NCA model 200-202, IV bolus, linear
trapezoidal linear interpolation).
[0587] The calculated pharmacokinetic parameters included at least
the following: AUCinf_pred, AUClast, AUC_% extrapol, AUC_%
Back_Ext_pred, Cmax, Tmax, CI_pred, Vss_pred, t1/2 (HL_Lambda_z).
The results are shown in Table 20 and FIGS. 33 and 34:
TABLE-US-00020 TABLE 20 Parameter Unit ALE058 ALE109 ALE0126 ALE129
ALE133 AUCINF_pred h*(nmol/L) 4261 10980 11145 12986 11909 AUClast
h*(nmol/L) 4253 10740 10726 12281 11246 Cmax nmol/L 255 328 337 295
295 Tmax h 0.083 0.083 0.083 0.083 0.083 Cl_pred L/(h*kg) 0.00297
0.00115 0.00114 0.00097 0.00105 Vss_pred L/kg 0.052 0.047 0.053
0.052 0.058 HL_Lambda_z h 20.0 31.5 36.7 41.4 41.1 AUC_%
Extrap_pred (%) 0 2 4 5 6 AUC_% Back_Ext_pred (%) 1 0 0 0 0
[0588] Results and Conclusions
[0589] The results demonstrated that ALE109 has improved
pharmacokinetic properties for systemic administration as compared
to the precursor molecule ALE058. In the mono-exponential
elimination phase of the serum concentration time profile, ALE109
serum concentrations declined with a half-life of 31.5 hours,
whereas ALE058 showed a half-life of 20 hours. Moreover, the
further engineered binding proteins ALE126, ALE129 and ALE133
displayed even more extended half-lives, when compared to ALE109,
i.e. half-lives of 36.7 hours, 41.4 hours and 41.1 hours,
respectively.
Example 15: In Vivo Evaluation of Therapeutic Efficacy of Two
Multi-Specific Binding Proteins, ALE049 and ALE109, in a Roborovski
Dwarf Hamster Model
[0590] In this study, a Roborovski dwarf hamster model was used to
evaluate the efficacy of two multi-specific binding proteins of the
invention as potential antiviral agents against SARS-CoV-2. The
Roborovski dwarf hamster model is a valuable non-transgenic rodent
model for SARS-CoV-2 research due to its high sensitivity to
SARS-CoV-2 infections, as indicated by severe clinical signs (e.g.
body weight loss or body temperature drop), viral replication in
both the upper and lower respiratory tract and histopathological
changes (Trimpert et al., Cell Reports 33, 108488, Dec. 8,
2020).
[0591] Thus, the objective of this study was to investigate the
therapeutic potential of ALE049 and ALE109 to inhibit or prevent
body weight loss, replication of SARS-CoV-2 in the upper and lower
respiratory tract and histopathological changes.
[0592] The tested binding proteins ALE049 and ALE109 are serum
half-life extended with domains that bind to human serum albumin
(HSA) (as well as to hamster serum albumin) to support long-acting
activity. In vitro data demonstrated potent inhibition of
SARS-CoV-2 virus infection in cell culture titration experiments by
both binding proteins.
[0593] The study design provided that 5 groups of 6 animals each
were used and treatment with tested binding protein was given
either at 0, 6 or 24 hours after inoculation with SARS-CoV-2. The
study design is illustrated in FIG. 35 (ALE049 is also called
MP0420 in this Example). Animals were treated by intraperitoneal
(i.p.) administration, which served as a safe and reproducible
alternative for intravenous administration. Animals in group 1 were
treated with ALE049 at 20 mg/kg at 0 h and animals in groups 2 to 4
were treated with ALE109 at 20 mg/kg at time points 0, 6 or 24 h
post-infection, respectively. Animals in the control group (group
5) were treated at time 0 h with a placebo (i.e. vehicle of tested
proteins only). Infection with SARS-CoV-2 was performed via the
intranasal (i.n.) route, for which the dose and route of infection
were based on results from earlier (model development) studies.
Animals were weighed and temperatures were measured daily. Three
animals for each group were euthanized on day 3 and 5
post-infection, respectively, to perform necropsy. Viral load in
lung and throat tissue was determined by qPCR or virus titration
and counting the plaque forming units (PFUs). Histopathological
changes in selected tissues were assessed after euthanasia.
[0594] Materials
[0595] Formulation buffer and all test and control item
formulations were prepared on the day of administration and were
aliquoted into appropriate volumes for each group and stored at
4.degree. C. until administration. The volume of the test/control
item administered was 100 .mu.L per animal and adjusted to the
animal's body weight measured on the administration day. The
infection material was SARS-CoV-2, strain
BetaCoV/Germany/BavPat1/2020.
[0596] Animals
[0597] Roborovski dwarf hamsters (Phodopus roborovskii), age 6-9
weeks, with a body weight range at the start of the study on day -2
of 20-25 gram, were used.
[0598] Procedures
[0599] Anaesthesia and Analgesia
[0600] For infections and prior to euthanasia, animals were
anesthetized by the injection of medetomidine, midazolam, and
butorphanol at doses of 0.15, 2.0 and 2.5 mg/kg, respectively.
Following infection, anaesthesia was antagonized with 0.15 mg/kg
atipamezole.
[0601] Intraperitoneal Administration
[0602] For intraperitoneal administration the animal was fixed by
grasping the neck skin and the back skin between thumb and fingers.
Subsequently, the hand was turned over so that the animal rests
with its back in the palm of the hand. The head of the animal was
kept downwards to prevent injection/damage in/of the organs and the
needle was inserted left of the median line in the groin area,
between the 4th and the 5th mammary gland/nipple. Finally, the
needle was removed in a smooth motion.
[0603] Intranasal Administration
[0604] For intranasal administration the animals were held on their
back and the inoculum (20 .mu.l) was equally divided over both
nostrils using a pipette. Animals were held on their back until the
complete inoculum was inhaled after which they were placed back in
the cage to recover.
[0605] Sampling for Histology
[0606] Histopathological analysis from selected tissues was
performed for all animals euthanized at experimental or humane
endpoints (i.e. day 2, 5 and 7). After fixation with 4% formalin
for a minimum of 48 hours, sections from lung and throat were
embedded in paraffin and the tissue sections were stained for
histological examination.
[0607] End-Point Serum Samples
[0608] Serum samples on day 2, 5 and 7 post-infection were
collected during euthanasia and immediately transferred to
appropriate tubes containing a clot activator.
[0609] Virological Analysis
[0610] Detection of Viral RNA
[0611] RNA was extracted from nasal washes and tracheal swabs with
the RTP DNA/RNA Virus Mini Kit (Stratec, Birkenfeld, Germany)
according to the manufacturer's instructions. The innuPREP Virus
DNA/RNA Kit (Analytic Jena, Jena, Germany) was used for RNA
extractions from tissue samples. Viral RNA was quantified using a
one-step RT qPCR reaction with the NEB Luna Universal Probe
One-Step RT-qPCR (New England Biolabs, Ipswich, Mass., USA) and the
2019-nCoV RT-qPCR primers and probe (E_Sarbeco) on a StepOnePlus
RealTime PCR System (Thermo Fisher Scientific, Waltham, Mass., USA)
according to the manufacturer's instructions. Viral RNA copies were
then normalized to cellular RPL18 as previously described. Standard
curves for absolute quantification were generated from serial
dilutions of SARS-CoV-2 RNA obtained from a full-length virus
genome cloned as a bacterial artificial chromosome and propagated
in E. coli.
[0612] Detection of Replication Competent Virus
[0613] Duplicate 10-fold serial dilutions were used to determine
the virus titers in confluent layers of Vero E6 cells (SARS-CoV-2
titration on Vero E6 cells). To this end, serial dilutions of the
samples (lung tissue homogenates) were made and incubated on Vero
E6 monolayers for 2 hours at 37 degrees. Cells were washed and
overlaid with semi-solid cell culture medium containing 1.5% Avicel
and incubated for 48 h at 37 degrees after which plates were fixed
with 4% formalin and stained with 0.75% crystal violet for plaque
counting.
[0614] Histopathology
[0615] Histopathological analysis from selected tissues was
performed for all animals euthanized due to reaching an
experimental or humane endpoint. After fixation with 4% formalin
for 48 hours, sections from lungs were embedded in paraffin and the
tissue sections were stained for histological examination.
[0616] Results
[0617] The aim of this study was to assess the therapeutic
potential of binding proteins ALE049 and ALE109 in a COVID-19
Roborovski dwarf hamster model. For this assessment, the hamsters
were treated therapeutically with 20 mg/kg of binding protein at 0,
6 or 24 hours after the SARS-CoV-2 intranasal challenge with 105
PFUs per animal.
[0618] All 24 animals, treated with a binding protein of the
invention at either 0, 6 or 24 hours post-infection, survived until
the day of sacrifice (i.e. day 3 or 5), while 5 out of 6 animals
from the placebo group had to be taken out of the study prior to
the study endpoints by day 3 due to severe clinical symptoms and
body weight loss. Average of body weights was determined in each of
the five study groups. The placebo group showed a steady decrease
in body weight until the timepoint at day 3. After this timepoint
only one animal from the placebo group could be taken forward to
day 5 for further evaluation. All test protein-treated groups
demonstrated no or only minor body weight losses. When comparing
the various timepoints for treatment or when comparing ALE049 with
ALE109, no significant differences were observed in terms of
clinical symptoms or body weight loss (see FIG. 36). Generally,
there seemed to be some variation in the response of the individual
animals to either the viral infection or the treatment which led to
a relatively wide spread in body weight loss.
[0619] Measurement of viral titers in lung by live virus titration
of lung homogenate and plaque counting demonstrated that, already
at day 3, a reduction in the live virus could be observed (FIG.
37A). This was especially pronounced for the timepoint where the
treatment was initiated directly after the viral challenge (0 h
timepoint). Still, also the treatment injections with ALE109
administered at 6 h or 24 h after the viral challenge showed a
considerable reduction in the load of infectious virus already at
day 3. This effect seemed to be even more pronounced for the 3
animals remaining at day 5 where only 5 out of 12 binding
protein-treated animals had detectable infectious virus remaining
in the lung homogenates (FIG. 37B). Reduction of viral RNA genome
copies as detected by qPCR seemed to be considerable slower than
the elimination of infectious virus. At day 3, only 1 out of 3
animals for each of the 0 h time points showed a reduction of viral
RNA in the lungs (FIG. 37C). On viral genome level, more pronounced
differences between the binding protein-treated groups and the
placebo group occurred only at day 5 post infection, where again a
trend for better reduction of viral genomic RNA could be observed
for the earlier time points of the treatment (FIG. 37D). When
comparing ALE049 and ALE109 at the 0 h time point, a trend for
better virus elimination could be observed for ALE049.
[0620] The histopathological assessment for various parameters in
different tissues was scored with a ranking from 0 (no obvious
histopathological signs) to 4 (most severe histopathological
signs). All scores were averaged for the different treatment groups
and categorized into four sets: i) inflammation, ii) blood vessels,
iii) alveoli, and iv) bronchi. The sum graphs for all the averaged
parameters are provided in FIGS. 38A to 38D. Generally, in all four
categories, clear differences were observed between the binding
protein-treated hamsters and the placebo-treated hamsters.
According to the histopathological assessment, all binding protein
treatments had strongest effects on the reduction of tissue damage
in bronchi (FIG. 38D), alveoli (FIG. 38C) and blood vessels (FIG.
38B) and lowest impact on the reduction of inflammatory cells (FIG.
38A), when compared to the placebo group. The group treated with
ALE109 at the timepoint 6 h after viral infection indicated the
lowest reduction of inflammation and tissue damage amongst all
binding protein-treated groups.
CONCLUSIONS
[0621] At viral inoculation of 105 PFUs, the Roborovsky dwarf
hamster model is a well-suited COVID-19 disease model, in which
non-treated animals generally develop strong clinical symptoms
reaching criteria for euthanasia. The therapeutic treatment of the
animals with either ALE049 at 0 hours after the viral challenge or
ALE109 at 0, 6 or 24 hours after the viral challenge, led to
significant reductions of severe clinical symptoms, comparable for
all binding protein treatment groups, such that none of the 24
binding protein-treated animals reached euthanasia criteria prior
to the official sacrifice time points at day 3 or 5, while for the
6 placebo-treated animals, 2 animals at day 2 and another 3 animals
at day 3 developed strong clinical symptoms and had to be taken out
of the study, with only one placebo-treated animal remaining on
study until day 5.
[0622] In terms of viral load for infectious virus or viral genome
copies, a clear reduction was observed for all binding protein
treatment groups. This reduction increased from day 3 to day 5 and
the treatment groups where the therapy was given earlier seemed to
respond with a more pronounced reduction. When comparing ALE049
with ALE109, administered at 0 h, the ALE049 treatment group
responded slightly better, with respect to the rather low number of
animals per treatment group.
[0623] Histopathological findings in the lungs showed a clear
reduction of pathological scores for all binding protein treatment
groups when compared to the placebo group. These findings seem to
be independent of the therapeutic regimens tested in this
study.
[0624] In conclusion, both ALE049 and ALE109 demonstrated
therapeutic potential against SARS-CoV-2 infections, using a
Roborovsky dwarf hamster model.
[0625] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification. The
embodiments within the specification provide an illustration of
embodiments of the invention and should not be construed to limit
the scope of the invention. The skilled artisan readily recognizes
that many other embodiments are encompassed by the invention. All
publications, patents, and GenBank sequences cited in this
disclosure are incorporated by reference in their entirety. To the
extent the material incorporated by reference contradicts or is
inconsistent with this specification, the specification will
supersede any such material. The citation of any references herein
is not an admission that such references are prior art to the
present invention.
[0626] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
951126PRTArtificial SequenceSpike protein binding domain 1Gly Ser
Asp Leu Gly Asn Lys Leu Leu Asp Ala Ala Trp Val Gly Gln1 5 10 15Asp
Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala 20 25
30Lys Asp Trp Gln Gly Glu Thr Pro Leu His Leu Ala Ala Thr Lys Gly
35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
Asn 50 55 60Ala Lys Asp Val Val Gly Tyr Thr Pro Leu His Val Ala Ala
Ser Gln65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Val His Gly Tyr Thr Pro Ala
Asp Leu Ala Ala Gln 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val
Leu Gln Lys Ala Ala 115 120 1252159PRTArtificial SequenceSpike
protein binding domain 2Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala
Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Arg Glu Gly Trp Thr Pro Leu
His Leu Ala Ala His Gln Gly 35 40 45His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Val Phe Gly Arg
Thr Pro Leu His Leu Ala Ala Trp Val65 70 75 80Gly His Leu Glu Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp
Val Ser Gly Ala Thr Pro Leu His Pro Ala Ala Ile 100 105 110Tyr Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 115 120
125Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala
130 135 140Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala145 150 1553159PRTArtificial SequenceSpike protein binding
domain 3Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly
Gln1 5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala 20 25 30Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala
Gln Glu Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp Val Asn 50 55 60Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp Ile65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Val Ser Gly Ala
Thr Pro Leu His Ala Ala Ala Leu 100 105 110His Gly His Leu Glu Ile
Val Glu Val Leu Leu Asn Ala Gly Ala Asp 115 120 125Val Asn Ala Gln
Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135 140Arg Ala
Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala145 150
1554159PRTArtificial SequenceSpike protein binding domain 4Gly Ser
Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10 15Leu
Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
30Lys Asp Lys Ile Gly Val Thr Pro Leu His Ile Ala Ala Glu Val Gly
35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
Asn 50 55 60Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala
Trp Ser65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Val Lys Ala
Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Ile Ala Gly Ala Thr Pro Leu
His Ala Ala Ala Leu 100 105 110Phe Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Val Gly Ala Asp 115 120 125Val Asn Ala Gln Asp Lys Ser
Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135 140Arg Ala Gly His Gln
Asp Ile Ala Glu Val Leu Gln Lys Ala Ala145 150 1555159PRTArtificial
SequenceSpike protein binding domain 5Gly Ser Asp Leu Gly Lys Lys
Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Glu
Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Gln Glu Gly
Ile Thr Pro Leu His Val Ala Ala His Gln Gly 35 40 45His Leu Glu Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp
Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Arg65 70 75 80Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90
95Asn Ala Lys Asp His Ala Gly Ala Thr Pro Leu His Ala Ala Ala Leu
100 105 110Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp 115 120 125Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala
Asp Leu Ala Ala 130 135 140Arg Ala Gly His Gln Asp Ile Ala Glu Val
Leu Gln Lys Ala Ala145 150 1556159PRTArtificial SequenceSpike
protein binding domain 6Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala
Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Arg Glu Gly Ile Thr Pro Leu
His Leu Ala Ala Gln His Gly 35 40 45His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Val Trp Gly Arg
Thr Pro Leu His Leu Ala Ala Trp Gln65 70 75 80Gly His Leu Glu Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp
Leu Ala Gly Ala Thr Pro Leu His Val Ala Ala Leu 100 105 110Tyr Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 115 120
125Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala
130 135 140Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala145 150 1557126PRTArtificial SequenceSpike protein binding
domain 7Gly Ser Asp Leu Gly Leu Lys Leu Leu Thr Ala Ala Lys Gln Gly
Gln1 5 10 15Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val
Asn Ala 20 25 30Lys Asp Tyr Arg Gly Leu Thr Pro Leu His Tyr Ala Ala
Ala Ile Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp Val Asn 50 55 60Ala Lys Asp Gln Tyr Gly Ala Thr Pro Leu His
Val Ala Ala Tyr Ile65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Phe Ser Gly Ser
Thr Pro Ala Asp Leu Ala Ala Glu 100 105 110Glu Gly His Glu Asp Ile
Ala Glu Val Leu Gln Lys Ala Ala 115 120 1258126PRTArtificial
SequenceSpike protein binding domain 8Gly Ser Asp Leu Gly Trp Lys
Leu Leu Trp Ala Ala Gln Val Gly Gln1 5 10 15Asp Asp Glu Val Arg Ile
Leu Leu Ala Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Asp Leu Gly
Trp Thr Pro Leu His Ile Ala Ala Trp Val Gly 35 40 45His Leu Glu Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp
Phe Thr Gly Arg Thr Pro Leu His His Ala Ala Thr Glu65 70 75 80Gly
His Leu Glu Ile Val Lys Val Leu Leu Lys Ala Gly Ala Asp Val 85 90
95Asn Ala Gln Asp Asp Val Gly His Thr Pro Ala Asp Leu Ala Ala Leu
100 105 110Trp Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
115 120 1259126PRTArtificial SequenceSpike protein binding domain
9Gly Ser Asp Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala Gly Gln1 5
10 15Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn
Ala 20 25 30Lys Asp Val Phe Gly Gln Thr Pro Leu His Val Ala Ala Val
Ala Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn 50 55 60Ala Lys Asp Ser Ile Gly Tyr Thr Pro Leu His His
Ala Ala Arg Val65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Trp Ile Gly Ile Thr
Pro Ala Asp Leu Ala Ala Phe 100 105 110Glu Gly His Glu Asp Ile Ala
Glu Val Leu Gln Lys Ala Ala 115 120 12510126PRTArtificial
SequenceSpike protein binding domain 10Gly Ser Asp Leu Gly Gln Lys
Leu Leu His Ala Ala Gln Tyr Gly Gln1 5 10 15Asp Asp Glu Val Arg Ile
Leu Leu Ala Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Lys Gln Gly
Asn Thr Pro Leu His Ile Ala Ala Phe His Gly 35 40 45His Leu Glu Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp
Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala Trp65 70 75 80Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90
95Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro Ala Asp Leu Ala Ala Glu
100 105 110Ser Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
115 120 12511159PRTArtificial SequenceSpike protein binding domain
11Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1
5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala 20 25 30Lys Asp Glu Thr Gly Phe Thr Pro Leu His Val Ala Ala Glu
Lys Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn 50 55 60Ala Lys Asp His Phe Gly Phe Thr Pro Leu His Leu
Val Ser Glu Trp65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Ser Tyr Gly Trp Thr
Pro Leu His Val Ala Ala Ile 100 105 110Leu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp 115 120 125Val Asn Ala Gln Asp
Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135 140Arg Ala Gly
His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala145 150
15512784PRTArtificial SequenceMulti-specific binding protein 12Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu
Gly Ile Thr Pro 325 330 335Leu His Leu Ala Ala Gln His Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Gln Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Leu Ala Gly Ala385 390 395 400Thr
Pro Leu His Val Ala Ala Leu Tyr Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Asn Lys Leu Leu Asp
Ala Ala Trp Val Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu
Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Trp 500 505 510Gln Gly Glu
Thr Pro Leu His Leu Ala Ala Thr Lys Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Val Gly Tyr Thr Pro Leu His Val Ala Ala Ser Gln Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Val His Gly Tyr Thr Pro Ala Asp Leu
Ala Ala Gln Ala Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp 690 695 700Ile Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Val Ser
Gly Ala Thr Pro Leu His Ala Ala Ala 725 730 735Leu His Gly His Leu
Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala
Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78013784PRTArtificial SequenceMulti-specific binding protein 13Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr
Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu
Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala
Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala
Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105
110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser
115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr
Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu
Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr
Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg Asn Gly His
Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200 205Ala Asp Val
Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu 210 215 220Ala
Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala225 230
235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala
Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val
Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala Ala Arg Ala Gly
Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315 320Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Lys Ile Gly Val Thr Pro 325 330 335Leu His
Ile Ala Ala Glu Val Gly His Leu Glu Ile Val Glu Val Leu 340 345
350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Trp Gly Arg Thr
355 360 365Pro Leu His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val
Glu Val 370 375 380Leu Val Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
Ile Ala Gly Ala385 390 395 400Thr Pro Leu His Ala Ala Ala Leu Phe
Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu Lys Val Gly Ala
Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys Thr Pro Ala Asp
Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440 445Glu Val Leu
Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr 450 455 460Pro
Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp465 470
475 480Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp
Glu 485 490 495Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Lys Asp Arg 500 505 510Glu Gly Trp Thr Pro Leu His Leu Ala Ala His
Gln Gly His Leu Glu 515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala Lys Asp 530 535 540Val Phe Gly Arg Thr Pro Leu
His Leu Ala Ala Trp Val Gly His Leu545 550 555 560Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 565 570 575Asp Val
Ser Gly Ala Thr Pro Leu His Pro Ala Ala Ile Tyr Gly His 580 585
590Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
595 600 605Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg
Ala Gly 610 615 620His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
Gly Ser Pro Thr625 630 635 640Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr Pro Thr 645 650 655Pro Thr Gly Ser Asp Leu Gly
Asn Lys Leu Leu Asp Ala Ala Trp Val 660 665 670Gly Gln Asp Asp Glu
Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val 675 680 685Asn Ala Lys
Asp Trp Gln Gly Glu Thr Pro Leu His Leu Ala Ala Thr 690 695 700Lys
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp705 710
715 720Val Asn Ala Lys Asp Val Val Gly Tyr Thr Pro Leu His Val Ala
Ala 725 730 735Ser Gln Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala 740 745 750Asp Val Asn Ala Gln Asp Val His Gly Tyr Thr
Pro Ala Asp Leu Ala 755 760 765Ala Gln Ala Gly His Glu Asp Ile Ala
Glu Val Leu Gln Lys Ala Ala 770 775 78014817PRTArtificial
SequenceMulti-specific binding protein 14Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Ile Gly Val Thr Pro
325 330 335Leu His Ile Ala Ala Glu Val Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Ser Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Val Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Ile Ala Gly Ala385 390 395 400Thr Pro Leu His Ala
Ala Ala Leu Phe Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Val Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala
Gly Gln Leu Asp Glu 485 490 495Val Arg Glu Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Arg 500 505 510Glu Gly Ile Thr Pro Leu His
Leu Ala Ala Gln His Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Val Trp Gly
Arg Thr Pro Leu His Leu Ala Ala Trp Gln Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
565 570 575Asp Leu Ala Gly Ala Thr Pro Leu His Val Ala Ala Leu Tyr
Gly His 580 585 590Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala 595 600 605Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp
Leu Ala Ala Arg Ala Gly 610 615 620His Gln Asp Ile Ala Glu Val Leu
Gln Lys Ala Ala Gly Ser Pro Thr625 630 635 640Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 645 650 655Pro Thr Gly
Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala 660 665 670Gly
Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala Gln
690 695 700Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Lys Asp Val Trp Gly Arg Thr Pro
Leu His Leu Ala Ala 725 730 735Trp Ile Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn Ala Lys Asp Val
Ser Gly Ala Thr Pro Leu His Ala Ala 755 760 765Ala Leu His Gly His
Leu Glu Ile Val Glu Val Leu Leu Asn Ala Gly 770 775 780Ala Asp Val
Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu785 790 795
800Ala Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala
805 810 815Ala15817PRTArtificial SequenceMulti-specific binding
protein 15Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu
His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150
155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu
His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val
Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala
Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val
Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala
Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265
270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro
275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys
Lys Leu 290 295 300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val
Arg Glu Leu Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys
Asp Arg Glu Gly Ile Thr Pro 325 330 335Leu His Leu Ala Ala Gln His
Gly His Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His
Leu Ala Ala Trp Gln Gly His Leu Glu Ile Val Glu Val 370 375 380Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Leu Ala Gly Ala385 390
395 400Thr Pro Leu His Val Ala Ala Leu Tyr Gly His Leu Glu Ile Val
Glu 405 410 415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp
Lys Ser Gly 420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly
His Gln Asp Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser
Pro Thr Pro Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Lys Lys
Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu 485 490 495Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg 500 505
510Glu Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu Gly His Leu Glu
515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Lys Asp 530 535 540Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp
Ile Gly His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys 565 570 575Asp Val Ser Gly Ala Thr Pro
Leu His Ala Ala Ala Leu His Gly His 580 585 590Leu Glu Ile Val Glu
Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala 595 600 605Gln Asp Lys
Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly 610 615 620His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr625 630
635 640Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr 645 650 655Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala
Ala Arg Ala 660 665 670Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys
Ala Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Arg Glu Gly Ile Thr
Pro Leu His Leu Ala Ala Gln 690 695 700His Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys
Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala 725 730 735Trp Gln
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745
750Asp Val Asn Ala Lys Asp Leu Ala Gly Ala Thr Pro Leu His Val Ala
755 760 765Ala Leu Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly 770 775 780Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr
Pro Ala Asp Leu785 790 795 800Ala Ala Arg Ala Gly His Gln Asp Ile
Ala Glu Val Leu Gln Lys Ala 805 810 815Ala16784PRTArtificial
SequenceMulti-specific binding protein 16Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu
His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150
155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu
His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val
Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala
Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val
Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala
Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265
270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro
275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Leu
Lys Leu 290 295 300Leu Thr Ala Ala Lys Gln Gly Gln Asp Asp Glu Val
Arg Ile Leu Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn Ala Lys
Asp Tyr Arg Gly Leu Thr Pro 325 330 335Leu His Tyr Ala Ala Ala Ile
Gly His Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Gln Tyr Gly Ala Thr 355 360 365Pro Leu His
Val Ala Ala Tyr Ile Gly His Leu Glu Ile Val Glu Val 370 375 380Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Phe Ser Gly Ser385 390
395 400Thr Pro Ala Asp Leu Ala Ala Glu Glu Gly His Glu Asp Ile Ala
Glu 405 410 415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro
Thr Thr Pro 420 425 430Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro
Thr Gly Ser Asp Leu 435 440 445Gly Lys Lys Leu Leu Gln Ala Ala Arg
Ala Gly Gln Leu Asp Glu Val 450 455 460Arg Glu Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala Lys Asp Arg Glu465 470 475 480Gly Lys Thr Pro
Leu His Val Ala Ala Gln Glu Gly His Leu Glu Ile 485 490 495Val Glu
Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505
510Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Ile Gly His Leu Glu
515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Lys Asp 530 535 540Val Ser Gly Ala Thr Pro Leu His Ala Ala Ala Leu
His Gly His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Asn Ala
Gly Ala Asp Val Asn Ala Gln 565 570 575Asp Lys Ser Gly Lys Thr Pro
Ala Asp Leu Ala Ala Arg Ala Gly His 580 585 590Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630
635 640Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn 645 650 655Ala Lys Asp Arg Glu Gly Ile Thr Pro Leu His Leu Ala
Ala Gln His 660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr
Pro Leu His Leu Ala Ala Trp 690 695 700Gln Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys
Asp Leu Ala Gly Ala Thr Pro Leu His Val Ala Ala 725 730 735Leu Tyr
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745
750Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala
755 760 765Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys
Ala Ala 770 775 78017751PRTArtificial SequenceMulti-specific
binding protein 17Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala
Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His
Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile
Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His
Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro
Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135
140Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala
Ala145 150 155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu
Lys Ala Gly Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His
Thr Pro Leu His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile
Val Glu Val Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly
Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250
255Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys
260 265 270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro 275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu
Gly Trp Lys Leu 290 295 300Leu Trp Ala Ala Gln Val Gly Gln Asp Asp
Glu Val Arg Ile Leu Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn
Ala Lys Asp Asp Leu Gly Trp Thr Pro 325 330 335Leu His Ile Ala Ala
Trp Val Gly His Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Phe Thr Gly Arg Thr 355 360 365Pro
Leu His His Ala Ala Thr Glu Gly His Leu Glu Ile Val Lys Val 370 375
380Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asp Val Gly
His385 390 395 400Thr Pro Ala Asp Leu Ala Ala Leu Trp Gly His Glu
Asp Ile Ala Glu 405 410 415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr
Pro Thr Pro Thr Thr Pro 420 425 430Thr Pro Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Gly Ser Asp Leu 435 440 445Gly Lys Lys Leu Leu Gln
Ala Ala Arg Ala Gly Gln Leu Asp Glu Val 450 455 460Arg Glu Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Ile465 470 475 480Gly
Val Thr Pro Leu His Ile Ala Ala Glu Val Gly His Leu Glu Ile 485 490
495Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
500 505 510Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Ser Gly His
Leu Glu 515 520 525Ile Val Glu Val Leu Val Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp 530 535 540Ile Ala Gly Ala Thr Pro Leu His Ala Ala
Ala Leu Phe Gly His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu
Lys Val Gly Ala Asp Val Asn Ala Gln 565 570 575Asp Lys Ser Gly Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His 580 585 590Gln Asp Ile
Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr
Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615
620Thr Gly Ser Asp Leu Gly Asn Lys Leu Leu Asp Ala Ala Trp Val
Gly625 630 635 640Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly
Ala Asp Val Asn 645 650 655Ala Lys Asp Trp Gln Gly Glu Thr Pro Leu
His Leu Ala Ala Thr Lys 660 665 670Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Val Val
Gly Tyr Thr Pro Leu His Val Ala Ala Ser 690 695 700Gln Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val
Asn Ala Gln Asp Val His Gly Tyr Thr Pro Ala Asp Leu Ala Ala 725 730
735Gln Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 740
745 75018784PRTArtificial SequenceMulti-specific binding protein
18Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1
5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg
Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr
Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala
Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro
Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro
Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155
160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala
165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His
Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu
Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly
Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn
Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala
Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala
Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280
285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu
290 295 300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu
Leu Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg
Glu Gly Lys Thr Pro 325 330 335Leu His Val Ala Ala Gln Glu Gly His
Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala
Ala Trp Ile Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys
Ala Gly Ala Asp Val Asn Ala Lys Asp Val Ser Gly Ala385 390 395
400Thr Pro Leu His Ala Ala Ala Leu His Gly His Leu Glu Ile Val Glu
405 410 415Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys
Ser Gly 420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His
Gln Asp Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro
Thr Pro Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro
Thr Pro Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Lys Lys Leu
Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu 485 490 495Val Arg Glu
Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg 500 505 510Glu
Gly Ile Thr Pro Leu His Leu Ala Ala Gln His Gly His Leu Glu 515 520
525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
530 535 540Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Gln Gly
His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys 565 570 575Asp Leu Ala Gly Ala Thr Pro Leu His
Val Ala Ala Leu Tyr Gly His 580 585 590Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala 595 600 605Gln Asp Lys Ser Gly
Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly 610 615 620His Gln Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr625 630 635
640Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
645 650 655Pro Thr Gly Ser Asp Leu Gly Leu Lys Leu Leu Thr Ala Ala
Lys Gln 660 665 670Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala
Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Tyr Arg Gly Leu Thr Pro
Leu His Tyr Ala Ala Ala 690 695 700Ile Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp
Gln Tyr Gly Ala Thr Pro Leu His Val Ala Ala 725 730 735Tyr Ile Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp
Val Asn Ala Gln Asp Phe Ser Gly Ser Thr Pro Ala Asp Leu Ala 755 760
765Ala Glu Glu Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
770 775 78019751PRTArtificial SequenceMulti-specific binding
protein 19Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu
His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150
155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu
His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Ile
Gly Val Thr Pro 325 330 335Leu His Ile Ala Ala Glu Val Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Ser Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Val Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Ile Ala Gly Ala385 390 395 400Thr
Pro Leu His Ala Ala Ala Leu Phe Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Lys Val Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Asn Lys Leu Leu Asp
Ala Ala Trp Val Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu
Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Trp 500 505 510Gln Gly Glu
Thr Pro Leu His Leu Ala Ala Thr Lys Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Val Gly Tyr Thr Pro Leu His Val Ala Ala Ser Gln Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Val His Gly Tyr Thr Pro Ala Asp Leu
Ala Ala Gln Ala Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Trp Lys Leu Leu Trp Ala Ala Gln Val Gly625 630 635 640Gln
Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Asp Leu Gly Trp Thr Pro Leu His Ile Ala Ala Trp Val
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Phe Thr Gly Arg Thr Pro Leu His
His Ala Ala Thr 690 695 700Glu Gly His Leu Glu Ile Val Lys Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Gln Asp Asp Val
Gly His Thr Pro Ala Asp Leu Ala Ala 725 730 735Leu Trp Gly His Glu
Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 740 745
75020784PRTArtificial SequenceMulti-specific binding protein 20Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu
Gly Lys Thr Pro 325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Ile Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr
Pro Leu His Ala Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Gln
Ala Ala Arg Ala Gly Gln Leu Asp Glu 485 490 495Val Arg Glu Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg 500 505 510Glu Gly Ile
Thr Pro Leu His Leu Ala Ala Gln His Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Gln Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys 565 570 575Asp Leu Ala Gly Ala Thr Pro Leu His Val
Ala Ala Leu Tyr Gly His 580 585 590Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala 595 600 605Gln Asp Lys Ser Gly Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly 610 615 620His Gln Asp Ile
Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr625 630 635 640Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 645 650
655Pro Thr Gly Ser Asp Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala
660 665 670Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Val Phe Gly Gln Thr Pro Leu His
Val Ala Ala Val 690 695 700Ala Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Ser Ile
Gly Tyr Thr Pro Leu His His Ala Ala 725 730 735Arg Val Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala 755 760 765Ala
Phe Glu Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78021784PRTArtificial SequenceMulti-specific binding protein 21Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly His Lys Leu 290 295
300Leu Leu Ala Ala Gln Ala Gly Gln Asp Asp Glu Val Arg Ile Leu
Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Val Phe
Gly Gln Thr Pro 325 330 335Leu His Val Ala Ala Val Ala Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Ser Ile Gly Tyr Thr 355 360 365Pro Leu His His Ala Ala
Arg Val Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Trp Ile Gly Ile385 390 395 400Thr
Pro Ala Asp Leu Ala Ala Phe Glu Gly His Glu Asp Ile Ala Glu 405 410
415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
420 425 430Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser
Asp Leu 435 440 445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln
Leu Asp Glu Val 450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Arg Glu465 470 475 480Gly Lys Thr Pro Leu His Val
Ala Ala Gln Glu Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Trp Gly Arg
Thr Pro Leu His Leu Ala Ala Trp Ile Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Ser Gly Ala Thr Pro Leu His Ala Ala Ala Leu His Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu
Ala Ala Arg Ala Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Arg Glu Gly Ile Thr Pro Leu His Leu Ala Ala Gln His
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp 690 695 700Gln Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Leu Ala
Gly Ala Thr Pro Leu His Val Ala Ala 725 730 735Leu Tyr Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala
Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78022751PRTArtificial SequenceMulti-specific binding protein 22Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asn Lys Leu 290 295
300Leu Asp Ala Ala Trp Val Gly Gln Asp Asp Glu Val Arg Ile Leu
Leu305 310 315
320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Trp Gln Gly Glu Thr Pro
325 330 335Leu His Leu Ala Ala Thr Lys Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Val Gly Tyr Thr 355 360 365Pro Leu His Val Ala Ala Ser Gln Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Val His Gly Tyr385 390 395 400Thr Pro Ala Asp Leu
Ala Ala Gln Ala Gly His Glu Asp Ile Ala Glu 405 410 415Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro 420 425 430Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu 435 440
445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val
450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
Arg Glu465 470 475 480Gly Ile Thr Pro Leu His Leu Ala Ala Gln His
Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp Gln Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Leu Ala Gly
Ala Thr Pro Leu His Val Ala Ala Leu Tyr Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala
Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly His Lys
Leu Leu Leu Ala Ala Gln Ala Gly625 630 635 640Gln Asp Asp Glu Val
Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Val Phe Gly Gln Thr Pro Leu His Val Ala Ala Val Ala 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Ser Ile Gly Tyr Thr Pro Leu His His Ala Ala Arg
690 695 700Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Trp Ile Gly Ile Thr Pro
Ala Asp Leu Ala Ala 725 730 735Phe Glu Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75023751PRTArtificial
SequenceMulti-specific binding protein 23Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly His Lys Leu 290 295 300Leu Leu Ala
Ala Gln Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu305 310 315
320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Val Phe Gly Gln Thr Pro
325 330 335Leu His Val Ala Ala Val Ala Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ser
Ile Gly Tyr Thr 355 360 365Pro Leu His His Ala Ala Arg Val Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Trp Ile Gly Ile385 390 395 400Thr Pro Ala Asp Leu
Ala Ala Phe Glu Gly His Glu Asp Ile Ala Glu 405 410 415Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro 420 425 430Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu 435 440
445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val
450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
Arg Glu465 470 475 480Gly Ile Thr Pro Leu His Leu Ala Ala Gln His
Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp Gln Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Leu Ala Gly
Ala Thr Pro Leu His Val Ala Ala Leu Tyr Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala
Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Asn Lys
Leu Leu Asp Ala Ala Trp Val Gly625 630 635 640Gln Asp Asp Glu Val
Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Trp Gln Gly Glu Thr Pro Leu His Leu Ala Ala Thr Lys 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Val Val Gly Tyr Thr Pro Leu His Val Ala Ala Ser
690 695 700Gln Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Val His Gly Tyr Thr Pro
Ala Asp Leu Ala Ala 725 730 735Gln Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75024751PRTArtificial
SequenceMulti-specific binding protein 24Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Ile Thr Pro
325 330 335Leu His Leu Ala Ala Gln His Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Gln Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Leu Ala Gly Ala385 390 395 400Thr Pro Leu His Val
Ala Ala Leu Tyr Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala
Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln Thr Pro Leu His
Val Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Ser Ile Gly
Tyr Thr Pro Leu His His Ala Ala Arg Val Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala Ala Phe Glu
Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Gln Lys
Leu Leu His Ala Ala Gln Tyr Gly625 630 635 640Gln Asp Asp Glu Val
Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Lys Gln Gly Asn Thr Pro Leu His Ile Ala Ala Phe His 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala
690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro
Ala Asp Leu Ala Ala 725 730 735Glu Ser Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75025784PRTArtificial
SequenceMulti-specific binding protein 25Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Lys Thr Pro
325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Ile Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr Pro Leu His Ala
Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala
Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505
510Phe Gly Gln Thr Pro Leu His Val Ala Ala Val Ala Gly His Leu Glu
515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Lys Asp 530 535 540Ser Ile Gly Tyr Thr Pro Leu His His Ala Ala Arg
Val Gly His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln 565 570 575Asp Trp Ile Gly Ile Thr Pro
Ala Asp Leu Ala Ala Phe Glu Gly His 580 585 590Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630
635 640Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn 645 650 655Ala Lys Asp Glu Thr Gly Phe Thr Pro Leu His Val Ala
Ala Glu Lys 660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 675 680 685Asn Ala Lys Asp His Phe Gly Phe Thr
Pro Leu His Leu Val Ser Glu 690 695 700Trp Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys
Asp Ser Tyr Gly Trp Thr Pro Leu His Val Ala Ala 725 730 735Ile Leu
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745
750Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala
755 760 765Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys
Ala Ala 770 775 78026751PRTArtificial SequenceMulti-specific
binding protein 26Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala
Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His
Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile
Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His
Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro
Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135
140Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala
Ala145 150 155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu
Lys Ala Gly Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His
Thr Pro Leu His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile
Val Glu Val Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly
Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250
255Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys
260 265 270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro 275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu
Gly Gln Lys Leu 290 295 300Leu His Ala Ala Gln Tyr Gly Gln Asp Asp
Glu Val Arg Ile Leu Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn
Ala Lys Asp Lys Gln Gly Asn Thr Pro 325 330 335Leu His Ile Ala Ala
Phe His Gly His Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Gln Trp Gly Leu Thr 355 360 365Pro
Leu His Leu Ala Ala Ala Trp Gly His Leu Glu Ile Val Glu Val 370 375
380Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asn Tyr Gly
Gln385 390 395 400Thr Pro Ala Asp Leu Ala Ala Glu Ser Gly His Glu
Asp Ile Ala Glu 405 410 415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr
Pro Thr Pro Thr Thr Pro 420 425 430Thr Pro Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Gly Ser Asp Leu 435 440 445Gly His Lys Leu Leu Leu
Ala Ala Gln Ala Gly Gln Asp Asp Glu Val 450 455 460Arg Ile Leu Leu
Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Val Phe465 470 475 480Gly
Gln Thr Pro Leu His Val Ala Ala Val Ala Gly His Leu Glu Ile 485 490
495Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ser
500 505 510Ile Gly Tyr Thr Pro Leu His His Ala Ala Arg Val Gly His
Leu Glu 515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp 530 535 540Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala
Ala Phe Glu Gly His Glu545 550 555 560Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro Thr 565 570 575Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr 580 585 590Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln 595 600 605Leu
Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 610 615
620Lys Asp Arg Glu Gly Ile Thr Pro Leu His Leu Ala Ala Gln His
Gly625 630 635 640His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp Val Asn 645 650 655Ala Lys Asp Val Trp Gly Arg Thr Pro Leu
His Leu Ala Ala Trp Gln 660 665 670Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Leu Ala
Gly Ala Thr Pro Leu His Val Ala Ala Leu 690 695 700Tyr Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val
Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 725 730
735Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 740
745 75027784PRTArtificial SequenceMulti-specific binding protein
27Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1
5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg
Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr
Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala
Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro
Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro
Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155
160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala
165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His
Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu
Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly
Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn
Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala
Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala
Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280
285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu
290 295 300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu
Leu Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu
Thr Gly Phe Thr Pro 325 330 335Leu His Val Ala Ala Glu Lys Gly His
Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp His Phe Gly Phe Thr 355 360 365Pro Leu His Leu Val
Ser Glu Trp Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys
Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Tyr Gly Trp385 390 395
400Thr Pro Leu His Val Ala Ala Ile Leu Gly His Leu Glu Ile Val Glu
405 410 415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys
Ser Gly 420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His
Gln Asp Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro
Thr Pro Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro
Thr Pro Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly His Lys Leu
Leu Leu Ala Ala Gln Ala Gly Gln Asp Asp Glu 485 490 495Val Arg Ile
Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Phe
Gly Gln Thr Pro Leu His Val Ala Ala Val Ala Gly His Leu Glu 515 520
525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
530 535 540Ser Ile Gly Tyr Thr Pro Leu His His Ala Ala Arg Val Gly
His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Gln 565 570 575Asp Trp Ile Gly Ile Thr Pro Ala Asp
Leu Ala Ala Phe Glu Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu
Gln Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro
Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser
Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635
640Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn
645 650 655Ala Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala
Gln Glu 660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro
Leu His Leu Ala Ala Trp 690 695 700Ile Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp
Val Ser Gly Ala Thr Pro Leu His Ala Ala Ala 725 730 735Leu His Gly
His Leu Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala 740 745 750Asp
Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760
765Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
770 775 78028784PRTArtificial SequenceMulti-specific binding
protein 28Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala
Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu
His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150
155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu
His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val
Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala
Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val
Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala
Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265
270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro
275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys
Lys Leu 290 295 300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val
Arg Glu Leu Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys
Asp Gln Glu Gly Ile Thr Pro 325 330 335Leu His Val Ala Ala His Gln
Gly His Leu Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His
Leu Ala Ala Trp Arg Gly His Leu Glu Ile Val Glu Val 370 375 380Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp His Ala Gly Ala385 390
395 400Thr Pro Leu His Ala Ala Ala Leu Ser Gly His Leu Glu Ile Val
Glu 405 410 415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp
Lys Ser Gly 420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly
His Gln Asp Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser
Pro Thr Pro Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Asn Lys
Leu Leu Asp Ala Ala Trp Val Gly Gln Asp Asp Glu 485 490 495Val Arg
Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Trp 500 505
510Gln Gly Glu Thr Pro Leu His Leu Ala Ala Thr Lys Gly His Leu Glu
515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Lys Asp 530 535 540Val Val Gly Tyr Thr Pro Leu His Val Ala Ala Ser
Gln Gly His Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln 565 570 575Asp Val His Gly Tyr Thr Pro
Ala Asp Leu Ala Ala Gln Ala Gly His 580 585 590Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630
635 640Gln Leu Asp Glu
Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650 655Ala Lys
Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu 660 665
670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala
Ala Trp 690 695 700Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Val Ser Gly Ala
Thr Pro Leu His Ala Ala Ala 725 730 735Leu His Gly His Leu Glu Ile
Val Glu Val Leu Leu Asn Ala Gly Ala 740 745 750Asp Val Asn Ala Gln
Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala Arg Ala
Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78029784PRTArtificial SequenceMulti-specific binding protein 29Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asn Lys Leu 290 295
300Leu Asp Ala Ala Trp Val Gly Gln Asp Asp Glu Val Arg Ile Leu
Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Trp Gln
Gly Glu Thr Pro 325 330 335Leu His Leu Ala Ala Thr Lys Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Val Gly Tyr Thr 355 360 365Pro Leu His Val Ala Ala
Ser Gln Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Val His Gly Tyr385 390 395 400Thr
Pro Ala Asp Leu Ala Ala Gln Ala Gly His Glu Asp Ile Ala Glu 405 410
415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
420 425 430Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser
Asp Leu 435 440 445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln
Leu Asp Glu Val 450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Arg Glu465 470 475 480Gly Trp Thr Pro Leu His Leu
Ala Ala His Gln Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Arg
Thr Pro Leu His Leu Ala Ala Trp Val Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Ser Gly Ala Thr Pro Leu His Pro Ala Ala Ile Tyr Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu
Ala Ala Arg Ala Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Gln Glu Gly Ile Thr Pro Leu His Val Ala Ala His Gln
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp 690 695 700Arg Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp His Ala
Gly Ala Thr Pro Leu His Ala Ala Ala 725 730 735Leu Ser Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala
Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78030817PRTArtificial SequenceMulti-specific binding protein 30Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu
Gly Lys Thr Pro 325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Ile Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr
Pro Leu His Ala Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Gln
Ala Ala Arg Ala Gly Gln Leu Asp Glu 485 490 495Val Arg Glu Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln 500 505 510Glu Gly Ile
Thr Pro Leu His Val Ala Ala His Gln Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Arg Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys 565 570 575Asp His Ala Gly Ala Thr Pro Leu His Ala
Ala Ala Leu Ser Gly His 580 585 590Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala 595 600 605Gln Asp Lys Ser Gly Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly 610 615 620His Gln Asp Ile
Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr625 630 635 640Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 645 650
655Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala
660 665 670Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Arg Glu Gly Ile Thr Pro Leu His
Leu Ala Ala Gln 690 695 700His Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Val Trp
Gly Arg Thr Pro Leu His Leu Ala Ala 725 730 735Trp Gln Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Lys Asp Leu Ala Gly Ala Thr Pro Leu His Val Ala 755 760 765Ala
Leu Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 770 775
780Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp
Leu785 790 795 800Ala Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val
Leu Gln Lys Ala 805 810 815Ala31817PRTArtificial
SequenceMulti-specific binding protein 31Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Ile Thr Pro
325 330 335Leu His Leu Ala Ala Gln His Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Gln Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Leu Ala Gly Ala385 390 395 400Thr Pro Leu His Val
Ala Ala Leu Tyr Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala
Gly Gln Leu Asp Glu 485 490 495Val Arg Glu Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Arg 500 505 510Glu Gly Lys Thr Pro Leu His
Val Ala Ala Gln Glu Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Val Trp Gly
Arg Thr Pro Leu His Leu Ala Ala Trp Ile Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
565 570 575Asp Val Ser Gly Ala Thr Pro Leu His Ala Ala Ala Leu His
Gly His 580 585 590Leu Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala
Asp Val Asn Ala 595 600 605Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp
Leu Ala Ala Arg Ala Gly 610 615 620His Gln Asp Ile Ala Glu Val Leu
Gln Lys Ala Ala Gly Ser Pro Thr625 630 635 640Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 645 650 655Pro Thr Gly
Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala 660 665 670Gly
Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Gln Glu Gly Ile Thr Pro Leu His Val Ala Ala His
690 695 700Gln Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Val Trp
Gly Arg Thr Pro Leu His Leu Ala Ala 725 730 735Trp Arg Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Lys Asp His Ala Gly Ala Thr Pro Leu His Ala Ala 755 760 765Ala
Leu Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly 770 775
780Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp
Leu785 790 795 800Ala Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val
Leu Gln Lys Ala 805 810 815Ala32784PRTArtificial
SequenceMulti-specific binding protein 32Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Leu Lys Leu 290 295 300Leu Thr Ala
Ala Lys Gln Gly Gln Asp Asp Glu Val Arg Ile Leu Leu305 310 315
320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Arg Gly Leu Thr Pro
325 330 335Leu His Tyr Ala Ala Ala Ile Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln
Tyr Gly Ala Thr 355 360 365Pro Leu His Val Ala Ala Tyr Ile Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Phe Ser Gly Ser385 390 395 400Thr Pro Ala Asp Leu
Ala Ala Glu Glu Gly His Glu Asp Ile Ala Glu 405 410 415Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro 420 425 430Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu 435 440
445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val
450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
Arg Glu465 470 475 480Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu
Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp Ile Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Val Ser Gly
Ala Thr Pro Leu His Ala Ala Ala Leu His Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala
Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Lys Lys
Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln Leu Asp Glu Val
Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Gln Glu Gly Ile Thr Pro Leu His Val Ala Ala His Gln 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp
690 695 700Arg Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Lys Asp His Ala Gly Ala Thr Pro
Leu His Ala Ala Ala 725 730 735Leu Ser Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn Ala Gln Asp Lys
Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala Arg Ala Gly His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78033784PRTArtificial SequenceMulti-specific binding protein 33Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Trp Lys Leu 290 295
300Leu Trp Ala Ala Gln Val Gly Gln Asp Asp Glu Val Arg Ile Leu
Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Leu
Gly Trp Thr Pro 325 330 335Leu His Ile Ala Ala Trp Val Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Phe Thr Gly Arg Thr 355 360 365Pro Leu His His Ala Ala
Thr Glu Gly His Leu Glu Ile Val Lys Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Asp Val Gly His385 390 395 400Thr
Pro Ala Asp Leu Ala Ala Leu Trp Gly His Glu Asp Ile Ala Glu 405 410
415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
420 425 430Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser
Asp Leu 435 440 445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln
Leu Asp Glu Val 450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Gln Glu465 470 475 480Gly Ile Thr Pro Leu His Val
Ala Ala His Gln Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Trp Gly Arg
Thr Pro Leu His Leu Ala Ala Trp Arg Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540His Ala Gly Ala Thr Pro Leu His Ala Ala Ala Leu Ser Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu
Ala Ala Arg Ala Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Arg Glu Gly Ile Thr Pro Leu His Leu Ala Ala Gln His
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp 690 695 700Gln Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Leu Ala
Gly Ala Thr Pro Leu His Val Ala Ala 725 730 735Leu Tyr Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala
Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78034784PRTArtificial SequenceMulti-specific binding protein 34Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu
Gly Ile Thr Pro 325 330 335Leu His Leu Ala Ala Gln His Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Gln Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Leu Ala Gly Ala385 390 395 400Thr
Pro Leu His Val Ala Ala Leu Tyr Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Gln Lys Leu Leu His
Ala Ala Gln Tyr Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu
Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Lys 500 505 510Gln Gly Asn
Thr Pro Leu His Ile Ala Ala Phe His Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala Trp Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Asn Tyr Gly Gln Thr Pro Ala Asp Leu
Ala Ala Glu Ser Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Glu Thr Gly Phe Thr Pro Leu His Val Ala Ala Glu Lys
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp His Phe Gly Phe Thr Pro Leu His
Leu Val Ser Glu 690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp Ser Tyr
Gly Trp Thr Pro Leu His Val Ala Ala 725 730 735Ile Leu Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala
Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu
Gln Lys Ala Ala 770 775 78035751PRTArtificial
SequenceMulti-specific binding protein 35Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Lys Thr Pro
325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Ile Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr Pro Leu His Ala
Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly Gln Lys Leu Leu His Ala Ala Gln Tyr
Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Lys 500 505 510Gln Gly Asn Thr Pro Leu His
Ile Ala Ala Phe His Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Gln Trp Gly
Leu Thr Pro Leu His Leu Ala Ala Ala Trp Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Asn Tyr Gly Gln Thr Pro Ala Asp Leu Ala Ala Glu Ser
Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Gln Lys
Leu Leu His Ala Ala Gln Tyr Gly625 630 635 640Gln Asp Asp Glu Val
Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Lys Gln Gly Asn Thr Pro Leu His Ile Ala Ala Phe His 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala
690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro
Ala Asp Leu Ala Ala 725 730 735Glu Ser Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75036784PRTArtificial
SequenceMulti-specific binding protein 36Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly Ile Thr Pro
325 330 335Leu His Val Ala Ala His Gln Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Arg Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp His Ala Gly Ala385 390 395 400Thr Pro Leu His Ala
Ala Ala Leu Ser Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala
Gly Gln Leu Asp Glu 485 490 495Val Arg Glu Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Arg 500 505 510Glu Gly Ile Thr Pro Leu His
Leu Ala Ala Gln His Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Val Trp Gly
Arg Thr Pro Leu His Leu Ala Ala Trp Gln Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
565 570 575Asp Leu Ala Gly Ala Thr Pro Leu His Val Ala Ala Leu Tyr
Gly His 580 585 590Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala 595 600 605Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp
Leu Ala Ala Arg Ala Gly 610 615 620His Gln Asp Ile Ala Glu Val Leu
Gln Lys Ala Ala Gly Ser Pro Thr625 630 635 640Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr 645 650 655Pro Thr Gly
Ser Asp Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala 660 665 670Gly
Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Val Phe Gly Gln Thr Pro Leu His Val Ala Ala Val
690 695 700Ala Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Lys Asp Ser Ile Gly Tyr Thr Pro
Leu His His Ala Ala 725 730 735Arg Val Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn Ala Gln Asp Trp
Ile Gly Ile Thr Pro Ala Asp Leu Ala 755 760 765Ala Phe Glu Gly His
Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78037784PRTArtificial SequenceMulti-specific binding protein 37Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly His Lys Leu 290 295
300Leu Leu Ala Ala Gln Ala Gly Gln Asp Asp Glu Val Arg Ile Leu
Leu305 310 315 320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Val Phe
Gly Gln Thr Pro 325 330 335Leu His Val Ala Ala Val Ala Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Ser Ile Gly Tyr Thr 355 360 365Pro Leu His His Ala Ala
Arg Val Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Gln Asp Trp Ile Gly Ile385 390 395 400Thr
Pro Ala Asp Leu Ala Ala Phe Glu Gly His Glu Asp Ile Ala Glu 405 410
415Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
420 425 430Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser
Asp Leu 435 440 445Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln
Leu Asp Glu Val 450 455 460Arg Glu Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Arg Glu465 470 475 480Gly Lys Thr Pro Leu His Val
Ala Ala Gln Glu Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Trp Gly Arg
Thr Pro Leu His Leu Ala Ala Trp Ile Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Val Ser Gly Ala Thr Pro Leu His Ala Ala Ala Leu His Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala Asp
Val Asn Ala Gln 565 570 575Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu
Ala Ala Arg Ala Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Gln Glu Gly Ile Thr Pro Leu His Val Ala Ala His Gln
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His
Leu Ala Ala Trp 690 695 700Arg Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Lys Asp His Ala
Gly Ala Thr Pro Leu His Ala Ala Ala 725 730 735Leu Ser Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn
Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala
Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78038784PRTArtificial SequenceMulti-specific binding protein 38Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly
His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120
125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr
130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu
Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu
Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser
His Thr Pro Leu His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala
Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn
Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235
240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp
245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu
Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser
Asp Leu Gly His Lys Leu 290 295 300Leu Leu Ala Ala Gln Ala Gly Gln
Asp Asp Glu Val Arg Ile Leu Leu305 310 315 320Ala Ala Gly Ala Asp
Val Asn Ala Lys Asp Val Phe Gly Gln Thr Pro 325 330 335Leu His Val
Ala Ala Val Ala Gly His Leu Glu Ile Val Glu Val Leu 340 345 350Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Ile Gly Tyr Thr 355 360
365Pro Leu His His Ala Ala Arg Val Gly His Leu Glu Ile Val Glu Val
370 375 380Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Trp Ile
Gly Ile385 390 395 400Thr Pro Ala Asp Leu Ala Ala Phe Glu Gly His
Glu Asp Ile Ala Glu 405 410 415Val Leu Gln Lys Ala Ala Gly Ser Pro
Thr Pro Thr Pro Thr Thr Pro 420 425 430Thr Pro Thr Pro Thr Thr Pro
Thr Pro Thr Pro Thr Gly Ser Asp Leu 435 440 445Gly Lys Lys Leu Leu
Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val 450 455 460Arg Glu Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu465 470 475
480Gly Ile Thr Pro Leu His Leu Ala Ala Gln His Gly His Leu Glu Ile
485 490 495Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
Asp Val 500 505 510Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Gln
Gly His Leu Glu 515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp 530 535 540Leu Ala Gly Ala Thr Pro Leu His
Val Ala Ala Leu Tyr Gly His Leu545 550 555 560Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln 565 570 575Asp Lys Ser
Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His 580 585 590Gln
Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro 595 600
605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro
610 615 620Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg
Ala Gly625 630 635 640Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala
Gly Ala Asp Val Asn 645 650 655Ala Lys Asp Gln Glu Gly Ile Thr Pro
Leu His Val Ala Ala His Gln 660 665 670Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala Asp Val 675 680 685Asn Ala Lys Asp Val
Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp 690 695 700Arg Gly His
Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp705 710 715
720Val Asn Ala Lys Asp His Ala Gly Ala Thr Pro Leu His Ala Ala Ala
725 730 735Leu Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala 740 745 750Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro
Ala Asp Leu Ala 755 760 765Ala Arg Ala Gly His Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 770 775 78039751PRTArtificial
SequenceMulti-specific binding protein 39Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly Ile Thr Pro
325 330 335Leu His Val Ala Ala His Gln Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Arg Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp His Ala Gly Ala385 390 395 400Thr Pro Leu His Ala
Ala Ala Leu Ser Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala
Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln Thr Pro Leu His
Val Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Ser Ile Gly
Tyr Thr Pro Leu His His Ala Ala Arg Val Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala Ala Phe Glu
Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Gln Lys
Leu Leu His Ala Ala Gln Tyr Gly625 630 635 640Gln Asp Asp Glu Val
Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Lys Gln Gly Asn Thr Pro Leu His Ile Ala Ala Phe His 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala
690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro
Ala Asp Leu Ala Ala 725 730 735Glu Ser Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75040784PRTArtificial
SequenceMulti-specific binding protein 40Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Ile Thr Pro
325 330 335Leu His Leu Ala Ala Gln His Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Gln Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Leu Ala Gly Ala385 390 395 400Thr Pro Leu His Val
Ala Ala Leu Tyr Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala
Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln Thr Pro Leu His
Val Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Ser Ile Gly
Tyr Thr Pro Leu His His Ala Ala Arg Val Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala Ala Phe Glu
Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Lys Lys
Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln Leu Asp Glu Val
Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Glu Thr Gly Phe Thr Pro Leu His Val Ala Ala Glu Lys 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp His Phe Gly Phe Thr Pro Leu His Leu Val Ser Glu
690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Lys Asp Ser Tyr Gly Trp Thr Pro
Leu His Val Ala Ala 725 730 735Ile Leu Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn Ala Gln Asp Lys
Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala Arg Ala Gly His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78041751PRTArtificial SequenceMulti-specific binding protein 41Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe
Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu
Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr
Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu 290 295 300Leu His
Ala Ala Gln Tyr Gly Gln Asp Asp Glu Val Arg Ile Leu Leu305 310 315
320Ala Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Gln Gly Asn Thr Pro
325 330 335Leu His Ile Ala Ala Phe His Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln
Trp Gly Leu Thr 355 360 365Pro Leu His Leu Ala Ala Ala Trp Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Asn Tyr Gly Gln385 390 395 400Thr Pro Ala Asp Leu
Ala Ala Glu Ser Gly His Glu Asp Ile Ala Glu 405 410 415Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro 420 425 430Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu 435 440
445Gly His Lys Leu Leu Leu Ala Ala Gln Ala Gly Gln Asp Asp Glu Val
450 455 460Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp
Val Phe465 470 475 480Gly Gln Thr Pro Leu His Val Ala Ala Val Ala
Gly His Leu Glu Ile 485 490 495Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Ser 500 505 510Ile Gly Tyr Thr Pro Leu His
His Ala Ala Arg Val Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp 530 535 540Trp Ile Gly
Ile Thr Pro Ala Asp Leu Ala Ala Phe Glu Gly His Glu545 550 555
560Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr
565 570 575Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr 580 585 590Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala
Arg Ala Gly Gln 595 600 605Leu Asp Glu Val Arg Glu Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala 610 615 620Lys Asp Gln Glu Gly Ile Thr Pro
Leu His Val Ala Ala His Gln Gly625 630 635 640His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Arg 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp His Ala Gly Ala Thr Pro Leu His Ala Ala Ala Leu
690 695 700Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro
Ala Asp Leu Ala Ala 725 730 735Arg Ala Gly His Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75042784PRTArtificial
SequenceMulti-specific binding protein 42Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu Thr Gly Phe Thr Pro
325 330 335Leu His Val Ala Ala Glu Lys Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp His
Phe Gly Phe Thr 355 360 365Pro Leu His Leu Val Ser Glu Trp Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Ser Tyr Gly Trp385 390 395 400Thr Pro Leu His Val
Ala Ala Ile Leu Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly His Lys Leu Leu Leu Ala Ala Gln Ala
Gly Gln Asp Asp Glu 485 490 495Val Arg Ile Leu Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln Thr Pro Leu His
Val Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Ser Ile Gly
Tyr Thr Pro Leu His His Ala Ala Arg Val Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln
565 570 575Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala Ala Phe Glu
Gly His 580 585 590Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Lys Lys
Leu Leu Gln Ala Ala Arg Ala Gly625 630 635 640Gln Leu Asp Glu Val
Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Arg Glu Gly Ile Thr Pro Leu His Leu Ala Ala Gln His 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp
690 695 700Gln Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Lys Asp Leu Ala Gly Ala Thr Pro
Leu His Val Ala Ala 725 730 735Leu Tyr Gly His Leu Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala 740 745 750Asp Val Asn Ala Gln Asp Lys
Ser Gly Lys Thr Pro Ala Asp Leu Ala 755 760 765Ala Arg Ala Gly His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 770 775
78043703PRTArtificial SequenceTarget domain of spike protein 43Val
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser1 5 10
15Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val
20 25 30Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val
Thr 35 40 45Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
Arg Phe 50 55 60Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
Ala Ser Thr65 70 75 80Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe
Gly Thr Thr Leu Asp 85 90 95Ser Lys Thr Gln Ser Leu Leu Ile Val Asn
Asn Ala Thr Asn Val Val 100 105 110Ile Lys Val Cys Glu Phe Gln Phe
Cys Asn Asp Pro Phe Leu Gly Val 115 120 125Tyr Tyr His Lys Asn Asn
Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140Tyr Ser Ser Ala
Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe145 150 155 160Leu
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170
175Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His
180 185 190Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
Ala Leu 195 200 205Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
Thr Arg Phe Gln 210 215 220Thr Leu Leu Ala Leu His Arg Ser Tyr Leu
Thr Pro Gly Asp Ser Ser225 230 235 240Ser Gly Trp Thr Ala Gly Ala
Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255Pro Arg Thr Phe Leu
Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270Ala Val Asp
Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285Lys
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg 290 295
300Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
Leu305 310 315 320Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
Ala Ser Val Tyr 325 330 335Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
Val Ala Asp Tyr Ser Val 340 345 350Leu Tyr Asn Ser Ala Ser Phe Ser
Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365Pro Thr Lys Leu Asn Asp
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380Phe Val Ile Arg
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr385 390 395 400Gly
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 405 410
415Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
420 425 430Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
Lys Pro 435 440 445Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
Gly Ser Thr Pro 450 455 460Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
Phe Pro Leu Gln Ser Tyr465 470 475 480Gly Phe Gln Pro Thr Asn Gly
Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495Val Leu Ser Phe Glu
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505 510Lys Lys Ser
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe 515 520 525Asn
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535
540Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
Ala545 550 555 560Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
Thr Pro Cys Ser 565 570 575Phe Gly Gly Val Ser Val Ile Thr Pro Gly
Thr Asn Thr Ser Asn Gln 580 585 590Val Ala Val Leu Tyr Gln Asp Val
Asn Cys Thr Glu Val Pro Val Ala 595 600 605Ile His Ala Asp Gln Leu
Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620Ser Asn Val Phe
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His625 630 635 640Val
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650
655Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Gly Gly
660 665 670Gly Ser Gly Gly Gly Ser His His His His His His His His
His His 675 680 685Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu
Trp His Glu 690 695 700441209PRTArtificial SequenceTarget domain of
spike protein 44Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
Thr Asn Ser1 5 10 15Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe
Arg Ser Ser Val 20 25 30Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe
Phe Ser Asn Val Thr 35 40 45Trp Phe His Ala Ile His Val Ser Gly Thr
Asn Gly Thr Lys Arg Phe 50 55 60Asp Asn Pro Val Leu Pro Phe Asn Asp
Gly Val Tyr Phe Ala Ser Thr65 70 75 80Glu Lys Ser Asn Ile Ile Arg
Gly Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95Ser Lys Thr Gln Ser Leu
Leu Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110Ile Lys Val Cys
Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 115 120 125Tyr Tyr
His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135
140Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro
Phe145 150 155 160Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys
Asn Leu Arg Glu 165 170 175Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe
Lys Ile Tyr Ser Lys His 180 185 190Thr Pro Ile Asn Leu Val Arg Asp
Leu Pro Gln Gly Phe Ser Ala Leu 195 200 205Glu Pro Leu Val Asp Leu
Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln 210 215 220Thr Leu Leu Ala
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser225 230 235 240Ser
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250
255Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp
260 265 270Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
Thr Leu 275 280 285Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr
Ser Asn Phe Arg 290 295 300Val Gln Pro Thr Glu Ser Ile Val Arg Phe
Pro Asn Ile Thr Asn Leu305 310 315 320Cys Pro Phe Gly Glu Val Phe
Asn Ala Thr Arg Phe Ala Ser Val Tyr 325 330 335Ala Trp Asn Arg Lys
Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 340 345 350Leu Tyr Asn
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365Pro
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375
380Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
Thr385 390 395 400Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
Asp Phe Thr Gly 405 410 415Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
Asp Ser Lys Val Gly Gly 420 425 430Asn Tyr Asn Tyr Leu Tyr Arg Leu
Phe Arg Lys Ser Asn Leu Lys Pro 435 440 445Phe Glu Arg Asp Ile Ser
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 450
455 460Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
Tyr465 470 475 480Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
Tyr Arg Val Val 485 490 495Val Leu Ser Phe Glu Leu Leu His Ala Pro
Ala Thr Val Cys Gly Pro 500 505 510Lys Lys Ser Thr Asn Leu Val Lys
Asn Lys Cys Val Asn Phe Asn Phe 515 520 525Asn Gly Leu Thr Gly Thr
Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535 540Leu Pro Phe Gln
Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala545 550 555 560Val
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser 565 570
575Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln
580 585 590Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro
Val Ala 595 600 605Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val
Tyr Ser Thr Gly 610 615 620Ser Asn Val Phe Gln Thr Arg Ala Gly Cys
Leu Ile Gly Ala Glu His625 630 635 640Val Asn Asn Ser Tyr Glu Cys
Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650 655Ala Ser Tyr Gln Thr
Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val 660 665 670Ala Ser Gln
Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn 675 680 685Ser
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr 690 695
700Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr
Ser705 710 715 720Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr
Glu Cys Ser Asn 725 730 735Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr
Gln Leu Asn Arg Ala Leu 740 745 750Thr Gly Ile Ala Val Glu Gln Asp
Lys Asn Thr Gln Glu Val Phe Ala 755 760 765Gln Val Lys Gln Ile Tyr
Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly 770 775 780Phe Asn Phe Ser
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg785 790 795 800Ser
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala 805 810
815Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg
820 825 830Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
Pro Pro 835 840 845Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser
Ala Leu Leu Ala 850 855 860Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly
Ala Gly Ala Ala Leu Gln865 870 875 880Ile Pro Phe Ala Met Gln Met
Ala Tyr Arg Phe Asn Gly Ile Gly Val 885 890 895Thr Gln Asn Val Leu
Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe 900 905 910Asn Ser Ala
Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser 915 920 925Ala
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu 930 935
940Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser
Ser945 950 955 960Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val
Glu Ala Glu Val 965 970 975Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu
Gln Ser Leu Gln Thr Tyr 980 985 990Val Thr Gln Gln Leu Ile Arg Ala
Ala Glu Ile Arg Ala Ser Ala Asn 995 1000 1005Leu Ala Ala Thr Lys
Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg 1010 1015 1020Val Asp
Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser1025 1030
1035 1040Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
Ala Gln 1045 1050 1055Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys
His Asp Gly Lys Ala 1060 1065 1070His Phe Pro Arg Glu Gly Val Phe
Val Ser Asn Gly Thr His Trp Phe 1075 1080 1085Val Thr Gln Arg Asn
Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn 1090 1095 1100Thr Phe
Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn1105 1110
1115 1120Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
Glu Glu 1125 1130 1135Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro
Asp Val Asp Leu Gly 1140 1145 1150Asp Ile Ser Gly Ile Asn Ala Ser
Val Val Asn Ile Gln Lys Glu Ile 1155 1160 1165Asp Arg Leu Asn Glu
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp 1170 1175 1180Leu Gln
Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Ala His1185 1190
1195 1200His His His His His His His His His 120545462PRTArtificial
SequenceTarget domain of spike protein 45Arg Val Gln Pro Thr Glu
Ser Ile Val Arg Phe Pro Asn Ile Thr Asn1 5 10 15Leu Cys Pro Phe Gly
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30Tyr Ala Trp Asn
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45Val Leu Tyr
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60Ser Pro
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp65 70 75
80Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
Thr 100 105 110Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
Lys Val Gly 115 120 125Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
Lys Ser Asn Leu Lys 130 135 140Pro Phe Glu Arg Asp Ile Ser Thr Glu
Ile Tyr Gln Ala Gly Ser Thr145 150 155 160Pro Cys Asn Gly Val Glu
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175Tyr Gly Phe Gln
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190Val Val
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200
205Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly
210 215 220Gly Gly Ser Gly Gly Gly Ser Pro Lys Ser Ser Asp Lys Thr
His Thr225 230 235 240Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe 245 250 255Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro 260 265 270Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val 275 280 285Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val305 310 315
320Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
325 330 335Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser 340 345 350Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro 355 360 365Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val 370 375 380Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly385 390 395 400Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440
445Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455
4604610PRTArtificial SequenceHis-tag 46Met Arg Gly Ser His His His
His His His1 5 1047126PRTArtificial SequenceHuman serum albumin
binding domain 47Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala
Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His
Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile
Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His
Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120
12548126PRTArtificial SequenceHuman serum albumin binding domain
48Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1
5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg
Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
Ala Ala Ala Asp65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr
Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala
Glu Val Leu Gln Lys Ala Ala 115 120 12549126PRTArtificial
SequenceHuman serum albumin binding domain 49Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val
Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr
Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu
Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala
Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asp Ala65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala 115 120 1255033PRTArtificial SequenceConsensus
sequenceVARIANT2,3,5,12,13,14,16,17,18,21,25,26,33x" denotes any
amino acid 50Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu Ala Xaa Xaa
Xaa Gly Xaa1 5 10 15Xaa Xaa Leu Val Xaa Val Leu Leu Xaa Xaa Gly Ala
Asp Val Asn Ala 20 25 30Xaa5133PRTArtificial SequenceConsensus
sequenceVARIANT2,3,5,13,14,26,33x" denotes any amino acid x" in
position 26 is selected from the group consisting of asparagine,
histidine, or tyrosine 51Asp Xaa Xaa Gly Xaa Thr Pro Leu His Leu
Ala Ala Xaa Xaa Gly His1 5 10 15Leu Glu Ile Val Glu Val Leu Leu Lys
Xaa Gly Ala Asp Val Asn Ala 20 25 30Xaa5224PRTArtificial
SequencePT-rich linker 52Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
Thr Pro Thr Pro Thr Thr1 5 10 15Pro Thr Pro Thr Pro Thr Gly Ser
205322PRTArtificial SequencePT-rich linker 53Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr1 5 10 15Pro Thr Pro Thr
Pro Thr 20545PRTArtificial SequenceConsensus GS
linkerVARIANT1..5[Gly-Gly-Gly-Gly-Ser]n, wherein n is 1, 2, 3, 4,
5, or 6 54Gly Gly Gly Gly Ser1 55532PRTArtificial SequenceN-cap
55Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1
5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn
Ala 20 25 305632PRTArtificial SequenceN-cap 56Gly Ser Asp Leu Gly
Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val
Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25
305732PRTArtificial SequenceN-cap 57Gly Ser Asp Leu Gly Lys Lys Leu
Leu Gln Ala Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Ile Leu
Leu Ala Ala Gly Ala Asp Val Asn Ala 20 25 305828PRTArtificial
SequenceC-cap 58Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala
Arg Ala Gly1 5 10 15His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
20 2559977PRTArtificial SequenceTarget domain of spike protein
59Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn1
5 10 15His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
Asn 20 25 30Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn
Asn Ala 35 40 45Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr
Leu Ala Gln 50 55 60Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val
Lys Leu Gln Leu65 70 75 80Gln Ala Leu Gln Gln Asn Gly Ser Ser Val
Leu Ser Glu Asp Lys Ser 85 90 95Lys Arg Leu Asn Thr Ile Leu Asn Thr
Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Asn Pro Asp
Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asn Glu
Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140Leu Trp Ala
Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg145 150 155
160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr
Glu Val 180 185 190Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln
Leu Ile Glu Asp 195 200 205Val Glu His Thr Phe Glu Glu Ile Lys Pro
Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met
Asn Ala Tyr Pro Ser Tyr Ile Ser225 230 235 240Pro Ile Gly Cys Leu
Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr
Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn
Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280
285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp
Pro Gly305 310 315 320Asn Val Gln Lys Ala Val Cys His Pro Thr Ala
Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Leu Met Cys Thr
Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met
Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ala Gln Pro Phe
Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val
Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His385 390 395
400Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val
Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp
Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Asp Gln Trp Met Lys
Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu
Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp
Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490
495Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu
500 505 510Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp
Ile Ser 515 520 525Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met
Leu Arg Leu Gly 530 535 540Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu
Asn Val Val Gly Ala Lys545 550 555 560Asn Met Asn Val Arg Pro Leu
Leu Asn Tyr Phe Glu Pro Leu Phe Thr 565 570 575Trp Leu Lys Asp Gln
Asn Lys Asn Ser Phe Val Gly Trp Ser Thr Asp 580 585 590Trp Ser Pro
Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser
Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615
620Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
Lys625 630 635 640Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp
Val Arg Val Ala 645 650 655Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe
Phe Val Thr Ala Pro Lys 660 665 670Asn Val Ser Asp Ile Ile Pro Arg
Thr Glu Val Glu Lys Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile
Asn Asp Ala Phe Arg Leu Asn Asp Asn Ser 690 695 700Leu Glu Phe Leu
Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln Pro705 710 715 720Pro
Val Ser Gly Gly Gly Ser Gly Gly Gly Ser Pro Lys Ser Ser Asp 725 730
735Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
740 745 750Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile 755 760 765Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu 770 775 780Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His785 790 795 800Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 805 810 815Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 820 825 830Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 835 840 845Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 850 855
860Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu865 870 875 880Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 885 890 895Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val 900 905 910Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp 915 920 925Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His 930 935 940Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro945 950 955 960Gly
Lys Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His 965 970
975Glu60756PRTArtificial SequenceTarget domain of spike protein
60Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe Asn1
5 10 15His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
Asn 20 25 30Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn
Asn Ala 35 40 45Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr
Leu Ala Gln 50 55 60Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val
Lys Leu Gln Leu65 70 75 80Gln Ala Leu Gln Gln Asn Gly Ser Ser Val
Leu Ser Glu Asp Lys Ser 85 90 95Lys Arg Leu Asn Thr Ile Leu Asn Thr
Met Ser Thr Ile Tyr Ser Thr 100 105 110Gly Lys Val Cys Asn Pro Asp
Asn Pro Gln Glu Cys Leu Leu Leu Glu 115 120 125Pro Gly Leu Asn Glu
Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg 130 135 140Leu Trp Ala
Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg145 150 155
160Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
165 170 175Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr
Glu Val 180 185 190Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln
Leu Ile Glu Asp 195 200 205Val Glu His Thr Phe Glu Glu Ile Lys Pro
Leu Tyr Glu His Leu His 210 215 220Ala Tyr Val Arg Ala Lys Leu Met
Asn Ala Tyr Pro Ser Tyr Ile Ser225 230 235 240Pro Ile Gly Cys Leu
Pro Ala His Leu Leu Gly Asp Met Trp Gly Arg 245 250 255Phe Trp Thr
Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys Pro 260 265 270Asn
Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln 275 280
285Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp
Pro Gly305 310 315 320Asn Val Gln Lys Ala Val Cys His Pro Thr Ala
Trp Asp Leu Gly Lys 325 330 335Gly Asp Phe Arg Ile Leu Met Cys Thr
Lys Val Thr Met Asp Asp Phe 340 345 350Leu Thr Ala His His Glu Met
Gly His Ile Gln Tyr Asp Met Ala Tyr 355 360 365Ala Ala Gln Pro Phe
Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe His 370 375 380Glu Ala Val
Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His385 390 395
400Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn Glu
405 410 415Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val
Gly Thr 420 425 430Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp
Met Val Phe Lys 435 440 445Gly Glu Ile Pro Lys Asp Gln Trp Met Lys
Lys Trp Trp Glu Met Lys 450 455 460Arg Glu Ile Val Gly Val Val Glu
Pro Val Pro His Asp Glu Thr Tyr465 470 475 480Cys Asp Pro Ala Ser
Leu Phe His Val Ser Asn Asp Tyr Ser Phe Ile 485 490 495Arg Tyr Tyr
Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala Leu 500 505 510Cys
Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile Ser 515 520
525Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu Gly
530 535 540Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly
Ala Lys545 550 555 560Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe
Glu Pro Leu Phe Thr 565 570 575Trp Leu Lys Asp Gln Asn Lys Asn Ser
Phe Val Gly Trp Ser Thr Asp 580 585 590Trp Ser Pro Tyr Ala Asp Gln
Ser Ile Lys Val Arg Ile Ser Leu Lys 595 600 605Ser Ala Leu Gly Asp
Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met Tyr 610 615 620Leu Phe Arg
Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu Lys625 630 635
640Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val Ala
645 650 655Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala
Pro Lys 660 665 670Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu
Lys Ala Ile Arg 675 680 685Met Ser Arg Ser Arg Ile Asn Asp Ala Phe
Arg Leu Asn Asp Asn Ser 690 695 700Leu Glu Phe Leu Gly Ile Gln Pro
Thr Leu Gly Pro Pro Asn Gln Pro705 710 715 720Pro Val Ser Gly Gly
Gly Ser Gly Gly Gly Ser His His His His His 725 730 735His His His
His His Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile 740 745 750Glu
Trp His Glu 755611209PRTArtificial SequenceTarget domain of spike
protein 61Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr
Asn Ser1 5 10 15Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
Ser Ser Val 20 25 30Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe
Ser Asn Val Thr 35 40 45Trp Phe His Ala Ile His Val Ser Gly Thr Asn
Gly Thr Lys Arg Phe 50 55 60Asp Asn Pro Val Leu Pro Phe Asn Asp Gly
Val Tyr Phe Ala Ser Thr65 70 75 80Glu Lys Ser Asn Ile Ile Arg Gly
Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95Ser Lys Thr Gln Ser Leu Leu
Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110Ile Lys Val Cys Glu
Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 115 120 125Tyr Tyr His
Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140Tyr
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe145 150
155 160Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg
Glu 165 170 175Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
Ser Lys His 180 185 190Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln
Gly Phe Ser Ala Leu 195 200 205Glu Pro Leu Val Asp Leu Pro Ile Gly
Ile Asn Ile Thr Arg Phe Gln 210 215 220Thr Leu Leu Ala Leu His Arg
Ser Tyr Leu Thr Pro Gly Asp Ser Ser225 230 235 240Ser Gly Trp Thr
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255Pro Arg
Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265
270Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu
275 280 285Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
Phe Arg 290 295 300Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
Ile Thr Asn Leu305 310 315 320Cys Pro Phe Gly Glu Val Phe Asn Ala
Thr Arg Phe Ala Ser Val Tyr 325 330 335Ala Trp Asn Arg Lys Arg Ile
Ser Asn Cys Val Ala Asp Tyr Ser Val 340 345 350Leu Tyr Asn Ser Ala
Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365Pro Thr Lys
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380Phe
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr385 390
395 400Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
Gly 405 410 415Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
Val Gly Gly 420 425 430Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
Ser Asn Leu Lys Pro 435 440 445Phe Glu Arg Asp Ile Ser Thr Glu Ile
Tyr Gln Ala Gly Ser Thr Pro 450 455 460Cys Asn Gly Val Glu Gly Phe
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr465 470 475 480Gly Phe Gln Pro
Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495Val Leu
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505
510Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe
515 520 525Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
Lys Phe 530 535 540Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp
Thr Thr Asp Ala545 550 555 560Val Arg Asp Pro Gln Thr Leu Glu Ile
Leu Asp Ile Thr Pro Cys Ser 565 570 575Phe Gly Gly Val Ser Val Ile
Thr Pro Gly Thr Asn Thr Ser Asn Gln 580 585 590Val Ala Val Leu Tyr
Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala 595 600 605Ile His Ala
Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620Ser
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His625 630
635 640Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile
Cys 645 650 655Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
Arg Ser Val 660 665 670Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser
Leu Gly Ala Glu Asn 675 680 685Ser Val Ala Tyr Ser Asn Asn Ser Ile
Ala Ile Pro Thr Asn Phe Thr 690 695 700Ile Ser Val Thr Thr Glu Ile
Leu Pro Val Ser Met Thr Lys Thr Ser705 710 715 720Val Asp Cys Thr
Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn 725 730 735Leu Leu
Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu 740 745
750Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala
755 760 765Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
Gly Gly 770 775 780Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys
Pro Ser Lys Arg785 790 795 800Ser Phe Ile Glu Asp Leu Leu Phe Asn
Lys Val Thr Leu Ala Asp Ala 805 810 815Gly Phe Ile Lys Gln Tyr Gly
Asp Cys Leu Gly Asp Ile Ala Ala Arg 820 825 830Asp Leu Ile Cys Ala
Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro 835 840 845Leu Leu Thr
Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala 850 855 860Gly
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln865 870
875 880Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly
Val 885 890 895Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
Asn Gln Phe 900 905 910Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu
Ser Ser Thr Ala Ser 915 920 925Ala Leu Gly Lys Leu Gln Asp Val Val
Asn Gln Asn Ala Gln Ala Leu 930 935 940Asn Thr Leu Val Lys Gln Leu
Ser Ser Asn Phe Gly Ala Ile Ser Ser945 950 955 960Val Leu Asn Asp
Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val 965 970 975Gln Ile
Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr 980 985
990Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
995 1000 1005Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln
Ser Lys Arg 1010 1015 1020Val Asp Phe Cys Gly Lys Gly Tyr His Leu
Met Ser Phe Pro Gln Ser1025 1030 1035 1040Ala Pro His Gly Val Val
Phe Leu His Val Thr Tyr Val Pro Ala Gln 1045 1050 1055Glu Lys Asn
Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala 1060 1065
1070His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe
1075 1080 1085Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr
Thr Asp Asn 1090 1095 1100Thr Phe Val Ser Gly Asn Cys Asp Val Val
Ile Gly Ile Val Asn Asn1105 1110 1115 1120Thr Val Tyr Asp Pro Leu
Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu 1125 1130 1135Leu Asp Lys
Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly 1140 1145
1150Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile
1155 1160 1165Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser
Leu Ile Asp 1170 1175 1180Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr
Ile Lys Trp Pro Ala His1185 1190 1195 1200His His His His His
His
His His His 120562256PRTArtificial SequenceTarget domain of spike
protein 62Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
Thr Asn1 5 10 15Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
Ala Ser Val 20 25 30Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
Ala Asp Tyr Ser 35 40 45Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
Lys Cys Tyr Gly Val 50 55 60Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
Thr Asn Val Tyr Ala Asp65 70 75 80Ser Phe Val Ile Arg Gly Asp Glu
Val Arg Gln Ile Ala Pro Gly Gln 85 90 95Thr Gly Lys Ile Ala Asp Tyr
Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110Gly Cys Val Ile Ala
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125Gly Asn Tyr
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140Pro
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr145 150
155 160Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
Ser 165 170 175Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
Tyr Arg Val 180 185 190Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
Ala Thr Val Cys Gly 195 200 205Pro Lys Lys Ser Thr Asn Leu Val Lys
Asn Lys Cys Val Asn Phe Gly 210 215 220Gly Gly Ser Gly Gly Gly Ser
His His His His His His His His His225 230 235 240His Gly Leu Asn
Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu 245 250
25563908PRTArtificial SequenceTarget domain of spike protein 63Val
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser1 5 10
15Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val
20 25 30Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val
Thr 35 40 45Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
Arg Phe 50 55 60Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
Ala Ser Thr65 70 75 80Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe
Gly Thr Thr Leu Asp 85 90 95Ser Lys Thr Gln Ser Leu Leu Ile Val Asn
Asn Ala Thr Asn Val Val 100 105 110Ile Lys Val Cys Glu Phe Gln Phe
Cys Asn Asp Pro Phe Leu Gly Val 115 120 125Tyr Tyr His Lys Asn Asn
Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140Tyr Ser Ser Ala
Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe145 150 155 160Leu
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170
175Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His
180 185 190Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
Ala Leu 195 200 205Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
Thr Arg Phe Gln 210 215 220Thr Leu Leu Ala Leu His Arg Ser Tyr Leu
Thr Pro Gly Asp Ser Ser225 230 235 240Ser Gly Trp Thr Ala Gly Ala
Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255Pro Arg Thr Phe Leu
Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270Ala Val Asp
Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285Lys
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg 290 295
300Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
Leu305 310 315 320Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
Ala Ser Val Tyr 325 330 335Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
Val Ala Asp Tyr Ser Val 340 345 350Leu Tyr Asn Ser Ala Ser Phe Ser
Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365Pro Thr Lys Leu Asn Asp
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380Phe Val Ile Arg
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr385 390 395 400Gly
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 405 410
415Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
420 425 430Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
Lys Pro 435 440 445Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
Gly Ser Thr Pro 450 455 460Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
Phe Pro Leu Gln Ser Tyr465 470 475 480Gly Phe Gln Pro Thr Asn Gly
Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495Val Leu Ser Phe Glu
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505 510Lys Lys Ser
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe 515 520 525Asn
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535
540Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
Ala545 550 555 560Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
Thr Pro Cys Ser 565 570 575Phe Gly Gly Val Ser Val Ile Thr Pro Gly
Thr Asn Thr Ser Asn Gln 580 585 590Val Ala Val Leu Tyr Gln Asp Val
Asn Cys Thr Glu Val Pro Val Ala 595 600 605Ile His Ala Asp Gln Leu
Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620Ser Asn Val Phe
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His625 630 635 640Val
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650
655Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala Asp
660 665 670Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr
Cys Pro 675 680 685Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe 690 695 700Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val705 710 715 720Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe 725 730 735Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 740 745 750Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 755 760 765Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 770 775
780Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala785 790 795 800Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg 805 810 815Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly 820 825 830Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro 835 840 845Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 850 855 860Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln865 870 875 880Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 885 890
895Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 900
90564681PRTArtificial SequenceTarget domain of spike protein 64Val
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser1 5 10
15Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val
20 25 30Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val
Thr 35 40 45Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys
Arg Phe 50 55 60Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
Ala Ser Thr65 70 75 80Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe
Gly Thr Thr Leu Asp 85 90 95Ser Lys Thr Gln Ser Leu Leu Ile Val Asn
Asn Ala Thr Asn Val Val 100 105 110Ile Lys Val Cys Glu Phe Gln Phe
Cys Asn Asp Pro Phe Leu Gly Val 115 120 125Tyr Tyr His Lys Asn Asn
Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140Tyr Ser Ser Ala
Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe145 150 155 160Leu
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170
175Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His
180 185 190Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser
Ala Leu 195 200 205Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile
Thr Arg Phe Gln 210 215 220Thr Leu Leu Ala Leu His Arg Ser Tyr Leu
Thr Pro Gly Asp Ser Ser225 230 235 240Ser Gly Trp Thr Ala Gly Ala
Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255Pro Arg Thr Phe Leu
Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270Ala Val Asp
Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285Lys
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg 290 295
300Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
Leu305 310 315 320Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
Ala Ser Val Tyr 325 330 335Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
Val Ala Asp Tyr Ser Val 340 345 350Leu Tyr Asn Ser Ala Ser Phe Ser
Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365Pro Thr Lys Leu Asn Asp
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380Phe Val Ile Arg
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr385 390 395 400Gly
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 405 410
415Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
420 425 430Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
Lys Pro 435 440 445Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
Gly Ser Thr Pro 450 455 460Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
Phe Pro Leu Gln Ser Tyr465 470 475 480Gly Phe Gln Pro Thr Asn Gly
Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495Val Leu Ser Phe Glu
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505 510Lys Lys Ser
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe 515 520 525Asn
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535
540Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
Ala545 550 555 560Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile
Thr Pro Cys Ser 565 570 575Phe Gly Gly Val Ser Val Ile Thr Pro Gly
Thr Asn Thr Ser Asn Gln 580 585 590Val Ala Val Leu Tyr Gln Asp Val
Asn Cys Thr Glu Val Pro Val Ala 595 600 605Ile His Ala Asp Gln Leu
Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620Ser Asn Val Phe
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His625 630 635 640Val
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650
655Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala His
660 665 670His His His His His His His His His 675
68065730PRTArtificial SequenceTarget domain of spike protein 65Gln
Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr1 5 10
15Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser
20 25 30Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
Asn 35 40 45Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly
Thr Lys 50 55 60Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val
Tyr Phe Ala65 70 75 80Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp
Ile Phe Gly Thr Thr 85 90 95Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile
Val Asn Asn Ala Thr Asn 100 105 110Val Val Ile Lys Val Cys Glu Phe
Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125Gly Val Tyr Tyr His Lys
Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140Arg Val Tyr Ser
Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln145 150 155 160Pro
Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170
175Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser
180 185 190Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly
Phe Ser 195 200 205Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile
Asn Ile Thr Arg 210 215 220Phe Gln Thr Leu Leu Ala Leu His Arg Ser
Tyr Leu Thr Pro Gly Asp225 230 235 240Ser Ser Ser Gly Trp Thr Ala
Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255Leu Gln Pro Arg Thr
Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270Thr Asp Ala
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285Thr
Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295
300Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
Thr305 310 315 320Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
Arg Phe Ala Ser 325 330 335Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
Asn Cys Val Ala Asp Tyr 340 345 350Ser Val Leu Tyr Asn Ser Ala Ser
Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365Val Ser Pro Thr Lys Leu
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380Asp Ser Phe Val
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly385 390 395 400Gln
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410
415Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
420 425 430Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
Asn Leu 435 440 445Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
Gln Ala Gly Ser 450 455 460Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
Cys Tyr Phe Pro Leu Gln465 470 475 480Ser Tyr Gly Phe Gln Pro Thr
Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495Val Val Val Leu Ser
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510Gly Pro Lys
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525Asn
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys 530 535
540Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
Thr545 550 555 560Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu
Asp Ile Thr Pro 565 570 575Cys Ser Phe Gly Gly Val Ser Val Ile Thr
Pro Gly Thr Asn Thr Ser 580 585 590Asn
Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro 595 600
605Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser
610 615 620Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile
Gly Ala625 630 635 640Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile
Pro Ile Gly Ala Gly 645 650 655Ile Cys Ala Ser Tyr Gln Thr Gln Thr
Asn Ser Pro Asp Pro Asp Tyr 660 665 670Lys Asp Asp Asp Asp Lys Ala
Gly Pro Gly Trp Ser His Pro Gln Phe 675 680 685Glu Lys Gly Gly Gly
Ser Gly Gly Gly Ser Gly Gly Gly Ser Trp Ser 690 695 700His Pro Gln
Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly705 710 715
720Gly Ser Trp Ser His Pro Gln Phe Glu Lys 725
73066227PRTArtificial SequenceTarget domain of spike protein 66Thr
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala1 5 10
15Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
20 25 30Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
Tyr 35 40 45Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
Val Tyr 50 55 60Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
Ile Ala Pro65 70 75 80Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
Lys Leu Pro Asp Asp 85 90 95Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
Asn Asn Leu Asp Ser Lys 100 105 110Val Gly Gly Asn Tyr Asn Tyr Leu
Tyr Arg Leu Phe Arg Lys Ser Asn 115 120 125Leu Lys Pro Phe Glu Arg
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 130 135 140Ser Thr Pro Cys
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu145 150 155 160Gln
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 165 170
175Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
180 185 190Cys Gly Pro Gly Gly Gly Ser Trp Ser His Pro Gln Phe Glu
Lys Gly 195 200 205Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Trp
Ser His Pro Gln 210 215 220Phe Glu Lys22567539PRTArtificial
SequenceTarget domain of spike protein 67Ser Val Ala Ser Gln Ser
Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala1 5 10 15Glu Asn Ser Val Ala
Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn 20 25 30Phe Thr Ile Ser
Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys 35 40 45Thr Ser Val
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys 50 55 60Ser Asn
Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg65 70 75
80Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
85 90 95Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
Phe 100 105 110Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser
Lys Pro Ser 115 120 125Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn
Lys Val Thr Leu Ala 130 135 140Asp Ala Gly Phe Ile Lys Gln Tyr Gly
Asp Cys Leu Gly Asp Ile Ala145 150 155 160Ala Arg Asp Leu Ile Cys
Ala Gln Lys Phe Asn Gly Leu Thr Val Leu 165 170 175Pro Pro Leu Leu
Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu 180 185 190Leu Ala
Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala 195 200
205Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
210 215 220Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile
Ala Asn225 230 235 240Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp
Ser Leu Ser Ser Thr 245 250 255Ala Ser Ala Leu Gly Lys Leu Gln Asp
Val Val Asn Gln Asn Ala Gln 260 265 270Ala Leu Asn Thr Leu Val Lys
Gln Leu Ser Ser Asn Phe Gly Ala Ile 275 280 285Ser Ser Val Leu Asn
Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala 290 295 300Glu Val Gln
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln305 310 315
320Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
325 330 335Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly
Gln Ser 340 345 350Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu
Met Ser Phe Pro 355 360 365Gln Ser Ala Pro His Gly Val Val Phe Leu
His Val Thr Tyr Val Pro 370 375 380Ala Gln Glu Lys Asn Phe Thr Thr
Ala Pro Ala Ile Cys His Asp Gly385 390 395 400Lys Ala His Phe Pro
Arg Glu Gly Val Phe Val Ser Asn Gly Thr His 405 410 415Trp Phe Val
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr 420 425 430Asp
Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val 435 440
445Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
450 455 460Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp
Val Asp465 470 475 480Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val
Val Asn Ile Gln Lys 485 490 495Glu Ile Asp Arg Leu Asn Glu Val Ala
Lys Asn Leu Asn Glu Ser Leu 500 505 510Ile Asp Leu Gln Glu Leu Gly
Lys Tyr Glu Gln Tyr Ile Lys Trp Pro 515 520 525Ala His His His His
His His His His His His 530 53568521PRTArtificial
SequenceMulti-specific binding protein 68Gly Ser Asp Leu Gly Lys
Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Arg Glu
Gly Ile Thr Pro Leu His Leu Ala Ala Gln His Gly 35 40 45His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Gln65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Lys Asp Leu Ala Gly Ala Thr Pro Leu His Val Ala Ala
Leu 100 105 110Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp 115 120 125Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro
Ala Asp Leu Ala Ala 130 135 140Arg Ala Gly His Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly145 150 155 160Ser Pro Thr Pro Thr Pro
Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro 165 170 175Thr Pro Thr Pro
Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala 180 185 190Ala Arg
Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val
210 215 220Ala Ala Gln Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Lys Asp Val Trp Gly
Arg Thr Pro Leu His 245 250 255Leu Ala Ala Trp Ile Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys 260 265 270Ala Gly Ala Asp Val Asn Ala
Lys Asp Val Ser Gly Ala Thr Pro Leu 275 280 285His Ala Ala Ala Leu
His Gly His Leu Glu Ile Val Glu Val Leu Leu 290 295 300Asn Ala Gly
Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro305 310 315
320Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu
325 330 335Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro
Thr Pro 340 345 350Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser
Asp Leu Gly Lys 355 360 365Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln
Leu Asp Glu Val Arg Glu 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Gln Glu Gly Ile385 390 395 400Thr Pro Leu His Val
Ala Ala His Gln Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Trp Gly 420 425 430Arg
Thr Pro Leu His Leu Ala Ala Trp Arg Gly His Leu Glu Ile Val 435 440
445Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp His Ala
450 455 460Gly Ala Thr Pro Leu His Ala Ala Ala Leu Ser Gly His Leu
Glu Ile465 470 475 480Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Lys 485 490 495Ser Gly Lys Thr Pro Ala Asp Leu Ala
Ala Arg Ala Gly His Gln Asp 500 505 510Ile Ala Glu Val Leu Gln Lys
Ala Ala 515 52069455PRTArtificial SequenceMulti-specific binding
protein 69Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala
Gly Gln1 5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala 20 25 30Lys Asp Gln Glu Gly Ile Thr Pro Leu His Val Ala
Ala His Gln Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala
Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Val Trp Gly Arg Thr Pro Leu
His Leu Ala Ala Trp Arg65 70 75 80Gly His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp His Ala Gly
Ala Thr Pro Leu His Ala Ala Ala Leu 100 105 110Ser Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp 115 120 125Val Asn Ala
Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135 140Arg
Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly145 150
155 160Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr
Pro 165 170 175Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly His Lys Leu
Leu Leu Ala 180 185 190Ala Gln Ala Gly Gln Asp Asp Glu Val Arg Ile
Leu Leu Ala Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Val Phe
Gly Gln Thr Pro Leu His Val 210 215 220Ala Ala Val Ala Gly His Leu
Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val
Asn Ala Lys Asp Ser Ile Gly Tyr Thr Pro Leu His 245 250 255His Ala
Ala Arg Val Gly His Leu Glu Ile Val Glu Val Leu Leu Lys 260 265
270Ala Gly Ala Asp Val Asn Ala Gln Asp Trp Ile Gly Ile Thr Pro Ala
275 280 285Asp Leu Ala Ala Phe Glu Gly His Glu Asp Ile Ala Glu Val
Leu Gln 290 295 300Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
Pro Thr Pro Thr305 310 315 320Pro Thr Thr Pro Thr Pro Thr Pro Thr
Gly Ser Asp Leu Gly Gln Lys 325 330 335Leu Leu His Ala Ala Gln Tyr
Gly Gln Asp Asp Glu Val Arg Ile Leu 340 345 350Leu Ala Ala Gly Ala
Asp Val Asn Ala Lys Asp Lys Gln Gly Asn Thr 355 360 365Pro Leu His
Ile Ala Ala Phe His Gly His Leu Glu Ile Val Glu Val 370 375 380Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Trp Gly Leu385 390
395 400Thr Pro Leu His Leu Ala Ala Ala Trp Gly His Leu Glu Ile Val
Glu 405 410 415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp
Asn Tyr Gly 420 425 430Gln Thr Pro Ala Asp Leu Ala Ala Glu Ser Gly
His Glu Asp Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala 450
45570477DNAArtificial SequenceNucleic acid encoding spike protein
binding domain 70ggcagcgatt tgggtaaaaa gctgctgcag gccgcgagag
cgggccagtt ggacgaggtg 60cgtgagctgt tgaaagcggg tgcagacgtt aatgctaaag
accgcgaggg tatcactccg 120ttgcatctgg cggcacagca cggtcatctt
gagatcgtag aagtcttgtt gaaagctggt 180gctgacgtca atgccaagga
tgtttggggt cgtactccac tgcatctcgc ggcgtggcag 240ggtcaccttg
agattgtcga ggttttgctg aaagcgggcg ccgacgtcaa tgcgaaagat
300ctggcaggcg cgacccctct gcatgttgcg gcgctgtacg gtcacctgga
aatcgttgaa 360gtcctcttga aggcgggtgc ggatgtaaac gcgcaggaca
agagcggtaa aacgccggcc 420gatctggcag cacgcgccgg tcaccaagat
atcgcagaag tgctgcaaaa ggctgcg 47771477DNAArtificial SequenceNucleic
acid encoding spike protein binding domain 71ggtagcgacc tgggcaagaa
actgctgcaa gccgcacgtg ccggtcagct ggacgaagtg 60cgtgagttgt tgaaggctgg
cgctgacgtg aacgcaaaag accgtgaggg taagacgccg 120ttacacgtgg
cagcgcaaga aggccacctg gagattgttg aagtgctgct caaagctggc
180gcagacgtca acgctaaaga tgtgtggggt cgcactccgc tgcatctggc
cgcgtggatt 240ggtcatttgg aaattgtgga agttctgctc aaggccggcg
ctgatgtcaa cgccaaagat 300gtgagcggtg cgaccccgct gcacgcagcg
gcgctgcacg gtcacttgga aatcgtggag 360gttctgttga acgccggtgc
tgatgttaac gcgcaagata aatcgggtaa gactccggcg 420gatctggcgg
ctcgtgcggg tcaccaggat attgccgaag ttttgcagaa agccgct
47772477DNAArtificial SequenceNucleic acid encoding spike protein
binding domain 72ggtagcgact tgggtaagaa actgttgcag gcagcgcgcg
cgggtcaact ggacgaggtt 60cgtgagcttt tgaaagccgg tgcggacgtt aacgcgaagg
atcaagaagg cattacccca 120ctgcacgtgg cggcacatca gggtcatctg
gagatcgttg aggttctgct gaaggccgga 180gcggatgtca acgcgaaaga
cgtttggggc cgtaccccat tgcacctggc ggcgtggcgc 240ggtcacctcg
aaatcgtcga agtgttactg aaagctgggg cagatgtgaa cgccaaggac
300cacgcgggtg cgacgccgct gcatgcggca gcgctgagcg gccatctgga
aattgtcgaa 360gtcctgctga aagccggcgc agatgttaat gcccaggata
aatccggtaa gaccccggca 420gacctggcag cgcgtgcggg ccaccaagac
attgccgagg ttctgcaaaa agccgcg 477731563DNAArtificial
SequenceNucleic acid encoding multi-specific binding protein
73ggcagcgatt tgggtaaaaa gctgctgcag gccgcgagag cgggccagtt ggacgaggtg
60cgtgagctgt tgaaagcggg tgcagacgtt aatgctaaag accgcgaggg tatcactccg
120ttgcatctgg cggcacagca cggtcatctt gagatcgtag aagtcttgtt
gaaagctggt 180gctgacgtca atgccaagga tgtttggggt cgtactccac
tgcatctcgc ggcgtggcag 240ggtcaccttg agattgtcga ggttttgctg
aaagcgggcg ccgacgtcaa tgcgaaagat 300ctggcaggcg cgacccctct
gcatgttgcg gcgctgtacg gtcacctgga aatcgttgaa 360gtcctcttga
aggcgggtgc ggatgtaaac gcgcaggaca agagcggtaa aacgccggcc
420gatctggcag cacgcgccgg tcaccaagat atcgcagaag tgctgcaaaa
ggctgcgggt 480agcccaaccc caacgccgac cactcctacc cctaccccaa
ccaccccaac tccaactccg 540actggtagcg acctgggcaa gaaactgctg
caagccgcac gtgccggtca gctggacgaa 600gtgcgtgagt tgttgaaggc
tggcgctgac gtgaacgcaa aagaccgtga gggtaagacg 660ccgttacacg
tggcagcgca agaaggccac ctggagattg ttgaagtgct gctcaaagct
720ggcgcagacg tcaacgctaa agatgtgtgg ggtcgcactc cgctgcatct
ggccgcgtgg 780attggtcatt tggaaattgt ggaagttctg ctcaaggccg
gcgctgatgt caacgccaaa 840gatgtgagcg gtgcgacccc gctgcacgca
gcggcgctgc acggtcactt ggaaatcgtg 900gaggttctgt tgaacgccgg
tgctgatgtt aacgcgcaag ataaatcggg taagactccg 960gcggatctgg
cggctcgtgc gggtcaccag gatattgccg aagttttgca gaaagccgct
1020ggttctccta cgccgacccc gacgacgccg actccaaccc cgaccacgcc
gacgcctacc 1080ccgaccggta gcgacttggg taagaaactg ttgcaggcag
cgcgcgcggg tcaactggac 1140gaggttcgtg agcttttgaa agccggtgcg
gacgttaacg cgaaggatca agaaggcatt 1200accccactgc acgtggcggc
acatcagggt catctggaga tcgttgaggt tctgctgaag 1260gccggagcgg
atgtcaacgc gaaagacgtt tggggccgta ccccattgca cctggcggcg
1320tggcgcggtc acctcgaaat cgtcgaagtg ttactgaaag ctggggcaga
tgtgaacgcc 1380aaggaccacg cgggtgcgac gccgctgcat gcggcagcgc
tgagcggcca tctggaaatt 1440gtcgaagtcc tgctgaaagc cggcgcagat
gttaatgccc aggataaatc cggtaagacc 1500ccggcagacc tggcagcgcg
tgcgggccac caagacattg ccgaggttct gcaaaaagcc 1560gcg
1563742451DNAArtificial SequenceNucleic acid encoding
multi-specific binding protein
74ggatccgatc tgggcaaaaa gttgctggaa gcggcacgtg caggccagga cgatgaagtt
60cgcgagctgt taaaagcagg cgcagatgtt aatgcgaagg actatttcag ccacacgccg
120ttgcatttgg cggcacgtaa cggccacctg aaaatcgtgg aagtgctgtt
aaaggctggt 180gccgatgtga atgccaaaga ttttgcgggt aaaaccccgt
tacacttggc ggcgaacgag 240ggtcatttag agatcgtcga ggtcctgctt
aaggctggcg cggacgtaaa tgcgcaagac 300atcttcggca aaaccccggc
tgacattgcg gcggatgcgg gccacgaaga tatcgccgaa 360gtgttgcaaa
aggcagcggg cagcccgacc ccaaccccta cgaccccgac tcctacgcct
420acgactccga ctccgacccc tacgggtagc gatctgggta agaagctgct
ggaagccgca 480cgcgcaggcc aagacgacga agtccgtgag ctcctgaagg
caggcgccga tgtcaatgca 540aaagactact tttctcatac gccactgcac
ctggcagccc gtaacggtca tctgaagatt 600gtagaagtgt tgctgaaggc
gggcgcagat gttaatgcaa aggactttgc gggcaagacc 660ccgttgcacc
ttgcggcgaa tgagggtcac ttagagattg tggaggtcct gttgaaggcc
720ggtgccgacg tgaatgcaca agacattttc ggcaagacgc cggcagacat
cgcagcggac 780gcgggtcacg aggacatcgc tgaagttctg cagaaagctg
cgggttcccc gacgccaacg 840cctaccacgc ctacgccaac tcctaccacc
ccgaccccga cgccgaccgg cagcgatttg 900ggtaaaaagc tgctgcaggc
cgcgagagcg ggccagttgg acgaggtgcg tgagctgttg 960aaagcgggtg
cagacgttaa tgctaaagac cgcgagggta tcactccgtt gcatctggcg
1020gcacagcacg gtcatcttga gatcgtagaa gtcttgttga aagctggtgc
tgacgtcaat 1080gccaaggatg tttggggtcg tactccactg catctcgcgg
cgtggcaggg tcaccttgag 1140attgtcgagg ttttgctgaa agcgggcgcc
gacgtcaatg cgaaagatct ggcaggcgcg 1200acccctctgc atgttgcggc
gctgtacggt cacctggaaa tcgttgaagt cctcttgaag 1260gcgggtgcgg
atgtaaacgc gcaggacaag agcggtaaaa cgccggccga tctggcagca
1320cgcgccggtc accaagatat cgcagaagtg ctgcaaaagg ctgcgggtag
cccaacccca 1380acgccgacca ctcctacccc taccccaacc accccaactc
caactccgac tggtagcgac 1440ctgggcaaga aactgctgca agccgcacgt
gccggtcagc tggacgaagt gcgtgagttg 1500ttgaaggctg gcgctgacgt
gaacgcaaaa gaccgtgagg gtaagacgcc gttacacgtg 1560gcagcgcaag
aaggccacct ggagattgtt gaagtgctgc tcaaagctgg cgcagacgtc
1620aacgctaaag atgtgtgggg tcgcactccg ctgcatctgg ccgcgtggat
tggtcatttg 1680gaaattgtgg aagttctgct caaggccggc gctgatgtca
acgccaaaga tgtgagcggt 1740gcgaccccgc tgcacgcagc ggcgctgcac
ggtcacttgg aaatcgtgga ggttctgttg 1800aacgccggtg ctgatgttaa
cgcgcaagat aaatcgggta agactccggc ggatctggcg 1860gctcgtgcgg
gtcaccagga tattgccgaa gttttgcaga aagccgctgg ttctcctacg
1920ccgaccccga cgacgccgac tccaaccccg accacgccga cgcctacccc
gaccggtagc 1980gacttgggta agaaactgtt gcaggcagcg cgcgcgggtc
aactggacga ggttcgtgag 2040cttttgaaag ccggtgcgga cgttaacgcg
aaggatcaag aaggcattac cccactgcac 2100gtggcggcac atcagggtca
tctggagatc gttgaggttc tgctgaaggc cggagcggat 2160gtcaacgcga
aagacgtttg gggccgtacc ccattgcacc tggcggcgtg gcgcggtcac
2220ctcgaaatcg tcgaagtgtt actgaaagct ggggcagatg tgaacgccaa
ggaccacgcg 2280ggtgcgacgc cgctgcatgc ggcagcgctg agcggccatc
tggaaattgt cgaagtcctg 2340ctgaaagccg gcgcagatgt taatgcccag
gataaatccg gtaagacccc ggcagacctg 2400gcagcgcgtg cgggccacca
agacattgcc gaggttctgc aaaaagccgc g 245175751PRTArtificial
SequenceMulti-specific binding protein 75Gly Ser Asp Leu Gly Lys
Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe
Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala
Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala
Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg Ala Gly Gln Asp Asp
Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170 175Asp Val Asn Ala
Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala 180 185 190Ala Arg
Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu
210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln Asp Ile Phe Gly
Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala Gly His Glu Asp
Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala
Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315
320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu Gly Lys Thr Pro
325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu Glu Ile Val Glu
Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val
Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala Trp Ile Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr Pro Leu His Ala
Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys
Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Leu Ala Ala Arg Ala
Gly Gln Leu Asp Glu 485 490 495Val Arg Ile Leu Leu Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln Thr Pro Leu His
Val Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535 540Ser Ile Gly
Tyr Thr Pro Leu His His Ala Ala Arg Val Gly His Leu545 550 555
560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys
565 570 575Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala Ala Phe Glu
Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly
Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp Leu Gly Gln Lys
Leu Leu His Ala Ala Gln Ala Gly625 630 635 640Gln Leu Asp Glu Val
Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650 655Ala Lys Asp
Lys Gln Gly Asn Thr Pro Leu His Ile Ala Ala Phe His 660 665 670Gly
His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 675 680
685Asn Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala
690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp705 710 715 720Val Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro
Ala Asp Leu Ala Ala 725 730 735Glu Ser Gly His Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 740 745 75076126PRTArtificial SequenceSpike
protein binding domain 76Gly Ser Asp Leu Gly Lys Lys Leu Leu Leu
Ala Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Ile Leu Leu Lys
Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Val Phe Gly Gln Thr Pro
Leu His Val Ala Ala Val Ala Gly 35 40 45His Leu Glu Ile Val Glu Val
Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Ser Ile Gly
Tyr Thr Pro Leu His His Ala Ala Arg Val65 70 75 80Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys
Asp Trp Ile Gly Ile Thr Pro Ala Asp Leu Ala Ala Phe 100 105 110Glu
Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120
12577126PRTArtificial SequenceSpike protein binding domain 77Gly
Ser Asp Leu Gly Gln Lys Leu Leu His Ala Ala Gln Ala Gly Gln1 5 10
15Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Lys Gln Gly Asn Thr Pro Leu His Ile Ala Ala Phe His
Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala
Ala Ala Trp65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro
Ala Asp Leu Ala Ala Glu 100 105 110Ser Gly His Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala 115 120 125782253PRTArtificial
SequenceNucleic acid encoding multi-specific binding protein 78Gly
Gly Ala Thr Cys Cys Gly Ala Cys Cys Thr Gly Gly Gly Thr Ala1 5 10
15Ala Ala Ala Ala Gly Cys Thr Gly Cys Thr Gly Gly Ala Gly Gly Cys
20 25 30Ala Gly Cys Gly Cys Gly Thr Gly Cys Cys Gly Gly Thr Cys Ala
Ala 35 40 45Gly Ala Cys Gly Ala Cys Gly Ala Gly Gly Thr Thr Cys Gly
Cys Gly 50 55 60Ala Ala Thr Thr Gly Cys Thr Thr Ala Ala Ala Gly Cys
Gly Gly Gly65 70 75 80Thr Gly Cys Ala Gly Ala Cys Gly Thr Cys Ala
Ala Cys Gly Cys Cys 85 90 95Ala Ala Ala Gly Ala Thr Thr Ala Thr Thr
Thr Cys Thr Cys Thr Cys 100 105 110Ala Thr Ala Cys Cys Cys Cys Gly
Thr Thr Gly Cys Ala Thr Thr Thr 115 120 125Ala Gly Cys Cys Gly Cys
Gly Cys Gly Thr Ala Ala Thr Gly Gly Cys 130 135 140Cys Ala Thr Cys
Thr Gly Ala Ala Gly Ala Thr Cys Gly Thr Cys Gly145 150 155 160Ala
Gly Gly Thr Cys Cys Thr Cys Thr Thr Gly Ala Ala Gly Gly Cys 165 170
175Ala Gly Gly Cys Gly Cys Gly Gly Ala Thr Gly Thr Cys Ala Ala Thr
180 185 190Gly Cys Gly Ala Ala Gly Gly Ala Thr Thr Thr Thr Gly Cys
Gly Gly 195 200 205Gly Cys Ala Ala Ala Ala Cys Gly Cys Cys Gly Cys
Thr Gly Cys Ala 210 215 220Cys Thr Thr Ala Gly Cys Gly Gly Cys Gly
Ala Ala Cys Gly Ala Gly225 230 235 240Gly Gly Thr Cys Ala Thr Thr
Thr Ala Gly Ala Ala Ala Thr Cys Gly 245 250 255Thr Thr Gly Ala Ala
Gly Thr Cys Cys Thr Gly Thr Thr Ala Ala Ala 260 265 270Ala Gly Cys
Gly Gly Gly Cys Gly Cys Cys Gly Ala Thr Gly Thr Gly 275 280 285Ala
Ala Thr Gly Cys Gly Cys Ala Gly Gly Ala Thr Ala Thr Thr Thr 290 295
300Thr Cys Gly Gly Thr Ala Ala Ala Ala Cys Gly Cys Cys Gly Gly
Cys305 310 315 320Gly Gly Ala Cys Ala Thr Thr Gly Cys Gly Gly Cys
Ala Gly Ala Thr 325 330 335Gly Cys Gly Gly Gly Thr Cys Ala Thr Gly
Ala Ala Gly Ala Thr Ala 340 345 350Thr Cys Gly Cys Ala Gly Ala Ala
Gly Thr Cys Cys Thr Gly Cys Ala 355 360 365Gly Ala Ala Gly Gly Cys
Ala Gly Cys Ala Gly Gly Cys Ala Gly Cys 370 375 380Cys Cys Thr Ala
Cys Ala Cys Cys Thr Ala Cys Gly Cys Cys Gly Ala385 390 395 400Cys
Thr Ala Cys Gly Cys Cys Thr Ala Cys Gly Cys Cys Gly Ala Cys 405 410
415Thr Cys Cys Gly Ala Cys Thr Ala Cys Cys Cys Cys Gly Ala Cys Thr
420 425 430Cys Cys Gly Ala Cys Cys Cys Cys Gly Ala Cys Cys Gly Gly
Ala Thr 435 440 445Cys Ala Gly Ala Cys Cys Thr Gly Gly Gly Thr Ala
Ala Ala Ala Ala 450 455 460Gly Cys Thr Gly Cys Thr Gly Gly Ala Gly
Gly Cys Ala Gly Cys Gly465 470 475 480Cys Gly Thr Gly Cys Cys Gly
Gly Thr Cys Ala Ala Gly Ala Cys Gly 485 490 495Ala Cys Gly Ala Gly
Gly Thr Thr Cys Gly Cys Gly Ala Ala Thr Thr 500 505 510Gly Cys Thr
Thr Ala Ala Ala Gly Cys Gly Gly Gly Thr Gly Cys Ala 515 520 525Gly
Ala Cys Gly Thr Cys Ala Ala Cys Gly Cys Cys Ala Ala Ala Gly 530 535
540Ala Thr Thr Ala Thr Thr Thr Cys Thr Cys Thr Cys Ala Thr Ala
Cys545 550 555 560Cys Cys Cys Gly Thr Thr Gly Cys Ala Thr Thr Thr
Ala Gly Cys Cys 565 570 575Gly Cys Gly Cys Gly Thr Ala Ala Thr Gly
Gly Cys Cys Ala Thr Cys 580 585 590Thr Gly Ala Ala Gly Ala Thr Cys
Gly Thr Cys Gly Ala Gly Gly Thr 595 600 605Cys Cys Thr Cys Thr Thr
Gly Ala Ala Gly Gly Cys Ala Gly Gly Cys 610 615 620Gly Cys Gly Gly
Ala Thr Gly Thr Cys Ala Ala Thr Gly Cys Gly Ala625 630 635 640Ala
Gly Gly Ala Thr Thr Thr Thr Gly Cys Gly Gly Gly Cys Ala Ala 645 650
655Ala Ala Cys Gly Cys Cys Gly Cys Thr Gly Cys Ala Cys Thr Thr Ala
660 665 670Gly Cys Gly Gly Cys Gly Ala Ala Cys Gly Ala Gly Gly Gly
Thr Cys 675 680 685Ala Thr Thr Thr Ala Gly Ala Ala Ala Thr Cys Gly
Thr Thr Gly Ala 690 695 700Ala Gly Thr Cys Cys Thr Gly Thr Thr Ala
Ala Ala Ala Gly Cys Gly705 710 715 720Gly Gly Cys Gly Cys Cys Gly
Ala Thr Gly Thr Gly Ala Ala Thr Gly 725 730 735Cys Gly Cys Ala Gly
Gly Ala Thr Ala Thr Thr Thr Thr Cys Gly Gly 740 745 750Thr Ala Ala
Ala Ala Cys Gly Cys Cys Gly Gly Cys Gly Gly Ala Cys 755 760 765Ala
Thr Thr Gly Cys Gly Gly Cys Ala Gly Ala Thr Gly Cys Gly Gly 770 775
780Gly Thr Cys Ala Thr Gly Ala Ala Gly Ala Thr Ala Thr Cys Gly
Cys785 790 795 800Ala Gly Ala Ala Gly Thr Cys Cys Thr Gly Cys Ala
Gly Ala Ala Gly 805 810 815Gly Cys Ala Gly Cys Ala Gly Gly Thr Thr
Cys Cys Cys Cys Gly Ala 820 825 830Cys Cys Cys Cys Thr Ala Cys Gly
Cys Cys Ala Ala Cys Gly Ala Cys 835 840 845Thr Cys Cys Gly Ala Cys
Cys Cys Cys Ala Ala Cys Thr Cys Cys Ala 850 855 860Ala Cys Gly Ala
Cys Cys Cys Cys Thr Ala Cys Cys Cys Cys Gly Ala865 870 875 880Cys
Cys Cys Cys Gly Ala Cys Cys Gly Gly Ala Thr Cys Ala Gly Ala 885 890
895Cys Cys Thr Gly Gly Gly Thr Ala Ala Ala Ala Ala Ala Cys Thr Gly
900 905 910Cys Thr Gly Cys Ala Ala Gly Cys Ala Gly Cys Ala Cys Gly
Thr Gly 915 920 925Cys Ala Gly Gly Thr Cys Ala Gly Cys Thr Gly Gly
Ala Thr Gly Ala 930 935 940Ala Gly Thr Thr Cys Gly Thr Gly Ala Ala
Cys Thr Gly Cys Thr Gly945 950 955 960Ala Ala Ala Gly Cys Ala Gly
Gly Cys Gly Cys Cys Gly Ala Thr Gly 965 970 975Thr Thr Ala Ala Thr
Gly Cys Ala Ala Ala Ala Gly Ala Thr Ala Gly 980 985 990Ala Gly Ala
Gly Gly Gly Cys Ala Ala Gly Ala Cys Cys Cys Cys Gly 995 1000
1005Cys Thr Gly Cys Ala Thr Gly Thr Gly Gly Cys Thr Gly Cys Thr Cys
1010 1015 1020Ala Ala Gly Ala Gly Gly Gly
Thr Cys Ala Cys Cys Thr Gly Gly Ala1025 1030 1035 1040Ala Ala Thr
Thr Gly Thr Thr Gly Ala Ala Gly Thr Thr Cys Thr Gly 1045 1050
1055Cys Thr Gly Ala Ala Ala Gly Cys Cys Gly Gly Thr Gly Cys Ala Gly
1060 1065 1070Ala Thr Gly Thr Thr Ala Ala Thr Gly Cys Ala Ala Ala
Ala Gly Ala 1075 1080 1085Thr Gly Thr Gly Thr Gly Gly Gly Gly Cys
Ala Gly Ala Ala Cys Cys 1090 1095 1100Cys Cys Gly Cys Thr Gly Cys
Ala Thr Cys Thr Gly Gly Cys Thr Gly1105 1110 1115 1120Cys Thr Thr
Gly Gly Ala Thr Cys Gly Gly Thr Cys Ala Cys Cys Thr 1125 1130
1135Gly Gly Ala Ala Ala Thr Thr Gly Thr Thr Gly Ala Ala Gly Thr Thr
1140 1145 1150Cys Thr Gly Cys Thr Gly Ala Ala Ala Gly Cys Cys Gly
Gly Thr Gly 1155 1160 1165Cys Ala Gly Ala Thr Gly Thr Thr Ala Ala
Thr Gly Cys Ala Ala Ala 1170 1175 1180Ala Gly Ala Thr Gly Thr Gly
Thr Cys Thr Gly Gly Cys Gly Cys Thr1185 1190 1195 1200Ala Cys Cys
Cys Cys Gly Cys Thr Gly Cys Ala Thr Gly Cys Gly Gly 1205 1210
1215Cys Thr Gly Cys Thr Cys Thr Gly Cys Ala Cys Gly Gly Thr Cys Ala
1220 1225 1230Cys Cys Thr Gly Gly Ala Ala Ala Thr Thr Gly Thr Thr
Gly Ala Ala 1235 1240 1245Gly Thr Thr Cys Thr Gly Cys Thr Gly Ala
Ala Cys Gly Cys Cys Gly 1250 1255 1260Gly Thr Gly Cys Ala Gly Ala
Thr Gly Thr Thr Ala Ala Cys Gly Cys1265 1270 1275 1280Ala Cys Ala
Gly Gly Ala Thr Ala Ala Ala Ala Gly Cys Gly Gly Thr 1285 1290
1295Ala Ala Ala Ala Cys Cys Cys Cys Thr Gly Cys Cys Gly Ala Thr Cys
1300 1305 1310Thr Gly Gly Cys Ala Gly Cys Thr Cys Gly Cys Gly Cys
Cys Gly Gly 1315 1320 1325Thr Cys Ala Thr Cys Ala Gly Gly Ala Thr
Ala Thr Thr Gly Cys Thr 1330 1335 1340Gly Ala Ala Gly Thr Gly Cys
Thr Gly Cys Ala Gly Ala Ala Gly Gly1345 1350 1355 1360Cys Ala Gly
Cys Ala Gly Gly Cys Ala Gly Cys Cys Cys Cys Ala Cys 1365 1370
1375Gly Cys Cys Ala Ala Cys Thr Cys Cys Thr Ala Cys Ala Ala Cys Cys
1380 1385 1390Cys Cys Cys Ala Cys Ala Cys Cys Thr Ala Cys Ala Cys
Cys Gly Ala 1395 1400 1405Cys Gly Ala Cys Gly Cys Cys Gly Ala Cys
Ala Cys Cys Gly Ala Cys 1410 1415 1420Thr Cys Cys Ala Ala Cys Cys
Gly Gly Ala Thr Cys Ala Gly Ala Cys1425 1430 1435 1440Cys Thr Gly
Gly Gly Thr Ala Ala Ala Ala Ala Ala Thr Thr Gly Thr 1445 1450
1455Thr Ala Thr Thr Gly Gly Cys Cys Gly Cys Thr Cys Gly Cys Gly Cys
1460 1465 1470Gly Gly Gly Cys Cys Ala Gly Cys Thr Gly Gly Ala Cys
Gly Ala Gly 1475 1480 1485Gly Thr Ala Cys Gly Thr Ala Thr Cys Thr
Thr Ala Thr Thr Gly Ala 1490 1495 1500Ala Gly Gly Cys Thr Gly Gly
Gly Gly Cys Ala Gly Ala Cys Gly Thr1505 1510 1515 1520Cys Ala Ala
Thr Gly Cys Gly Ala Ala Gly Gly Ala Cys Gly Thr Thr 1525 1530
1535Thr Thr Thr Gly Gly Ala Cys Ala Gly Ala Cys Thr Cys Cys Thr Cys
1540 1545 1550Thr Thr Cys Ala Thr Gly Thr Gly Gly Cys Cys Gly Cys
Cys Gly Thr 1555 1560 1565Gly Gly Cys Cys Gly Gly Thr Cys Ala Thr
Cys Thr Gly Gly Ala Gly 1570 1575 1580Ala Thr Thr Gly Thr Cys Gly
Ala Ala Gly Thr Ala Thr Thr Ala Thr1585 1590 1595 1600Thr Ala Ala
Ala Gly Gly Cys Thr Gly Gly Thr Gly Cys Ala Gly Ala 1605 1610
1615Thr Gly Thr Ala Ala Ala Thr Gly Cys Thr Ala Ala Ala Gly Ala Thr
1620 1625 1630Thr Cys Thr Ala Thr Cys Gly Gly Ala Thr Ala Cys Ala
Cys Ala Cys 1635 1640 1645Cys Gly Thr Thr Gly Cys Ala Thr Cys Ala
Thr Gly Cys Ala Gly Cys 1650 1655 1660Thr Cys Gly Cys Gly Thr Thr
Gly Gly Ala Cys Ala Thr Thr Thr Ala1665 1670 1675 1680Gly Ala Gly
Ala Thr Cys Gly Thr Cys Gly Ala Gly Gly Thr Cys Thr 1685 1690
1695Thr Gly Cys Thr Gly Ala Ala Ala Gly Cys Ala Gly Gly Thr Gly Cys
1700 1705 1710Cys Gly Ala Cys Gly Thr Thr Ala Ala Thr Gly Cys Cys
Ala Ala Gly 1715 1720 1725Gly Ala Thr Thr Gly Gly Ala Thr Cys Gly
Gly Gly Ala Thr Cys Ala 1730 1735 1740Cys Cys Cys Cys Ala Gly Cys
Gly Gly Ala Thr Cys Thr Thr Gly Cys1745 1750 1755 1760Ala Gly Cys
Ala Thr Thr Thr Gly Ala Gly Gly Gly Thr Cys Ala Cys 1765 1770
1775Cys Ala Gly Gly Ala Thr Ala Thr Thr Gly Cys Thr Gly Ala Ala Gly
1780 1785 1790Thr Thr Cys Thr Gly Cys Ala Gly Ala Ala Gly Gly Cys
Ala Gly Cys 1795 1800 1805Ala Gly Gly Thr Thr Cys Gly Cys Cys Gly
Ala Cys Cys Cys Cys Ala 1810 1815 1820Ala Cys Cys Cys Cys Thr Ala
Cys Cys Ala Cys Thr Cys Cys Ala Ala1825 1830 1835 1840Cys Gly Cys
Cys Gly Ala Cys Gly Cys Cys Thr Ala Cys Cys Ala Cys 1845 1850
1855Thr Cys Cys Ala Ala Cys Ala Cys Cys Ala Ala Cys Ala Cys Cys Ala
1860 1865 1870Ala Cys Gly Gly Gly Ala Thr Cys Ala Gly Ala Cys Cys
Thr Gly Gly 1875 1880 1885Gly Thr Cys Ala Ala Ala Ala Gly Thr Thr
Gly Thr Thr Ala Cys Ala 1890 1895 1900Cys Gly Cys Gly Gly Cys Thr
Cys Ala Ala Gly Cys Gly Gly Gly Ala1905 1910 1915 1920Cys Ala Ala
Thr Thr Ala Gly Ala Cys Gly Ala Gly Gly Thr Cys Cys 1925 1930
1935Gly Thr Ala Thr Thr Cys Thr Gly Cys Thr Thr Ala Ala Ala Gly Cys
1940 1945 1950Cys Gly Gly Gly Gly Cys Thr Gly Ala Thr Gly Thr Ala
Ala Ala Thr 1955 1960 1965Gly Cys Ala Ala Ala Ala Gly Ala Cys Ala
Ala Ala Cys Ala Gly Gly 1970 1975 1980Gly Cys Ala Ala Cys Ala Cys
Ala Cys Cys Cys Thr Thr Ala Cys Ala1985 1990 1995 2000Thr Ala Thr
Cys Gly Cys Gly Gly Cys Ala Thr Thr Cys Cys Ala Thr 2005 2010
2015Gly Gly Ala Cys Ala Thr Cys Thr Gly Gly Ala Gly Ala Thr Thr Gly
2020 2025 2030Thr Gly Gly Ala Ala Gly Thr Ala Cys Thr Gly Cys Thr
Gly Ala Ala 2035 2040 2045Ala Gly Cys Cys Gly Gly Gly Gly Cys Ala
Gly Ala Thr Gly Thr Cys 2050 2055 2060Ala Ala Cys Gly Cys Thr Ala
Ala Ala Gly Ala Cys Cys Ala Ala Thr2065 2070 2075 2080Gly Gly Gly
Gly Ala Thr Thr Gly Ala Cys Cys Cys Cys Cys Cys Thr 2085 2090
2095Thr Cys Ala Thr Thr Thr Gly Gly Cys Cys Gly Cys Thr Gly Cys Cys
2100 2105 2110Thr Gly Gly Gly Gly Cys Cys Ala Thr Thr Thr Gly Gly
Ala Gly Ala 2115 2120 2125Thr Thr Gly Thr Ala Gly Ala Gly Gly Thr
Ala Cys Thr Thr Cys Thr 2130 2135 2140Gly Ala Ala Gly Gly Cys Gly
Gly Gly Gly Gly Cys Thr Gly Ala Thr2145 2150 2155 2160Gly Thr Thr
Ala Ala Thr Gly Cys Cys Cys Ala Gly Gly Ala Thr Ala 2165 2170
2175Ala Cys Thr Ala Thr Gly Gly Gly Cys Ala Ala Ala Cys Thr Cys Cys
2180 2185 2190Thr Gly Cys Gly Gly Ala Thr Cys Thr Gly Gly Cys Gly
Gly Cys Thr 2195 2200 2205Gly Ala Ala Thr Cys Thr Gly Gly Gly Cys
Ala Cys Cys Ala Ala Gly 2210 2215 2220Ala Thr Ala Thr Thr Gly Cys
Thr Gly Ala Ala Gly Thr Thr Cys Thr2225 2230 2235 2240Gly Cys Ala
Gly Ala Ala Gly Gly Cys Gly Gly Cys Ala 2245 225079455PRTArtificial
SequenceMulti-specific binding protein 79Gly Ser Asp Leu Gly Lys
Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg
Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala 20 25 30Lys Asp Arg Glu
Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu Gly 35 40 45His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys
Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala Ala Trp Ile65 70 75
80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val
85 90 95Asn Ala Lys Asp Val Ser Gly Ala Thr Pro Leu His Ala Ala Ala
Leu 100 105 110His Gly His Leu Glu Ile Val Glu Val Leu Leu Asn Ala
Gly Ala Asp 115 120 125Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro
Ala Asp Leu Ala Ala 130 135 140Arg Ala Gly His Gln Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly145 150 155 160Ser Pro Thr Pro Thr Pro
Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro 165 170 175Thr Pro Thr Pro
Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Leu Ala 180 185 190Ala Arg
Ala Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly 195 200
205Ala Asp Val Asn Ala Lys Asp Val Phe Gly Gln Thr Pro Leu His Val
210 215 220Ala Ala Val Ala Gly His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Lys Asp Ser Ile Gly
Tyr Thr Pro Leu His 245 250 255His Ala Ala Arg Val Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys 260 265 270Ala Gly Ala Asp Val Asn Ala
Lys Asp Trp Ile Gly Ile Thr Pro Ala 275 280 285Asp Leu Ala Ala Phe
Glu Gly His Gln Asp Ile Ala Glu Val Leu Gln 290 295 300Lys Ala Ala
Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr305 310 315
320Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys
325 330 335Leu Leu His Ala Ala Gln Ala Gly Gln Leu Asp Glu Val Arg
Ile Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys
Gln Gly Asn Thr 355 360 365Pro Leu His Ile Ala Ala Phe His Gly His
Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Lys Asp Gln Trp Gly Leu385 390 395 400Thr Pro Leu His Leu
Ala Ala Ala Trp Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asn Tyr Gly 420 425 430Gln
Thr Pro Ala Asp Leu Ala Ala Glu Ser Gly His Gln Asp Ile Ala 435 440
445Glu Val Leu Gln Lys Ala Ala 450 45580477DNAArtificial
SequenceNucleic acid encoding spike protein binding domain
80ggatcagacc tgggtaaaaa actgctgcaa gcagcacgtg caggtcagct ggatgaagtt
60cgtgaactgc tgaaagcagg cgccgatgtt aatgcaaaag atagagaggg caagaccccg
120ctgcatgtgg ctgctcaaga gggtcacctg gaaattgttg aagttctgct
gaaagccggt 180gcagatgtta atgcaaaaga tgtgtggggc agaaccccgc
tgcatctggc tgcttggatc 240ggtcacctgg aaattgttga agttctgctg
aaagccggtg cagatgttaa tgcaaaagat 300gtgtctggcg ctaccccgct
gcatgcggct gctctgcacg gtcacctgga aattgttgaa 360gttctgctga
acgccggtgc agatgttaac gcacaggata aaagcggtaa aacccctgcc
420gatctggcag ctcgcgccgg tcatcaggat attgctgaag tgctgcagaa ggcagca
47781378DNAArtificial SequenceNucleic acid encoding spike protein
binding domain 81ggatcagacc tgggtaaaaa attgttattg gccgctcgcg
cgggccagct ggacgaggta 60cgtatcttat tgaaggctgg ggcagacgtc aatgcgaagg
acgtttttgg acagactcct 120cttcatgtgg ccgccgtggc cggtcatctg
gagattgtcg aagtattatt aaaggctggt 180gcagatgtaa atgctaaaga
ttctatcgga tacacaccgt tgcatcatgc agctcgcgtt 240ggacatttag
agatcgtcga ggtcttgctg aaagcaggtg ccgacgttaa tgccaaggat
300tggatcggga tcaccccagc ggatcttgca gcatttgagg gtcaccagga
tattgctgaa 360gttctgcaga aggcagca 37882378DNAArtificial
SequenceNucleic acid encoding spike protein binding domain
82ggatcagacc tgggtcaaaa gttgttacac gcggctcaag cgggacaatt agacgaggtc
60cgtattctgc ttaaagccgg ggctgatgta aatgcaaaag acaaacaggg caacacaccc
120ttacatatcg cggcattcca tggacatctg gagattgtgg aagtactgct
gaaagccggg 180gcagatgtca acgctaaaga ccaatgggga ttgacccccc
ttcatttggc cgctgcctgg 240ggccatttgg agattgtaga ggtacttctg
aaggcggggg ctgatgttaa tgcccaggat 300aactatgggc aaactcctgc
ggatctggcg gctgaatctg ggcaccaaga tattgctgaa 360gttctgcaga aggcggca
378831365DNAArtificial SequenceNucleic acid encoding multi-specific
binding protein 83ggatcagacc tgggtaaaaa actgctgcaa gcagcacgtg
caggtcagct ggatgaagtt 60cgtgaactgc tgaaagcagg cgccgatgtt aatgcaaaag
atagagaggg caagaccccg 120ctgcatgtgg ctgctcaaga gggtcacctg
gaaattgttg aagttctgct gaaagccggt 180gcagatgtta atgcaaaaga
tgtgtggggc agaaccccgc tgcatctggc tgcttggatc 240ggtcacctgg
aaattgttga agttctgctg aaagccggtg cagatgttaa tgcaaaagat
300gtgtctggcg ctaccccgct gcatgcggct gctctgcacg gtcacctgga
aattgttgaa 360gttctgctga acgccggtgc agatgttaac gcacaggata
aaagcggtaa aacccctgcc 420gatctggcag ctcgcgccgg tcatcaggat
attgctgaag tgctgcagaa ggcagcaggc 480agccccacgc caactcctac
aacccccaca cctacaccga cgacgccgac accgactcca 540accggatcag
acctgggtaa aaaattgtta ttggccgctc gcgcgggcca gctggacgag
600gtacgtatct tattgaaggc tggggcagac gtcaatgcga aggacgtttt
tggacagact 660cctcttcatg tggccgccgt ggccggtcat ctggagattg
tcgaagtatt attaaaggct 720ggtgcagatg taaatgctaa agattctatc
ggatacacac cgttgcatca tgcagctcgc 780gttggacatt tagagatcgt
cgaggtcttg ctgaaagcag gtgccgacgt taatgccaag 840gattggatcg
ggatcacccc agcggatctt gcagcatttg agggtcacca ggatattgct
900gaagttctgc agaaggcagc aggttcgccg accccaaccc ctaccactcc
aacgccgacg 960cctaccactc caacaccaac accaacggga tcagacctgg
gtcaaaagtt gttacacgcg 1020gctcaagcgg gacaattaga cgaggtccgt
attctgctta aagccggggc tgatgtaaat 1080gcaaaagaca aacagggcaa
cacaccctta catatcgcgg cattccatgg acatctggag 1140attgtggaag
tactgctgaa agccggggca gatgtcaacg ctaaagacca atggggattg
1200accccccttc atttggccgc tgcctggggc catttggaga ttgtagaggt
acttctgaag 1260gcgggggctg atgttaatgc ccaggataac tatgggcaaa
ctcctgcgga tctggcggct 1320gaatctgggc accaagatat tgctgaagtt
ctgcagaagg cggca 136584766PRTArtificial SequenceMulti-specific
binding protein 84Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala
Arg Ala Gly Gln1 5 10 15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala 20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His
Leu Ala Ala Arg Asn Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu Ala Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile
Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His
Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro
Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135
140Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala
Ala145 150 155 160Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu
Lys Ala Gly Ala 165 170 175Asp Val Asn Ala Lys Asp Tyr Phe Ser His
Thr Pro Leu His Leu Ala 180 185 190Ala Arg Asn Gly His Leu Lys Ile
Val Glu Val Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys
Asp Phe Ala Gly Lys Thr Pro Leu His Leu 210 215 220Ala Ala Asn Glu
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly
Ala Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250
255Ile Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys
260 265 270Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr
Pro Thr Pro Thr Pro 275 280 285Thr Thr Pro Thr Pro Thr Pro Thr Gly
Ser Asp Leu Gly Lys Lys Leu 290 295 300Leu Gln Ala Ala Arg Ala Gly
Gln Leu Asp Glu Val Arg Glu Leu Leu305 310 315 320Lys Ala Gly Ala
Asp Val Asn Ala Lys Asp Arg Glu Gly Lys Thr Pro 325 330 335Leu His
Val Ala Ala Gln Glu Gly His Leu Glu Ile Val Glu Val Leu 340 345
350Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val Trp Gly Arg Thr
355 360 365Pro Leu His Leu Ala Ala Trp Ile Gly His Leu Glu Ile Val
Glu Val 370 375 380Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp
Val Ser Gly Ala385 390 395 400Thr Pro Leu His Ala Ala Ala Leu His
Gly His Leu Glu Ile Val Glu 405 410 415Val Leu Leu Asn Ala Gly Ala
Asp Val Asn Ala Gln Asp Lys Ser Gly 420 425 430Lys Thr Pro Ala Asp
Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala 435 440 445Glu Val Leu
Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr 450 455 460Pro
Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp465 470
475 480Leu Gly Lys Lys Leu Leu Leu Ala Ala Arg Ala Gly Gln Leu Asp
Glu 485 490 495Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Lys Asp Val 500 505 510Phe Gly Gln Thr Pro Leu His Thr Ala Ala Val
Ala Gly His Leu Glu 515 520 525Ile Val Glu Val Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala Lys Asp 530 535 540Lys Val Gly Tyr Thr Pro Leu
His Leu Ala Ala Gln Ser Gly His Leu545 550 555 560Glu Ile Val Glu
Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys 565 570 575Asp Val
Val Gly Val Thr Pro Ala Asp Leu Ala Ala Phe Glu Gly His 580 585
590Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
595 600 605Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro 610 615 620Thr Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr625 630 635 640Gly Ser Asp Leu Gly Gln Lys Leu Leu
His Ala Ala Gln Ala Gly Gln 645 650 655Leu Asp Glu Val Arg Ile Leu
Leu Lys Ala Gly Ala Asp Val Asn Ala 660 665 670Lys Asp Lys Gln Gly
Asn Thr Pro Leu His Ile Ala Ala Phe His Gly 675 680 685His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn 690 695 700Ala
Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala Trp705 710
715 720Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val 725 730 735Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro Ala Asp Leu
Ala Ala Glu 740 745 750Ser Gly His Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala 755 760 76585126PRTArtificial SequenceSpike protein
binding domain 85Gly Ser Asp Leu Gly Lys Lys Leu Leu Leu Ala Ala
Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala 20 25 30Lys Asp Val Phe Gly Gln Thr Pro Leu His
Thr Ala Ala Val Ala Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Lys Val Gly Tyr Thr
Pro Leu His Leu Ala Ala Gln Ser65 70 75 80Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Val
Val Gly Val Thr Pro Ala Asp Leu Ala Ala Phe 100 105 110Glu Gly His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 115 120
125862298DNAArtificial SequenceNucleic acid encoding multi-specific
binding protein 86ggatccgacc tgggtaaaaa gctgctggag gcagcgcgtg
ccggtcaaga cgacgaggtt 60cgcgaattgc ttaaagcggg tgcagacgtc aacgccaaag
attatttctc tcataccccg 120ttgcatttag ccgcgcgtaa tggccatctg
aagatcgtcg aggtcctctt gaaggcaggc 180gcggatgtca atgcgaagga
ttttgcgggc aaaacgccgc tgcacttagc ggcgaacgag 240ggtcatttag
aaatcgttga agtcctgtta aaagcgggcg ccgatgtgaa tgcgcaggat
300attttcggta aaacgccggc ggacattgcg gcagatgcgg gtcatgaaga
tatcgcagaa 360gtcctgcaga aggcagcagg cagccctaca cctacgccga
ctacgcctac gccgactccg 420actaccccga ctccgacccc gaccggatca
gacctgggta aaaagctgct ggaggcagcg 480cgtgccggtc aagacgacga
ggttcgcgaa ttgcttaaag cgggtgcaga cgtcaacgcc 540aaagattatt
tctctcatac cccgttgcat ttagccgcgc gtaatggcca tctgaagatc
600gtcgaggtcc tcttgaaggc aggcgcggat gtcaatgcga aggattttgc
gggcaaaacg 660ccgctgcact tagcggcgaa cgagggtcat ttagaaatcg
ttgaagtcct gttaaaagcg 720ggcgccgatg tgaatgcgca ggatattttc
ggtaaaacgc cggcggacat tgcggcagat 780gcgggtcatg aagatatcgc
agaagtcctg cagaaggcag caggttcccc gacccctacg 840ccaacgactc
cgaccccaac tccaacgacc cctaccccga ccccgaccgg atcagacctg
900ggtaaaaaac tgctgcaagc agcacgtgca ggtcagctgg atgaagttcg
tgaactgctg 960aaagcaggcg ccgatgttaa tgcaaaagat agagagggca
agaccccgct gcatgtggct 1020gctcaagagg gtcacctgga aattgttgaa
gttctgctga aagccggtgc agatgttaat 1080gcaaaagatg tgtggggcag
aaccccgctg catctggctg cttggatcgg tcacctggaa 1140attgttgaag
ttctgctgaa agccggtgca gatgttaatg caaaagatgt gtctggcgct
1200accccgctgc atgcggctgc tctgcacggt cacctggaaa ttgttgaagt
tctgctgaac 1260gccggtgcag atgttaacgc acaggataaa agcggtaaaa
cccctgccga tctggcagct 1320cgcgccggtc atcaggatat tgctgaagtg
ctgcagaagg cagcaggcag ccccacgcca 1380actcctacaa cccccacacc
tacaccgacg acgccgacac cgactccaac cggatcagac 1440ctgggtaaaa
agttgttact tgcagctcgc gccggccaat tggacgaagt ccgcatttta
1500ctgaaggccg gtgcggatgt gaacgcgaaa gacgtgtttg ggcagacccc
tcttcacacc 1560gcagcagttg caggtcatct ggagatcgtg gaagtgcttt
taaaagcggg tgcggacgtc 1620aacgctaagg acaaagttgg gtacacgcca
ctgcacttag ctgcacagtc aggacatctt 1680gagattgtgg aggtcttgct
gaaggcaggc gcagatgtga acgctaagga tgtcgttggg 1740gttacgccgg
cggatttggc agctttcgag ggacaccagg acattgctga agttctgcag
1800aaggcagcag gttcgccgac cccaacccct accactccaa cgccgacgcc
taccactcca 1860acaccaacac caacgggatc accaactcca acaccgacca
ccccgacccc taccccaaca 1920ggatcagacc tgggtcaaaa gttgttacac
gcggctcaag cgggacaatt agacgaggtc 1980cgtattctgc ttaaagccgg
ggctgatgta aatgcaaaag acaaacaggg caacacaccc 2040ttacatatcg
cggcattcca tggacatctg gagattgtgg aagtactgct gaaagccggg
2100gcagatgtca acgctaaaga ccaatgggga ttgacccccc ttcatttggc
cgctgcctgg 2160ggccatttgg agattgtaga ggtacttctg aaggcggggg
ctgatgttaa tgcccaggat 2220aactatgggc aaactcctgc ggatctggcg
gctgaatctg ggcaccaaga tattgctgaa 2280gttctgcaga aggcggca
229887751PRTArtificial SequenceMulti-specific binding protein 87Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu
Gly Lys Thr Pro 325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Ile Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr
Pro Leu His Ala Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Leu
Ala Ala Arg Ala Gly Gln Leu Asp Glu 485 490 495Val Arg Ile Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln
Thr Pro Leu His Thr Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Lys Val Gly Tyr Thr Pro Leu His Leu Ala Ala Gln Ser Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys 565 570 575Asp Val Val Gly Val Thr Pro Ala Asp Leu
Ala Ala Phe Glu Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Asp
Leu Gly Gln Lys Leu Leu His Ala Ala Gln Ala Gly625 630 635 640Gln
Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn 645 650
655Ala Lys Asp Lys Gln Gly Asn Thr Pro Leu His Ile Ala Ala Phe His
660 665 670Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala
Asp Val 675 680 685Asn Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His
Leu Ala Ala Ala 690 695 700Trp Gly His Leu Glu Ile Val Glu Val Leu
Leu Lys Ala Gly Ala Asp705 710 715 720Val Asn Ala Gln Asp Asn Tyr
Gly Gln Thr Pro Ala Asp Leu Ala Ala 725 730 735Glu Ser Gly His Gln
Asp Ile Ala Glu Val Leu Gln Lys Ala Ala 740 745
75088759PRTArtificial SequenceMulti-specific binding protein 88Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln1 5 10
15Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn
Gly 35 40 45His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala
Ala Asn Glu65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro
Ala Asp Ile Ala Ala Asp 100 105 110Ala Gly His Glu Asp Ile Ala Glu
Val Leu Gln Lys Ala Ala Gly Ser 115 120 125Pro Thr Pro Thr Pro Thr
Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr 130 135 140Pro Thr Pro Thr
Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala145 150 155 160Arg
Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala 165 170
175Asp Val Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala
180 185 190Ala Arg Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Phe Ala Gly Lys Thr
Pro Leu His Leu 210 215 220Ala Ala Asn Glu Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Gln
Asp Ile Phe Gly Lys Thr Pro Ala Asp 245 250 255Ile Ala Ala Asp Ala
Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys 260 265 270Ala Ala Gly
Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 275 280 285Thr
Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu 290 295
300Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu
Leu305 310 315 320Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Glu
Gly Lys Thr Pro 325 330 335Leu His Val Ala Ala Gln Glu Gly His Leu
Glu Ile Val Glu Val Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Val Trp Gly Arg Thr 355 360 365Pro Leu His Leu Ala Ala
Trp Ile Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Val Ser Gly Ala385 390 395 400Thr
Pro Leu His Ala Ala Ala Leu His Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Asn Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly
420 425 430Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro
Thr Pro Thr Thr 450 455 460Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp465 470 475 480Leu Gly Lys Lys Leu Leu Leu
Ala Ala Arg Ala Gly Gln Leu Asp Glu 485 490 495Val Arg Ile Leu Leu
Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Val 500 505 510Phe Gly Gln
Thr Pro Leu His Thr Ala Ala Val Ala Gly His Leu Glu 515 520 525Ile
Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp 530 535
540Lys Val Gly Tyr Thr Pro Leu His Leu Ala Ala Gln Ser Gly His
Leu545 550 555 560Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn Ala Lys 565 570 575Asp Val Val Gly Val Thr Pro Ala Asp Leu
Ala Ala Phe Glu Gly His 580 585 590Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala Gly Ser Pro Thr Pro 595 600 605Thr Pro Thr Thr Pro Thr
Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro 610 615 620Thr Gly Ser Pro
Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys625 630 635 640Leu
Leu His Ala Ala Gln Ala Gly Gln Leu Asp Glu Val Arg Ile Leu 645 650
655Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Gln Gly Asn Thr
660 665 670Pro Leu His Ile Ala Ala Phe His Gly His Leu Glu Ile Val
Glu Val 675 680
685Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Trp Gly Leu
690 695 700Thr Pro Leu His Leu Ala Ala Ala Trp Gly His Leu Glu Ile
Val Glu705 710 715 720Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
Gln Asp Asn Tyr Gly 725 730 735Gln Thr Pro Ala Asp Leu Ala Ala Glu
Ser Gly His Gln Asp Ile Ala 740 745 750Glu Val Leu Gln Lys Ala Ala
75589455PRTArtificial SequenceMulti-specific binding protein 89Gly
Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10
15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu
Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala
Ala Trp Ile65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Val Ser Gly Ala Thr Pro
Leu His Ala Ala Ala Leu 100 105 110His Gly His Leu Glu Ile Val Glu
Val Leu Leu Asn Ala Gly Ala Asp 115 120 125Val Asn Ala Gln Asp Lys
Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135 140Arg Ala Gly His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly145 150 155 160Ser
Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro 165 170
175Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Leu Ala
180 185 190Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Val Phe Gly Gln Thr
Pro Leu His Thr 210 215 220Ala Ala Val Ala Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Lys
Asp Lys Val Gly Tyr Thr Pro Leu His 245 250 255Leu Ala Ala Gln Ser
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys 260 265 270Ala Gly Ala
Asp Val Asn Ala Lys Asp Val Val Gly Val Thr Pro Ala 275 280 285Asp
Leu Ala Ala Phe Glu Gly His Gln Asp Ile Ala Glu Val Leu Gln 290 295
300Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr305 310 315 320Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp
Leu Gly Gln Lys 325 330 335Leu Leu His Ala Ala Gln Ala Gly Gln Leu
Asp Glu Val Arg Ile Leu 340 345 350Leu Lys Ala Gly Ala Asp Val Asn
Ala Lys Asp Lys Gln Gly Asn Thr 355 360 365Pro Leu His Ile Ala Ala
Phe His Gly His Leu Glu Ile Val Glu Val 370 375 380Leu Leu Lys Ala
Gly Ala Asp Val Asn Ala Lys Asp Gln Trp Gly Leu385 390 395 400Thr
Pro Leu His Leu Ala Ala Ala Trp Gly His Leu Glu Ile Val Glu 405 410
415Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asn Tyr Gly
420 425 430Gln Thr Pro Ala Asp Leu Ala Ala Glu Ser Gly His Gln Asp
Ile Ala 435 440 445Glu Val Leu Gln Lys Ala Ala 450
45590463PRTArtificial SequenceMulti-specific binding protein 90Gly
Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln1 5 10
15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala
20 25 30Lys Asp Arg Glu Gly Lys Thr Pro Leu His Val Ala Ala Gln Glu
Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
Val Asn 50 55 60Ala Lys Asp Val Trp Gly Arg Thr Pro Leu His Leu Ala
Ala Trp Ile65 70 75 80Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Val Ser Gly Ala Thr Pro
Leu His Ala Ala Ala Leu 100 105 110His Gly His Leu Glu Ile Val Glu
Val Leu Leu Asn Ala Gly Ala Asp 115 120 125Val Asn Ala Gln Asp Lys
Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135 140Arg Ala Gly His
Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly145 150 155 160Ser
Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro 165 170
175Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Leu Ala
180 185 190Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys
Ala Gly 195 200 205Ala Asp Val Asn Ala Lys Asp Val Phe Gly Gln Thr
Pro Leu His Thr 210 215 220Ala Ala Val Ala Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala225 230 235 240Gly Ala Asp Val Asn Ala Lys
Asp Lys Val Gly Tyr Thr Pro Leu His 245 250 255Leu Ala Ala Gln Ser
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys 260 265 270Ala Gly Ala
Asp Val Asn Ala Lys Asp Val Val Gly Val Thr Pro Ala 275 280 285Asp
Leu Ala Ala Phe Glu Gly His Gln Asp Ile Ala Glu Val Leu Gln 290 295
300Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro
Thr305 310 315 320Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Pro
Thr Pro Thr Pro 325 330 335Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu
His Ala Ala Gln Ala Gly 340 345 350Gln Leu Asp Glu Val Arg Ile Leu
Leu Lys Ala Gly Ala Asp Val Asn 355 360 365Ala Lys Asp Lys Gln Gly
Asn Thr Pro Leu His Ile Ala Ala Phe His 370 375 380Gly His Leu Glu
Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val385 390 395 400Asn
Ala Lys Asp Gln Trp Gly Leu Thr Pro Leu His Leu Ala Ala Ala 405 410
415Trp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp
420 425 430Val Asn Ala Gln Asp Asn Tyr Gly Gln Thr Pro Ala Asp Leu
Ala Ala 435 440 445Glu Ser Gly His Gln Asp Ile Ala Glu Val Leu Gln
Lys Ala Ala 450 455 46091470PRTArtificial SequenceMulti-specific
binding protein 91Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala
Arg Ala Gly Gln1 5 10 15Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly
Ala Asp Val Asn Ala 20 25 30Lys Asp Arg Glu Gly Lys Thr Pro Leu His
Val Ala Ala Gln Glu Gly 35 40 45His Leu Glu Ile Val Glu Val Leu Leu
Lys Ala Gly Ala Asp Val Asn 50 55 60Ala Lys Asp Val Trp Gly Arg Thr
Pro Leu His Leu Ala Ala Trp Ile65 70 75 80Gly His Leu Glu Ile Val
Glu Val Leu Leu Lys Ala Gly Ala Asp Val 85 90 95Asn Ala Lys Asp Val
Ser Gly Ala Thr Pro Leu His Ala Ala Ala Leu 100 105 110His Gly His
Leu Glu Ile Val Glu Val Leu Leu Asn Ala Gly Ala Asp 115 120 125Val
Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala 130 135
140Arg Ala Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala
Gly145 150 155 160Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr
Pro Thr Thr Pro 165 170 175Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly
Lys Lys Leu Leu Leu Ala 180 185 190Ala Arg Ala Gly Gln Leu Asp Glu
Val Arg Ile Leu Leu Lys Ala Gly 195 200 205Ala Asp Val Asn Ala Lys
Asp Val Phe Gly Gln Thr Pro Leu His Thr 210 215 220Ala Ala Val Ala
Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala225 230 235 240Gly
Ala Asp Val Asn Ala Lys Asp Lys Val Gly Tyr Thr Pro Leu His 245 250
255Leu Ala Ala Gln Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys
260 265 270Ala Gly Ala Asp Val Asn Ala Lys Asp Val Val Gly Val Thr
Pro Ala 275 280 285Asp Leu Ala Ala Phe Glu Gly His Gln Asp Ile Ala
Glu Val Leu Gln 290 295 300Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro
Thr Thr Pro Thr Pro Thr305 310 315 320Pro Thr Thr Pro Thr Pro Thr
Pro Thr Gly Ser Pro Thr Pro Thr Pro 325 330 335Thr Thr Pro Thr Pro
Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu 340 345 350Leu His Ala
Ala Gln Ala Gly Gln Leu Asp Glu Val Arg Ile Leu Leu 355 360 365Lys
Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Gln Gly Asn Thr Pro 370 375
380Leu His Ile Ala Ala Phe His Gly His Leu Glu Ile Val Glu Val
Leu385 390 395 400Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln
Trp Gly Leu Thr 405 410 415Pro Leu His Leu Ala Ala Ala Trp Gly His
Leu Glu Ile Val Glu Val 420 425 430Leu Leu Lys Ala Gly Ala Asp Val
Asn Ala Gln Asp Asn Tyr Gly Gln 435 440 445Thr Pro Ala Asp Leu Ala
Ala Glu Ser Gly His Gln Asp Ile Ala Glu 450 455 460Val Leu Gln Lys
Ala Ala465 47092378DNAArtificial SequenceNucleic acid encoding
spike protein binding domain 92ggatcagacc tgggtaaaaa gttgttactt
gcagctcgcg ccggccaatt ggacgaagtc 60cgcattttac tgaaggccgg tgcggatgtg
aacgcgaaag acgtgtttgg gcagacccct 120cttcacaccg cagcagttgc
aggtcatctg gagatcgtgg aagtgctttt aaaagcgggt 180gcggacgtca
acgctaagga caaagttggg tacacgccac tgcacttagc tgcacagtca
240ggacatcttg agattgtgga ggtcttgctg aaggcaggcg cagatgtgaa
cgctaaggat 300gtcgttgggg ttacgccggc ggatttggca gctttcgagg
gacaccagga cattgctgaa 360gttctgcaga aggcagca 378932253DNAArtificial
SequenceNucleic acid encoding multi-specific binding protein
93ggatccgacc tgggtaaaaa gctgctggag gcagcgcgtg ccggtcaaga cgacgaggtt
60cgcgaattgc ttaaagcggg tgcagacgtc aacgccaaag attatttctc tcataccccg
120ttgcatttag ccgcgcgtaa tggccatctg aagatcgtcg aggtcctctt
gaaggcaggc 180gcggatgtca atgcgaagga ttttgcgggc aaaacgccgc
tgcacttagc ggcgaacgag 240ggtcatttag aaatcgttga agtcctgtta
aaagcgggcg ccgatgtgaa tgcgcaggat 300attttcggta aaacgccggc
ggacattgcg gcagatgcgg gtcatgaaga tatcgcagaa 360gtcctgcaga
aggcagcagg cagccctaca cctacgccga ctacgcctac gccgactccg
420actaccccga ctccgacccc gaccggatca gacctgggta aaaagctgct
ggaggcagcg 480cgtgccggtc aagacgacga ggttcgcgaa ttgcttaaag
cgggtgcaga cgtcaacgcc 540aaagattatt tctctcatac cccgttgcat
ttagccgcgc gtaatggcca tctgaagatc 600gtcgaggtcc tcttgaaggc
aggcgcggat gtcaatgcga aggattttgc gggcaaaacg 660ccgctgcact
tagcggcgaa cgagggtcat ttagaaatcg ttgaagtcct gttaaaagcg
720ggcgccgatg tgaatgcgca ggatattttc ggtaaaacgc cggcggacat
tgcggcagat 780gcgggtcatg aagatatcgc agaagtcctg cagaaggcag
caggttcccc gacccctacg 840ccaacgactc cgaccccaac tccaacgacc
cctaccccga ccccgaccgg atcagacctg 900ggtaaaaaac tgctgcaagc
agcacgtgca ggtcagctgg atgaagttcg tgaactgctg 960aaagcaggcg
ccgatgttaa tgcaaaagat agagagggca agaccccgct gcatgtggct
1020gctcaagagg gtcacctgga aattgttgaa gttctgctga aagccggtgc
agatgttaat 1080gcaaaagatg tgtggggcag aaccccgctg catctggctg
cttggatcgg tcacctggaa 1140attgttgaag ttctgctgaa agccggtgca
gatgttaatg caaaagatgt gtctggcgct 1200accccgctgc atgcggctgc
tctgcacggt cacctggaaa ttgttgaagt tctgctgaac 1260gccggtgcag
atgttaacgc acaggataaa agcggtaaaa cccctgccga tctggcagct
1320cgcgccggtc atcaggatat tgctgaagtg ctgcagaagg cagcaggcag
ccccacgcca 1380actcctacaa cccccacacc tacaccgacg acgccgacac
cgactccaac cggatcagac 1440ctgggtaaaa agttgttact tgcagctcgc
gccggccaat tggacgaagt ccgcatttta 1500ctgaaggccg gtgcggatgt
gaacgcgaaa gacgtgtttg ggcagacccc tcttcacacc 1560gcagcagttg
caggtcatct ggagatcgtg gaagtgcttt taaaagcggg tgcggacgtc
1620aacgctaagg acaaagttgg gtacacgcca ctgcacttag ctgcacagtc
aggacatctt 1680gagattgtgg aggtcttgct gaaggcaggc gcagatgtga
acgctaagga tgtcgttggg 1740gttacgccgg cggatttggc agctttcgag
ggacaccagg acattgctga agttctgcag 1800aaggcagcag gttcgccgac
cccaacccct accactccaa cgccgacgcc taccactcca 1860acaccaacac
caacgggatc agacctgggt caaaagttgt tacacgcggc tcaagcggga
1920caattagacg aggtccgtat tctgcttaaa gccggggctg atgtaaatgc
aaaagacaaa 1980cagggcaaca cacccttaca tatcgcggca ttccatggac
atctggagat tgtggaagta 2040ctgctgaaag ccggggcaga tgtcaacgct
aaagaccaat ggggattgac cccccttcat 2100ttggccgctg cctggggcca
tttggagatt gtagaggtac ttctgaaggc gggggctgat 2160gttaatgccc
aggataacta tgggcaaact cctgcggatc tggcggctga atctgggcac
2220caagatattg ctgaagttct gcagaaggcg gca 2253942277DNAArtificial
SequenceNucleic acid encoding multi-specific binding protein
94ggatccgacc tgggtaaaaa gctgctggag gcagcgcgtg ccggtcaaga cgacgaggtt
60cgcgaattgc ttaaagcggg tgcagacgtc aacgccaaag attatttctc tcataccccg
120ttgcatttag ccgcgcgtaa tggccatctg aagatcgtcg aggtcctctt
gaaggcaggc 180gcggatgtca atgcgaagga ttttgcgggc aaaacgccgc
tgcacttagc ggcgaacgag 240ggtcatttag aaatcgttga agtcctgtta
aaagcgggcg ccgatgtgaa tgcgcaggat 300attttcggta aaacgccggc
ggacattgcg gcagatgcgg gtcatgaaga tatcgcagaa 360gtcctgcaga
aggcagcagg cagccctaca cctacgccga ctacgcctac gccgactccg
420actaccccga ctccgacccc gaccggatca gacctgggta aaaagctgct
ggaggcagcg 480cgtgccggtc aagacgacga ggttcgcgaa ttgcttaaag
cgggtgcaga cgtcaacgcc 540aaagattatt tctctcatac cccgttgcat
ttagccgcgc gtaatggcca tctgaagatc 600gtcgaggtcc tcttgaaggc
aggcgcggat gtcaatgcga aggattttgc gggcaaaacg 660ccgctgcact
tagcggcgaa cgagggtcat ttagaaatcg ttgaagtcct gttaaaagcg
720ggcgccgatg tgaatgcgca ggatattttc ggtaaaacgc cggcggacat
tgcggcagat 780gcgggtcatg aagatatcgc agaagtcctg cagaaggcag
caggttcccc gacccctacg 840ccaacgactc cgaccccaac tccaacgacc
cctaccccga ccccgaccgg atcagacctg 900ggtaaaaaac tgctgcaagc
agcacgtgca ggtcagctgg atgaagttcg tgaactgctg 960aaagcaggcg
ccgatgttaa tgcaaaagat agagagggca agaccccgct gcatgtggct
1020gctcaagagg gtcacctgga aattgttgaa gttctgctga aagccggtgc
agatgttaat 1080gcaaaagatg tgtggggcag aaccccgctg catctggctg
cttggatcgg tcacctggaa 1140attgttgaag ttctgctgaa agccggtgca
gatgttaatg caaaagatgt gtctggcgct 1200accccgctgc atgcggctgc
tctgcacggt cacctggaaa ttgttgaagt tctgctgaac 1260gccggtgcag
atgttaacgc acaggataaa agcggtaaaa cccctgccga tctggcagct
1320cgcgccggtc atcaggatat tgctgaagtg ctgcagaagg cagcaggcag
ccccacgcca 1380actcctacaa cccccacacc tacaccgacg acgccgacac
cgactccaac cggatcagac 1440ctgggtaaaa agttgttact tgcagctcgc
gccggccaat tggacgaagt ccgcatttta 1500ctgaaggccg gtgcggatgt
gaacgcgaaa gacgtgtttg ggcagacccc tcttcacacc 1560gcagcagttg
caggtcatct ggagatcgtg gaagtgcttt taaaagcggg tgcggacgtc
1620aacgctaagg acaaagttgg gtacacgcca ctgcacttag ctgcacagtc
aggacatctt 1680gagattgtgg aggtcttgct gaaggcaggc gcagatgtga
acgctaagga tgtcgttggg 1740gttacgccgg cggatttggc agctttcgag
ggacaccagg acattgctga agttctgcag 1800aaggcagcag gttcgccgac
cccaacccct accactccaa cgccgacgcc taccactcca 1860acaccaacac
caacgggatc accgacccct accccaacag gatcagacct gggtcaaaag
1920ttgttacacg cggctcaagc gggacaatta gacgaggtcc gtattctgct
taaagccggg 1980gctgatgtaa atgcaaaaga caaacagggc aacacaccct
tacatatcgc ggcattccat 2040ggacatctgg agattgtgga agtactgctg
aaagccgggg cagatgtcaa cgctaaagac 2100caatggggat tgacccccct
tcatttggcc gctgcctggg gccatttgga gattgtagag 2160gtacttctga
aggcgggggc tgatgttaat gcccaggata actatgggca aactcctgcg
2220gatctggcgg ctgaatctgg gcaccaagat attgctgaag ttctgcagaa ggcggca
2277951365DNAArtificial SequenceNucleic acid encoding
multi-specific binding protein 95ggatcagacc tgggtaaaaa actgctgcaa
gcagcacgtg caggtcagct ggatgaagtt 60cgtgaactgc tgaaagcagg cgccgatgtt
aatgcaaaag atagagaggg caagaccccg 120ctgcatgtgg ctgctcaaga
gggtcacctg gaaattgttg aagttctgct gaaagccggt 180gcagatgtta
atgcaaaaga tgtgtggggc agaaccccgc tgcatctggc tgcttggatc
240ggtcacctgg aaattgttga agttctgctg aaagccggtg cagatgttaa
tgcaaaagat 300gtgtctggcg ctaccccgct gcatgcggct gctctgcacg
gtcacctgga aattgttgaa 360gttctgctga acgccggtgc agatgttaac
gcacaggata aaagcggtaa aacccctgcc 420gatctggcag ctcgcgccgg
tcatcaggat attgctgaag tgctgcagaa ggcagcaggc 480agccccacgc
caactcctac aacccccaca cctacaccga cgacgccgac accgactcca
540accggatcag acctgggtaa aaagttgtta cttgcagctc gcgccggcca
attggacgaa 600gtccgcattt tactgaaggc cggtgcggat gtgaacgcga
aagacgtgtt tgggcagacc 660cctcttcaca ccgcagcagt tgcaggtcat
ctggagatcg tggaagtgct tttaaaagcg 720ggtgcggacg tcaacgctaa
ggacaaagtt gggtacacgc cactgcactt agctgcacag 780tcaggacatc
ttgagattgt ggaggtcttg ctgaaggcag gcgcagatgt gaacgctaag
840gatgtcgttg gggttacgcc ggcggatttg gcagctttcg agggacacca
ggacattgct 900gaagttctgc agaaggcagc aggttcgccg accccaaccc
ctaccactcc aacgccgacg 960cctaccactc caacaccaac accaacggga
tcagacctgg gtcaaaagtt gttacacgcg 1020gctcaagcgg gacaattaga
cgaggtccgt attctgctta aagccggggc tgatgtaaat 1080gcaaaagaca
aacagggcaa cacaccctta catatcgcgg cattccatgg acatctggag
1140attgtggaag tactgctgaa agccggggca gatgtcaacg ctaaagacca
atggggattg 1200accccccttc atttggccgc tgcctggggc catttggaga
ttgtagaggt acttctgaag 1260gcgggggctg atgttaatgc ccaggataac
tatgggcaaa ctcctgcgga tctggcggct 1320gaatctgggc accaagatat
tgctgaagtt ctgcagaagg cggca 1365
* * * * *
References