US 2023/0101439 A1 · Engineered Polypeptides Derived From Variable Domain of Adenovirus Penton Base

Abstract

An engineered polypeptide derived from adenovirus pentane base protein. The polypeptide of the invention is based on the “upper” alpha-helical domain of the adenovirus pentane base as shown in the pentane base atomic structure, but it lacks essentially completely any amino acids of the beta-barrel sheet domain showing a jellyroll fold structure (the jellyroll fold domain). The polypeptide contains at least the large fragment of the alpha-helical domain of the pentane base, which fragment includes the RGD loop(s) and the VLP loop, and may contain also the second, short fragment of the alpha-helical domain of the adenovirus pentane base. The polypeptide of the invention provides a new scaffold for optimized presentation of peptidic entities such as oligopeptides, polypeptide sequences, protein domains, proteins and protein complexes as high affinity agents to target molecules.

Claims48

Dependent claims

An isolated engineered polypeptide comprising the amino acid stretches essentially corresponding to a first and a second fragment of the penton base protein of an adenovirus wherein the first fragment of the polypeptide is present between the first and second amino acid stretches forming the jellyroll fold domain in the full length penton base protein and wherein the second fragment of the polypeptide is present between the second and third fragments forming the jellyroll fold domain in the full length penton base, respectively, wherein the isolated engineered polypeptide lacks the amino acid stretches forming the jellyroll fold domain of the adenovirus penton base, wherein optionally the first and/or second fragments of the polypeptide contain(s) one or more heterologous modification(s).

An isolated engineered polypeptide comprising the large fragment of the alpha-helical domain of an adenovirus penton base protein which polypeptide lacks the small fragment of the alpha-helical domain and the jellyroll fold domain of the adenovirus penton base protein, wherein said large fragment optionally contains one or more heterologous modifications.

A method of identifying a binding sequence to a target molecule comprising the steps of:

(ia) preparing a library of vectors each containing a nucleotide sequence encoding a polypeptide having a candidate binding sequence in an expression cassette, each polypeptide encoded by said nucleotide sequence comprising a candidate binding sequence as a heterologous modification in one or more of RGD loop region and/or V loop and/or floor region and/or B loop and having the structure of the following general formula (I):
N-A-L-B-C (I)
wherein
A represents an amino acid stretch corresponding to the N-terminal amino acid stretch of the adenovirus penton base protein present between the first and the second amino acid stretch forming the jellyroll fold domain of the adenovirus penton base;
B represents an amino acid stretch corresponding to the C-terminal amino acid stretch of the adenovirus penton base protein inserted between the second and the third amino acid stretch forming the jellyroll fold domain of the adenovirus penton base protein;
L represents a chemical group selected from the group consisting of an amino acid, an oligopeptide and a polypeptide;
N may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide;
C:
may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide;
wherein fragment A and/or B contain(s) one or more heterologous modifications
wherein said one or more heterologous modifications is/are contained in the following sites:
the RGD loop region of fragment A;
and/or
the V-loop of fragment A;
and/or
the floor region having the sequence (from N- to C-terminal)
(SEQ ID NO:
21)
X1-X2-X3-X4-X5-X6-D-X7-X8-X9-S-Y-
N-X10-X11-X12-X13-X14-X15-X16
of fragment A, wherein
X1 is I or L, and is preferably I;
X2 is selected from the group consisting of K, Q and E, and is preferably Q;
X3 is P or A, and is preferably P;
X4 is selected from the group consisting of L, V and I, and is preferably L
X5 is selected from the group consisting of T, E, A, K and L, and is preferably E;
X6 is selected from the group consisting of E, K, T and Q, and is preferably K;
X7 is selected from the group consisting of S, P and D, and is preferably S;
X8 is selected from the group consisting of K, T and S, and is preferably K;
X9 is selected from the group consisting of K, S, N, G and D, and is preferably S;
X10 is L or V, and is preferably V;
X11 is I or L, and is preferably I;
X12 is selected from the group consisting of S, E and P, and is preferably E;
X13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid;
X14 is D or G, and is preferably D;
X15 is selected from the group consisting of S, K, Q and T, and is preferably K;
and
X16 is selected from the group consisting of T, N, I, K and M, and is preferably I;
and/or
the sequence (from N- to C-terminal) T-H-V-F-X17-R-F-P (SEQ ID NO:
22) of fragment B wherein X17 is D or N, and is preferably N;
wherein the candidate binding sequence encoded by the nucleotide sequence in each vector is different such that the vectors contain a randomized library of nucleotide sequences encoding randomized candidate binding sequences;
or
(ib) preparing a library of vectors each containing a nucleotide sequence encoding a polypeptide having a candidate binding sequence in an expression cassette, each polypeptide encoded by said nucleotide sequence comprising a candidate binding sequence as a heterologous modification in one or more of RGD loop region and/or V loop and/or floor region the polypeptide having from N- to C-terminal, the structure of the following general formula II:
N-A-C (II)
wherein
A represents an amino acid stretch corresponding to the N-terminal amino acid stretch of the adenovirus penton base present between the first and the second amino acid stretch forming the jellyroll fold domain of the adenovirus penton base;
N may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide;
C:
may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide;
wherein the fragment A comprises an amino acid sequence selected from the group consisting of the amino acid sequenoes according to the following table and amino acid sequences having an identity of at least 85% with the respective amino acid sequence shown in the following table:
Sequence
N-terminal
C-terminal
Sequence based
according to
amino acid
amino acid
on penton base
UniProt
SEQ ID
selected from
selected from
protomer of
Acc. No.
NO:
positions
positions
hAd3
Q2Y0H9
1
130 to 137
399 to 405
hAd2
P03276
2
130 to 137
426 to 432
hAd4
Q2KSF3
3
126 to 133
380 to 386
hAd5
P12538
4
130 to 137
426 to 432
hAd7
Q9JFT6
5
130 to 137
399 to 405
hAd11
D2DM93
6
130 to 137
416 to 422
hAd12
P36716
7
120 to 127
352 to 358
hAd17
F1DT65
8
117 to 124
371 to 377
hAd25
M0QUK0
9
125 to 133
389 to 395
hAd35
Q7T941
10
131 to 138
446 to 452
hAd37
Q912J1
11
117 to 124
373 to 379
hAd41
F8WQN4
12
125 to 135
362 to 368
gorAd
E5L3Q9
13
131 to 138
417 to 423
ChimpAd
G9G849
14
126 to 133
373 to 379
sAd18
H8PFZ9
15
128 to 135
354 to 360
sAd20
F6KSU4
16
127 to 134
359 to 365
sAd49
F2WTK5
17
128 to 135
357 to 363
rhAd51
A0A0A1EWW1
18
125 to 132
353 to 359
rhAd52
A0A0A1EWX7
19
125 to 132
351 to 357
rhAd53
A0A0A1EWZ7
20
126 to 133
352 to 358
wherein fragment A contains one or more heterologous modifications wherein said one or more heterologous modifications is/are contained in the following sites:
the RGD loop region of fragment A;
and/or
the V-loop of fragment A;
and/or
the floor region having the sequence (from N- to C-terminal)
(SEQ ID NO:
21)
X1-X2-X3-X4-X5-X6-D-X7-X8-X9-S-Y-
N-X10-X11-X12-X13-X14-X15-X16
of fragment A, wherein
X1 is I or L, and is preferably I;
X2 is selected from the group consisting of K, Q and E, and is preferably Q;
X3 is P or A, and is preferably P;
X4 is selected from the group consisting of L, V and I, and is preferably L
X5 is selected from the group consisting of T, E, A, K and L, and is preferably E;
X6 is selected from the group consisting of E, K, T and Q, and is preferably K;
X7 is selected from the group consisting of S, P and D, and is preferably S;
X8 is selected from the group consisting of K, T and S, and is preferably K;
X9 is selected from the group consisting of K, S, N, G and D, and is preferably S;
X10 is L or V, and is preferably V;
X11 is I or L, and is preferably I;
X12 is selected from the group consisting of S, E and P, and is preferably E;
X13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid;
X14 is D or G, and is preferably D;
X15 is selected from the group consisting of S, K, Q and T, and is preferably K;
and
X16 is selected from the group consisting of T, N, I, K and M, and is preferably I;
and/or
the sequence (from N- to C-terminal) T-H-V-F-X17-R-F-P (SEQ ID NO:
22) of fragment B wherein X17 is D or N, and is preferably N;
wherein the candidate binding sequence encoded by the nucleotide sequence in each vector is different such that the vectors contain a randomized library of nucleotide sequences encoding randomized candidate binding sequences
(ii) expressing the polypeptides encoded by the nucleotide sequences from the library of vectors of step (ia) or ib) in a host cell or a cell-free system, preferably cell free system;
(iii) contacting the polypeptides expressed in step (ii), optionally after purification from the host cells or the cell-free system, preferably cell free system, with the target molecule;
and
(iv) detecting which polypeptide(s) have/has bound to the target molecule.

201 more elements

Dependencies are inferred from the claim text.

Engineered Polypeptides Derived From Variable Domain of Adenovirus Penton Base

Life of this patent

Engineered Polypeptides Derived From Variable Domain of Adenovirus Penton Base

Life of this patent

Abstract

Claims48