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.

Claims

1. 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).

2. The polypeptide of claim 1 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, optionally, fragment A and/or B contain(s) one or more heterologous modifications.

3. The polypeptide of claim 2 wherein the fragment A comprises an amino acid sequence selected from the group consisting of the amino acid sequences 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: TABLE-US-00039 N-terminal C-terminal Sequence based Sequence amino acid amino acid on penton base according to SEQ ID selected from selected from protomer of UniProt 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 12 128 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, optionally, fragment A contains one or more heterologous modifications.

4. The polypeptide of claim 2 wherein the fragment B comprises an amino acid sequence selected from the group consisting of the amino acid sequences according to the following table and amino acid sequences having an identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table: TABLE-US-00040 Sequence Sequence N-terminal based on according to amino acid C-terminal amino penton base UniProt SEQ ID selected from acid selected protomer of Acc. No. NO: positions from positions hAd3 Q2Y0H9 1 441 to 444 491 to 494 hAd2 P03276 2 468 to 471 518 to 521 hAd4 Q2KSF3 3 422 to 445 465 to 468 hAd5 P12538 4 468 to 471 491 to 494 hAd7 Q9JFT6 5 441 to 444 464 to 467 hAd11 D2DM93 6 458 to 461 481 to 484 hAd12 P36716 7 394 to 398 418 to 421 hAd17 F1DT65 8 414 to 417 438 to 441 hAd25 M0QUK0 9 441 to 444 454 to 457 hAd35 Q7T941 10 498 to 501 521 to 524 hAd37 Q912J1 11 415 to 418 438 to 441 hAd41 F8WQN4 12 405 to 408 438 to 441 gorAd E5L3Q9 13 459 to 462 482 to 485 ChimpAd G9G849 14 421 to 424 444 to 457 sAd18 H8PFZ9 15 396 to 399 419 to 422 sAd20 F6KSU4 16 401 to 404 424 to 427 sAd49 F2WTK5 17 399 to 402 422 to 425 rhAd51 A0A0A1EWW1 18 395 to 398 418 to 421 rhAd52 A0A0A1EWX7 19 393 to 396 416 to 419 rhAd53 A0A0A1EWZ7 20 394 to 397 417 to 420 wherein, optionally, fragment B contains one or more heterologous modifications.

5. The polypeptide of claim 2 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) TABLE-US-00041 (SEQ ID NO: 21) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub.8-X.sub.9-S-Y- N-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16 of fragment A, wherein X.sub.1 is I or L, and is preferably I; X.sub.2 is selected from the group consisting of K, Q and E, and is preferably Q; X.sub.3 is P or A, and is preferably P; X.sub.4 is selected from the group consisting of L, V and I, and is preferably L X.sub.5 is selected from the group consisting of T, E, A, K and L, and is preferably E; X.sub.6 is selected from the group consisting of E, K, T and Q, and is preferably K; X.sub.7 is selected from the group consisting of S, P and D, and is preferably S; X.sub.8 is selected from the group consisting of K, T and S, and is preferably K; X.sub.9 is selected from the group consisting of K, S, N, G and D, and is preferably S; X.sub.10 is L or V, and is preferably V; X.sub.11 is I or L, and is preferably I; X.sub.12 is selected from the group consisting of S, E and P, and is preferably E; X.sub.13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid; X.sub.14 is D or G, and is preferably D; X.sub.15 is selected from the group consisting of S, K, Q and T, and is preferably K; and X.sub.16 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-X.sub.17-R-F-P (SEQ ID NO: 22) of fragment B wherein X.sub.17 is D or N, and is preferably N.

6. The polypeptide of claim 5 wherein the N-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00042 (SEQ ID NO: 23) X.sub.18-X.sub.19-X.sub.20-X.sub.21-X.sub.22-X.sub.23-X.sub.24-X.sub.25-X.sub.26 wherein X.sub.18 is selected from the group consisting of D, E and N, and is preferably D; X.sub.19 is selected from the group consisting of V, L, and I, and is preferably V; X.sub.20 is any amino acid, preferably selected from the group consisting of A, D, E, K, S, and T, and is more preferably T; X.sub.21 is any amino acid, preferably selected from the group consisting of A, D, E, and K, and is more preferably A; X.sub.22 is selected from the group consisting of F, Y, and W, and is preferably Y; X.sub.23 is selected from the group consisting of A, D, E, N, and Q, is preferably E or Q, and is more preferably E; X.sub.24 is any amino acid, preferably selected from the group consisting of A, D, E, N, and K, and is more preferably E; X.sub.25 is selected from the group consisting of S or T, and is preferably S; and X.sub.26 is any amino acid and constitutes the N-terminal amino acid of the RGD loop region

7. The polypeptide of claim 5 wherein the C-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00043 (SEQ ID NO: 24) X.sub.27-X.sub.28-X.sub.29-X.sub.30-X.sub.31-X.sub.32-X.sub.33-X.sub.34 Wherein X.sub.27 is any amino acid and constitutes the C-terminal amino acid of the second RGD loop; X.sub.28 is selected from the group consisting of I, L and V, and is preferably I; X.sub.29 is selected from the group consisting of D, E, K, N, Q, and V, is preferably Q or K, and is more preferably Q; X.sub.30 is selected from the group consisting of C, G and P, and is preferably P; X.sub.31 is selected from the group consisting of I, L and V, is preferably L or V and is more preferably L; X.sub.32 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; X.sub.33 is selected from the group consisting of D, E, S and T, is preferably E, or T, and is more preferably K; and X.sub.34 is selected from the group consisting of D and E, and is preferably D;

8. The polypeptide of claim 5, wherein the N-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00044 (SEQ ID NO: 25) X.sub.35-X.sub.36-X.sub.37-X.sub.38-X.sub.39-X.sub.40-X.sub.41-X.sub.42 wherein X.sub.35 is selected from the group consisting of F, Y, and W, and is preferably F; X.sub.38 is selected from the group consisting of H, K and R, and is preferably K; X.sub.37 is selected from the group consisting of A, V, I, and L, and is preferably A; X.sub.38 is selected from the group consisting of H, K, and R, and is preferably R; X.sub.39 is selected from the group consisting of A, V, I, and L, and is preferably V; X.sub.40 is selected from the group consisting of A, V, I, L and M, and is preferably M; X.sub.41 is selected from the group consisting of A, V, I, and L, and is preferably V; and X.sub.42 is any amino acid and constitutes the N-terminal amino acid of the V loop.

9. The polypeptide of claim 5, wherein the C-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00045 (SEQ ID NO: 26) X.sub.43-X.sub.44-X.sub.45-X.sub.46-X.sub.47-X.sub.48-X.sub.49 wherein X.sub.43 is any amino acid and constitutes the C-terminal amino acid of the V loop; X.sub.44 is selected from the group consisting of F, Y, and W, and is preferably Y; X.sub.45 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; X.sub.46 is selected from the group consisting of F, Y, and W, and is preferably W; X.sub.47 is selected from the group consisting of A, F, V, Y, and W, is preferably F or V and is more preferably F; X.sub.48 is selected from the group consisting of D, E, S and T, is preferably D or E and is more preferably E; and X.sub.49 is selected from the group consisting of F, Y, and W, and is preferably F.

10. The polypeptide of claim 2, wherein the heterologous modification is selected from the group consisting of one or more single amino acid mutations in comparison to the wildtype sequence of fragment A and/or B, one or more replacements of wildtype amino acid stretches by one or more heterologous amino acids and/or amino acid stretches one or more insertions of heterologous amino acid stretches, one or more deletions of one or more amino acids and one or more amino acid modifications as well as any combination(s) thereof.

11. The polypeptide of claim 2, wherein the heterologous modification provides a target specific binding entity.

12. The polypeptide of claim 11, wherein the target specific binding entity is selected from the group consisting of antigens, epitopes, CDRs, antibodies, antibody fragments such as an antigen binding (Fab) fragment, a Fab′ fragment, a F(ab′)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, a paratope, an alternative scaffold protein, and a fusion protein thereof, a toxin and a venom.

13. The polypeptide of claim 1 having one of the following amino acid sequences (from N-terminal to C-terminal): TABLE-US-00046 (SEQ ID NO: 27) MSYYHHHHHHDYDIPTTENLYFQGAMGSGIQPNVN EYMFSNKFKARVMVSRKAPEGVTVNDTYDHKEDIL KYEWFEFILPEGNFSATMTIDLMNNAIIDNYLEIG RQNGVLESDIGVKFDTRNFRLGWDPETKLIMPGVY TYEAFHPDIVLLPGCGVDFTESRLSNLLGIRKRHP FQEGFKIMYEDLEGGNIPALLDVTAYEESKKDTTT ETTTKKELKIQPLEKDSKSRSYNVLEDKINTAYRS WYLSYNYGNPEKGIRSWTLLTTSDVTCGANGDSGN PVFSKSFYNEQAVYSQQLRQATSLTHVFNRFPENQ IURPPAPTITTVSENVP (SEQ ID NO: 32) GAMGSGIQPNVNEYMFSNKFKARVMVSRKAPEGVT VNDTYDHKEDILKYEWFEFILPEGNFSATMTIDLM NNAIIDNYLEIGRQNGVLESDIGVKFDTRNFRLGW DPETKLIMPGVYTYEAFHPDIVLLPGCGVDFTESR LSNLLGIRKRHPFQEGFKIMYEDLEGGNIPALLDV TAYEESKKDTTTETTTKKELKIQPLEKDSKSRSYN VLEDKINTAYRSWYLSYNYGNPEKGIRSWTLLTTS DVTCGANGDSGNPVFSKSFYNEQAVYSQQLRQATS LTHVFNRFPENQILIRPPAPTITTVSENVP (SEQ ID NO: 27) MSYYHHHHHHDYDIPTTENLYFQGTIMHTNMPNVN EFMYSNKFKARVMVSRKAPEGVTVNDTYDHKEDIL EYEWVEFELPEGNFSVTMTIDLMNNAIIDNYLAVG RQNGVLESDIGVKFDTRNFRLGWDPVTELVMPGVY TNEAFHPDIVLLPGCGVDFTEMSYYHHHHHHDYDI PTTENLYFQGAMGSGIQPNVNEYMFSNKFKARVMV SRKAPEGVTVNDTYDHKEDILKYEWFEFILPEGNF SATMTIDLMNNAIIDNYLEIGRQNGVLESDIGVKF DTRNFRLGWDPETKLIMPGVYTYEAFHPDIVLLPG CGVDFTESRLSNLLGIRKRHPFQEGFKIMYEDLEG GNIPALLDVTAYEESKKDTTTETTTKKELKIQPLE KDSKSRSYNVLEDKINTAYRSWYLSYNYGNPEKGI RSWTLLTTSDVTCGANGDSGNPVFSKSFYNEQAVY SQQLRQATSLTHVFNRFPENQIURPPAPTITTVSE NVP (SEQ ID NO: 28) GAMGSGIQPNVNEYMFSNKFKARVMVSRKAPEGVT VNDTYDHKEDILKYEWFEFILPEGNFSATMTIDLM NNAIIDNYLEIGRQNGVLESDIGVKFDTRNFRLGW DPETKLIMPGVYTYEAFHPDIVLLPGCGVDFTESR LSNLLGIRKRHPFQEGFKIMYEDLEGGNIPALLDV TAYEESKKDTTTETTTKKELKIQPLEKDSKSRSYN VLEDKINTSRLSNLLGIRKRQPFQEGFQIMYEDLE GGNIPALLDVDAYEKSKKDTTTETTTKKELKIQPV EKDSKDRSYNVLPDKINTAYRSWYLAYNYGDPEKG VRSWTLLTTSDVTCGVEQAELLPVYSKSFFNEQAV YSQQLRAFTSLTHVFNRFPENQILVRPPAPTITTV SENVP (SEQ ID NO: 33) QGTIMHTNMPNVNEFMYSNKFKARVMVSRKAPEGV TVNDTYDHKEDILEYEWVEFELPEGNFSVTMTIDL MNNAIIDNYLAVGRQNGVLESDIGVKFDTRNFRLG WDPVTELVMPGVYTNEAFHPDIVLLPGCGVDFTES RLSNLLGIRKRQPFQEGFQIMYEDLEGGNIPALLD VDAYEKSKKDTTTETTTKKELKIQPVEKDSKDRSY NVLPDKINTAYRSWYLAYNYGDPEKGVRSWTLLTT SDVTCGVEQAELLPVYSKSFFNEQAVYSQQLRAFT SLTHVFNRFPENQILVRPPAPTITTVSENVP

14. 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.

15. The polypeptide of claim 14 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, optionally, fragment A contain one or more heterologous modifications.

16. The polypeptide of claim 15 wherein the fragment A comprises an amino acid sequence selected from the group consisting of the amino acid sequences 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: TABLE-US-00047 Sequence Sequence N-terminal C-terminal based on according to amino acid amino acid 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 12 128 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, optionally, fragment A contains one or more heterologous modifications.

17. The polypeptide of claim 15 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) TABLE-US-00048 (SEQ ID NO: 21) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub.8-X.sub.9-S-Y- N-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16 of fragment A, wherein X.sub.1 is I or L, and is preferably I; X.sub.2 is selected from the group consisting of K, Q and E, and is preferably Q; X.sub.3 is P or A, and is preferably P; X.sub.4 is selected from the group consisting of L, V and I, and is preferably L X.sub.5 is selected from the group consisting of T, E, A, K and L, and is preferably E; X.sub.6 is selected from the group consisting of E, K, T and Q, and is preferably K; X.sub.7 is selected from the group consisting of S, P and D, and is preferably S; X.sub.8 is selected from the group consisting of K, T and S, and is preferably K; X.sub.9 is selected from the group consisting of K, S, N, G and D, and is preferably S; X.sub.10 is L or V, and is preferably V; X.sub.11 is I or L, and is preferably I; X.sub.12 is selected from the group consisting of S, E and P, and is preferably E; X.sub.13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid; X.sub.14 is D or G, and is preferably D; X.sub.15 is selected from the group consisting of S, K, Q and T, and is preferably K; and X.sub.15 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-X.sub.17-R-F-P (SEQ ID NO: 22) of fragment B wherein X.sub.17 is D or N, and is preferably N.

18. The polypeptide of claim 17 wherein the N-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00049 (SEQ ID NO: 23) X.sub.18-X.sub.19-X.sub.20-X.sub.21-X.sub.22-X.sub.23-X.sub.24-X.sub.25-X.sub.26 wherein X.sub.18 is selected from the group consisting of D, E and N, and is preferably D; X.sub.19 is selected from the group consisting of V, L, and I, and is preferably V; X.sub.20 is any amino acid, preferably selected from the group consisting of A, D, E, K, S, and T, and is more preferably T; X.sub.21 is any amino acid, preferably selected from the group consisting of A, D, E, and K, and is more preferably A; X.sub.22 is selected from the group consisting of F, Y, and W, and is preferably Y; X.sub.23 is selected from the group consisting of A, D, E, N, and Q, is preferably E or Q, and is more preferably E; X.sub.24 is any amino acid, preferably selected from the group consisting of A, D, E, N, and K, and is more preferably E; X.sub.25 is selected from the group consisting of S or T, and is preferably S; and X.sub.26 is any amino acid and constitutes the N-terminal amino acid of the RGD loop region

19. The polypeptide of claim 17 wherein the C-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00050 (SEQ ID NO: 24) X.sub.27-X.sub.28-X.sub.29-X.sub.30-X.sub.31-X.sub.32-X.sub.33-X.sub.34 Wherein X.sub.27 is any amino acid and constitutes the C-terminal amino acid of the second RGD loop; X.sub.28 is selected from the group consisting of I, L and V, and is preferably I; X.sub.29 is selected from the group consisting of D, E, K, N, Q, and V, is preferably Q or K, and is more preferably Q; X.sub.30 is selected from the group consisting of C, G and P, and is preferably P; X.sub.31 is selected from the group consisting of I, L and V, is preferably L or V and is more preferably L; X.sub.32 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; X.sub.33 is selected from the group consisting of D, E, S and T, is preferably E, or T, and is more preferably K; and X.sub.34 is selected from the group consisting of D and E, and is preferably D;

20. The polypeptide of claim 17 wherein the N-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00051 (SEQ ID NO: 25) X.sub.35-X.sub.36-X.sub.37-X.sub.38-X.sub.39-X.sub.40-X.sub.41-X.sub.42 wherein X.sub.35 is selected from the group consisting of F, Y, and W, and is preferably F; X.sub.38 is selected from the group consisting of H, K and R, and is preferably K; X.sub.37 is selected from the group consisting of A, V, I, and L, and is preferably A; X.sub.38 is selected from the group consisting of H, K, and R, and is preferably R; X.sub.39 is selected from the group consisting of A, V, I, and L, and is preferably V; X.sub.40 is selected from the group consisting of A, V, I, L and M, and is preferably M; X.sub.41 is selected from the group consisting of A, V, I, and L, and is preferably V; and X.sub.42 is any amino acid and constitutes the N-terminal amino acid of the V loop.

21. The polypeptide of claim 17 wherein the C-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal): TABLE-US-00052 (SEQ ID NO: 26) X.sub.43-X.sub.44-X.sub.45-X.sub.46-X.sub.47-X.sub.48-X.sub.49 wherein X.sub.43 is any amino acid and constitutes the C-terminal amino acid of the V loop; X.sub.44 is selected from the group consisting of F, Y, and W, and is preferably Y; X.sub.45 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; X.sub.46 is selected from the group consisting of F, Y, and W, and is preferably W; X.sub.47 is selected from the group consisting of A, F, V, Y, and W, is preferably F or V and is more preferably F; X.sub.48 is selected from the group consisting of D, E, S and T, is preferably D or E and is more preferably E; and X.sub.49 is selected from the group consisting of F, Y, and W, and is preferably F.

22. The polypeptide of claim 15 wherein the heterologous modification is selected from the group consisting of one or more single amino acid mutations in comparison to the wildtype sequence of fragment A, one or more replacements of wildtype amino acid stretches by one or more heterologous amino acids and/or amino acid stretches one or more insertions of heterologous amino acid stretches, one or more deletions of one or more amino acids and one or more amino acid modifications as well as any combination(s) thereof.

23. The polypeptide of claim 15, wherein the heterologous modification provides a target specific binding entity.

24. The polypeptide of claim 23, wherein the target specific binding entity is selected from the group consisting of antigens, epitopes, CDRs, antibodies, antibody fragments such as an antigen binding (Fab) fragment, a Fab′ fragment, a F(ab′)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, a paratope, an alternative scaffold protein, and a fusion protein thereof, a toxin and a venom.

25. A nucleic acid encoding the polypeptide of claim 1.

26. A vector comprising the nucleic acid of claim 25.

27. The vector of claim 26 containing the nucleic acid of claim 25 within an expression cassette.

28. A recombinant host cell comprising the vector of claim 26.

29. A method for the production of a polypeptide comprising the step of culturing the host cell of claim 28 under conditions allowing the expression of said polypeptide.

30. The method of claim 29 further comprising the step of purifying the polypeptide from the cultured host cells.

31. An engineered adenovirus penton base protein comprising the polypeptide of claim 5 fused to the multimerization domain (jellyroll fold domain) of an adenovirus penton base protein.

32. The penton base protein of claim 31 having, from N- to C-terminal, the structure of the following general formula III:
D-A-E-B-F (III) wherein A and B are the fragments of the alpha-helical crown domain as defined in any one of claims 2 to 4, and D, E and F are the amino acid sequences of an adenovirus penton base forming the multimerization (jellyroll fold) domain, wherein one or more heterologous modifications is/are present in the floor region of fragment A and/or in the B loop of fragment B.

33. An engineered adenovirus penton base protein comprising the polypeptide of claim 15 fused to the multimerization domain (jellyroll fold domain) of an adenovirus penton base protein.

34. The penton base of claim 33 having, from N- to C-terminal, the structure of the following general formula IV:
D-A-E-Li-F (IV) wherein A is the large fragment of the alpha-helical crown domain as defined in any one of claims 15 or 16, and D, E and F are the amino acid sequences of an adenovirus penton base forming the multimerization (jellyroll fold) domain, wherein, optionally and preferably, one or more heterologous modifications is/are present in the floor region of fragment A, and wherein Li is a linker selected from peptides, oligopeptides, polypeptides, proteins and protein complexes.

35. The penton base of claim 32 wherein fragment D of general formula (III) or (IV) has the following consensus sequence (SEQ ID NO: 34):
(U).sub.1-47 PTJ.sub.1GRNSIRY SJ.sub.2J.sub.3x.sub.4PJ.sub.5J.sub.6DTT J.sub.7J.sub.8YLVDNKSA DIASLNYQND HSNFJ.sub.5TTVJ.sub.9Q NNDJ.sub.10J.sub.11PJ.sub.12EAJ.sub.13 TQTINJ.sub.14DJ.sub.15RS RWGJ.sub.16J.sub.17LKTIJ.sub.18 J.sub.19TZ.sub.1Z.sub.2Z.sub.3Z.sub.4Z.sub.5Z.sub.6Z.sub.7Z.sub.8 Z.sub.9Z.sub.10Z.sub.11Z.sub.12Z.sub.13Z.sub.14Z.sub.15 wherein: fragment D ends on the C-terminal side before Z.sub.1 at residue T or at an amino acid from Z.sub.1 to Z.sub.15 U is any or no amino acid J.sub.1 is E or G J.sub.2 is E or S J.sub.3 is L or V J.sub.4 is A or S J.sub.5 is L or Q J.sub.6 is Y or E J.sub.7 is R or K J.sub.8 is V or L J.sub.9 is V or I J.sub.10 is F or Y J.sub.11 is T or S J.sub.12 is A or T or I or G J.sub.13 is S or G J.sub.14 is F or L J.sub.15 is E or D J.sub.16 is A or G J.sub.17 is D or Q J.sub.18 is L or M J.sub.19 is H or R Z.sub.1, if present, is N Z.sub.2, if present, is M Z.sub.3, if present, is P Z.sub.4, if present, is N Z.sub.5, if present, is V or I Z.sub.6, if present, is N Z.sub.7, if present, is E or D Z.sub.8, if present, is Y or F Z.sub.9, if present, is M Z.sub.10, if present, is F or S or Y Z.sub.11, if present, is T or S Z.sub.12, if present, is S or N Z.sub.13, if present, is K Z.sub.14, if present, is F Z.sub.16, if present, is K.

36. The penton base of claim 35 wherein the fragment D comprises an amino acid sequence selected from the group consisting of the amino acid sequences of the following table and amino acid sequences having and identity of at least 85% with the respective amino acid sequence shown in the following table: TABLE-US-00053 Sequence N-terminal C-terminal Sequence based according to amino acid amino acid on penton base UniProt Acc. SEQ selected from selected from protomer of No. ID NO: positions positions hAd3 Q2Y0H9 1 1 to 48 129 to 144 hAd2 P03276 2 1 to 48 129 to 144 hAd4 Q2KSF3 3 1 to 44 125 to 140 hAd5 P12538 4 1 to 48 129 to 144 hAd7 Q9JFT6 5 1 to 48 129 to 144 hAd11 D2DM93 6 1 to 48 129 to 144 hAd12 P36716 7 1 to 38 119 to 134 hAd17 F1DT65 8 1 to 35 116 to 131 hAd25 M0QUK0 9 1 to 43 124 to 139 hAd35 Q7T941 10 1 to 49 130 to 145 hAd37 Q912J1 11 1 to 35 116 to 131 hAd41 F8WQN4 12 1 to 46 127 to 142 gorAd E5L3Q9 13 1 to 49 130 to 145 ChimpAd G9G849 14 1 to 44 125 to 140 sAd18 H8PFZ9 15 1 to 46 127 to 142 sAd20 F6KSU4 16 1 to 45 126 to 141 sAd49 F2WTK5 17 1 to 48 127 to 142 rhAd51 A0A0A1EWW1 18 1 to 43 124 to 139 rhAd52 A0A0A1EWX7 19 1 to 43 124 to 139 rhAd53 A0A0A1EWZ7 20 1 to 44 125 to 140

37. The penton base claim 32 wherein fragment E of general formula (III) or (IV) has the following sequence (SEQ ID NO: 35):
Z.sub.17Z.sub.18Z.sub.19Z.sub.20Z.sub.21Z.sub.22Z.sub.23Z.sub.24Z.sub.25Z.sub.26 Z.sub.27QVYWSLPDJ.sub.20 MJ.sub.21DPVTFRST J.sub.22QJ.sub.23J.sub.24NJ.sub.25PVVGJ.sub.26 ELZ.sub.28Z.sub.29Z.sub.30 wherein: fragment E begins on the N-terminal side at an amino acid from Z.sub.17 to Z.sub.27 or at amino acid Q after Z.sub.27; amino acid stretch B ends on the C-terminal side before Z.sub.28 at amino acid L or at an amino acid from Z.sub.28 to Z.sub.30; Z.sub.17, if present, is L or S Z.sub.18, if present, is T or P or C Z.sub.19, if present, is T or P Z.sub.20, if present, is P or S or A or R Z.sub.21, if present, is N or D Z.sub.22, if present, is G or V Z.sub.23, if present, is H or T Z.sub.24, if present, is C Z.sub.25, if present, is G Z.sub.26, if present, is A or V or S Z.sub.27, if present, is E or Q J.sub.20 is L or M J.sub.21 is Q or K J.sub.22 is Q or R or S J.sub.23 is V or I J.sub.24 is S or N J.sub.25 is Y or F J.sub.26 is A or V Z.sub.28, if present, is M or L Z.sub.29, if present, is P Z.sub.30, if present, is V or F.

38. The penton base of claim 37 wherein the fragment E comprises an amino acid sequence selected from the group consisting of the amino acid sequences of the following table and amino acid sequences having and identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table: TABLE-US-00054 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 1 398 to 409 440 to 443 hAd2 P03276 2 425 to 436 467 to 470 hAd4 Q2KSF3 3 379 to 390 421 to 444 hAd5 P12538 4 425 to 436 467 to 470 hAd7 Q9JFT6 5 398 to 409 440 to 443 hAd11 D2DM93 6 415 to 426 457 to 460 hAd12 P36716 7 351 to 362 393 to 397 hAd17 F1DT65 8 370 to 381 413 to 416 hAd25 M0QUK0 9 388 to 399 440 to 443 hAd35 Q7T941 10 445 to 456 497 to 500 hAd37 Q912J1 11 372 to 383 414 to 417 hAd41 F8WQN4 12 362 to 373 404 to 407 gorAd E5L3Q9 13 416 to 427 458 to 461 ChimpAd G9G849 14 372 to 383 420 to 423 sAd18 H8PFZ9 15 353 to 364 395 to 398 sAd20 F6KSU4 16 358 to 369 400 to 403 sAd49 F2WTK5 17 356 to 367 398 to 401 rhAd51 A0A0A1EWW1 18 352 to 363 394 to 397 rhAd52 A0A0A1EWX7 19 350 to 361 392 to 395 rhAd53 A0A0A1EWZ7 20 351 to 362 393 to 396

39. The penton base of claim 32 wherein fragment F of general formula (III) or (IV) has the following sequence (SEQ ID NO: 36):
Z.sub.31Z.sub.32Z.sub.33ALTDHGT LPLRSSIJ.sub.27GV QRVTJ.sub.28TDARR RTCPYVYKA LGIVJ.sub.30PJ.sub.31VLS SRTF wherein: fragment F begins on the N-terminal side at an amino acid from Z.sub.31 to Z.sub.33 or at amino acid A after Z.sub.33; Z.sub.31, if present, is N Z.sub.32, if present, is V Z.sub.33, if present, is P J.sub.27 is R or S or G J.sub.28 is V or I J.sub.29 is Y or H J.sub.30 is A or S J.sub.31 is R or K

40. The penton base of claim 39 wherein fragment F comprises an amino acid sequence selected from the group consisting of the amino acid sequences of the following table and amino acid sequences having and identity of at least 85% with the respective amino acid sequence shown in the following table: TABLE-US-00055 Sequence Sequence N-terminal C- based on according to amino terminal penton base UniProt SEQ acid selected amino acid protomer of Acc. No. ID NO: from positions position hAd3 Q2Y0H9 1 492 to 495 544 hAd2 P03276 2 519 to 522 571 hAd4 Q2KSF3 3 466 to 469 535 hAd5 P12538 4 492 to 495 571 hAd7 Q9JFT6 5 465 to 468 544 hAd11 D2DM93 6 482 to 485 561 hAd12 P36716 7 419 to 422 497 hAd17 F1DT65 8 438 to 441 517 hAd25 M0QUK0 9 455 to 458 534 hAd35 Q7T941 10 522 to 525 561 hAd37 Q912J1 11 439 to 442 519 hAd41 F8WQN4 12 439 to 432 508 gorAd E5L3Q9 13 483 to 486 875 ChimpAd G9G849 14 445 to 458 532 sAd18 H8PFZ9 15 420 to 423 508 sAd20 F6KSU4 16 425 to 428 512 sAd49 F2WTK5 17 423 to 426 511 rhAd51 A0A0A1EWW1 18 419 to 422 505 rhAd52 A0A0A1EWX7 19 417 to 420 503 rhAd53 A0A0A1EWZ7 20 418 to 421 504

41. A pentameric complex of the engineered adenovirus penton base protein according to claim 32.

42. A virus-like particle (VLP) comprising 12 pentameric complexes of claim 41.

43. The polypeptide of claim 1 for use as a medicament.

44. A pharmaceutical composition comprising the engineered adenovirus penton base protein of claim 32, optionally together with at least one pharmaceutically acceptable carrier, excipient and/or diluent.

45. A method for producing the VLP of claim 42 comprising the step of incubating a solution of a penton base according to claim 32 under conditions allowing the assembly of the polypeptide into a VLP.

46. The polypeptide of claim 14 for use in the treatment and/or prevention of an infectious disease, an immune disease, tumour or cancer.

47. 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) TABLE-US-00056 (SEQ ID NO: 21) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub.8-X.sub.9-S-Y- N-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16 of fragment A, wherein X.sub.1 is I or L, and is preferably I; X.sub.2 is selected from the group consisting of K, Q and E, and is preferably Q; X.sub.3 is P or A, and is preferably P; X.sub.4 is selected from the group consisting of L, V and I, and is preferably L X.sub.5 is selected from the group consisting of T, E, A, K and L, and is preferably E; X.sub.6 is selected from the group consisting of E, K, T and Q, and is preferably K; X.sub.7 is selected from the group consisting of S, P and D, and is preferably S; X.sub.8 is selected from the group consisting of K, T and S, and is preferably K; X.sub.9 is selected from the group consisting of K, S, N, G and D, and is preferably S; X.sub.10 is L or V, and is preferably V; X.sub.11 is I or L, and is preferably I; X.sub.12 is selected from the group consisting of S, E and P, and is preferably E; X.sub.13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid; X.sub.14 is D or G, and is preferably D; X.sub.15 is selected from the group consisting of S, K, Q and T, and is preferably K; and X.sub.16 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-X.sub.17-R-F-P (SEQ ID NO: 22) of fragment B wherein X.sub.17 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: TABLE-US-00057 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) TABLE-US-00058 (SEQ ID NO: 21) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub.8-X.sub.9-S-Y- N-X.sub.10-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-X.sub.16 of fragment A, wherein X.sub.1 is I or L, and is preferably I; X.sub.2 is selected from the group consisting of K, Q and E, and is preferably Q; X.sub.3 is P or A, and is preferably P; X.sub.4 is selected from the group consisting of L, V and I, and is preferably L X.sub.5 is selected from the group consisting of T, E, A, K and L, and is preferably E; X.sub.6 is selected from the group consisting of E, K, T and Q, and is preferably K; X.sub.7 is selected from the group consisting of S, P and D, and is preferably S; X.sub.8 is selected from the group consisting of K, T and S, and is preferably K; X.sub.9 is selected from the group consisting of K, S, N, G and D, and is preferably S; X.sub.10 is L or V, and is preferably V; X.sub.11 is I or L, and is preferably I; X.sub.12 is selected from the group consisting of S, E and P, and is preferably E; X.sub.13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid; X.sub.14 is D or G, and is preferably D; X.sub.15 is selected from the group consisting of S, K, Q and T, and is preferably K; and X.sub.16 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-X.sub.17-R-F-P (SEQ ID NO: 22) of fragment B wherein X.sub.17 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.

48. The method of claim 47 further comprising the step of determining the dissociation constant(s) (Kd) of the polypeptide(s) bound to the target molecule.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0259] FIG. 1 shows a schematic representation illustrating the use of the adenovirus penton base protein crown domain of the invention (the “ADDobody”) to generate high affinity mono- and/or multivalent binder molecules by selection/evolution from a randomized ADDobody library. An ADDobody library can be generated by randomizing a selection or all of the loops. From this ADDobody library, specific binders can be identified that can bind to any antigen by applying selection/evolution techniques, such as phage display, mRNA display, yeast display, baculovirus capsid display or ribosome display or others.

[0260] FIG. 2 shows a schematic representation illustrating the use of the adenovirus penton base protein crown domain of the invention (the “ADDobody”) to generate multivalent vaccines by reverse vaccinology. The crown is shown schematically on the extreme left. The crown domain is colored in green. Four distinct sites have been identified for insertion of heterologous peptide and polypeptide sequences in ADDobody (abbreviated here schematically as ‘loops’). Randomization of these loops yields an ADDobody library that can be utilized to identify loop sequences specifically bound by antibodies prevalent in sera of infected patients or immunized animals. Identification of such epitopic sequences can be used to generate mono- or multivalent ADDOmer superantigens for therapeutic purposes.

[0261] FIG. 3 shows (A) Dodecahedra formed by base proteins of certain Adenovirus serotypes represent a versatile scaffold. The high resolution cryo-EM structure (6HCR) is shown. The view is down a central penton axis. Each color stands for a different penton base protein (B) The ADDomer comprises 60 copies of the adenovirus penton base protein. The penton base protein has a two-domain architecture; a crown domain (top) and a multimerization domain (below). The multimerization domain adopts a jellyroll fold. The crown domain comprises exposed highly variable loops. The domains can be separated into two independent protein entities. The crown domain itself can be produced and purified in large quantity. In addition, the crown domain can be engineered to allow display of heterologous peptide sequences (see FIG. 1 and FIG. 2). (C) The crown domain is represented in this schematic view in FIGS. 1 and 2. Removal of the multimerization domain yields an autonomous protein domain according to the invention (the “ADDobody”) which can be produced in large quantity, purified and crystallized.

[0262] FIG. 4 shows a schematic representation of the construct pPROEX HTb ADDomer2_Head for expression of an ADDobody of the invention based on human Adenovirus serotype 3 (Adh3).

[0263] FIG. 5 shows a MonoQ elution profile of the ADDobody construct ADDomer2_Head

[0264] FIG. 6 shows a size exclusion chromatogram of the MonoQ-eluted ADDobody construct ADDomer2_Head

[0265] FIG. 7 shows a SDS polyacrylamide gel analysis of the peak fractions of the size exclusion chromatography according to FIG. 6.

[0266] FIG. 8 shows a SDS polyacryl amide gel analysis of a sample of the pooled peak fractions of the size exclusion chromatography according to FIG. 6 before and after freeze drying.

[0267] FIG. 9 shows an exemplary single crystal of the ADDobody construct ADDomer2_Head.

[0268] FIG. 10 shows top views of the X-ray solution structure of the ADDobody construct ADDomer2_Head.

[0269] FIG. 11 shows top views of the X-ray solution structure of the ADDobody construct ADDomer2_Head without unit cell.

[0270] FIG. 12 shows side views of the X-ray solution structure of the ADDobody construct ADDomer2_Head.

[0271] FIG. 13 shows side views of the X-ray solution structure of the ADDobody construct ADDomer2_Head without unit cell.

DETAILED DESCRIPTION OF THE INVENTION

[0272] The present invention Is further illustrated by the following non-limiting examples:

Example 1

[0273] Cloning, Expression and Purification of Adenovirus Penton Base Crown Domain of Human Adenovirus Serotype 3 (hAd3)

[0274] The nucleotide sequence coding for an ADDobody of the invention (named ADDomer2_Head) equipped with a His-Tag sequence

TABLE-US-00017 (SEQ ID NO: 27) MSYYHHHHHHDYDIPTTENLYFQGAMGSGIQPNVNEYMESNKFKARVMVS RKAPEGVTVNDTYDHKEDILKYEWFEFILPEGNFSATMTIDLMNNAIIDN YLEIGRQNGVLESDIGVKFDTRNFRLGWDPETKLIMPGVYTYEAFHPDIV LLPGCGVDFTESRLSNLLGIRKRHPFQEGFKIMYEDLEGGNIPALLDVTA YEESKKDTTTETTTKKELKIQPLEKDSKSRSYNVLEDKINTAYRSWYLSY NYGNPEKGIRSWTLLTTSDVTCGANGDSGNPVFSKSFYNEQAVYSQQLRQ ATSLTHVFNRFPENQILIRPPAPTITTVSENVP
was cloned in expression vector pProEx using the restriction sites shown in FIG. 4.

[0275] The resulting vector has the following sequence (ORF start and stop shown in bold, coding sequence in lower characters):

TABLE-US-00018 (SEQ ID NO: 31) GTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGG AAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCAC TCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAAT GAGCTGTTGACAATTAATCATCCGGTCCGTATAATCTGTGGAATTGTGAGCGGATAACAATT TCACACAGGAAACAGACCATGTCGTACTACCATCACCATCACCATCACGATTACGATATCCC AACGACCGAAAACCTGTATTTTCAGGGCGCCATGGGATCCGGAATTCaaccgaacgtgaacg aatatatgtttagcaacaaatttaaagcgcgcgtgatggtgagccgcaaagcgccggaaggc gtgaccgtgaacgatacctatgatcataaagaagatattctgaaatatgaatggtttgaatt tattctgccggaaggcaactttagcgcgaccatgaccattgatctgatgaacaacgcgatta ttgataactatctggaaattggccgccagaacggcgtgctggaaagcgatattggcgtgaaa tttgatacccgcaactttcgcctgggctgggatccggaaaccaaactgattatgccgggcgt gtatacctatgaagcgtttcatccggatattgtgctgctgccgggctgcggcgtggatttta ccgaaagccgcctgagcaacctgctgggcattcgcaaacgccatccgtttcaggaaggcttt aaaattatgtatgaagatctggaaggcggcaacattccggcgctgctggatgtgaccgcgta tgaagaaagcaaaaaagataccaccaccgaaaccaccaccaaaaaagaactgaaaattcagc cgctggaaaaagatagcaaaagccgcagctataacgtgctggaagataaaattaacaccgcg tatcgcagctggtatctgagctataactatggcaacccggaaaaaggcattcgcagctggac cctgctgaccaccagcgatgtgacctgcggcgcgaacggcgatagcggcaacccggtgttta gcaaaagcttttataacgaacaggcggtgtatagccagcagctgcgccaggcgaccagcctg acccatgtgtttaaccgctttccggaaaaccagattctgattcgcccgccggcgccgaccat taccaccgtgagcgaaaacgtgccgTGATAAGCGGCCGCTTTCGAATCTAGAGCCTGCAGTC TCGAGGCATGCGGTACCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATA CAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGC GGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTG TGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTC GAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAA ATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGC CCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGC GTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAA ACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAA CTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA GCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATA ACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT ACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTAT TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT AAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATC TGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATA TATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTT TTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTT TTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGAT AGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAG CAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTG CGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGC CGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCG GTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAATTTTGTTAAAATTCGCGT TAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTAT AAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACT ATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCAC TACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGG AACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAA GGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGC GCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC AGGCTGCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTACCA GTCACGTAGCGATATCGGAGTGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGC GCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCG CTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCA CCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATTTACGTT GACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCA ATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCT CTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAA AAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGC GGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGC AAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATG GTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGT CAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCT GCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATT TTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCA AATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGC ATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCC ATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCT GGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCG TTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCG CCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCT GCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAA GAAAAACCACCCTGGCACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTA ATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG TGAGTTAGCGCGAATTGATCTG

[0276] The polypeptide was produced and purified as follows:

Buffers and Stock Solutions

[0277] 1000× Ampicillin stock: 1 g Ampicillin dissolved in 10 ml dH.sub.2O, filtered through a 0.2 μm filter [0278] 1 M IPTG stock: 2.4 g IPTG is dissolved in 10 ml dH.sub.2O and filtered through a 0.2 μm filter. [0279] 10×PBS buffer stock: [0280] 80 g NaCl [0281] 2 g KCl [0282] 7.62 g Na.sub.2HPO.sub.4 [0283] 0.77 g KH.sub.2PO.sub.4 in 1 L ultrapure H.sub.2O, set pH 7.4 [0284] 10×PBS is diluted 1:10 before use to get 1×PBS.

[0285] [In the following text, the expression AD3Head is used for ADDobody]

[0286] The expression and purification protocol is described per 1 L culture. It should be upscaled accordingly if more expression cultures are required. Normally, 3-5 L expression culture for one big prep should be made, and the purified protein stored frozen.

1. BL21(DE3) Bacteria Transformation

[0287] pProEX_HTB_AD3Head plasmid encoding for the ADDomer head or ‘crown’ domain (the ADDobody) is transformed into BL21(DE3) competent cells and plated onto LB agarose plate containing ampicillin as selection marker

1.1. Plate Preparation:

[0288] LB agarose is warmed in microwave oven [0289] Bottle is chilled under running tap water [0290] 1000× Ampicillin stock is added (100 μl to 100 ml LB agarose)

1.2 Transformation

[0291] BL21(DE3) cells are put on ice for 20 min to thaw [0292] 5 μl of plasmid DNA (˜0.67 μg) is added to the cells and incubated on ice for 1 h [0293] Heat shock is performed for 45 s at 42° C. and tube is chilled on ice for 10 min [0294] 500 μl sterile LB is added to the tube [0295] Cells are incubated for 1 h at 37° C. in incubator rotating at 180 rpm [0296] 100 μl of cell culture is plated onto LB agarose plate containing ampicillin and incubated at 37° C. overnight [0297] The remaining 400 μl of cells are incubated overnight at 37° C. in incubator rotating at 180 rpm and kept as backup if re-plating is needed

2. Bacterial Preculture

[0298] Single colony is inoculated to sterile flask containing 50 ml dYT media with 50 μl ampicillin [0299] Incubated at 37° C. overnight rotating at 180 rpm

3. Induction of Expression Cultures

[0300] 50 ml preculture at OD 3-4 is inoculated to 1 L prewarmed dYT medium containing 1 ml ampicillin (1000× stock) [0301] Cultures are incubated at 37° C. in incubator rotating at 180 rpm [0302] OD measurement is taken hourly until it reaches 1 OD again (takes about 3 h) [0303] Preinduction sample is taken from each shaker flask (see below) [0304] Then culture is induced with 1 ml IPTG stock and incubated at 30° C. for 3 h. [0305] Postinduction sample is taken from each shaker flask (see box below) Run SDS-PAGE gel (12%)

4. Cell Harvesting

[0306] 1 L culture is distributed to two plastic bottles and precisely balanced for centrifugation [0307] Centrifuge at 3500 G for 20 min at 4° C. [0308] Supernatant is discarded and pellet is resuspended in 20 ml used media, then transferred to 50 ml falcon tubes [0309] Centrifuge at 3000 G for 10 min in table-top centrifuge [0310] Media is discarded and Falcon tubes tapped in paper towel to remove rest of media [0311] Pellet is flash-frozen in liquid N.sub.2, stored at −20° C. until purification

5. Batch Purification by IMAC Using TALON Resin

[0312] Buffers: [0313] PBS (1×) (see above) [0314] High salt wash buffer: pH 7.4 (set on ice at 4° C.) [0315] 50 mM TRIS->0.622 g Trizma base [0316] 1 M KCl->7.45 g KCl [0317] 5 mM imidazole->0.034 g imidazole in final volume [0318] 100 ml dH.sub.2O [0319] Elution buffer: pH 7.4 (set on ice at 4° C.) [0320] 200 mM imidazole->1.36 g in final volume 100 ml [0321] 1×PBS [0322] Measure harvested pellet weight, add 10 ml 1×PBS per gram pellet and resuspend. [0323] Sonicate cells two times for 5 min: pulse sonication 10 s on, 15 s off (on ice) [0324] Centrifuge at 3000 G for 10 min [0325] Transfer supernatant to new falcons and centrifuge again at 3000 G for 10 min [0326] Cleared supernatant is loaded on equilibrated TALON resin (see below).

5.2. Resin Equilibration (HisPur TALON Resin)

[0327] 1 ml ‘slurry’ is added to a 15 ml falcon and washed with 5 ml dH.sub.2O, centrifuged at 1000 rpm for 5 min, supernatant is discarded [0328] The slurry is washed with 5 ml 1×PBS, centrifuged at 1000 rpm for 5 min, supernatant is discarded

5.3. Binding and Washing Steps

[0329] Cleared lysate is loaded on top of the equilibrated resin and incubated overnight at 4° C. in the cold room with constant rotation. [0330] Next day, centrifuge at 1000 rpm for 5 min and collect flow through (FT) [0331] Add High Salt Wash Buffer (7 ml) to the resin, incubate for 20 min at 4° C. rotating in the cold room. [0332] Falcon is centrifuged at 1000 rpm for 5 min, supernatant is collected (HS). [0333] Washing: 7 ml 1×PBS is added to the resin, then centrifuged at 1000 rpm for 5 min, supernatant is collected (W1). [0334] Washing step with 1×PBS is repeated two more times and supernatant is collected (W2, W3). [0335] Resin is resuspended in 1 mL 1×PBS and transferred to a 1.5 ml Eppendorf tube. Centrifuged at 1000 rpm for 5 min, and supernatant is discarded.

5.4 Elution

[0336] 5 ml Elution buffer is added to the resin, incubated for 20 min at 4° C. in the cold room while rotating. [0337] Tube is centrifuged at 13.000 rpm for 5 min, supernatant is collected to fresh tube (E1). [0338] 5 ml Elution buffer is added to the resin, incubated for 20 min at 4° C. in the cold room while rotating. [0339] Tube is centrifuged at 13000 rpm for 5 min, supernatant is collected to fresh tube (E2). [0340] 5 ml Elution buffer is added to the resin, incubated for 20 min at 4° C. in the cold room while rotating. [0341] Tube is centrifuged at 13000 rpm for 5 min, supernatant is collected to fresh tube (E3). [0342] Resin is resuspended in 1 ml elution buffer (‘Resin’). 12 ul taken. [0343] Samples (12 μl+4 μl PGLB) from each step are run on SDS-PAGE gel (12%).

6. Dialysis and TEV Cleavage

[0344] Protein concentration is measured with Nanodrop (A280) prior to dialysis and TEV cleavage (use Elution Buffer as blank to correct for imidazol). [0345] Take 12 ul sample (Before diaysis' sample). [0346] 20 μl TEV protease is added [0347] Dialyse in dialysis bag in the cold room against 1×PBS with 2 mM □-MeSH (□-MeSH must be handled under the fume hood). [0348] Change to new beaker with fresh 1×PBS w. □-MeSH after 30 min. [0349] Continue dialysis overnight at 4° C. [0350] Take 12 ul sample (After dialysis' sample). [0351] Recover sample comprising ADDobody, measure concentration of protein against dialysis buffer as blank, determine volume of sample and insert in Table I (see Annex).

7. Removing Uncut Protein and TEV (Reverse TALON)

[0352] Dialysed sample is loaded to 1 ml equilibrated TALON resin (equilibrate resin as described previously) and incubated for 1 h at 4° C. rotating in the cold room. [0353] Concentration is measured with Nanodrop. 12 ul sample taken (‘Before conc’). [0354] Sample is concentrated using am Amicon Millipore Protein concentrator

[0355] (MWCO 10 kDa). Centrifugation is performed at 3000 G at 4° C. to 500 ul. [0356] Take 12u1 sample (‘After conc’). [0357] Measure concentration with Nanodrop. Determine volume of sample and insert into Table I (Annex). [0358] Samples (12 μl+4 μl PGLB) from each step are run on SDS-PAGE gel (12%).

8. Reverse Ion Exchange Chromatography (IEX)

[0359] Ion exchange chromatography is performed with AKTA Pure system using Mono Q 5/50 GL column. [Remark: This is a ‘reverse’ IEX as ADDobody drops through the column (FT), impurities in contrast, are expected to bind.] [0360] Buffer A: 1×PBS (freshly made and filtered) [0361] Buffer B: 1×PBS with 1 M NaCl [0362] (for 1 L Buffer B weigh out 58.44 g (1 mol) NaCl and add to 1 L 1×PBS dissolve by stirring). [0363] Delta column pressure: 4.00 MPa [0364] Column is equilibrated prior to injection in 1×PBS (15 ml, i.e. 15 column volumes) [0365] Flow rate 1 ml/min [0366] Samples are collected with fractionator, fraction volume is 1 ml. [0367] Column is washed with Buffer A at 1 ml/min flow-rate [0368] 500 μl sample is centrifuged at 13.000 rpm in table-top centrifuge 12 ul probe taken (IEX IN) [0369] Sample injected. [0370] Linear gradient applied: [0371] 0-100% B in 20 min [0372] 100% B for 5 min [0373] 0% B for 5 min [0374] (0% B for 5 min is for reequilibrating the column). [0375] Collect in 1 ml fractions. [0376] ADDobody elutes in FT 1-10 ml elution volume (and a minor peak around 20 ml elution volume.)
An exemplary elution profile is shown in FIG. 5. [0377] Combine fractions which comprise ADDobody. Measure concentration with Nanodrop against Buffer A as blank. Determine volume of sample and insert into Table I (Annex).

9. Size Exclusion Chromatography (SEC)

[0378] Buffer A: 1×PBS (freshly made and filtered) [0379] Fractions from ion exchange runs corresponding to the main peak are pooled and concentrated with Amicon Millipore protein concentrator (MWCO 10 kDa) as described previously. [0380] Important: Be careful not to overconcentrate, the protein might precipitate. If you use 3-5 L expression culture, you might need to use two concentrators, monitor concentration in steps and make sure you do not see precipitation. [0381] Size exclusion chromatography is performed of 500 ul aliquots of concentrated sample with AKTA Pure system using HiLoad 16/600 Superdex 200 pg column. [0382] Delta column pressure: 0.3 MPa [0383] Column is equilibrated prior to injection with 1×PBS to total volume 5 ml [0384] Flow rate: 1 ml/min [0385] Sample application: loop is emptied with 2 ml [0386] Sample are collected with fractionation, fraction volume: 1 ml [0387] 500 μl sample aliquots are centrifuged at 13.000 rpm in table-top centrifuge. [0388] Take 12 ul probe of each injection (SEC IN). [0389] Inject the rest of the 500 ul.

[0390] An exemplary chromatogram is shown in FIG. 6 and the SDS polyacryl amide gel analysis of the peak fractions are shown in FIG. 7.

10. Freezing and Storage

[0391] Fractions from SEC corresponding to the major peak are pooled. [0392] Measure concentration with Nanodrop against Buffer A as blank. Determine volume of sample and insert into Table I (Annex). [0393] Take 12 ul sample (‘ConcFreeze IN’) [0394] Concentrate to 5 mg/ml final concentration using Amicon Millipore protein concentrator (MWCO 10 kDa). [0395] Take 12 ul sample (‘ConcFreeze OUT’)

[0396] Protein is flash-frozen in 100 μl aliquots in low protein binding tubes and stored at −20° C.

[0397] A SDS polyacrylamide gel analysis of the pooled fractions before and after freezing is shown in FIG. 8 (before freezing: Conc. In; after freezing: Conc. out) shows that the ADDobody construct ADDomer2_Head is stable.

Example 2

[0398] Crystallization and X-Ray Structure Determination of ADDobody Construct ADDomer2_Head

[0399] The truncated (i.e. maturated) polypeptide ADDomer2_Head (the amino acid sequence according to SEQ ID NO: 27 lacking the His tag-containing sequence

TABLE-US-00019 (SEQ ID NO: 39) MSYYHHHHHHDYDIPTTENLYF) (SEQ ID NO: 28) GAMGSGIQPNVNEYMFSNKFKARVMVSRKAPEGVTVNDTYDHKEDILKYE WFEFILPEGNFSATMTIDLMNNAIIDNYLEIGRQNGVLESDIGVKFDTRN FRLGWDPETKLIMPGVYTYEAFHPDIVLLPGCGVDFTESRLSNLLGIRKR HPFQEGFKIMYEDLEGGNIPALLDVTAYEESKKDTTTETTTKKELKIQPL EKDSKSRSYNVLEDKINTAYRSWYLSYNYGNPEKGIRSWTLLTTSDVTCG ANGDSGNPVFSKSFYNEQAVYSQQLRQATSLTHVFNRFPENQILIRPPAP TITTVSENVP
was crystalized and subjected to X-ray diffraction.

[0400] Crystallization conditions: sitting drop, PEG3350 20% (w/v), 0.1 M citrate pH 5.5, protein concentration: 5 mg/ml, drop size: 0.5 ul protein and 0.5 ul reservoir.

[0401] A representative single crystal is shown in FIG. 9. The best crystals diffracted to approximately 4.5 Å.

[0402] The X-ray diffraction gave the following results:

[0403] Spacegroup P1

[0404] a=102.124 b=103.837 c=177.578 α=92.308 β=94.314 γ=112.047

[0405] The solution structure is shown in FIGS. 10 to 13.

[0406] The unit cell contains 20 ADDobodies present in 2 decamers.

[0407] Importantly, the floor region of fragment A and the B loop of fragment B of the ADDobody forms a flexible structure in the isolated ADDobody

Example 3

[0408] Cloning, Expression and Purification of Adenovirus Penton Base Crown Domain of Chimpanzee Adenovirus

[0409] The nucleotide sequence coding for a further ADDobody of the invention (named ChimpCrown) equipped with a His-Tag sequence

TABLE-US-00020 (SEQ ID NO: 28) MSYYHHHHHHDYDIPTTENLYFQGTIMHTNMPNVNEFMYSNKFKARVMVS RKAPEGVTVNDTYDHKEDILEYEWVEFELPEGNFSVTMTIDLMNNAIIDN YLAVGRQNGVLESDIGVKFDTRNFRLGWDPVTELVMPGVYTNEAFHPDIV LLPGCGVDFTESRLSNLLGIRKRQPFQEGFQIMYEDLEGGNIPALLDVDA YEKSKKDTTTETTTKKELKIQPVEKDSKDRSYNVLPDKINTAYRSWYLAY NYGDPEKGVRSWTLLTTSDVTCGVEQAELLPVYSKSFFNEQAVYSQQLRA FTSLTHVFNRFPENQILVRPPAPTITTVSENVP
was cloned in expression vector pProEx as outlined in Example 1

[0410] Expression and purification was carried out according to the protocol of Example 1.

Example 4

[0411] Cloning, Expression and Purification of Adenovirus Penton Base Crown Domain of Human Adenovirus Serotype 3 (hAd3) Containing a Heterologous Insertion

[0412] The nucleotide sequence coding for a modified ADDobody of the invention containing the major neutralizing epitope from Chikungunya virus STKDNFNVYKATRPYLAH (SEQ ID NO: 40) in the B loop of fragment B and replacing the sequence HVFNRF (SEQ ID NO: 41) in the wild-type fragment B of hAd3 penton base (equipped with a His-Tag sequence)

TABLE-US-00021 (SEQ ID NO: 42) MSYYHHHHHHDYDIPTTENLYFQGAMGSGIQPNVNEYMFSNKFKARVMVS RKAPEGVTVNDTYDHKEDILKYEWFEFILPEGNFSATMTIDLMNNAIIDN YLEIGRQNGVLESDIGVKFDTRNFRLGWDPETKLIMPGVYTYEAFHPDIV LLPGCGVDFTESRLSNLLGIRKRHPFQEGFKIMYEDLEGGNIPALLDVTA YEESKKDTTTETTTKKELKIQPLEKDSKSRSYNVLEDKINTAYRSWYLSY NYGNPEKGIRSWTLLTTSDVTCGANGDSGNPVFSKSFYNEQAVYSQQLRQ ATSLTSTKDNFNVYKATRPYLAHPENQILIRPPAPTITTVSENVP
was cloned in expression vector pProEx as outlined in Example 1 Expression and purification was carried out according to the protocol of Example 1.

[0413] In summary, the present invention is particularly directed to the following items: [0414] 1. An isolated engineered polypeptide comprising the amino acid stretches essentially corresponding to a first and a second fragment of the penton base 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 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 domain 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). [0415] 2. The polypeptide of item 1 having the structure of the following general formula (I):

N-A-L-B-C (I) [0416] wherein [0417] 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; [0418] B represents an amino acid stretch corresponding to the C-terminal amino acid stretch of the adenovirus penton base inserted between the second and the third amino acid stretch forming the jellyroll fold domain of the adenovirus penton base; [0419] L represents a chemical group selected from the group consisting of an amino acid, an oligopeptide and a polypeptide; [0420] N may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide; [0421] C: may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide; [0422] wherein, optionally, fragment A and/or B contain(s) one or more heterologous modifications. [0423] 3. The polypeptide according to any one of items 2 wherein the fragment A comprises an amino acid sequence selected from the group consisting of the amino acid sequences according to the following table and amino acid sequences having an identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table:

TABLE-US-00022 Sequence Sequence N-terminal C-terminal based on according to amino acid amino acid 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 12 128 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, optionally, fragment A contains one or more heterologous modifications. [0424] 4. The polypeptide of item 2 or 3 wherein the fragment B comprises an amino acid sequence selected from the group consisting of the amino acid sequences according to the following table and amino acid sequences having an identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table:

TABLE-US-00023 Sequence Sequence N-terminal C-terminal based on according to amino acid amino acid penton base UniProt SEQ ID selected from selected from protomer of Acc. No. NO: positions positions hAd3 Q2Y0H9 1 441 to 444 491 to 494 hAd2 P03276 2 468 to 471 518 to 521 hAd4 Q2KSF3 3 422 to 445 465 to 468 hAd5 P12538 4 468 to 471 491 to 494 hAd7 Q9JFT6 5 441 to 444 464 to 467 hAd11 D2DM93 6 458 to 461 481 to 484 hAd12 P36716 7 394 to 398 418 to 421 hAd17 F1DT65 8 414 to 417 438 to 441 hAd25 M0QUK0 9 441 to 444 454 to 457 hAd35 Q7T941 10 498 to 501 521 to 524 hAd37 Q912J1 11 415 to 418 438 to 441 hAd41 F8WQN4 12 405 to 408 438 to 441 gorAd E5L3Q9 13 459 to 462 482 to 485 ChimpAd G9G849 14 421 to 424 444 to 457 sAd18 H8PFZ9 15 396 to 399 419 to 422 sAd20 F6KSU4 16 401 to 404 424 to 427 sAd49 F2WTK5 17 399 to 402 422 to 425 rhAd51 A0A0A1EWW1 18 395 to 398 418 to 421 rhAd52 A0A0A1EWX7 19 393 to 396 416 to 419 rhAd53 A0A0A1EWZ7 20 394 to 397 417 to 420
wherein, optionally, fragment B contains one or more heterologous modifications.
5. The polypeptide according to any one of items 2 to 4 characterized in that 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: [0425] the RGD loop region of fragment A; and/or [0426] the V-loop of fragment A; and/or [0427] the floor region having the sequence (from N- to C-terminal)

TABLE-US-00024 (SEQ ID NO: 21) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub.8-X.sub.9-S-Y-N-X.sub.10-X.sub.11-X.sub.12- X.sub.13-X.sub.14-X.sub.15-X.sub.16 [0428] of fragment A, wherein [0429] X.sub.1 is I or L, and is preferably I; [0430] X.sub.2 is selected from the group consisting of K, Q and E, and is preferably Q; [0431] X.sub.3 is P or A, and is preferably P, [0432] X.sub.4 is selected from the group consisting of L, V and I, and is preferably L [0433] X.sub.5 is selected from the group consisting of T, E, A, K and L, and is preferably E; [0434] X.sub.6 is selected from the group consisting of E, K, T and Q, and is preferably K; [0435] X.sub.7 is selected from the group consisting of S, P and D, and is preferably 5, [0436] X.sub.8 is selected from the group consisting of K, T and S, and is preferably K; [0437] X.sub.9 is selected from the group consisting of K, S, N, G and D, and is preferably S, [0438] X.sub.10 is L or V, and is preferably V; [0439] X.sub.11 is I or L, and is preferably I; [0440] X.sub.12 is selected from the group consisting of S, E and P, and is preferably E; [0441] X.sub.13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid; [0442] X.sub.14 is D or G, and is preferably ID, [0443] X.sub.15 is selected from the group consisting of S, K, Q and T, and is preferably K; and [0444] X.sub.16 is selected from the group consisting of T, N, I, K and M, and is preferably I; and/or [0445] the sequence (from N- to C-terminal) T-H-V-F-X.sub.17-R-F-P (SEQ ID NO: 22) of fragment B wherein X.sub.17 is D or N, and is preferably N. [0446] 6. The polypeptide of item 5 wherein the N-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00025 (SEQ ID NO: 23) X.sub.18-X.sub.19-X.sub.20-X.sub.21-X.sub.22-X.sub.23-X.sub.24-X.sub.25-X.sub.26
wherein [0447] X.sub.18 is selected from the group consisting of D, E and N, and is preferably D; [0448] X.sub.19 is selected from the group consisting of V, L, and is preferably V; [0449] X.sub.20 is any amino acid, preferably selected from the group consisting of A, O, E, K, S, and T, and is more preferably T; [0450] X.sub.21 is any amino add; preferably selected from the group consisting of A, D, E, and K, and is more preferably A; [0451] X.sub.22 is selected from the group consisting of F, Y, and IN, and is preferably Y; [0452] X.sub.23 is selected from the group consisting of A, D, E, N, and Q, is preferably E or Q, and is more preferably E; [0453] X.sub.24 is any amino acid, preferably selected from the group consisting of A, O, E, N, and K, and is more preferably E; [0454] X.sub.25 is selected from the group consisting of S or T, and is preferably S; and [0455] X.sub.26 is any amino acid and constitutes the N-terminal amino acid of the RGD loop region [0456] 7. The polypeptide of item 5 or 6 wherein the C-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00026 (SEQ ID NO: 24) X.sub.27-X.sub.28-X.sub.29-X.sub.30-X.sub.31-X.sub.32-X.sub.33-X.sub.34 [0457] Wherein [0458] X.sub.27 is any amino add and constitutes the C-terminal amino acid of the second RGD loop; [0459] X.sub.28 is selected from the group consisting of i, L and V, and is preferably I; [0460] X.sub.29 is selected from the group consisting of D, E, K, N, Q, and V, is preferably Q or K, and is more preferably Q; [0461] X.sub.30 is selected from the group consisting of C, G and P, and is preferably P; [0462] X.sub.31 is selected from the group consisting of 1, L and V, is preferably L or V and is more preferably L; [0463] X.sub.32 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; [0464] X.sub.33 is selected from the group consisting of D, E, S and T, is preferably E, or T, and is more preferably K; and [0465] X.sub.34 is selected from the group consisting of D and E, and is preferably D; [0466] 8. The polypeptide according to any one of items 5 to 7 wherein the N-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00027 (SEQ ID NO: 25) X.sub.35-X.sub.36-X.sub.37-X.sub.38-X.sub.39-X.sub.40-X.sub.41-X.sub.42 [0467] wherein [0468] X.sub.35 is selected from the group consisting of F, Y, and W, and is preferably F; [0469] X.sub.36 is selected from the croup consisting of H, K and R, and is preferably K; [0470] X.sub.37 is selected from the group consisting of A, V, i, and L, and is preferably A; [0471] X.sub.36 is selected from the group consisting of H, K, and R, and is preferably R; [0472] X.sub.39 is selected from the group consisting of A, V, I, and L, and is preferably V; [0473] X.sub.40 is selected from the group consisting of A, V, I, L and M, and is preferably M; [0474] X.sub.41 is selected from the group consisting of A, V, I, and L, and is preferably V; and [0475] X.sub.42 is any amino acid and constitutes the N-terminal amino acid of the V loop. [0476] 9. The polypeptide according to any one of items 5 to 8 wherein the C-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00028 (SEQ ID NO: 26) X.sub.43-X.sub.44-X.sub.45-X.sub.46-X.sub.47-X.sub.48-X.sub.49 [0477] wherein [0478] X.sub.43 is any amino acid and constitutes the C-terminal amino acid of the V loop; [0479] X.sub.44 is selected from the group consisting of F, Y, and W, and is preferably Y; [0480] X.sub.45 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; [0481] X.sub.46 is selected from the group consisting of F, Y, and W, and is preferably W; [0482] X.sub.47 is selected from the group consisting of A, F, V, Y, and W, is preferably F or V and is more preferably F; [0483] X.sub.48 is selected from the group consisting of D, E, S and T, is preferably D or E and is more preferably E; and [0484] X.sub.49 is selected from the group consisting of F, Y, and VV, and is preferably F. [0485] 10. The polypeptide according to any one of items 2 to 9 wherein the heterologous modification is selected from the group consisting of one or more single amino acid mutations in comparison to the wildtype sequence of fragment A and/or B, one or more replacements of wildtype amino acid stretches by one or more heterologous amino acids and/or amino acid stretches one or more insertions of heterologous amino acid stretches, one or more deletions of one or more amino acids and one or more amino acid modifications as well as any combination(s) thereof. [0486] 11. The polypeptide according to any one of items 2 to 10, wherein the heterologous modification provides a target specific binding entity. [0487] 12. The polypeptide of item 11, wherein the target specific binding entity is selected from the group consisting of antigens, epitopes, CDRs, antibodies, antibody fragments such as an antigen binding (Fab) fragment, a Fab′ fragment, a F(ab′)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, a paratope, an alternative scaffold protein, and a fusion protein thereof, a toxin and a venom. [0488] 13. The polypeptide according to any one of items 1 to 3 having one of the following amino acid sequences (from N-terminal to C-terminal):

TABLE-US-00029 (SEQ ID NO: 27) MSYYHHHHHHDYDIPTTENLYFQGAMGSGIQPNVNEYMFSNKFKARVMVS RKAPEGVTVNDTYDHKEDILKYEWFEFILPEGNFSATMTIDLMNNAIIDN YLEIGRQNGVLESDIGVKFDTRNFRLGWDPETKLIMPGVYTYEAFHPDIV LLPGCGVDFTESRLSNLLGIRKRHPFQEGFKIMYEDLEGGNIPALLDVTA YEESKKDTTTETTTKKELKIQPLEKDSKSRSYNVLEDKINTAYRSWYLSY NYGNPEKGIRSWTLLTTSDVTCGANGDSGNPVFSKSFYNEQAVYSQQLRQ ATSLTHVFNRFPENQILIRPPAPTITTVSENVP (SEQ ID NO: 32) GAMGSGIQPNVNEYMFSNKFKARVMVSRKAPEGVTVNDTYDHKEDILKYE WFEFILPEGNFSATMTIDLMNNAIIDNYLEIGRQNGVLESDIGVKFDTRN FRLGWDPETKLIMPGVYTYEAFHPDIVLLPGCGVDFTESRLSNLLGIRKR HPFQEGFKIMYEDLEGGNIPALLDVTAYEESKKDTTTETTTKKELKIQPL EKDSKSRSYNVLEDKINTAYRSWYLSYNYGNPEKGIRSWTLLTTSDVTCG ANGDSGNPVFSKSFYNEQAVYSQQLRQATSLTHVFNRFPENQILIRPPAP TITTVSENVP (SEQ ID NO: 28) MSYYHHHHHHDYDIPTTENLYFQGTIMHTNMPNVNEFMYSNKFKARVMVS RKAPEGVTVNDTYDHKEDILEYEWVEFELPEGNFSVTMTIDLMNNAIIDN YLAVGRQNGVLESDIGVKFDTRNFRLGWDPVTELVMPGVYTNEAFHPDIV LLPGCGVDFTESRLSNLLGIRKRQPFQEGFQIMYEDLEGGNIPALLDVDA YEKSKKDTTTETTTKKELKIQPVEKDSKDRSYNVLPDKINTAYRSWYLAY NYGDPEKGVRSWTLLTTSDVTCGVEQAELLPVYSKSFFNEQAVYSQQLRA FTSLTHVFNRFPENQILVRPPAPTITTVSENVP (SEQ ID NO: 33) QGTIMHTNMPNVNEFMYSNKFKARVMVSRKAPEGVTVNDTYDHKEDILEY EWVEFELPEGNFSVTMTIDLMNNAIIDNYLAVGRQNGVLESDIGVKFDTR NFRLGWDPVTELVMPGVYTNEAFHPDIVLLPGCGVDFTESRLSNLLGIRK RQPFQEGFQIMYEDLEGGNIPALLDVDAYEKSKKDTTTETTTKKELKIQP VEKDSKDRSYNVLPDKINTAYRSWYLAYNYGDPEKGVRSWTLLTTSDVTC GVEQAELLPVYSKSFFNEQAVYSQQLRAFTSLTHVFNRFPENQILVRPPA PTITTVSENVP [0489] 14. 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. [0490] 15. The polypeptide of item 14 having, from N- to C-terminal, the structure of the following general formula II:

N-A-C (II) [0491] wherein [0492] 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; [0493] N may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide; [0494] C: may or may not be present, and, if present, represents a chemical group consisting of an amino acid, an oligopeptide and a polypeptide; [0495] wherein, optionally, fragment A contain one or more heterologous modifications. [0496] 16. The polypeptide of item 15 wherein the fragment A comprises an amino acid sequence selected from the group consisting of the amino acid sequences according to the following table and amino acid sequences having an identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table:

TABLE-US-00030 Sequence Sequence N-terminal C-terminal based on according to amino acid amino acid 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 12 128 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, optionally, fragment A contains one or more heterologous modifications. [0497] 17. The polypeptide of item 15 or 16 characterized in that fragment A contains one or more heterologous modifications wherein said one or more heterologous modifications is/are contained in the following sites: [0498] the RGD loop region of fragment A; and/or [0499] the V-loop of fragment A; and/or [0500] the floor region having the sequence (from N- to C-terminal)

TABLE-US-00031 (SEQ ID NO: 21) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-D-X.sub.7-X.sub.8-X.sub.9-S-Y-N-X.sub.10-X.sub.11-X.sub.12- X.sub.13-X.sub.14-X.sub.15-X.sub.16 [0501] of fragment A, wherein [0502] X.sub.1 is I or L, and is preferably I; [0503] X.sub.2 is selected from the group consisting of K, Q and E, and is preferably Q; [0504] X.sub.3 is P or A, and is preferably P, [0505] X.sub.4 is selected from the group consisting of L, V and I, and is preferably L [0506] X.sub.5 is selected from the group consisting of T, E, A, K and L, and is preferably E; [0507] X.sub.6 is selected from the group consisting of E, K, T and Q, and is preferably K; [0508] X.sub.7 is selected from the group consisting of S, P and D, and is preferably S; [0509] X.sub.8 is selected from the group consisting of K, T and S, and is preferably K; [0510] X.sub.9 is selected from the group consisting of K, S, N, G and D, and is preferably S; [0511] X.sub.10 is L or V, and is preferably V; [0512] X.sub.11 is I or L, and is preferably I; [0513] X.sub.12 is selected from the group consisting of S, E and P, and is preferably E; [0514] X.sub.13 is no amino acid (i.e. not present) or is N, and is preferably no amino acid; [0515] X.sub.14 is D or G, and is preferably ID, [0516] X.sub.15 is selected from the group consisting of S, K, Q and T, and is preferably K; and [0517] X.sub.16 is selected from the group consisting of T, N, I, K and M, and is preferably I; and/or [0518] the sequence (from N- to C-terminal) T-H-V-F-X.sub.17-R-F-P (SEQ ID NO: 22) of fragment B wherein X.sub.17 is D or N, and is preferably N. [0519] 18. The polypeptide of item 17 wherein the N-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00032 (SEQ ID NO: 23) X.sub.18-X.sub.19-X.sub.20-X.sub.21-X.sub.22-X.sub.23-X.sub.24-X.sub.25-X.sub.26
wherein) [0520] X.sub.18 is selected from the group consisting of D, E and N, and is preferably D; [0521] X.sub.19 is selected from the group consisting of V, L, and is preferably V; [0522] X.sub.20 is any amino acid, preferably selected from the group consisting of A, D, E, K, S, and T, and is more preferably T; [0523] X.sub.21 is any amino add, preferably selected from the group consisting of A, D, E, and K, and is more preferably A; [0524] X.sub.22 is selected from the group consisting of F, Y, and W, and is preferably Y; [0525] X.sub.23 is selected from the group consisting of A, D, E, N, and Q, is preferably E or Q, and is more preferably E; [0526] X.sub.24 is any amino acid, preferably selected from the group consisting of A, D, E, N, and K, and is more preferably E, [0527] X.sub.25 is selected from the group consisting of S or T, and is preferably S; and [0528] X.sub.26 is any amino acid and constitutes the N-terminal amino add of the RGD loop region [0529] 19. The polypeptide of item 17 or 18 wherein the C-terminus of the RGD loop region of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00033 (SEQ ID NO: 24) X.sub.27-X.sub.28-X.sub.29-X.sub.30-X.sub.31-X.sub.32-X.sub.33-X.sub.34 [0530] Wherein [0531] X.sub.27 is any amino add and constitutes the C-terminal amino add of the second RGD loop; [0532] X.sub.28 is selected from the group consisting of I, L and V, and is preferably I; [0533] X.sub.29 is selected from the group consisting of D, E, K, N, Q, and V, is preferably or K, and is more preferably Q; [0534] X.sub.30 is selected from the group consisting of C, G and P, and is preferably P; [0535] X.sub.31 is selected from the group consisting of I, L and V, is preferably L or V and is more preferably L; [0536] X.sub.32 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; [0537] X.sub.33 is selected from the group consisting of D, E, S and T, is preferably E, or T, and is more preferably K; and [0538] X.sub.34 is selected from the group consisting of 0 and E, and is preferably D; [0539] 20. The polypeptide according to any one of items 17 to 19 wherein the N-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00034 (SEQ ID NO: 25) X.sub.35-X.sub.36-X.sub.37-X.sub.38-X.sub.39-X.sub.40-X.sub.41-X.sub.42 [0540] wherein [0541] X.sub.35 is selected from the group consisting of F, Y, and W, and is preferably F; [0542] X.sub.36 is selected from the group consisting of H, K and R, and is preferably K; [0543] X.sub.37 is selected from the croup consisting of A, V, I, and L, and is preferably A; [0544] X.sub.38 is selected from the group consisting of H, K, and R, and is preferably R; [0545] X.sub.39 is selected from the group consisting of A, V, I, and L, and is preferably V; [0546] X.sub.40 is selected from the group consisting of A, V, I, L and M, and is preferably M; [0547] X.sub.41 is selected from the group consisting of A, V, I, and L, and is preferably V; and [0548] X.sub.42 is any amino acid and constitutes the N-terminal amino acid of the V loop. [0549] 21. The polypeptide according to any one of items 17 to 20 wherein the C-terminus of the V loop of fragment A is defined by the following sequence (from N-terminal to C-terminal):

TABLE-US-00035 (SEQ ID NO: 26) X.sub.43-X.sub.44-X.sub.45-X.sub.46-X.sub.47-X.sub.48-X.sub.49 [0550] wherein [0551] X.sub.43 is any amino acid and constitutes the C-terminal amino acid of the V loop; [0552] X.sub.44 is selected from the group consisting of F, Y, and W, and is preferably Y; [0553] X.sub.45 is selected from the group consisting of D, E, S and T, is preferably E or T and is more preferably E; [0554] X.sub.46 is selected from the group consisting of F, Y, and W, and is preferably W; [0555] X.sub.47 is selected from the group consisting of A, F, V, Y, and W, is preferably F or V and is more preferably F; [0556] X.sub.48 is selected from the group consisting of D, E, S and T, is preferably D or E and is more preferably E; and [0557] X.sub.49 is selected from the group consisting of F, Y, and IN, and is preferably F. [0558] 22. The polypeptide according to any one of items 15 to 21 wherein the heterologous modification is selected from the group consisting of one or more single amino acid mutations in comparison to the wildtype sequence of fragment A, one or more replacements of wildtype amino acid stretches by one or more heterologous amino acids and/or amino acid stretches one or more insertions of heterologous amino acid stretches, one or more deletions of one or more amino acids and one or more amino acid modifications as well as any combination(s) thereof. [0559] 23. The polypeptide according to any one of items 15 to 22, wherein the heterologous modification provides a target specific binding entity. [0560] 24. The polypeptide of item 23, wherein the target specific binding entity is selected from the group consisting of antigens, epitopes, CDRs, antibodies, antibody fragments such as an antigen binding (Fab) fragment, a Fab′ fragment, a F(ab″)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, a paratope, an alternative scaffold protein, and a fusion protein thereof, a toxin and a venom. [0561] 25. A nucleic acid encoding a polypeptide according to any one of the preceding items. [0562] 26. A vector comprising the nucleic acid of item 25. [0563] 27. The vector of item 26 containing the nucleic acid of item 15 within an expression cassette. [0564] 28. A recombinant host cell comprising the nucleic acid of item 24 or the vector of item 26 or 27. [0565] 29. A method for the production of a polypeptide according to any one of items 1 to 24 comprising the step of culturing the host cell of item 28 containing the vector of item 28 under conditions allowing the expression of said polypeptide. [0566] 30. The method of item 29 further comprising the step of purifying the polypeptide from the cultured host cells. [0567] 31. An engineered adenovirus penton base protein comprising the polypeptide according to any one of items 5 to 12 fused to the multimerization domain (jellyroll fold domain) of an adenovirus penton base protein. [0568] 32. The penton base protein of item 31 having, from N- to C-terminal, the structure of the following general formula III:

D-A-E-B-F (III) [0569] wherein A and B are the fragments of the alpha-helical crown domain as defined in any one of items 2 to 4, and D, E and F are the amino acid sequences of an adenovirus penton base forming the multimerization (jellyroll fold) domain, wherein one or more heterologous modifications is/are present in the floor region of fragment A and/or in the B loop of fragment B. [0570] 33. An engineered adenovirus penton base protein comprising the polypeptide according to any one of item 15 to 24 fused to the multimerization domain (jellyroll fold domain) of an adenovirus penton base protein. [0571] 34. The penton base of item 33 having, from N- to C-terminal, the structure of the following general formula IV:

D-A-E-Li-F (IV) [0572] wherein A is the large fragment of the alpha-helical crown domain as defined in any one of items 15 or 16, and D, E and F are the amino acid sequences of an adenovirus penton base forming the multimerization (jellyroll fold) domain, wherein, optionally and preferably, one or more heterologous modifications is/are present in the floor region of fragment A, and wherein Li is a linker selected from peptides, oligopeptides, polypeptides, proteins and protein complexes. [0573] 35. A penton base of item 32 or 34 wherein fragment D of general formula (III) or (IV) has the following consensus sequence (SEQ ID NO: 34):

(U).sub.1-47 PTJ.sub.1GRNSIRY SJ.sub.2J.sub.3x.sub.4PJ.sub.5J.sub.6DTT J.sub.7J.sub.BYLVDNKSA DIASLNYQND HSNFJ.sub.5TTVJ.sub.9Q NNDJ.sub.10J.sub.11PJ.sub.12EAJ.sub.13 TQT INJ.sub.14DJ.sub.15RS RWGJ.sub.16L-T.sub.17LKTIJ.sub.18 J.sub.19TZ.sub.1Z.sub.2Z.sub.3Z.sub.4Z.sub.5Z.sub.6Z.sub.7Z.sub.8 Z.sub.9Z.sub.10Z.sub.11Z.sub.12Z.sub.13Z.sub.14Z.sub.15 [0574] wherein: fragment D ends on the C-terminal side before Z.sub.1 at residue T or at an amino acid from Z.sub.1 to Z.sub.15 [0575] U is any or no amino acid [0576] J.sub.1 is E or G [0577] J.sub.2 is E or S [0578] J.sub.3 is L or V [0579] J.sub.4 is A or S [0580] J.sub.5 is L or Q [0581] J.sub.6 is Y or E [0582] J.sub.7 is R or K [0583] J.sub.8 is V or L [0584] J.sub.9 is V or I [0585] J.sub.10 is F or Y [0586] J.sub.11 is T or S [0587] J.sub.12 is A or T or I or G [0588] J.sub.13 is S or G [0589] J.sub.14 is F or L [0590] J.sub.15 is E or D [0591] J.sub.16 is A or G [0592] J.sub.17 is D or Q [0593] J.sub.18 is L or M [0594] J.sub.19 is H or R [0595] Z.sub.1, if present, is N [0596] Z.sub.2, if present, is M [0597] Z.sub.3, if present, is P [0598] Z.sub.4, if present, is N [0599] Z.sub.5, if present, is V or I [0600] Z.sub.6, if present, is N [0601] Z.sub.7, if present, is E or D [0602] Z.sub.8, if present, is Y or F [0603] Z.sub.9, if present, is M [0604] Z.sub.10, if present, is F or S or Y [0605] Z.sub.11, if present, is T or S [0606] Z.sub.12, if present, is S or N [0607] Z.sub.13, if present, is K [0608] Z.sub.14, if present, is F [0609] Z.sub.16, if present, is K. [0610] 36. The penton base of item 35 wherein the fragment D comprises an amino acid sequence selected from the group consisting of the amino acid sequences of the following table and amino acid sequences having and identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table:

TABLE-US-00036 Sequence Sequence N-terminal C-terminal based on according to amino acid amino acid penton base UniProt SEQ ID selected from selected from protomer of Acc. No. NO: positions positions hAd3 Q2Y0H9 1 1 to 48 129 to 144 hAd2 P03276 2 1 to 48 129 to 144 hAd4 Q2KSF3 3 1 to 44 125 to 140 hAd5 P12538 4 1 to 48 129 to 144 hAd7 Q9JFT6 5 1 to 48 129 to 144 hAd11 D2DM93 6 1 to 48 129 to 144 hAd12 P36716 7 1 to 38 119 to 134 hAd17 F1DT65 8 1 to 35 116 to 131 hAd25 M0QUK0 9 1 to 43 124 to 139 hAd35 Q7T941 10 1 to 49 130 to 145 hAd37 Q912J1 11 1 to 35 116 to 131 hAd41 F8WQN4 12 1 to 46 127 to 142 gorAd E5L3Q9 13 1 to 49 130 to 145 ChimpAd G9G849 14 1 to 44 125 to 140 sAd18 H8PFZ9 15 1 to 46 127 to 142 sAd20 F6KSU4 16 1 to 45 126 to 141 sAd49 F2WTK5 17 1 to 48 127 to 142 rhAd51 A0A0A1EWW1 18 1 to 43 124 to 139 rhAd52 A0A0A1EWX7 19 1 to 43 124 to 139 rhAd53 A0A0A1EWZ7 20 1 to 44 125 to 140 [0611] 37. The penton base according to any of items 32 to 36 fragment E of general formula (III) or (IV) has the following sequence (SEQ ID NO: 35):

Z.sub.17Z.sub.18Z.sub.19Z.sub.20Z.sub.21Z.sub.22Z.sub.23Z.sub.24Z.sub.25Z.sub.26 Z.sub.27QVYWSLPDJ.sub.20 MJ.sub.21DPVTFRST J.sub.22QJ.sub.23J.sub.24NJ.sub.25PVVGJ.sub.26 ELZ.sub.28Z.sub.29Z.sub.30 [0612] wherein: fragment E begins on the N-terminal side at an amino acid from Z.sub.17 to Z.sub.27 or at amino acid Q after Z.sub.27, [0613] amino acid stretch B ends on the C-terminal side before Z.sub.28 at amino acid L or at an amino acid from Z.sub.28 to Z.sub.30; [0614] Z.sub.17, if present, is L or S [0615] Z.sub.18, if present, is T or P or C [0616] Z.sub.19, if present, is T or P [0617] Z.sub.20, if present, is P or S or A or R [0618] Z.sub.21, if present, is N or D [0619] Z.sub.22, if present, is G or V [0620] Z.sub.23, if present, is H or T [0621] Z.sub.24, if present, is C [0622] Z.sub.25, if present, is G [0623] Z.sub.28, if present, is A or V or S [0624] Z.sub.27, if present, is E or Q [0625] J.sub.20 is L or M [0626] J.sub.21 is Q or K [0627] J.sub.22 is Q or R or S [0628] J.sub.23 is V or I [0629] J.sub.24 is S or N [0630] J.sub.25 is Y or F [0631] J.sub.28 is A or V [0632] Z.sub.28, if present, is M or L [0633] Z.sub.29, if present, is P [0634] Z.sub.30, if present, is V or F. [0635] 38. The penton base of item 37 wherein the fragment E comprises an amino acid sequence selected from the group consisting of the amino acid sequences of the following table and amino acid sequences having and identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table:

TABLE-US-00037 Sequence Sequence N-terminal C-terminal based on according to amino acid amino acid penton base UniProt SEQ ID selected from selected from protomer of Acc. No. NO: positions positions hAd3 Q2Y0H9 1 398 to 409 440 to 443 hAd2 P03276 2 425 to 436 467 to 470 hAd4 Q2KSF3 3 379 to 390 421 to 444 hAd5 P12538 4 425 to 436 467 to 470 hAd7 Q9JFT6 5 398 to 409 440 to 443 hAd11 D2DM93 6 415 to 426 457 to 460 hAd12 P36716 7 351 to 362 393 to 397 hAd17 F1DT65 8 370 to 381 413 to 416 hAd25 M0QUK0 9 388 to 399 440 to 443 hAd35 Q7T941 10 445 to 456 497 to 500 hAd37 Q912J1 11 372 to 383 414 to 417 hAd41 F8WQN4 12 362 to 373 404 to 407 gorAd E5L3Q9 13 416 to 427 458 to 461 ChimpAd G9G849 14 372 to 383 420 to 423 sAd18 H8PFZ9 15 353 to 364 395 to 398 sAd20 F6KSU4 16 358 to 369 400 to 403 sAd49 F2WTK5 17 356 to 367 398 to 401 rhAd51 A0A0A1EWW1 18 352 to 363 394 to 397 rhAd52 A0A0A1EWX7 19 350 to 361 392 to 395 rhAd53 A0A0A1EWZ7 20 351 to 362 393 to 396 [0636] 39. The penton base according to any one of items 32 to 38 wherein fragment F of general formula (III) or (IV) has the following sequence (SEQ ID NO: 36):

Z.sub.31Z.sub.32Z.sub.33ALTDHGT LPLRSSIJ.sub.27GV QRVTJ.sub.28TDARR RTCPYVYKA LGIVJ.sub.30PJ.sub.31VLS SRTF [0637] wherein: fragment F begins on the N-terminal side at an amino acid from Z.sub.31 to Z.sub.33 or at amino acid A after Z.sub.33; [0638] Z.sub.31, if present, is N [0639] Z.sub.32, if present, is V [0640] Z.sub.33, if present, is P [0641] J.sub.27 is R or S or G [0642] J.sub.28 is V or I [0643] J.sub.29 is Y or H [0644] J.sub.30 is A or S [0645] J.sub.31 is R or K [0646] 40. The penton base of item 39 wherein fragment F comprises an amino acid sequence selected from the group consisting of the amino acid sequences of the following table and amino acid sequences having and identity of at least 85%, more preferred at least 90%, even more preferred 95%, particularly preferred at least 98%, most preferred at least 99%, with the respective amino acid sequence shown in the following table:

TABLE-US-00038 Sequence Sequence N-terminal based on according to amino acid C-terminal penton base UniProt SEQ ID selected from amino acid protomer of Acc. No. NO: positions position hAd3 Q2Y0H9 1 492 to 495 544 hAd2 P03276 2 519 to 522 571 hAd4 Q2KSF3 3 466 to 469 535 hAd5 P12538 4 492 to 495 571 hAd7 Q9JFT6 5 465 to 468 544 hAd11 D2DM93 6 482 to 485 561 hAd12 P36716 7 419 to 422 497 hAd17 F1DT65 8 438 to 441 517 hAd25 M0QUK0 9 455 to 458 534 hAd35 Q7T941 10 522 to 525 561 hAd37 Q912J1 11 439 to 442 519 hAd41 F8WQN4 12 439 to 432 508 gorAd E5L3Q9 13 483 to 486 875 ChimpAd G9G849 14 445 to 458 532 sAd18 H8PFZ9 15 420 to 423 508 sAd20 F6KSU4 16 425 to 428 512 sAd49 F2WTK5 17 423 to 426 511 rhAd51 A0A0A1EWW1 18 419 to 422 505 rhAd52 A0A0A1EWX7 19 417 to 420 503 rhAd53 A0A0A1EWZ7 20 418 to 421 504 [0647] 41. A pentameric complex of the engineered adenovirus penton base protein according to any one of items 32 to 40. [0648] 42. A virus-like particle (VLP) comprising 12 pentameric complexes of item 41. [0649] 43. The polypeptide according to any one of items 1 to 24 or the VLP of item 42 for use as a medicament. [0650] 44. A pharmaceutical composition comprising a polypeptide according to any one of items 1 to 24 or the engineered adenovirus penton base protein according to any one of items 32 to 40 or the VLP of item 42, optionally together with at least one pharmaceutically acceptable carrier, excipient and/or diluent. [0651] 45. A method for producing the VLP of item 42 comprising the step of incubating a solution of a penton base according to any one of items 32 to 40 under conditions allowing the assembly of the polypeptide into a VLP. [0652] 46. The polypeptide according to any one of items 1 to 24 or the VLP of item 42 for use in the treatment and/or prevention of an infectious disease, an immune disease, tumor or cancer. [0653] 47. A method of identifying a binding sequence to a target molecule comprising the steps of: [0654] (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 as defined in any one of items 5 to 9, 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 [0655] (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 as defined in any one of items 17 to 21, 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 [0656] (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; [0657] (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 [0658] (iv) detecting which polypeptide(s) have/has bound to the target molecule. [0659] 48. The method of item 47 further comprising the step of determining the dissociation constant(s) (K.sub.d) of the polypeptide(s) bound to the target molecule.

Engineered Polypeptides Derived From Variable Domain of Adenovirus Penton Base

Inventors

Cpc classification

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C12N2710/10322

CHEMISTRY; METALLURGY

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C07K14/005

CHEMISTRY; METALLURGY

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C12N2710/10333

CHEMISTRY; METALLURGY

Classification Explorer

C12N15/1055

CHEMISTRY; METALLURGY

Classification Explorer

C12N2710/10323

CHEMISTRY; METALLURGY

Classification Explorer

C07K2319/21

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Classification Explorer

A61K38/00

HUMAN NECESSITIES

International classification

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C07K14/005

CHEMISTRY; METALLURGY

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C12N15/10

CHEMISTRY; METALLURGY

Abstract

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