PT85226B - PROCESS FOR THE PREPARATION OF NEW THROMBOLITIC PROTEINS - Google Patents

Abstract

A thrombolytic protein (I) having tissue plasminogen activator-type activity is characterised by a peptide sequence the same as that of human t-PA in which (a) Arg-275 is deleted or is replaced by a different amino acid and (b) at least one of the consensus N-linked glycosylation sites is modified to other than a consensus N-linked glycosylation site. Also claimed is a thrombolytic protein having tPA-type activity characterised by a peptide sequence the same as that of human t-PA,in which one or more amino acids in the region Gly-(-3) to Thr-91 are deleted or replaced with different.

Description

The present invention describes substances that have a plasminogen activating (t-PA) type action on tissues. More specifically, the present invention relates to recombinant thrombolytic proteins, a process for obtaining these proteins through cells treated by genetic processes and the therapeutic use of said substances as thrombolytic agents.

These proteins are active thrombolytic agents which are considered to have an improved fribrinolytic profile compared to natural human t-PA. This fact is evident in the presence of increased affinity for fibrin, decreased reactivity with t-PA inhibitors, a faster rate of thrombolysis, increased fibrinolytic activity and / or extended biological life span. It is also considered that the proteins of the present invention can be more conveniently prepared in a more homogeneous form than is natural human t-PA. For these proteins, an improved total pharmacokinetic profile is considered.

The structure of natural human t-PA can be considered to have an amino terminal (N-) of about 91 amino acid residues, two regions called

”Kringle ^M and, at the carboxylic terminal, a domain of the type

rhin protease. The N- terminal has been found to contain several subdomains that play functional roles, among others, in fibrin binding and in vivo protein clearance. Recently, it has been described the recovery of another form of t-PA that lacks the N-natural terminal and the first “Kringle” region, see published European patent application N? 019092O (published on October 8, 1986). According to this report, the truncated form of t-PA, which starts with Ala-160 of natural human t-PA, is active from a fibrinolytic point of view.

As described in detail below, the present invention provides novel human t-PA protein analogs that retain both natural human t-PA Kringle regions, but which contain N-terminal modifications. While certain aspects and modifications involve losses in the N- terminal, the first Kringle region ”remains intact and the loss in the N- terminal is never greater than 94 amino acids. Most aspects involve loss (s) or substitution (¿^ Due to the fact that they have most of the structure of natural human t-PAs, it is found that the proteins of the present invention selectively retain more desirable biological activities of t-PAs natural human protein and less immunogenicity than more strongly modified analogs of t-PA, therefore, the proteins of the present invention are considered to have better fibrinolytic and pharmacokinetic profiles compared to natural human t-PA and truncated t-PA in the Ala-160 region,

as well as other modified forms of t-PA

Spinal polypeptide of natural human t-PA also includes four Asn consensus regions linked to glycosylation sites. Two of these sites have been found to be typically glycosylated in the t-PA of mammalian cells derived from melanoma, i.e., Asn-117 and Asn-MiS. The Asn-184 region is glycosylated at times, while the Asn-218 region is not normally glycosylated. T-PA derived from mammalian melanoma cells, for example, Bowes cells, is also referred to herein as natural or native human t-PA.

The present invention, as mentioned above, involves new protein analogs of human t-PA that have thrombolytic activity of the type of t-PA. Proteins from

the present invention differs from human t-PA in its structure in that they contain changes in the peptide sequence (i) up to three Asn-linked glycosisation sites present in the natural t-PA; (ii) at the N- terminal of the proteins, which correspond to the 94 amino acids of the N-mature terminal of natural t-PA; and / or (iii) at the proteolytic fission site. between the Arg-275 and Ile-276 regions. These aspects of the proteins of the present invention are described in more detail below. Despite the various modifications, numerous amino acids remain as shown in Table 1 through the sequences represented by a one-letter code.

A. Modifications to the N- terminal

One aspect of this invention concerns proteins

in those that are characterized by the loss of one to 94 amino acids in the peptide region between Gly - (- 3) or Ser-1 through Thr-91, in relation to natural human t-PA. In aspect, p. ex. Cys-51 through Asp-87 are eliminated in natural t-PA. In other specific aspects, from Cys-6 to Ser-50 and Cys-6 to Ile-86, respectively, are eliminated.

More conservative modifications are present, in other aspects, in the N-terminal region of the proteins. For example, certain proteins of the present invention contain one or more amino acid deletions or substitutions in one or more of the subregions. more discrete options:

region in up until region in up until 1 Gly - (- 3) Gln-3 7 Cys-34 Cys-43 2 Go-4 Lys-10 8 His-44 Ser-50 3 Thr-11 His-18 9 Cys-51 Cys-62 4 Gln-19 Leu-22 10 Gln-63 Val-72 5 Arg-23 Arg-27 11 Cys-73 Cys-84 6 Ser-28 Tyr-33 12 Glu-85 Thr-91

These and other changes in the amino terminal between Gly - (- 3) and Thr-91 are described in detail below.

B, Modifications to glycosylation sites linked to nitrogen atoms

The protein variants of the present invention may also contain no carbohydrate radicals attached to nitrogen atoms or may be only partially glycosylated.

related to natural human t-PA. A partially glycosylated protein as used herein, means a protein that contains fewer carbohydrate radicals attached to nitrogen atoms than the fully glycosylated natural human t-PA protein. This absence of glycosylation or only partial glycosylation results from the substitution of amino acids or the elimination of amino acids at one or more glycosylation recognition sites attached to nitrogen atoms present in the natural t-PA molecule. Check. it has been found that proteins, variants of the present invention, encompass those modifications at one or more N-linked glycosylation sites, retain th-PA-type thrombolytic activity with greater fibrinolytic activity in certain cases, can be produced more rapidly in a more homogeneous form than native t-PA and, in many cases, have longer half-lives than natural t-PA.

It is now accepted that glycosylation recognition sites linked to nitrogen atoms include three tripeptide sequences that are specifically recognized by appropriate cell glycosylation enzymes. These tripeptide sequences are either asparagine-X-trionine or asparagine-X-serine, where the symbol X usually represents any amino acid. Their location in the t-PA peptide sequence is shown in Table 1. A variety of amino acid substitutions or deletions at one or more of the positions on the glycosylation recognition sites result in modified non-glycosylated sequences. As an example ^Asn 117 and Asn. _ς i, from t-PA were both substituted1 <í4

with the case of

Thr in one case and with Gin in another. At least in the double substitution of Gin, the resulting glycoprotein (α ^χη ₁₁₇ cattle at

Gln ^ y ^) will contain only one hydrocarbon radical li (in the Asn ^^ g region) instead of two or three radiazoto quays, as is the case in the case of natural t-PA. Those skilled in the art will find that analogous glycoproteins that have the same monoglycosylations in Asn ^ g can be prepared from amino acids by eliminating or replacing other amino acids at positions 117 θ or by substituting one or more amino acids and / or by elimination in other positions at the respective glycosylation recognition sites, eg, in the medium of one or more

S ^er n9 and Ser ^ g ^, as mentioned above, and / or by substitution or preferably by elimination at one of the X · 'positions of the tripeptide sites. In other cases the positions

Gin. The bonate variants linked to

117, 184 and 448, Asn are replaced by the resulting ones will not contain radicals to hydrocarbon atoms, preferably to the two or three radicals existing in the case of natural t-PA. In other cases potential glycosylation sites have been modified individually eg, by replacing Asn, eg with Gin, in position 117, in the presently preferred aspect in position 184 and in another aspect in position 448, in yet another case . The present invention encompasses such non-glycosylated, mono-glycosylated, diglycosylated and triglycosylated variants of t-PA.

Exemplary modifications in one or more of the three glycosylation sequences linked to an N atom represented by R _q , R _o and R_, as seen in X A. J several aspects

aspects of the present invention are represented below:

Examples of modifications of glycosylation sites linked to an N atom

^R 1 ^R 2 ^R 3 (t.s. ,) (Asn To be To be) (Asn Gly Ser) (Asn Arg Thr) I U To be To be V Gly Ser V Arg Thr II Asn W To be Asn X Ser Asn Y Thr III Asn To be Z Asn Gly Z Asn Arg U IV Asn W Z Asn X Z Asn Y U V - U * - * - - * - SAW - Asn * - * * - * (x VII U s (* - - * - - . X VIII Asn * - * * - * » AND IX - - - - _ - - - - J · * - and --- = an Link peptide * = any amino acid u = any amino acid except Asn, Thr or To be V = 11 II 11 Asn, or a Link peptide w = II II II Being, or a Link peptide X = II 11 II Gly, or a Link peptide Y = II II 11 Arg, or a Link peptide z = II II 11 Thr or Ser or a li peptide generation

t.s. = wild type i.e., before mutagenesis

C. Modifications at the Arg-275 / lle-27b cleavage sites.

In one aspect of the present invention, variants are eventually modified at the proteolytic cleavage site between Arg-275 and Ile-276 by virtue of the elimination of Arg-275 ^or the replacement of any other amino acid, preferably an amino acid other than lysine or histidine, for Arginine. In several aspects of this invention Thr is especially preferred for the substitution of the Arg-275 amino acid. The proteolytic cleavage in Arg-275 of natural t-PA produces a molecule called a double chain as it is known in the art. The proteins of the present invention, which are characterized by changes in this cleavage site, can be prepared more easily, in a more homogeneous way than the corresponding protein, without modification of the cleavage site and, probably, more importantly, may have a better fibrinolytic and pharmacokinetic profile.

The present invention, therefore, provides a family of new, thrombolytic proteins related to human t-PA. These proteins include analogs of t-PA characterized by various modifications or associations of modifications, as mentioned above, which may also contain other variations, for example, allyl variations or addional deletions, substitution or insertions of amino acids that still function. thrombolytic activity until the DNA encoding those proteins (prior to the modifications of the present invention) is still able to hybridize to a DNA sequence encoding human t-PA under restricted conditions. This family comprises several genera of proteins .

- Q -r '/

L l

One of the protein genera is characterized by a peptide sequence equal to or substantially the same as the human t-PA peptide sequence in which Arg-275 has been deleted or replaced by a different amino acid, preferably different from lysine or histidine, and at least at least one or more of the consensus-linked Asn glycosylation sites is either deleted or modified by another to another consensus-linked Asn glycosylation sequence. Examples of proteins of this kind are shown in Table 1 below. As used herein the phrase characterized by a peptide sequence equal to or substantially the same as the human t-PA peptide sequence, means the human t-PA peptide sequence or a peptide sequence encoded by a DNA sequence encoding the t -PA human or the DNA sequence capable of hybridizing to that under strict hybridization conditions.

-10 /

Table 1: Exemplary proteins that contain changes in Arg-275 and at least one glycosylation site linked to an N « ^{* 98} atom

-3 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCE1D TRATCYEDQG

ISYRGTWSTA ESGAECTNW- R ^ ALAQKPYS GRRPDAIRLG LGNHNYCRNP

148 DRDSKPWCYV FKAGKYSSEF CSTPACSEGN SDCYFG-R ² A YRGTHSLTES

198 GASCLPWNSM ILIGKVYTAQ NPSAQALGLG KHNYCRNPDG DAKPWCHVLK

248 NRRLTWEYCD VPSCSTCGLR QYSQPQFJIK GGLFADIASH PWQAAIFAKH

298 RRSPGERFLC GGILISSCWI LSAAHCFQER FPPHHLTVIL GRTYRWPGE

348 EEQKFEVEKY IVHKEFDDDT YDNDIALLQL KSDSSRCAQE SSWRTVCLP

398 PADLQLPDWT ECELSGYGKH EALSPFYSER LKEAHVRLYP SSRCTSQHLL

448 ”R ³ VTDNMLC AGDTRSGGPQ ANLHDACQGD SGGPLVCLND GRMTLVGIIS

498 WGLGCGQKDV PGVYTKVTNY LDWIRDNMRP

Compound

Compound ^R 1 ^R 2 ^r 3

wt NSS NGS NRT 1-1 QSS NGS NRT 1-2 NSS QGS NRT 1-3 NSS NGS QRT 1-4 QSS QGS NRT 1-5 QSS NGS -RT 1-6 NSS -GS QRT 1-7 QSS -GS -RT 1-8 - - 1-9 N-Q N-S N-T 1-10 N— N— N— 1-11 —Q -S - _T

^R 2 ^R 3

1-12 NSA NGS NRT 1-13 NSS NGA NRT 1-14 NSS NGS NRA 1-15 NSA NGA .NRT 1-16 NSA NGS NRA 1-17 NSV NGS NRT 1-18 NSS NGV NRV 1-19 TSS NGS NRT 1-20 TSS TGS NRT 1-21 TSS TGS QRT

J = s other than Arg, preferably different from Arg, His or Lys. R., R _o and R „each represent independently formed elements in the group consisting of a peptide, amino acid, dipeptide or tripeptide bond and, at least between R,, Rg and R„ are different from sequences of consensual nitrogen-linked glycosylation; ^ - ”and ---” = a peptide bond.

Indicates the site of an elimination of amino acids

In a second genus of proteins, they are characterized by a peptide sequence equal to or substantially the same as the human t-PA peptide sequence, in which 15 or more amino acids are deleted in the N-terminal region of

Gly - (- 3) or Ser-1 through Thr-91 and where (a) one or more Asn-linked glycosylation sites are usually deleted or otherwise modified to a different consensus-linked Asn-linked glycosylation site, and / or (b) Arg-275 and eventually eliminated or replaced by a different amino acid, preferably different from lysine or histidine. Exemplary proteins of this aspect of the present invention are shown below.

Illustrative proteins with N-terminal deletions

The following proteins have a peptide sequence represented in Table 1 in which R ^, Rg and R- represent the wt tripeptide sequences, but where the N (Gly - (- 3) through Thr-91) terminals are replaced with:

N-terminal sequence compound

D-l

GARSYQVI -------------------------------------------- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

D-2

GARSYQ --------------------------------------------- SEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

D-3

GARSYQVI —------------------------------------------------ ------------------------------- D TRAT

D-4

--------------------------------------, - D _TRAT

D-5

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCIISVP VKS ------------------------------------- TRAT

Indicates the site of an elimination of amino acids

This genus includes a subgenus of proteins in which 15 or more amino acids with an N-terminus are deleted as mentioned before and one or more of the glycosylation sites linked to Asn are deleted or in any way modified into another linked glycosylation sequence Asn, as you mentioned before. It also includes a sub-genus of compounds in which 15 or more amino acids are eliminated from the Gly - (- 3) or Ser-1 region through Thr-91, and the Arg 275 region is eliminated or replaced with a different amino acid, preferably that don't be lysine or histidine. Other examples of this kind are characterized by an elimination of 15 to about 94 amino acids from the Gly - (- 3) or Ser-1 to Thr-91 region, elimination or modification of one or more of the glycosylation sites linked to Asn ( see eg the table on page 7) θ elimination of Arg-275 or its replacement with a different amino acid. Tables 2 and 2.5 show examples of proteins belonging to this sub-genus.

This genus also includes examples where elimination at the nitrogen-linked terminus includes an elimination of 15 or more amino acids from the region between Ser-1 and Ser-50. They also include a subgenus of proteins in which 15 or more of the amino acids are deleted from the Ser-1 region to the Ser-50 region and one or more glycosylation sites are modified as described above. Another sub-genus includes proteins with a deletion of 15 or more amino acids from the Ser-1 region to the Ser-50 region where Arg-275 is deleted or substituted by other amino acids. Additionally, they include a sub-genus with an elimination of 15 or more amino acids from the Ser-1 region to the Ser-50 region and in which both of (a) one or more d

- 13 /, glycosylation and (b) Arg-275 are eventually modified, as described above. In Table 3 below, as well as in Tables 2 and 2._5, examples of these proteins from these sub-genera are mentioned.

Table 2: Exeplicative proteins with an elimination of 1 to 9 ^; t amino acids at the nitrogen-linked terminal and eventual modification to either one or both of the Arg-275 θ at least at one of the glycosylation sites linked to the nitrogen atom.

Terminal

compound 2 Th R _o Rg íi mi η o (w) R. X ’C’ U ' Τ ’· L · NGS NRT G ~ (~ 3) to T-91 2-0 R 1; SS NGS NRT X 2-1 R QSS NGS NRT X 2-2 P, NSS QGS NRT * 2-3 R NSS NGS QRT X 2-4 R NSS QGS QRT * 2-5 R QSS NGS QRT 2-6 R QSS QGS NRT X 2-7 R QSS QGS QRT * 2-8 R QSS NGS -RT * 2-9 R NSS -GS QRT X 2-10 R QSS -GS ”P <T * 2-11 P - - - * 2-12 - t »S _t f- c t.s. * 2-13 G t. s. t.s. t.s. THE 2-14 THE t.s. t.s. t.s. * 2-15 T t.s. t.s. t.s. X 2-16 Λ QSS NGS NRT 2-17 NSS QGS NRT X 2-18 NSS NGS QRT * 2-19 £ NSS QGS ‘ QRT THE 2-2 0 QSS NGS QRT * 2-21 £ QSS QGS NRT * 2-22 QSS QGS QRT * 2-23. £ QSS NGS "RT * 2-24 41 7Γ NSS -GS QRT * 2-2 5 QSS -GS -RT * 2-26 41 - - - *

--Z

Table 2 (Cont.) Terminal compound J -1 2, connected to N 2-27 R U Ser Ser ts ts * 2-28 μ U Set Ser t. s. t.s. λ 2-29 R Asn W Ser t.S. t.s. x 2-30 Asn W Ser f. s. t.s. • y 2-31 R Asn Ser z t.s. t.s. X 2-3 2 .Asn. To be z t.s. t.s. x · 2-33 R Asn V? z t.s. t.s. X 2-3 4 £ Asn W z t.s. t.s. X 2-35 P. U Λ t.s. t.s. X 2-36 41 u THE t.s. t.s. X 2-37 R - Asn THE t.s. t.s. X 2-38 - Asn THE t.s. t.s. X 2-39  P U THE t.s. t.s. X 2-40 = U ^Λ THE t.s. t.s. X 2-41 R Asn ^Λ - t.s. t.s. X 2-42 r Asn ^Λ - t.s. t.s. λ 2-4 3 R. - t.s. t.s. * 2-44 X - t.s. t.s. X 0-Scratch indicates a different te at least in wild-type mine, see below in Table 2 terminal attached to an atom of an amino acid for which illustrative examples .5 and in tables 3 to 5 and N which is in reverse reference 6-1J;

represents a peptide bond or an amino acid other than arginine (R); represents a peptide bond;

U represents any amino acid except Asn, Thr or Ser; K represents a peptide bond or any amino acid except Ser; Z represents a peptide bond or any amino acid except Thr or Ser; * represents any harmful ami; and t.s. represents a wild type terminal.

Table 2.5í Representation of N-terminals with an elimination of 1 to 94 amino acids

Terminal N designation

..... i £ -

GARSYQVI— VKSCSEPRCF

NGGTCQQALY

--- WLRPVLR FSDFVCQCPE

SNRVEYCWCN SGRAQCHSVP GFAGKCCEID TRAT

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SN ------- GFAGKCCEID • GAR -------------------------- VKSCSEPRCF NGGTCQQALY FSDFVCQCPE

-NRVEYCWCN GFAGKCCEID

TRAT

SGRAQCHSVP TRAT

GAR ------------------------------ VEYCWCN

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID

SGRAQCHSVP TRAT

GAR ------ VKSCSEPRCF

NGGTCQQALY

FSDFVCQCPE

---- EYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVI— VKSCSEPRCF

--- TQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRV ------------ HSVP

GFAGKCCEID TRAT

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

----------------- _S yp

GFAGKCCEID TRAT

GARSYQVI-R VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEY -------- GFAGKCCEID TRAT

GARSYQVI— VKSCSEPRCF —KTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

GFAGKCCEID —RAQ-HSVP TRAT

GARSYQVICR VKS ------ DEKTQMIYQQ ---- CQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVC --- SNRVEYCWCN ------- _EID

SGRAQCHSVP TRAT

GARSYQVICVKSCSEPRCF

----------------------------- _N

NGGTCQQALY FSDFVCQCPE GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVI ------------------------------------- p _R CF NGGTCQQALY FSDFVCQCPE GFAGKCCEID

TRAT

14GARSYQVICR DEK -------------------- VEYCWCN

VKSCSEPRCF-NGGTCQQALY FSDFVCQCPE GFAGKCCEID

SGRAQCHSVP

TRAT (continued --->)

GARSYQVICR DEKTQMIYQQ H --------------- CWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

-------------------- HQSWLRPVLR SNRVEY-WCN SGRAQ -----

--- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRC ----------------- QCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN VKSC ------------------------ PE GFAGKCCEID

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN VKSCSEPRC ---------------- VCQCPE GFAGKCCEID

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS ----------------------- Q _C p _E GFAGKCCEID TRAT

GARSYQVI ------------------------------------------

--- CSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVI -----------------------------------------

--------------------------------------- D TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKS ------------------------------------- TRAT

424

425

426

427

428

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV --- CSEPRCF NGGTCQQALY FSDFVCQ ---------- EID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPV-R SNR-EYCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQ ---------- EID TRAT

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ HQSWLRP — R SNR — YCWCN SGRAQCHSVP NGGTCQQALY FSDFVCQ ---------- EID TRAT

GARSYQVICR DEKTQMIYQVKSCSEPRCF NGGTCQQALY

HQSWLRPV-R SNR-EYCWCN SGRAQCHSVP FSDFVCQ ---------- EID TRAT

GARSYQVICR DEKTQMI — Q HQSWLRP — R SNR — YCWCN SGRAQCHSVP VKSCSEPRCF NGGTCQQALY FSDFVCQ ---------- EID TRAT

The specific proteins of the present invention can be identified by a 3-part compound designation comprising a number of compounds in Table 2 followed by an N designation and then the position identification 275 · For example, compound N5 2 -ll / N-6 / Arg refers to a protein in which the three glycosylation nozzles have been eliminated ("2-11", see table 2), C-6 to K-10 and E-32 to C-U3 were eliminated (terminal N N-6) and Arg-275 remained.

Table 3ί Examples of proteins with an elimination of 15 to about 45 amino acids in the region from Ser-1 to Ser-50 and a modification in one or both of (a) Arg-275 and (b) of at least one site glycosylation linked to the nitrogen atom (for general sequence, see table 1)

The illustrative proteins are as defined in Table 2 but with the following N-terminal replacing a wild-type sequence from Gly - (- 3) to Thr-91:

Terminal N designation

GAR --------------- QQ

VKSCSEPRCF NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARS --------------- Q

VKSCSEPRCF NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT garsy ———---------- VKSCSEPRCF NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQ ------------- VKSCSEPRCF NGGTCQQALY

-QSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID 'TRAT

SGRAQCHSVP

GARSYQV --------------- SWLRPVLR

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVI— VKSCSEPRCF

------------- WLRPVLR NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICVKSCSEPRCF

LRPVLR

NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

--------------- RPVLR NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

D --------------- PVLR

NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

FROM --------------- COME

NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

DEK --------------- LR

NGGTCQQALY FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

DEKT --------------- R

NGGTCQQALY FSDFVCQCPE

Table 3 (Cont.)

GARSYQVICR DEKTQ --------------- SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQM --------------- NRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMI --------------- RVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIY --------------- VEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQ --------------- EYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ --------------- YCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE. GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ H --------------- CWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQ --------’------- WCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQS --------------- CN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSW --------------- N SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWL --------------- SGRAQCHSVP. VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLR --------------- GRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRP --------------- RAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPV --------------- AQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVL --------------- QCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR --------------- CHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT /

X *

Table 3 (Cont.)

DEKTQMIYQQ

NGGTCQQALY

S --------------- HSVP

GFAGKCCEID TRAT

52

GARSYQVICR VKSCSEPRCF

HQSWLRPVLR FSDFVCQCPE

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SN --------------- SVP

GFAGKCCEID TRAT

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNR ----------- GFAGKCCEID TRAT

VP

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRV ---------- GFAGKCCEID TRAT

P

GARSYQVICR VKSCSEPRCF

HQSWLRPVLR

GARSYQVICR -KSCSEPRCF

GARSYQVICR —SCSEPRCF

GARSYQVICR --- CSEPRCF

GARSYQVICR ---- SEPRCF

GARSYQVICR ----- EPRCF

GARSYQVICR ------ PRCF

GARSYQVICR

------- r _CF

GARSYQVICR -------- _CF

GARSYQVICR --------- _F

GARSYQVICR

GARSYQVICR

DEKTQMIYQQ

NGGTCQQALY.FSDFVCQCPE

DEKTQMIYQQ HQSWLRPVLR

NGGTCQQALY FSDFVCQCPE

DEKTQMIYQQ HQSWLRPVLR NGGTCQQALY

FSDFVCQCPE

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ -GGTCQQALY

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

SNRVE --------- GFAGKCCEID TRAT

SNRVEY -------- GFAGKCCEID. TRAT

SNRVEYC ------- GFAGKCCEID TRAT

SNRVEYCW— GFAGKCCEID

SNRVEYCWCGFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

TRAT

TRAT

TRAT

S --- TRAT

SG— TRAT

SGR—

TRAT

SGRA · TRAT

SGRAQTRAT.

SGRAQC --- TRAT

Table 3 (Con t.)

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCH ---

------------ GTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHS—

-------------- TCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSV-

------ CQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

--------------- QQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

V --------------- QALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VK --------------- ALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKS --------------- LY FSDFVCQCPE GFAGKCCEID TRAT

With regard to the modified N terminals, referred to in

dro 3, it should be understood that more than 15 amino acids can be eliminated from the S-1 to S-50 region. When they have been eliminated from the multiple amino acids then, they can be adjacent to each other or separated by one or more amino acids. In the compounds referred to with these N-termini, the size of the elimination is indicated by Δ n where n represents the number of amino acids eliminated. For example, terminal N number 24 in which 15 amino acids have been deleted as shown in table 3, can be denoted by N-24 Δ 15. When 16 amino acids are eliminated, eg from Sl to Q-16 the N terminal for N-24 Δ 16, etc.

When there are combinations of eliminations, eg, when S-l,

Y-2 and 1-5 are eliminated, the N terminal can be referred to as ’’ ΝΔ1,2,5. As indicated in the text following Table 2.5, the specific compounds were designated by a 3-part code comprising the number of a compound in Table 2 followed by an N-terminal designation, eg from Table 2.5 θ then the identification of the position 275 »0 compound 2-26 / N-24A15, N-5OA15 / - designates the protein in which the three glycosylation sites R-275, Sl to Y-15 and R-27 to A- 41 were eliminated.

The present invention also encompasses a genus of proteins in which one or more, e.g. 1 to 45 amino acids are eliminated from the Cys-51 to Thr-91 region. In a sub-genus, one or more amino acids are also eliminated in the Gly - (- 3) or Ser-1 to Ser-50 region. In certain cases, proteins of this kind are further modified in such a way that Arg-275 ® is eliminated or replaced by a different amino acid, preferably different from lysine or histidine. In other cases, proteins of this genus, alternatively or in addition to the Arg-275 modification, are modified in such a way that (a) one or more N-linked glycosylation sites are abolished as described above and / or ( b) one or more amino acids are eliminated in the Gly - (- 3) region up to Ser-50. Examples of proteins from these sub-genera are similar to those referred to in Tables 2 and 3, but contain an N-terminus like those referred to in Table 4, below

(*

Table 4: Examples of proteins with an elimination of 1 to 41 amino acids from the Cys-51 to Thr-91 region (See Table 1 for the general sequence)

The illustrative proteins are as defined in Table 2, but with the N-terminus replacing the wild-type sequence from Gly - (- 3) to Thr-91:

End designation

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS ------------------------------------- TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS ----------- CQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVIR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVC ----------- EID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRC ----------------- QCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN VKSC ------: --------------.---- PE GFAGKCCEID

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN VKSCSEPRC --------------- VCQCPE GFAGKCCEID

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP VKS ----------------------- QCPE GFAGKCCEID TRAT

GARSYQVIC -----------------------------------

------ p _RCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVI ------------------------------------------

--------------------------------------- _{D TRA} T

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKS ------------------------------------- TRAT

GARSYQVI -------------- SWLRPVLR SN -------------

VK ------------- QQALY FSDFVCQCPE GFAGKCCEID TRAT

CHSVP

Table 4 (Cont.)

GARSYQVICR DEKTQMIYQQ VKS-SEPRCF NGGTCQQALY

GARSYQVICR DEKTQMIYQQ VKSC-EPRCF NGGTCQQALY

GARSYQVICR DEKTQMIYQQ VKSCS-PRCF NGGTCQQALY

GARSYQVICR DEKTQMIYQQ VKSCSE-RCF NGGTCQQALY

GARSYQVICR DEKTQMIYQQ 'VKSCSEP-CF NGGTCQQALY

GARSYQVICR DEKTQMIYQQ

VKSCSEPR-F NGGTCQQALY

GARSYQVICR DEKTQMIYQQ

VKSCSEPRC- NGGTCQQALY

GARSYQVICR DEKTQMIYQQ

VKSCSEPRCF -GGTCQQALY

GARSYQVICR DEKTQMIYQQ

VKSCSEPRCF N-GTCQQALY

GARSYQVICR DEKTQMIYQQ

VKSCSEPRCF NG-TCQQALY

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGG-CQQALY

GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGT-QQALY

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTC-QALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQ-ALY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQ-LY FSDFVCQCPE GFAGKCCEID TRAT

GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQA-Y FSDFVCQCPE GFAGKCCEID TRAT

Table 4 (Cont.)

I 95

100 • ¹⁰¹

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALDEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR -SDFVCQCPE

HQSWLRPVLR F-DFVCQCPE

HQSWLRPVLR FS-FVCQCPE

HQSWLRPVLR FSD-VCQCPE

HQSWLRPVLR FSDF-CQCPE

HQSWLRPVLR FSDFV-QCPE

HQSWLRPVLR FSDFVC-CPE

HQSWLRPVLR FSDFVCQ-PE

HQSWLRPVLR FSDFVCQCPSNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP

TRAT

102 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE -FAGKCCEID TRAT

103 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE G-AGKCCEID TRAT ¹⁰⁴

104 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQALY FSDFVCQCPE GF-GKCCEID TRAT

Table 4 (Cont.)

105 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP NGGTCQQALY FSDFVCQCPE GFA-KCCEID TRAT 106 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR FSDFVCQCPE SNRVEYCWCN GFAG-CCEID SGRAQCHSVP TRAT. 107 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR FSDFVCQCPE SNRVEYCWCN GFAGK-CEID SGRAQCHSVP TRAT 108 GARSYQVICR VKS CSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR FSDFVCQCPE SNRVEYCWCN GFAGKC-EID SGRAQCHSVP TRAT 109 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR FSDFVCQCPE SNRVEYCWCN GFAGKCC-ID SGRAQCHSVP TRAT 110 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR FSDFVCQCPE SNRVEYCWCN GFAGKCCE-D SGRAQCHSVP TRAT 111 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR FSDFVCQCPE SNRVEYCWCN GFAGKCCEI- SGRAQCHSVP TRAT 312 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQAL- HQSWLRPVLR —DFVCQCPE SNRVEYCWCN GFAGKCCEID SGRAQCHSVP TRAT 313 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQALY HQSWLRPVLR —DFVCQCPE SNRVEYCWCN GFAGKCCEID SGRAQCHSVP TRAT 314 GARSYQVICR VKSCSEPRCF DEKTQMIYQQ NGGTCQQAL- HQSWLRPVLR -SDFVCQCPE SNRVEYCWCN GFAGKCCEID SGRAQCHSVP TRAT

315 GARSYQVI -----------------------------------------

--- CSEPRCF NGGTCQQAL- FSDFVCQCPE GFAGKCCEID TRAT

316 GARSYQVI ------------- _: ----------------------------

--- CSEPRCF NGGTCQQALY -SDFVCQCPE GFAGKCCEID TRAT

317 GARSYQVI -----------------------------------------

--- CSEPRCF NGGTCQQALY F-DFVCQCPE GFAGKCCEID TRAT

318 GARSYQVI ------------------------------------------

--- CSEPRCF NGGTCQQALY —DFVCQCPE GFAGKCCEID TRAT

319 GARSYQVI ------------------------------------------

--- CSEPRCF NGGTCQQAL- —DFVCQCPE GFACKCCEID TRAT

In Table 4, mentioned above, several protein sub-classes are described. For example proteins with the elimination of]. a37 amino acids (individually, consecutively or in combination), from Cys-51 to Asp-87 are described as well as proteins that contain an elimination of 1 to 37 amino acids from Cys-51 to Arg-87 and an elimination of one or more amino- acids in the Gly region - (- 3) to Cys-51 (see N-76). With regard to compounds containing an N-terminus chosen from the N-termini of N-76 through N-11, it should be understood that one or more amino acids may be eliminated. For example, as shown in Table 4, the ^{N-0 wherein} amino acid 77 was deleted may refer to Ν-77Δ1. When six amino acids were eliminated, for example, from

S-52 through F-57, terminal N is called Ν-77Δ 6 ^U , etc. Specific proteins are designated as shown in Tables 2 and 3. Examples are shown below in which the three glycosylation sites and

Arg-275 have been eliminated:

Compound designation

Eliminations

2-26 / N-75 / 2-26 / Ν-74Δ6 / 2-27 / Ν-74Δ1, Ν-76Δ6 / C-51 to D-87

C-51 to C-56

C-51, E-53 to N-58

The following are compounds in which there are no changes in the glycosylation sites, but which contain several eliminations:

ον |

Compound designation

Λ

2-0 / N-424 / Arg

2-0 / N-425 / Arg

2-O / N-426 / Arg

2-0 / N-427 / Arg

2-0 / N-428 / Arg

2-0 / N-63 2, N-92 3 / Arg

2-0 / N-63 l, N-92 2 / Arg

2-0 / N-63 l, N-92 1 / Arg »

Illustrative compounds with but which are also

Modification: elimination of:

P-Λγ to S-50

L-26 & V-31

V-25, L-26, V-31 & E-32

Q-17, L-26 & V-31

Y-15, Q-16, V-25, L-26, V-31 & E-32

R-40, A-41, Y-67, F-68 & S-69 R-4o, Y-67 & F-68

R-4th & Y-67 same deletions referred to in the first glycoside site replaced by modified cosylation attached to a nitrogen atom and in which of the Asn-117 by Gin:

2-l / N-424 / Arg

2-1 / N-425 Arg

2-l / N-426 / Arg

2-l / N-427 / Arg

2-1 / N-428 / Arg

2-1 / N-63 2, N-92 3 / Arg

2-1 / N-63 l, N-92 2 / Arg

2-1 / N-63 l, N-92 1 / Arg

The following are illustrative compounds with the same deletions represented above, but which are also modified at all three glycosylation sites here by replacing Asn with Gin:

2-7 / N-424 / Arg

2-7 / Ν-425 / Arg

2-7 / N-U26 / Arg

2-7 / N-427 / Arg

2-7 / N-428 / Arg

2-7 / N-63 2, N-92 3 / Arg

2-7 / N-63 l, N-92 2 / Arg

2-7 / N-63 Ι, Ν-92 1 / Arg

Thus, the present invention further includes a subgenus of proteins in which one or more deletions of less than about 20 amino acids are present in the region from Gly - (- 3) to Thr-91. The proteins of this sub-genus can also be modified in the Arg-275 region and / or in one or more glycosylation sites linked to Asn. Examples of proteins of this sub-genus are similar to those described in Table 2 through 4, but instead substitute the wild-type N-terminal for an N-terminal as described in Table 5, below. Other examples of compounds of this sub-genre are also referred to earlier by their 3-part code code designations.

In another genus, proteins are characterized by a peptide sequence substantially equal to the human t-PA peptide sequence in which different amino acids are replaced by 1 to 94 of the amino acids in the Gly - (- 3) or Ser-1 to Thr- 91. This genus includes compounds characterized by the replacement of one or more amino acids with the N-termini referred to above and by modifications to Arg-275 as described above. It also includes the sub-genus of compounds characterized by the aforementioned substitutions before one or more N-terminal amino acids and modifications as described above at one or more of the linked glycosylation sites

consensus to Asn. Yet another sub-genus of these cases is characterized by the substitution of one or more amino acids with the N-terminus and modifications, as mentioned above, in both Arg-275 θ in one or more of the glycosylation sites linked to N. In certain cases one or more amino acids are substituted in the Gly - (- 3) or Ser-1 region up to Ser-50 with or without modifications in Arg-275 and / or one or more of the glycosylation sites linked to N. In other cases one or more amino acids are substituted in the region from Cys-51 to Thr-91, still with or without the modification in Arg-275 and / or in one or more of the glycosylation sites linked to N. In another case, from one to about eleven, preferably one to about six amino acids are substituted. in one or more of the following regions, possibly with other changes as mentioned above.

Region in The region in The 1 Gly - (- 3) G.ln-3 7 Cys-34 Cys-43 2 Val-4 Lys-10 8 His-44 Ser-50 3 Thr-11 His-18 9 Cys-51 Cys-62 4 Gln-19 Leu-22 10 Gln-63 Val-72 5 Arg-23 Arg-27 11 Cys-73 Cys-84 6 Ser-28 Tyr-33 12 Glu-85 Thr-91

In another aspect of the present invention one or more amino acids are substituted in one or more of the following regions:

R-7 to S-20, W-21 to Y-33, N-37 to Q-42 and H-44 to S-50.

In another aspect of the set, the N-terminal is still modified by replacing one to about eleven, preferably from

one to about six amino acids in one or more of the regions defined above and further modified by deleting from one to 93, preferably from 1 to about 45, and more preferably from 1 to about 15 amino acids.

Illustrative amino acid substitutions are shown in Table 6-e and in Examples 6-B, proteins are described in the examples. From the substitution of R-40, A-41 and Q-42, represented in Table 6-A , S represents a preferred substitution for R-40 and V and L are preferably substitutions for A-41 and Q-42, respectively. It should be noted that the proteins of the present invention that incorporate the substitutions identified by R-40, A-41 and Q-42, in Table 6-A, are presently preferred, alone or in other aspects of sub-genres of this aspect , in combination with other substitution (s) ^and / ^or deletions at the N-terminus and / or modifications at R-275 and / or at least one of the glycosylation sites. they are modified by substitutions rather than deletions, retain more of the conformation of natural t-PA and selectively retain more of their desired biological activities.

Table 5i Examples of proteins with one or more deletions of less than 20 amino acids in the Gly - (- 3) to Thr-91 region (See Table 1 for general sequence)

Illustrative proteins as defined in Table 2, but with the N-terminals replacing the Wild Type sequence from Gly - (- 3) to Thr-91i

Material designation

112

GARSYQVICR VKSCSEPRCF

NGGTCQQALY

-QSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

113

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

GFAGKCCEID

-GRAQCHSVP TRAT

114

GARSYQVICR VKS ------ DEKTQMIYQQ ---- CQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

115

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDF ----- SNRVEYCWCN ----- CCEID

SGRAQCHSVP TRAT

116

GARSYQVICR VKS ------- GGTCQQALY

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

117

GARSYQVICR VKSCSEPRCF

NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

GFAGKCCEID

SGRAQCHSVP TRAT

118

GARSYQVICR VKSCSEPRCF

NGGTCQQALY

HQSWLRPVLR FSDFV ---- SNRVEYCWCN ----- CCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSD ------ • GKCCEID

SGRAQCHSVP TRAT

GARSYQVICR VKSCS ---- DEKTQMIYQQ ---- CQQALY

HQSWLRPVLR FSDFVCQCPE

GFAGKCCEID

SGRAQCHSVP TRAT

GARSYQVICR DEKTQMIYQQ VKSCS ------ GGTCQQALY

HQSWLRPVLR FSDFV ---- SNRVEYCWCN ----- CCEID

SGRAQCHSVP TRAT

119

120

121

Table 5 (Cont'd) and, with reference to tables 2.5, 3 and 4:

N-l N-2 n-6 up until N-ll N-15 up until N-17 Ν-24Δ1 up until Δ19 Ν-27Δ1 up until Δ19 Ν-Λ4Δ1 up until Δ19 Ν-73Δ1 up until Δ19 Ν-95Δ1 up until Δ19 Ν111Δ1 up until Δ5

Table 6-There Representative substitutions of type amino acids

wild replacement t.s. replacement C-6 δ ', Τ, δ ..... õr a R-7 S _Z T _Z Q _Z N _Z G _Z H _Z D or K • S-52 G _Z A _Z Q _Z L _Z V _Z I or D-8 lt II E-53 S _Z T _Z Q _Z N _Z G, H _Z D 0 E-9 II π P-54 A _Z G _Z Y _Z D or S K-10 II II ’C-56 S _Z T _Z G or A T-ll N, S, L, G or A R-55 S _Z T _Z Q _Z N, G _Z H _Z D or T-ll N _Z S _Z L, G or A F-57 Y _Z I _Z W _Z H _Z D or R Q-12 N, S, L, G, A or T N-58 G _Z A _Z Q _Z L _Z V _Z I or T M-13 II II G-59 A _Z S _Z T _Z D _Z V or P 1-14 II II G-èO II II Q-16 II II T-61 N _Z S _Z L _Z G or A Q-17 H II C-62 S _Z T _Z G or A Q-63 N _Z S _Z L _Z G _Z A or T H-18 II II q-64 II II R-23 S _Z T _Z Q _Z N _Z G _Z H _Z D or X A-65 G _Z S _Z T, H _Z N or Q R-27 II II * L-66 N _Z S _Z L _Z G _Z A or T R-30 II II 'L-67 Y _Z I _Z W _Z H _Z D or R V-31 II II F-68 II H E-32 S, T, Q, N, G, H, D or X S-69 G _Z A, Q _Z L _Z V _Z I or T Y — 33 F, S, H or L D-70 S _Z T _Z Q _Z N _Z G _Z H _Z D or C-34 S / T / G or A W-35 T _z V _z Ior Q C-36 S _Z T _Z G or A F-71 Y _Z I _Z W _Z H _Z D or R N-37 G _Z A _Z Q _Z L _Z V _Z I or T V-72 N _Z S _Z L _Z G _Z A or T C-73 S _Z TG or A S-38 n Π Q-74 N _Z S _Z L _Z G or A C-75 S _z T, G or A G-39 A _Z S _Z T _Z D _Z V or P P-76 A _Z G _Z Y _Z D or S R-40 S _Z T _Z N _Z G _Z K or D E-77 S _Z T _Z Q, N _Z G _Z H _Z D or A-41 G _Z S _Z T _Z H _Z N or Q  G-78 A _Z S _Z T _Z D _Z V or P Q-42 N _Z S, L _Z G _Z A or T F-79 Y _Z I _Z W _Z H _Z D or R H-44 II I! A-80 G _Z S _Z T _Z H _Z N or Q ’S-45 G, A _Z Q _Z L _Z V _Z I or T G-81 A _Z S _Z T _Z D _Z V or P V-46 N _Z S _Z L _Z G _Z A orT X-82 S _Z T _Z Q, N _Z G _Z H _Z D or P-47 A _Z G _Z Y _Z D or S C-83 S _z T, G or A V-48 N _Z J _Z L _Z G _Z A or T C-84 S, T _z G or A K-49 S _Z T _Z Q _Z N _Z G _Z H _Z D orK E-85 S _Z T _Z Q _Z N _Z G _Z H _Z D or S-50 G, A _z Q _z L, V _z I orT 1-86 N _Z S _Z L _Z G _Z A or T C-51 S _z T, G or A D-87 . S _Z T _Z Q _Z N _Z G _Z H _Z D or  T-88 N _Z S _Z L _Z G or A R-89 S _Z T _Z N, G or D A-90 G _Z S _Z T _Z H _Z N or Q T-91 N _Z S _Z L _Z G or A

-34Picture é-Bí Example of proteins that contain substitutions of one or more amino acids in the Gly region - (- 3) until

Thr-91 (see table 1 for the general sequence)

Illustrative proteins as defined in Table 2, but with the N-terminus replacing the wild type sequence of

Gly - (- 3) to Thr-91

Terminal designation N * JL · -

122 GARGYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

123 GARSFQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

124 GARSYNVICR DEKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

125 GARSYQJICR DEKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

126 GARSYQVSCR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

127 GARSYQVISR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

128 GARSYQVICT DEKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

129 GARSYQVICR NEKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

130 GARSYQVICR DQKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

131 GARSYQVICR DETTQMIYQQ

VKSCSEPRCF NGGTCQQALY

132 GARSYQVICR DEKAQMIYQQ

VKSCSEPRCF NGGTCQQALY

133 GARSYQVICR DEKTLMIYQQ VKSCSEPRCF NGGTCQQALY

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

Table 6-B (Cont.)

134 GARSYQVICR DEKTQGIYQQ VKSCSEPRCF NGGTCQQALY

135 GARSYQVICR DEKTQMAYQQ VKSCSEPRCF NGGTCQQALY

136 GARSYQVICR DEKTQMISQQ VKSCSEPRCF NGGTCQQALY

137 GARSYQVICR DEKTQMIYLQ

VKSCSEPRCF NGGTCQQALY

138 GARSYQVICR DEKTQMIYQL

VKSCSEPRCF NGGTCQQALY

139 GARSYQVICR DEKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

140 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

141 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

142 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

143 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

144 GARSYQVICR DEKTQMIYQQ

VKSCSEPRCF NGGTCQQALY

145 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

146 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

147 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

148 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

149 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

150 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

NQSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HDSWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQNWLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSYLRPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWERPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLGPVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRTVLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPYLR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVDR SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLS SNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR PNRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

HQSWLRPVLR SDRVEYCWCN SGRAQCHSVP FSDFVCQCPE GFAGKCCEID TRAT

Table 6-B (Cont.)

151

152

153

154

155

156

157

158

159

160

163

164

165

166

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR

FSDFVCQCPE

SNSVEYCWCN GFAGKCCEID

SNRLEYCWCN GFAGKCCEID

SNRVSYCWCN GFAGKCCEID

SNRVESCWCN GFAGKCCEID

SNRVEYSWCN GFAGKCCEID

SNRVEYCTCN

GFAGKCCEID

SNRVEYCWTN GFAGKCCEID

SNRVEYCWCD GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

PGRAQCHSVP TRAT

SARAQCHSVP TRAT

SGSAQCHSVP TRAT

SGRVQCHSVP TRAT

SGRALCHSVP TRAT

SGRAQTHSVP TRAT

SGRAQCSSVP TRAT

SGRAQCHIVP

TRAT

-) 7Picture 6-B (Cont ♦)

167

168

169

170

171 ¹⁷²

173

174

175

176

177

178 »

179

180

181

182

183

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR NKSCSEPRCF

GARSYQVICR VDSCSEPRCF

GARSYQVICR

VKVCSEPRCF

GARSYQVICR VTSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR

VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEGRCF

GARSYQVICR VKSCSEPRCF

GARSYQVICR VKSCSEPRCF

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

NEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQAKY

DEKTQMIYQQ

NGGTCQQALA

DEKTQMIYQQ

NGGTCQQALG

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

DEKTQMIYQQ NGGTCQQALY

DEKTQMIYQQ

NGGTCQQALY

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR GSDFVCQCPE

HQSWLRPVLR ASDFVCQCPE

HQSWLRPVLR FSFFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR

FSDFVCQCPE

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN

GFAGKCCEID

SNRVEYCWCN GFAGHCCEID

SNRVEYCWCN

GFAGKCCEID

SGRAQCHSNP

TRAT

SGRAQCHSVD TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP

TRAT

Table 6-B (Cont.)

SGRAQCHSVP

TRAT

184 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 185 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 186 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 187 GARSYQVICR DEKTQMIYQQ VKSCSEPTCF NGGTCQQALY 188 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 189 GARSYQVICR DEKTQMIYQQ VKSCSEPHCF NGGTCQQALY 190 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF VGGTCQQALY 191 GARSYQVICR DEKTQMIYQQ VKSCSTPRCF NGGTCQQALY 192 GARSYQVICR DEKTQMIYQQ VKSCSQPRCF NGGTCQQALY 193 GARSYQVICR DEKTQMIYQQ VHSCSEPRCF NGGTCQQALY 194 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 195 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 196 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 197 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 198 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 199 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQALY 200 GARSYQVICR DEKTQMIYQQ VKSCSEPRCF NGGTCQQELY

HQSWLRPVLR HSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPT

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FIDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSSFVCQCPE

HQSWLRPVLR

RSDFVCQCPE

HQSWLRPVLR ISDFVCQCPE

HQSWLRPVLR PSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

HQSWLRPVLR FSDFVCQCPE

SNRVEYCWCN GFAGNCCEID

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

SNRVEYCWCN GFAGKCCEIH

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SNRVEYCWCN GFAGKCCEID

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT.

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRAQCHSVP TRAT

SGRALCHSVP TRAT

HQSWLRPVLR

FSDFVCQCPE

SGRAQCHSVP

TRAT

Table 6-B (Con t.)

201 GARSYQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPRCF NGGTCQQANY FSDFVCQCPE GFAGKCCEID TRAT

201 GARSVQVICR DEKTQMIYQQ HQSWLRPVLR SNRVEYCWCN SGRAQCHSVP

VKSCSEPSCF NGGTCQQALY FSDFVCQCPE GFAGKCCEID TRAT

The proteins of the present invention that incorporate a substitution (M amino acids can be designated by a three-part code as mentioned above. It must be understood, of course, that more than one wild type amino acid can be substituted. When they are designated compounds with an N-terminus indicate the number of substitutions by ns where n represents the number of amino acids substituted, eg with amino acid substitution such as (but not limited to)

a) described in table 6-A. For example, the N-terminal N-122sl means N-terminal 122, as referred to in Table 6-B, while the N-terminal N-122s4 means that the N-terminal where Sl is replaced by G and the following three wild-type amino acids are replaced by other amino acids. The proteins found in this case, which contain multiple amino acid substitutions, can be designated by a series of N-terminal designations that indicate specific substitutions, as follows:

Illustrative compounds with multiple N-terminal substitutions with Asn-117 replaced by Gin and Arg-275 replaced by

Thr:

£ compound

2-16 / N-128, Ν-130 / Thr substitutions:

ts substitute

R-7

E-9

2-16 / Ν-129, Ν-130 / Thr D-8 E-9 N Q 2-16 / Ν-130, Ν-131 / Thr E-9 Q K-10 T 2-16 / N-131, N-153 / Thr K-10 T E-32 s 2-16 / N-161, N-165 / Thr R-40 s H-44 s 2-1S / N-159, Ν-161 / Thr S-38 P R-40 s 2-16 / N-161, Ν-163 / Thr R-40 s Q-42 L 2-16 / N-161 ₁ N-17 2 / Thr R-40 s K-49 T 2-16 / N-138, Ν-142 / Thr Q-17 L W-21 Y 2-16 / N-133, Ν-138 / Thr Q-12 L Q-17 L 2-16 / N-128, Ν-133 / Thr R-7 T Q-12 L 2-16 / N-131, N-16 2 / Thr K-10 T A-41 V 2-16 / N-165, Ν-170 / Thr H-44 s K-49 D 2-16 / N-143, Ν-146 / Thr L-22 AND V-25 Y 2-16 / N-173, N-165 / Thr E-32 Q H-44 s 2-16 / N-165, Ν-172 / Thr H-44 s K-49 T 2-16 / N-14 8, Ν-151 / Thr R-27 s R-30 s 2-16 / N-143, Ν-153 / Thr L-22 AND E-32 s 2-16 / N-161, N-170 / Thr R-40 s K-49 D 2-16 / N-153, Ν-161 / Thr E-3 2 s R-40 s 2-16 / N-129, Ν-131 / Thr D-8 N K-10 T

(Cont.)

2-16 / N-131, N-143 / Thr K-10 L-22 T E 2-16 / N-12 8, N-131 / Thr R-7 T K-10 T 2-16 / N-128, N-161 / Thr 5-7 T R-40 s 2-16 / N-175, N-176 / Thr L-66 K Y-67 THE 2-16 / N-176, N-178 / Thr Y-67 THE F-68 G 2-16 / N-177, N-170 / Thr Y-67 G D-70 F 2-16 / N-177, Ν-179 / Thr Y-67 G F-68 THE 2-16 / N-181, N-182 / Thr p-54 G K-82 H 2-16 / N-202, Ν-183 / Thr R-55 s K-82 Q 2-16 / N-185, N-184 / Thr E-77 T K-82 N 2-16 / N-184, N-18 6 / Thr K-82 N D-87 H 2-16 / N-187, N-186 / Thr R-55 T D-87 H 2-16 / N-188, Ν-201 / Thr L-66 N D-70 s 2-16 / N-18 9, N-19 O / Thr R-55 H N-58 V 2-16 / N-191, N-18 9 / Thr E-53 T R-55 H 2-16 / N-193, Ν-192 / Thr K-49 H E-53 Q

/>

2 -16 / Ν-161, Ν-175 / Thr R-40 s L-66 K 2-16 / Ν-148, Ν-194 / Thr R-27 s F-68 H 2-16 / Ν-131, Ν-184 / Thr K-10 T F-68 H 2-16 / Ν-174, Ν-195 / Thr D-8 N F-68 I 2-16 / Ν-144, Ν-195 / Thr R-23 G F-68 I 2-16 / N-151, N-196 / Thr R-30 s F-68 P 2-16 / N-161, N-198 / Thr R-40 s S-69 I 2-16 / Ν-199, Ν-200. / Thr Q-42 L A-65 AND 2-16 / N-161, N-197 / Thr R-40 s F-68 R

- ^ 3- / (..

A sub-genus of particular interest is characterized by the substitution of one or more from Y-67 to S-69, with the possible elimination and / or substitution of one or more amino acids from Gly - (- 3) to L-66, with or without modification, as described above, at one or more glycosylation sites and / or Arg-275 »

One aspect of the present invention relates to proteins that contain at least one sugar radical called a carbohydrate complex characteristic of mammalian glycoprotein. As exemplified in great detail below, such glycoproteins called a carbohydrate complex can be produced by expressing a DNA molecule that encodes the desired polypeptide sequence in host mammalian cells. The appropriate mammalian host cells and methods for transformation, culture, amplification, drilling and product production and purification are known to those skilled in the art. See, eg, Gething and Sambrook, Nature, 293; 19 ^ 1; P. 620-625 or, alternatively, Kaufman et al .; Molecular and Cellular BicLogy; 5. (7); 1985; P. 1750-1759 or Howley et al .; American Patent No. 4,419,446.

Another aspect of the present invention involves variants of t-PA as defined above, in which a carbohydrate radical is a treated form of the oligosaccharide linked to the initial dololicol, characteristic of insect cells that produce glycoproteins, as opposed to a substituent carbohydrate complex, characteristic of mammalian glycoproteins including mammalian-derived t-PA. The glycosylation of such an insect cell type is referred to here

- 44 L as a high mannose carbohydrate for simplicity. In the present specification, the carbohydrate complex and the high mannose carbohydrate have the meaning defined by Kornfeld et al .; Ann. Rev. Biochem; 54; 1985; P. 631-64. Variants of high mannose according to the present invention are characterized by a polypej? varicide, as described above, containing at least one glycosylation site linked to an occupied nitrogen optimum. Such variants can be produced by expressing a DNA sequence encoding the variant in cells of a host insect. Suitable insect host cells as well as methods and materials for transforming / transfecting, culturing insect cells, probing and producing the product and purifying it useful in practicing this aspect of the present invention, are known in the art. The glycoproteins thus produced also differ from natural t-PA and t-PA produced to date by recombinant engineering techniques in mammalian cells in which the variants of this aspect of the present invention should not contain, in the carbohydrate radicals, as sialic acid or galactose substituents or other protein modifications characteristic of mammalian-derived glycoproteins.

Proteins of the present invention that do not contain carbohydrate radicals attached to a nitrogen atom can also be produced by expressing a DNA molecule that encodes the desired variant, e.g. compounds from 1 to 6 to 1 to 11, shown in Table 1, in mammalian host cells, insects, bacterial yeasts

eukaryotic host cells are presently preferred. As mentioned above, the appropriate host cells for insects or mammals, as well as the appropriate host cells for yeast and bacteria, as well as the methods and materials for transformation / transfection, cell culture, probing and product production and their purify. use in the practice of the present invention, are also known in the art.

In addition, as it should be clear to those skilled in the art, the present invention also contemplates other variants of t-PA, characterized, instead of eliminating 1 or more amino acids in the region Gli-3 or Ser 1 through Thr 91, by a or more amino acid substitutions in that region especially in the Arg 7 to Ser 50 region or by an association of eli. minations and substitution. The cDNAs encoding these compounds can be prepared easily, e.g., by methods very similar to the mutagenesis procedures described herein using appropriate mutagenic oligonucleotics. The cDNAs may eventually undergo mutagenesis in 1 or more of the codons represented by R _Q and R ^ and / or Arg-275 θ can be inserted into expression vectors expressed in host cells by the methods described herein. These proteins are considered to share the advantageous pharmacokinetic properties of other compounds of the present invention and possibly prevent undesirable antigenicity after administration in pharmaceutical preparations analogous to those described above.

As will be evident from the above, all of the variants of the present invention are prepared using

using recombination techniques using DNA sequences encoding analogs that may also contain few or no potential glycosylation sites relative to natural human t-PA and / or elimination or substitution of Arg-275. Such DNA sequences can be produced by conventional site-directed mutagenesis of DNA sequences encoding t-PA.

The DNA sequences encoding t-PA have been colonized and characterized. See, e.g., D. Pennica et al., Nature ”(London) 301; 19θ3; P. 214 and R. Kaufman et al.,; Mol. Cell Biol ”; 5 (?); 1985? P. 1750. A clone, ATCC 39891, which complicates a t-ΡΛ analogue with thrombolytic activity is unique in that it contains a remainder of Met at position 245 ^{and a} substitution for Vai. Normally, the DNA sequence encodes a main sequence that is treated, that is, recognized and deleted by the host cell, followed by the amino acid remains of the protein as full length, which starts with Gly.Ala. Arg.Ser.Tyr.Gin ... .According to the medium and the host cell in which the DNA sequence is expressed, the protein obtained in this way can start with an amino terminal Gly.Ala.Arg. or be further treated in such a way that the first three amino acid residues are eliminated by proteolytic enzymes. In the latter case, the mature protein has an amino terminal that comprises:

Ser.Tyr.Gin.Leu .... Variants of t-PA that have either of these arnine termini also have thrombolytic activity and are encompassed by the present invention. The variants according to the present invention also include proteins that

have Met.24.5 or Val 2 ^ 15, as well as other variants, eg, allelic variations or other eliminations or substitutions of amino acids that still retain thrombolytic activity.

The present invention also encompasses compounds, as mentioned above, which contain even more modifications in the polypeptide domain between Asn-218 and Thr-220. Specifically, compounds in these conditions are further characterized by an amino acid other than Asn or a peptide bond at position 218 and / or an amino acid other than Pro or a peptide bond at position 219 and / or an amino acid other than Ser or Thr or a peptide bond at position 220. The compounds in these conditions therefore do not have the consensus N-linked glycosylation site that is not normally glycosylated in t-PA produced by mammalian cells derived from melanoma.

As mentioned above, DNA sequences encoding individual variants of the present invention can be produced by conventional site-directed mutagenesis of a DNA sequence encoding a human t-PA or its analogs or variants. Such methods of mutagenesis include the M13 system of ZOLLER and SMITH; Nucleic Acids Res ”: 10; 1982; p, 6487-6500; Methods Enzimol ”; 100; 1983; P. 468-500; Edna; 2, 1984; P. 479-488, using single-stranded DNA and the method of MORINAGA et. al., Bio / technology; Jul. 1984; P. 636-639, using hetero duplex DNA. Table 7 shows several examples of oligonucleotides, used according to the methods of the present invention, to make /

./

eliminations at the N-terminus or to convert an asparagine residue to threonine or glutainin, e.g. ex .. It should be understood, in fact, that the DNA encoding each of the glycoproteins of the present invention, can be produced in an analogous manner by one skilled in the art, by means of site-directed mutagenesis using an oligonucleotide (s) chosen in an appropriate way. Expression of DNA by conventional means in a mammalian host cell system, yeast, spleen, bacteria or insects, produces the desired variant. Mammalian expression systems and variants obtained from this node are presently preferred.

The mammalian cell expression vectors described herein can be synthesized by techniques well known to those skilled in the art. Vector components such as replicas of bacteria, selected genes, reinforcers, promoters and equivalents can be obtained from natural sources or synthesized by known methods. See Kaufman et al.,; J. Mol Biol ”; 159 «19 ^ 2; P. 51-5--1} Kaufman} Proc. Natl. Acad, Sei. ”; 82, 19θ5} Ρ · 689-693.

Established cell lines, which include transformed cell lines, are appropriate hosts. Normal diploid cells, or strains of cells derived from in vitro culture of primary tissues, as well as primary explants (which include relatively undifferentiated cells such as cells from the hematopoietic branch) are also suitable. Candidate cells do not need to be genotypically deficient in the selection gene as much as it acts dominantly.

Preferred host cells should be

confirmed mammalian cell lines

For stable integration of vector DNA into chromosomal DNA and for subsequent amplification of integrated vector DNA, both by conventional methods, the presently preferred cells are CHO (Chinese hamster ovary) cells. Alternatively, the

Vector DNA can include all or part of the bovine papilloma virus genome (Lusky et al.,; Cell ”; 36 1984; p, 391-401) and be incorporated into cell lines such as C127 mouse cells, under the form of a stable episomic element. Other usable mammalian cell lines include HeLa cells, monkey COS-1 cells, mouse L-929 cells, Balb-c or NIII or Swiss mouse 3T3 cell lines, HaK hamster cell lines or BIIK and the like but are not limited to these.

Stable transformants are then probed for expression of the product by standard immunological or enzymatic assays. The presence of the DNA encoding the variant proteins can be detected by usual processes, such as South-blotting.

Transient expression of the DNA encoding the variants for several days after the intervention of the DNA expression vector in appropriate host cells, such as monkey COS-1 cells, is measured without selection by assaying protein activity or immunology in a culture medium.

In the case of bacterial suspension, the DNA that differs from the variant can be further modified to contain different codes for pox bacterial expression.

and preferably linked in an original way in the chain to a nucleotide sequence encoding secret main polypeptide that allows bacterial expression, secretion and processing of the mature protein variant, also by those known to those skilled in the art. The compounds expressed in host cells of mammals, insects, yeasts or bacteria, can then be recovered, purified and / or characterized with respect to physico-chemical, biochemical, and / or clinical parameters, all also by known methods.

These compounds have been found to bind to monoclonal antibodies targeting human t-PA and can thus be recognized and / or purified by immunoaffinity chromatography using these antibodies. In addition, these compounds have t-PA-like enzymatic activity, that is, the compounds of the present invention effectively activate plasminogen in the presence of fibrin to cause fibrinolysis, as assessed by means of an indirect assay using the chromogenic substrate. plasmin S-2251 as is known in the art.

The present invention also encompasses compositions for trornbolytic therapy that comprise a therapeutically effective amount of a variant mentioned above in admixture with a pharmaceutically acceptable carrier for parenteral application. Such a composition can be used in the same way as described for human t-PA and should be useful in humans or lower animals, such as dogs, cats, and other mammals known to be subject to thrombotic cardio-vascular problems. It is considered that

compositions should be used to treat and desirably to prevent thrombotic problems. The exact dose and method of administration should be determined by the attending physician, according to the potency and pharmacokinetic profile of the particular compound, as well as according to various factors that modify the action of the drugs, e.g. eg, body weight, sex, diet, time of administration, association of drugs, sensitivity reactions and severities of the particular case.

The following examples are given to illustrate aspects of the present invention. It is to be understood that these examples are illustrative and that the present invention is not considered to be limited but by the content indicated in the claims only.

In each of the examples involving the expression of insect cells, the nuclear polyhedrosis virus used was the L-l variant of Autographa Calii. ornica, and the insect cell line used was the spodoptera frugiperda TPLB-SF21 cell line (Vaughn, J.L. et al .; ”In Vitro; 13, 1977, P. 213-217). Virus and cell manipulations were performed according to the detailed description in the literature (Pennock G.D., et al., Supra; Miller, T). W. Safer, P., and Miller, L.K., Genetic Engineering, Vol. 8, pages 277-398; J.K. Sethow and A. Hollaender, ed. Plenum Press, 1986). The M13 PF vectors, <npl8 and mpll, can be purchased commercially from New England Biolabs. However, those skilled in the art to whom the present invention relates will appreciate that, as defined above, other viruses, strains, host cells, promoters and vectors may be used in the practice of practicing each aspect of this invention. contain relevant cDNA. The DNA manipulations performed are, unless otherwise specified herein, according to the methods Maniatis et al., Molecular Cloning; A Laboratory Manual (Cold Spring Harbor, NY 1982).

Table 7: Examples of Oligonucleotides for Mutagenesis

At the.

Sequence

Mutation

1. ACC AAC TGG ACC AGC AGC GCG

2. CTAC TTT GGG ACT GGG TCA GC

3. GTGCACCAACTGGCAGAGCAGCGCGTTGGC

4. CAACTGGCAGAGCAGCG ^As ”1L7 ^Asn 184 '

---- i> Tlir ---> Thr

Asn. _Ί _----> Gln (# 3) *

5. ACTGCTACTTTGGGCAGGGGTCAGCCTACC ^Asn 184 * Gin

6.

CTTTGGGCAGGGGTCAG (# 5) *

7. CATTTACTTCAGAGAACAGTC

Gin

8.

GGA GCC AGA TCT TAC CAA GTG ATC TGCAAGC (A FBR)

GAG CCA AGG TGT TTC AAC GGG GGC

9.

TGATC TGÇ ^ IGC GAG CC (# 8) *

10. AGA GGA GCC AGA TCT TAC CAA GTC

ATG ^ GAT ACC AGG GCC ACG TGC TAC GAG (AFBR / EGF)

11.

CAA GTG ATC4GAT ACC AG (# 10) *

12. TCA GTG CCT GTC AAA AGT ^ ACC AGG GCC

ACG TGC TAC (Δ EGF)

13. CTC AAA AGT ^ ACC AGG G (# 12) *

14. GC CAG CCT CAG TTT ^ ATC AAA GGA GGG C (R-275 del)

15. _______CT CAG TTT ACC ATC AAA G ______________ (..... T-275)

MA mutation indicated in parentheses used for probing (when a probing oligonocleotide is not indicated the same oligonocleotide

gonudeotide is used for mutagenesis and probing) 'Codons for amino acid substitution are underlined, A indicates elimination site. Those skilled in the art will appreciate that oligonucleotides can be rapidly constructed for use in the elimination of one or more amino acids or insertion of a different amino acid (ie, substitution) at a desired site by removing the codon (1) or replacing the codes for the substitution of the amino acid in the oligonucleotide respectively. Other mutagenesis oligonucleotides can be designated based on a sequence of approximately twenty to fifty nucleotides distributed at the desired site, with replacement or elimination of the original codon (1) according to the desired modification.

Plasmid derivations

Mutation of ΛΠΝ in the codons for the various amino acids was performed using appropriate restriction fragments of cDNA in M13 plasmids by the method of Zo-.ler and Smith. Deletions in the cDNA are performed by loop mutagenesis using an appropriate restriction fragment, e.g., the Saci fragment, from cADX either in the M13 vectors or by the heteroduplex loop in the pSVPA4 plasmid.

plasmid pSVPA4 was constructed to allow expression of t-ΡΛ glycoprotein in mammalian cells. This plasmid was constructed by first eliminating the ADX encoding the SV40 T polypeptide from the pspLT5 plasmid (Zhu, Z et al.j J. Virology; 51; 1984; ρ. 1? Θ-18θ), which was performed by digestion total with Xho 1 followed by endo-

partial Eam-Hl restriction nuclease. The SV40 T polypeptide encoding the region in pspLT5 has been replaced by the sequence

encoding human t-PA by ligating a cohesive Sall / BamHI t-PA fragment. encoding restriction fragment isolated by digesting the plasmid J2O5 (ATCC No. 3 ^ 568) with Sal I and Bam HI, to XIIol / BamHI main count the vector pre PSP LT5 stopped as described above. Consequently, t-ΡΛ should be transcribed into this vector under the control of the main promoter SV4O when introduced into mammalian cells. This final construction is called pSVPA4.

plasmid pLDSG is an amplifiable vector for the expression of t-PA in mammalian cells, such as CHO cells. The plasmid pLDSG contains a DHFR mouse DNA transcription unit using the main adenovirus type 2 (MLP) anterior promoter, the monkey 40 virus (SV4o) enhancer and the origin of replication, the SV'tO anterior promoter (in the same orientation as the MLP adenovirus), a gene encoding tetracycline resistance and a DNA encoding human t-PA (Met-245) in its own orientation with respect to the type 2 adenovirus MLP, The pLDSG preparation from pCVSVL2 (ATCC No. 39813) and a t-PA encoding DNA has been described in detail as well as the co-transformation and amplification of pLDSG in CHO cells. Kaufman et al .; Mol. And Cell. Bxo; _5 (?) J 1985í p. 1750-1759.

plasmid pWGSM is identical to plasmid pLDSG except as regards the inserted cDNA encoding human t-PA VAL-245. The pWSGM plasmid or plasmids work

Equally equivalent can be prepared from pLDSG using site-directed mutagenesis. In this critical memory pWGSM or pLDSG is used interchangeably, although, as previously indicated, the former should produce Val-245 proteins and the latter Met-245 proteins.

pIVPA / 1 (ATCC No. 39891) is a baculoviral translocation vector that contains a cDNA encoding t-PA. PIVPA / 1 and its mutagenic derivatives are used to insert the desired DNA into the baculoviral zenoma as the cDNA will be under transcriptional control of the baculoviral polyhedrin promoter.

Heteroduplex mutagenesis

Mutagenesis via hetero-duplex DNA of specific areas in the t-PA expression plasmid, pSVPA'l, involves the following phases:

Preparation of ampicillin-sensitive pSVPAU DNA

1. Plasmid pSVPA4 (15 JAg) was completely linearized with Pvul. This mixture was extracted with phenol / chloroform and the DNA precipitated using two volumes of ethanol in the presence of 0.1 molar NaCl.

2. DNA was resuspended in a solution consisting of 21 μl of water, 1 μl of a dNTB solution (containing 2 mM of dATP, dGTP, dTTP, dCTP), 2.5 μl of a cutting buffer translation 10 (TRIS-lel 0.5M pH 7.5,

- 56 / ί * / s

i 0.1 M MgSO4, 10 mM DTT, 500 pg / ml) and 0.5 µl (2 units) of large fragments of DNA polymerase (New England Biolabs). This mixture was incubated at room temperature for thirty minutes and then extracted with phenol / chloroform followed by ethanol precipitation, as described above.

3. The precipitated DNA was resuspended in 0.2 µg / μΐ by adding 75 µl of water.

Preparation of ampicillus-resistant pSVPA4 DNA. at.

1. The pSVPA4 plasmid (15 pg) was digested with Saci which cuts this plasmid twice in the sequence encoding t-PA to produce two restriction fragments, a 1.4 Kbp fragment of t-PA encoding the fragment constraint plus the main vector. Then the restriction digest was added 1 µl (28 units) of calf intestinal alkaline phosphatase (Boehringer Mannheim) followed by incubation at 37 ° D for five minutes. The two plates were separated by impregnating this mixture on 0.7% agarose gel. The restriction fragment of the main vector was removed from the extracted gel by adsorption on silica dioxide at a temperature of 4 ° C, followed by elution with 50 mM TRIS / 1 mM EDTA at a temperature of 37 ° C for 30 minutes. The eluted DNA was adjusted to a final concentration of 0.2 pg / pl.

K »- * /

- Hello

Circularization of the heteroduplex

1. 6 µl (1.2 µg) of ampicillin-sensitive pSVPA4 DNA was mixed with 6 µl (1.2 µg) of ampicillin-resistant pSVPA4 DNA.

2. An equal volume (12 µl) of 0.4 m sodium hydroxide was added. Incubate at room temperature for 10 minutes.

3. 4.5 volumes (108 zul) of 0.1 M Cl-Tris pH 7.5 20 mM HCL were added slowly.

4. 50 picomoles (5Ail) of phosphorylated mutagenic oligonucleotide were added to 45 Ail of heteroduplex mixture.

5. This mixture was incubated at 68 ° C for two hours and then cooled slowly to room temperature.

Mutagenesis

1. Each reaction medium for mutagenesis was. adjusted to the following concentrations by adding .il to the heteroduplex mixtures of 2 mM MgCl / 0.2 mM ATP / 10 µm dATP, dTTP, dGTP, dCTP / 4 mM DTT / 40 units / ml of Klenow fragments, E. coli polymerized DNA I. (B.R.L.), 2000 units / ml T4 bound DNA (N.E.B.). The mixture was incubated at room temperature for two hours.

2. The reaction medium was then extracted with phenol / chloroform followed by ethanol precipitation. The precepted DNA was resuspended in 12/11 of 50 mM Tris-Cl / 1 mM

EDTA. 4 µl were used to transform the competent HB1O1 bacteria.

3. Ampicillin-resistant colonies were given with 1 x 10 ^ cpm / nil of a probing oligonucleotide labeled with P or 5 x SSC, O, 1A SDS, 5N Denhardt reagent and 100 µg / ml sperm DNA. denatured salmon.

4. The filters were washed with 5 æC SSC, with 1% SDS at a temperature of 5Â ° below the melting temperature of the calculated oligonucleotide probe.

5. DNA was prepared from positive hybridization clones and analyzed initially by digestion with different restriction enzymes and electrophoresis on an agarose gel. DNA was transferred to nitro-cellulose filters and the filters were prepared and hybridized to probes to ensure that the mutagenic oligonucleotide was introduced into the correct fragment.

6. The DNA was then transformed again into E, coli and ampicillin resistant colonies were probed for hybridization for oligonucleotide probing.

7. Final mutations were confirmed by DNA sequencing (Sanger).

Preparation of cDNAs that have undergone mutagenesis; method MI3 schematic restriction map below illustrates the cDNA encoding human t-PA (referred to above) with the specific endonuclease cleavage sites indicated (below):

. ATG— τ 1 ^{1 2} 1 - I ³ 1 ~ T ~ Sac Bgl Nar Eco Eco 1 1 Sac Apa Xma I II I IR IR I I I

(Smal) ATG initiation codon and the ciDX regions encoding (a) the fractions that would be represented by R ₁ , R _o and R „that are indicated. Thus, N-terminal mutagenesis can be performed using a Saci fragment or the BglTl / Xarl fragment, for example. Mutagenesis in the Arg-275 region and / or in the regions represented by R and / or R ^, can be carried out using, for example, the Saci fragment or the Bglll / Sacl fragment. Mutagenesis in the region represented by

can be performed using an EcoRl / XmaT or EcoRl / Apal fragment. The choice of the restriction fragment should be determined based on the suitability of using particular vectors for mutagenesis and / or for the construction of the expression vector. Generally, the cDNA restriction fragment to be mutagenized can be excised from the full-length cABN present, eg, from pWGSX, pIVPA / l or pSVPA4, using the indicated endonuclease enzyme (s) and, the then, mutagenized, e.g., with the oligonucleotides shown in Table 7 or other oligonucleotides designated for the desired mutagenesis.

Examples of cADX fragments that have undergone mutagenesis and that can be prepared from this fear are shown in Table 8.

Table 8: Examples of Mutagenized (I) cDNA Fragments *

| ACT- Sac ΐ Bgl ΐ Xar 1 ^R 2 J Eco 1 Eco í Sac I II I IR IR I

(II)

X *

ATG

Sac

I

I

Bgl

II

I

Xar

I ⁿ í

Eco

IR

Sac

I (III) t

Sac

I

ATG í

Sac

I

ATG · x

(IV)

't | ^JV ] Λ I 1 Bgl Nar Eco Eco Sac II I IR IR I X r T) X T?

I

Bgl

II ² 1

Ecc Eco

RI RI

I

Eco

IR

Sac * R ³ 1

Apa

I

Xma (Signal)

I (V) asterisk indicates the site of mutagenesis; fragments of

t cDNAs from one to four are prepared by digestion of pWGSM or pSVPA4 with Saci, by inserting this fragment into the vector> 113 which causes mutagenesis with the desired oligonudcotide (s) and Saci digestion of the mutated M13 / t-PA DNA; alternatively 1-4 can be excised from the mutagenized M13 / t-PA with BglII and Saci and the fragment Eglll / Sacl that encodes the poptidic domain that covers the N, R ^, Rg and Arg-275 terminal can be inserted in the pTVPA digested with Dllll / / Saci; the cDNA V fragment is prepared as described in Example 2, below.

After mutagenesis, the fragment, with or without posterior mutagenesis, can then be excised from the vector> 113 θ linked to an expression vector containing the partial or total cDNA previously cleaved with the same enzyme (s) as used for excision of the vector> '13 from the mutagenized fragment. Through this method the total cDNA they are. freu a mutagenesis as desired, can be regrouped using one or more fragments that have mutagenized in the form of restriction fragment cartridges.

CDNAs encoding the following representative compounds (see table p. 11 and tables 2.0, 2.5 and 3) can be prepared from the mutagenized fragments re

presented in painting 8 as follows: Compound Via D-6 (The) fragment 1 of the linked mutagenized cDNA D-l (prepared using oligonucleotides D-3 dos / = 8, 10 or 12) in digested pSVPA4

/ *

2-l / N-23 / Arg

2-1 / N-22 / Arg

2-2 / N-23 / Arg

2-2 / N-21 / Arg

2-2 / N-22 / Aig

2-3 / N-23 / Arg with Saci or fragment I excised from

N13 / t-PA mutagenized as the fragment

Bglll / Sacl and inserted into BIVITl / SacI digested pIVPA / l.

(b) ligated mutagenized cDNA fragment II (prepared using oligonucleotides / 8, 10 or 12 and then NO 3 oligonucleotide) in Saci-digested pSVPA4 or the excised fragment of mutated M13 / t-PA as the fragment Bglll / Sacl and inserted into pTVPA / 1 digested with Eglll / Sacl.

(c) ligated mutagenized cDNA fragment III (prepared using oligonucleotides 8, 10 or 12 and oligonucleotide 5) in Saci digested pSVPA4 or excised fragment from mutagenized M13 / t-PA as the Bgl II / Saci fragment and it is inserted into the pIVPA / l digested in Bglll / Sacl.

(d) digested mutagenized pIVPA / l or pSVP_'J 'produced by method (a) with EcoRl (by partial digestion) and Xmal (Smal) or Apal (by total digestion) to eliminate the wild type coding region by binding to this mutated 5 4e cDNA fragment (prepared using the

oligonucleotide n? 7) in the form of the EcoRl / Apa I or EcoRl / Xmal (Smal) fragment,

- 63 2-4 / N-23 / Arg

2-4 / N-21 / Arg

2-4 / N-22 / Arg

I

2-5 / N-23 / Arg

2-5 / N-21 / Arg

2-5 / N-22 / Arg

2-6 / N-23 / Arg? -6 / N-21 / Arg

2-6 / N222 / Arg (e) digested mutagenized pIVPA or pSVPA prepared as indicated in íc) with EcoRI (so partial digests) and XmaT (Smal) or Apal (total digestion) to eliminate the wild-type coding region represented R ^ and ligate it with the V cDNA fragment (prepared using the NS 7 oligonucleotide) as an EcoR1 / Apal or EcoR1 / Xmal (Smal) fragment.

(f) digested mutagenized pIVPA or pSVPA4 prepared by route (b) with EcoRI (partial digestion) and XmaT (Smal) or Apal (total digestion) and attached to this fragment

V mutagenized cDNA (prepared using N ~ 7 oligonucleotide) as the EcoR1 / Apal oxy EcoR1 / Xmal (Smal) fragment.

(g) IV fragment of ligated mutagenized cDNA (prepared using oligonucleotides # 8, 10 or 12, and oligonucleotides # 3 and 5) in Saci-digested pSVPA-l or excised IV fragment of M13 / / tP A like the fragment

Bgl Tl / SacT and bind it with pivpa / 1 digested with Bglll / Sacl.

- 64 - / /

/.

? -7 / N_23 / Arg

2-7 / N-21 / Arg

2-7 / N-22 / Arg (h) fragment IV of the bound mutagenized cDNA (prepared using oligonucleotides N-2 · 8, 10 or 12 oligonucleotides N? 3 and 5) to pSVPA4 digested with Saci prepared by the routes indicated in (d), (e) or (f) or ligate the IV fragment thus obtained as a Bglll / / Saci fragment in the BglII / SacT-digested pIVPA produced by the routes indicated in (d), (e), of (f).

The pIVPA plasmids. or pSVPA4 for, in addition to being used as expression vectors, they can also be used as deposits in the construction of cDNAs that exhibit any desired exchange of mutagenized sites. Thus, the plasmid pIVPA / Δ or pSVPA4 / A, mutagenized (via 2113 or heteroduplex) containing a desired modification in the cDNA region encoding the ^r1 region, the N-terminus can be digested with NarI (partial) and Xmal (Smal) ( total) to eliminate the cDNA region that encodes the protein domain comprised in the regions represented by ΓΙ _χ , R _o and R ^. If desired, a second sinks, mutagenized pIVPA or pSVPA4, (via 'Ί.3 or heteroduplex) in any association of Arg-275 regions or represented by encoded ones can then be digested with NarI (total) and Xmal (Smal) (total) and the Narl / Xmal fragment (Smal) can

then be identified, isolated and linked to pIVPA / Δ or *

pSVPA4 / & digested with Nar I / Xmal (Smal). This use of the Narl / Xmal (Smal) restriction fragment Cassette, p. ex. , allows the construction of the desired inactagenated qWNs in pIVPA or pSVPA4.0 mutagenized cDNA can then be transferred, e.g. e.g., as a Cassette ' ¹ of Bglll / Xmal restriction fragments for pKGSM digested with BglII / XmaT for expression in mammals, if desired.

EXAMPLES

Example 1

Preparation of variants of Glnll7 eliminations

A. Preparation of Gln-117 truncated cDNA

CDNA molecules that encode the polypeptide sequence of compounds 2-1 / N-21 / Arg, 2-1 / / N-22 / Arg and 2-1 / N-23 / Arg were prepared using the directed mutagenesis method oligonucleotides, according to de Zoller and Smith. Specifically, the RF M13 / t-PA mutagenesis vector containing the t-PA gene is constructed from the t-PA expression plasmid pSVPA4. The RF M13 / Í-PA was constructed first by digestion of pSVPA4 complete with the restriction Saci endonucltisis. The approximately 1,436 base pair (bp) Saci fragment encodes a large portion of the t-PA lipeptide sequence and includes the nucleotide sequences that encode the consensus N-linked glycosylation sites spanning asparagines 117,184 and 218. This fragment 1,436 bp (later referred to as 1.4 Kpb) was purified

by preparative agarose gel electrophoresis. The Saci fragment of the t-PA DNA, obtained as a Saci fragment, referred to above, ligated to a double stranded M13 mp 18 RF DNA vector, which had been previously digested with Saci. The ligation mixture was used to transform the competent transforming bacterial JM 101 cells. M13 plates containing DNA derived from t-PA produced from the transformed cells were identified and isolated by analytical DNA restriction analysis and / or by plate hybridization. Radioactively labeled oligonucleotides (approximately 17 m of polaridack p: sitiva) derived from Saci restriction sites of t-PA encoding the nucleotide sequence described in Table 1 were used as probes when filter hybridization was used to detect plaque hybridization. viruses that contained t-PA DNA. All oligonucleotides were prepared by automated synthesis with a DNA synthesizer from Biosystems Applied, according to the manufacturer's instructions,

Several of the positive plates detected by restriction analysis or hybridization analysis were then cloned by conventional plate purification. The purified M13 / t-PA bacteriophage, obtained from the plaque purification process, was used to infect JM101 cells. These infected cells produce double-stranded cytoplasmic M13 / t-PA plasmid DNA (”RF ⁿ ). Infected cells also produce bacteriophages in a culture medium containing single-stranded DNA complementary to the fragment

-6? “

Saci with 1.4 kbp of t-PA or with M13 DNA.

Single stranded DNA was purified from the M13 / t-PA containing the phage isolated from the culture medium. This single stranded M13 / t-PA DNA was used as a template in a mutagenesis reaction, according to the method of Zoller and Smith, using the n? 3 in Table 7 · This mutagenesis changes the Asn codon to a Gin codon at position 117 of the subsequently obtained DNA coding strand, by changing the DNA sequence from AAC "to" CAG ". Following the mutagenesis reaction the DNA was transformed into bacterial strain J.M101. To identify the mutagenized cDNAs, the transformant plates were probed by DNA hybridization using oligonucleotide n? 4 of Table 7, radiolabelled. All of the oligonucleotide examples recorded in Table 7 have positive polarity, that is, they represent fractions of a DNA strand that is more difficult than a non-coding strand. All positive hybridization plates were further purified by subsequent secondary infections of JM1O1 cells with the M13 phage that contained the mutated DNA.

Plasmid RF M13 / t-PA was purified from JM 101 cells infected with purified M13 phage which contained the mutagenized t-PA cDNA. The 'RF M13 / t-PA plasmid obtained in this way contains the mutated Gin 117 Saci restriction fragment of t-PA DNA. This mutagenized restriction fragment can then be further mutagenized by the method of Zoller and Smith but using the oligonucleotides

described below. The oligonucleotides described below were designed to induce a deletion (’ΐοορ out) in the cDNA region encoding the N-terminal domain.

Elimination mutagenesis 1: 0 oligonucleotide n? 8 of the table induced a deletion of cDNA encoding Cys-6 through Ser-50 inclusive. Following this second mutagenesis reaction, the DNA is transformed into JM101 cells. To identify the mutagenized cDNAs, the transformant plates were probed as mentioned above, but using the oligonucleotide labeled with? 9 of Table 7. The positive hybridization plates can be further purified by subsequent secondary infections of JM1O1 cells with the M13 phage containing the twice-mutated t-PA cDNA. The cDNA prepared as described above, which contains this mutagenized restriction fragment, encodes compound 2-1 / N-21 / Arg in which Ile-5 is covalently linked to Cys-51 by a tanidic link.

Elimination mutagenesis 2: Oligonucleotide n? 10 of Table 7 induced an elimination of cDNA encoding Cys-6 through Ile-86 inclusive. Following this second mutagenesis reaction, the DNA is transformed into JM101 cells. To identify the mutagenized cDNAs, the transformant plates were probed, as mentioned above, but using oligonucleotides with the? 11 of Table 7 radiolabelled. The positive hybridization plates can be further purified by subsequent secondary infections of J.M101 cells with the M13 phage containing the twice-mutated t-PA cDNA.

cDNA prepared as described below, which contains this mutagenized fragment, encodes the compound 2-1 / N-22 / Arg in which Ile 5 is linked by a covalent bond to Asp 87 by means of a peptide bond.

Elimination mutagenesis 3: Oligonucleotide n? 12 of Table 7 can be used to generate a cDNA deletion encoding Cys-51 through Asp 87, inclusive.

Following this second mutagenesis reaction, the DNA is transformed into JM1O1 cells. To identify the mutagenized cDNAs, the transformant plates are analyzed, as described above but using oligonucleotide n? 13 of Table 7 radiolabeled. The positive hybridization plates can then be purified by subsequent secondary infections of JM1O1 cells with M13 phage containing twice-mutagenized t-PA cDNA. The cDNA prepared as described below, which contains this mutagenized restriction fragment, encodes the compound 2-1 / N-23 / Arg in which Ser 50 is covalently linked to Thr88 via a peptide bond.

Each of these mutagenized restriction fragments can be linked back to the mammalian expression vector pSVPAU as a Saci cassette by methods analogous to those described in example no. 3 B or prepared for insertion into the pIVPA / 1 insect cell expression vector (ATCC No. 39,891) as a Bglll / Sacl cassette derived from RF DNA Μ / 13 / t-PA.

B. Preparation of vectors used for the expression of high mannose Gln117 elimination variants

RF M13 / purified t-PA containing the modified and truncated t-PA cDNA, prepared as described above, can be digested with the restriction endonuclease BglII and Saci. The approximately 1.2 Kbp Bg11 / Sacl restriction fragment was purified by conventional preparative gel electrophoresis. The Bglll / Sacl fragment obtained in this way constitutes a mutagenized cassette which lacks a 5 'θ 3' fraction of the DNA encoding the amino and carboxylic terminal of the displaced protein.

insect expression vector pIVPA / 1 (ATCC No. 39,891) contains a wild-type t-PA cDNA operatively inserted into the polyhedrin promoter in conjunction with bacu lovirus flanking DNA sequences. The pIVPA / 1 was digested with BglII and Saci thus allowing the excision of a t-PA coding region that covers the N and R1 and R2 terminals. The Bglll / Sacl cassettes containing the mutagenized N-terminal modified t-PA cDNA fragments can each be linked to the DNA of the pTVPA / 1 expression vector that has been previously co-broken down below digestion with BglII and Saci. The resulting plasmids, pIVPA / 1 FBR, Gin 117, pIVPA / 4 FBR / d EGF, Gin 117, pIVPA / 4 EGF, GLN 117 must contain the mutated cDNAs encoding compounds 2-1 / N-21 / Arg, 2-1 / N-22 / / Arg and 2-1 / N-23 / Arg, respectively, now linked in an operational way to the polyhedrin promoter. The nucleus sequence

of each mutagenized cDNA insertion can be confirmed by supercoil sequencing with plasmid as substrate. See, p. E.Y. Chen et al .; DNA '*; 4 (2); 1985; P. I65-I7O.

B. Introduction of mutagenized cDNA in insect viruses.

Each of the pIVPA plasmids containing the cDNAs can be introduced into the insect virus by co-transfection with wild-type AcNPV into SPODOPTERA cells. 1 pg of purified California Autograph NPV DNA and 10 pg of the desired pIVPA DNA are introduced into Spodoptera cells growing in tissue culture plates by the calcium phosphate transfection process (Potter, KN and Miller, LK , "J. Invertebr. Path"; 36; 19θ0; p. 431-432). The joint introduction of these DNAs in the cells results in a double recombination between the plasmid pIVPA (which contains the mutagenized cDNAs) and the viral DNA in the homologous regions between the two; that is, the genetic region of the progenic virus polyhedrin from the recombination that occurred contains the insertion of mutated cDNA from the pIVPA plasmid.

Isolation of the virus containing the protein encoded nucleotide sequence of the present invention.

The progenic virus present in the medium after the transfected cells was plated in a single layer of fresh cells at different dilutions. Plates are analyzed and recombinants are collected based on the minus-PIB phenotype as follows: a virus that has lost its polyhedrin penis which, as it is a virus that contains a mutagenized cDNA, does not produce PIBs. Plates that are deficient in PIB are chosen, excised and enlarged in fresh cells. The supernatant of these cells is then analyzed for t-PA type enzyme activity. The results of positive tests indicate that the glycoprotein is in fact being pro. produced. An alternative method of purifying the virus via the protocol, which includes plaque pulling, differs slightly from the steps described above and is described below. The progeny virus from the transfection is placed in cell culture plates at an appropriate dilution. A nitrocellulose replica of the cell monolayer and virus plates is prepared. The upper agarose layer of the plate is reserved as a virus source, after which the results of the following phases are obtained.

The nitrocellulose filter with radioactive DNA fragments representative of the gene is introduced into a viral chromosome. Those that contain the foreign gene are marked as positive. The hybridized probe is removed. The filter is re-probed with radioactive DNA representative of a fraction of the viral chromosome eliminated by substitution with the foreign DNA. Positives can be marked among those who still have a polyhedrin gene.

The hybridized probe is removed. The filter is re-probed with a radioactive DNA fragment that it identifies. will display viral plaques regarding the status of the polyhedrin gene. One of the appropriate fragments can be the fragment

EcoRI 1. These are labeled with the progenic virus. Plates are chosen that are positive for the foreign gene DNA probe and negative for the polyhedrin gene probe and positive for the viral DNA probe. These are strong candidates for the desired genotype.

C. Production and characterization of mannose glycoprotein

Antibodies were used to demonstrate the presence of variant proteins in the extracellular medium of infected cells. Recombination viruses, prepared as mentioned above, are used to infect cells growing in the usual TC-100 nutrient salt solution (Gibco) but instead of the standard medium supplemented with 10% fetal calf serum, enrichment was used. with egg yolk (for a total volume of 1%, Scott Biologicals).

Previous experiments have shown a protein closer to integrity under these conditions. The supernatant part of the infected cells was fractionated on an affinity column that carried a monoclonal antibody bound to a natural human t-PA. The protein retained in a specific way by the column was eluted and analyzed for the enzymatic activity of t-PA. A fixed number of units of activity was separated from this and from the preparation of control t-PA on acrylamide gel. This gel was then stained with a silver reagent to highlight a protein model. This revealed that the virus, after infection of the

insect cells, leads to the extracellular production of a protein that has t-PA-like activity.

For better characterization, a radiolabelled protein was first produced by incubating spodoptera frugiperda cells infected with the virus (m. O. I = 1) for forty-eight hours. The culture plates were then washed with a medium deficient in methionine. Then, the methionine-deficient medium, supplemented with methionine S35, was added to the culture plates. Cell cultures were incubated for four hours. The overlying part containing the radiolabeled glycoprotein can be analyzed by SDS-PAGE (7.5%) against wild type, (ie, glycosylated over the entire length) of t-PA produced in an analogous way in cells of insect and mammalian t-PA produced p. eg, by the method of R. Kaufman et al .; Mol. Cell. Bioll '; £ (7); 1985; P. 1750, but in the presence of tunicamycin (not glycosylated). The partially glycosylated truncated proteins produced in example 1 must have increased gel mobility relative to the full glycosylated analog and the full-length non-glycosylated analog.

Example 2

Preparation of other proteins of the present invention

A. Preparation of other cDNAs

The mutagenesis methods of Example 1 can

V ...

be used with other synthetic oligonucleotides usually prepared that modify the DNA sequence of the original t-PA to produce modified proteins in the N-terminal region and / or possibly at the modified N-linked and / or Arg-linked glycosylation sites 275 with alteration (ode ^) of appropriate codon as previously described. See, p. ex. Preparation of Mutagized cDNAs: M13 Method ”and pathways a) through h), supra.

For example, cDNA encoding compounds

D-6, D-1 and D-3 can be prepared using the Saci restriction fragment in M13 / t-PA followed by mutagenesis with oligonucleotides n-s 8, 10 and 12 respectively but not with oligonucleotide n? 3. The Arg-275 region, for example, can be deleted or replaced by Thr, using oligonucleotides 14 or 15 respectively. Vector construction, transfection and expression can be performed as in example 1 for insect cells or as described below in example 3 for mammalian cells. Single-stranded DNA produced from the mutagenesis of the vector M13 (RF M13 / t-PA), prepared according to that described in example 1, can also be used as a template to mutagenize at a specific site, in Arg-275 and / or at the glycosylation site (s) R ^ or Rg or both. The region encoding the consensus tripeptide spanning Asn-218 can be mutagenized in a similar way. To prepare for multiple protein modifications at these sites, an iterative process can be used. For example, following the identification and purification of the M13 phage that contains a modified site, the single-stranded DNA that contains this site

The modified protein can be purified from the phage and used as a matrix to initiate a second mutagenesis pathway at the Rg site and / or the Arg-275 region. This process can be repeated until the desired modifications are achieved. Thus, cDNA encoding compounds 2-2 / N-23 / Arg, 2-2 / N-21 / Arg and 2-2 / N-22 / Arg can be prepared by the method described in example 1 but with substitution of the oligonucleotide of mutagenesis 5 by the oligo. nucleotide n? 3 θ oligonucleotide n? 6 of probing by oligonucleotide n? 4. The cDNA encoding compounds 2-6 / N-21 / / Arg, 2-6 / N-22 / Arg and 2-6 / N-23 / Arg can be prepared by double mutagenesis of the Saci fragment, as if described in example 1 and by additional mutagenesis and probing with oligonucleotides 5 and 6. Vector construction, transfection and expression are performed as in example 1 for insect cells or as described below for mammalian cells. See points a) to h), above.

RF mutagenesis vector M13 / t-PA should not contain the DNA sequence encoding R3, the N-linked glycosylation site of t-PA closest to the carboxyl terminus of the protein. Therefore, in order to make DNA modifications at that site, a new M13 / t-PA mutagenesis RF vector designated M13 / t-PA: R1-XmaT was constructed. This vector was constructed by digesting M13 mp 11 of the complete M13 vector with EcoRI and Xmal. The R13 / Xmal digested M13 vector was ligated to an EcoR1 / XmaT t-PA restriction fragment (approximately 439 bp, hereinafter referred to as 0.4 Kbp) that encodes the polypeptide region that encompasses the

glycosylation site R3. This 0.4 Kbp restriction fragment was purified following digestion of the plasmid pWGSM with EcoRI and Xmal. The mammalian expression pWGSM plasmid, which encodes the t-PA gene, is identical in the 439 bp EcoR1 / Xmal fragment. the plasmid pLDSG described by Kaufman et al .; «Mol. Cell Biol; j>; 1985? P. 1750-1759). The ligation mixture was used to transform bacterial JM 101 cells into competent ones. Several plaques were recovered and analyzed for the presence of the 0.4 kbp, t-PA EcoR1 / Xmal fragment by standard analysis of DNA restriction fragments. Double stranded M13 RF DNA was purified from cells containing the 0.4 kbp t-PA fragment. This DNA was called the RF mutagenesis vector M13 / t-PA: IR-Xmal. As previously indicated in example IA, this vector, when transformed into the competent JM101 cells, can be used to construct the M13 / t-PA / IR-Xmal phage from which M13 / t-PA DNA can be purified : Single-stranded Rl-Xmal. This single-stranded DNA can be used as a template in the site-directed mutagenesis reaction to modify the t-PA DNA at the N-linked glycosylation site R3.

The modified R3 coding sequences can be used to replace the wild type R3 sequences present in the modified pIVPA / 1 prepared according to example 1 (truncated and / or modified in RI and / or R2) or in the plasmid DNA pIVPA / 1 wild type. This can be carried out first by means of total Sacl / Apal digestion of the M13 / t-PA mutagenesis plasmid vector: R3 modified R1 / Xmal and 165 base pair modified R3 isolation of the t-PA restriction fragment produced in this way. This pTVPA / 1 insect expression vector or the modified pIVPA / 1 plasmid DNA, e.g. ex. as in example 1, it can be fully digested in an identical manner with Saci and Apal to eliminate the 165 base pair wild-type t-PA restriction fragment encoding the unmodified R3 site. Binding of the purified insect expression vector without the 165 base pair fragment to the 165 base pair modified R3 produces a new insect expression vector.

Expression of this vector produces a modified truncated protein at the R3 site, as well as at any or all of the other N-linked glycosylation sites present in natural t-PA and / or Arg-275.

The pIVPA plasmid containing the modified cDNA can also be used to generate the modified t-PA cDNA fragment Bglll / / APal that encompasses the deletion region in the N-terminal domain, as well as the region that encodes R1, R2 and R3 or the Narl / XmaT fragment that covers R1, R2 and R3. Any of these fragments can be inserted into expression vectors such as pSVPA / 4 or pWGSM as described in Example 3.

Example 3

Preparation of D-6, D-1 and D-3 compounds in mammalian cells

A. Preparation of cDNA.

The cDNA molecules encoding the polypeptide sequences of compounds D-6, D1 and D-3 were prepared using the mutagenic oligonucleotides n® 8, 10 and 12, respectively and the Saci fragment of the t-PA cDNA as a matrix by the method of M13 of example 1 or by heteroduplex mutagenesis (Moranaga Heteroduplex Mutagenesis pr <> tocolj both supra). The mutants chosen by DNA hybridization using probe oligonucleotides 9, 11 and 13, respectively, were confirmed by DNA sequence analysis to be correct in the modified DNA sequence.

B. Preparation of the modified t-PA vector.

Each modified cDNA prepared in Example IA (Δ, Gin 117) or 3A (Δ) was first eliminated from the RF mutagenesis vector M13 / t-PA by total digestion of the vector with Saci. The approximately 1.4 kph restriction fragment from each mutagenized cDNA was purified by gel electrophoresis and then ligated with pSVPA4 as follows. First, pSVPA4 was digested with Saci to eliminate the 1.4 kbp wild-type t-PA restriction fragment. The remaining fraction of SacI-digested pSVPA4 was then ligated into the 1.4 Kbp restriction fragment of the mutagenized cDNA.

This link can be produced in two orientations by the inserted fragment. The appropriate orientation in each case can be identified using EcoRI and PvuII enzymes in a conventional analytical restriction enzyme analysis.

This substitution allows the Saci fragment to be used as a cassette fragment between the RF mutant vector M13 / t-PA and the mammalian expiession vector pSVPA4. The modified M13 Sacl fragments (truncated and optionally modified at R ^ and / or R ₉₎ can be inserted into pSVPA4 DNA digested with SacI which was previously or subsequently modified if desired. Alternatively, the

DNA previously mediated in * 1A and / juR ^ can be excised from vectors such as pIVPA or pSVPA4 as a fragment

Narl / Apal or Narl / Xmal. The fragment obtained in this way can then be digested in vectors such as pSVPA4 or pWGSM previously digested with NarI (partial) and Apal or Xmal (total). By this method any combination of N-terminal eliminations and / or glycosylation substitution and mutagenesis and / or Arg-275 mutagenesis can be obtained.

C. Transfection of COS cells (SV40 from transformed African green monkey kidney)

COS-1 cells (ATCC CRL 1650) were transfected according to the method of Lopata, M.A, et al., * Nucl. Acids Res .; 12; 1984; P. 5707-5717 with the vectors prepared in example 3B, i.e., modified pSVPA4. 0 medium containing

serum was replaced with serum-free medium 24 hours after transfection and the conditioned medium was analyzed for the presence of plasmogenic activation activity using the chromium substrate S-2251 or for the presence of the t-PA antigen by ELISA method 48 hours and 7 [2 hours post-transfection.

D. Viral propagation in CV1 cells (African green monkey kidney).

The protein and carbohydrate complex modified by infection of CV1 cells (ATCC CCL 70) can be produced with viral SV4O from stock propagated in the manner described by Gething and Sambrook ('• Nature'; 293; 1981; P · 620 -625). This production was performed first by complete digestion of pSVPA4 with BamH1 restriction endonuclease to eliminate the short vector pxf3 from the SV40 viral DNA. Prior to transfection of this DNA into CV1 cells, along with the helper virus SV40-RINS-pBR322 DNA (described below). The SV40 / t-PA DNA linearized with BamH1 is circularized by ligation with DNA in diluted concentrations of 1 pg / ml. This process was repeated with the insulin containing SV40-RINS-pBR 322 of the SV40 vector (Horowitz M et al., 1982, Eucaryotic viral vectors, pp. 47-53, Cold Spring Harbor Laboratory). The short bacterial vector pBR322 was eliminated in SV40-RINS-pBR322 by total digestion with EcoRI. The viral SV40-linearized insulin DNA was then circulated by binding to a DNA with a concentration of 1 µg / ml. It is necessary, to transfect the

- 82 / $

’V.

# · CV-1 cells with circularly linked pSVPA4 and SV4O-rlNS DNA, in equimolar amounts in order to generate viral stocks. The SV40-rINS was used to provide late SV4O proteins ”while pSVPA4 provides fast SV4O proteins” needed for the production of virus DNA and also encodes the proteins of the present invention. Consequently, when the cells are transfected with the two DNA's as described by Gething and Sambrook, the SV4O virus is produced, which contains DNA from any of the viral vectors. Subsequent infection with extended CV1 pro duced virus protein having an activity of t-PA type that can be parsed 7 ² hours post infection as described in Example 3 C.

Example 4

Preparation of other proteins

The cDNAs encoding various proteins of the present invention were prepared by the methods described in examples 1, 2 and 3. The Bg11 / Xmal restriction fragment cassette can then be excised from the pIVPA vector or the pSVPA4 vector containing the cDNA encoding the truncated protein with or without modification at one or more glycosylation sites. The excised Bglll / Xmal fragment can then be ligated to Bglll / Xmal cut pSVPA4 or pWGSM for introduction into mammalian cells. The expression of such cDNAs in mammalian host cells, e.g. ex. by the method described

- 83 r (in example 3 or by the method of Kaufman et al., Supra (CHO host cells) or by the method of HOWLEY et al., U.S. Patent No. 4,419,446 (1983) (BPV expression systems ) produces the mammalian deprived truncated proteins corresponding _o Thus, the cDNAs encoding compounds 2-1 / N-21 / Arg (AFBR, Gin 117) and 2-1 / N-22 / Arg (AFBR / / EGF, Gin 117) were prepared and inserted into pSVPA as described above The cDNA encoding 2-1 / N-23 / / Arg (Δ EGF, Gin 117) was prepared using mutagenesis oligonucleotide # 12 and oligonucleotide no. 13 (table 7), but using the heteroduplex method, described above, with previously mutagenized pSVPA4 at position 117 (as mentioned above), as a matrix. Similarly, the cDNA encoding the D1 (Δ FBR) and D-3 (ÚEGF-FBR) were prepared by M13 mutagenesis, as mentioned above, and inserted as a Saci fragment into SacS digested pSVPA4. The cDNA encoding compound D-6 (Δ EGF) was prepared by the heteroduplex method described above, using pSVPA4 as the matrix and the mutagenic oligonucleotide n? 12, and probe oligonucleotide n? 13.

To prepare the cDNAs encoding proteins for amplification and expression in mammalian cells, the cDNA containing pSVPA4 or pIVPA was excised as a Bglll / Xmal fragment and ligated into purified Bglll / Xmal digested pWGSM. In either case, the resulting pWGSM vector was introduced into CHO cells and amplified by the Kaufman method, supra. The transformed and enlarged CHO cells

produce the compounds D-6, Dl, D-3, 2-l / N-23 / Arg, 2-1 / / N-21 / Arg and 2-1 / N-22 / Arg, respectively, which have been eliminated in the culture medium by human t-PA specific antibodies. The compounds can then be collected and purified by immunoaffinity chromatography.

Example 5 Example 4 can be repeated using cDNA encoding N-terminal and / or R-275 modified proteins with or without RI and R2 and / or R3 modification, to produce the desired protein in CHO cells. CDNAs can pre. stop as previously mentioned. Thus, cDNAs encoding compounds 2-7 / N-23 / Arg, 2-7 / N-21 / Arg and 2-7 / N-22 / / Arg, are prepared in pIVPA, as described in the example 2. The cDNAs can then be eliminated as the Bglll / Xmal fragment and ligated with pWGSM previously digested with Bglll / Xmal and the resulting vector, transformed and amplified in CHO cells, as in example 4, to produce compounds 2- 7 / N-23 / Arg, 2-7 / N-21 / Arg and 2 -? / N-22 / Arg.

Claims (13)

Claims 1. - Process for the preparation of a thrombolytic protein that has plasminogen activating type activity, containing a peptide sequence substantially identical to the human t-PA peptide sequence with the exception of: a) Arg-275 is cut off or replaced by another amino acid, and b) at least one of the amino-glycosylation consensus sites is modified to a different site, characterized in that a host cell containing a DNA molecule encoding said protein is actuatedly linked to a control sequence expression capable of allowing the host cell to produce the protein. 2. - Process for the preparation of a thrombolytic protein that has plasminogen activating activity, containing a peptide sequence substantially identical to the human t-PA peptide sequence, with the exception of 15 or more amino acids included in the region between aGly- ( -3) and Thr-91 have been eliminated, characterized by the cultivation of a host cell that contains a DNA molecule that encodes said protein in an actuating way to an expression control sequence, allowing the host cell to produce the protein. Process according to claim 2, characterized in that the thrombolytic protein contains an elimination of 15 or more of the amino acids included in the region between Gly - (- 3) or Ser-1 and Ser-50. 4. A process according to claim 3, characterized in that the thrombolytic protein contains an elimination of one or more of the amino acids in the region between Cys-51 and Thr-91. 5. - Process for the preparation of a thrombolytic protein which has plasminogen activating activity, containing a peptide sequence substantially identical to the human t-PA peptide sequence, with the exception of one or more of the amino acids in the region between Cys- 51 and Thr-91 are eliminated, characterized by the cultivation of a host cell containing a DNA molecule, which encodes said protein, linked in an active way to an expression control sequence in order to allow the cell host to produce the protein. 6. - Process for the preparation of a thrombolytic protein that has plasminogen activator-type activity, containing a peptide sequence substantially identical to the human t-PA peptide sequence, with the exception of one or more of the amino acids in the region defined by Gly - (- 3) or Ser-1 and Thr-91 are replaced by different amino acids, characterized by the fact that a host cell containing a DNA molecule, which encodes said protein, is cultivated, linked in an active way to a sequence expression control to allow the host cell to produce the protein. 7. A process according to claim 6, characterized in that the thrombolytic protein contains an elimination of one or more amino acids in the region between Gly - (- 3) or Ser-1 and Thr-91. 8. A process according to claim 6, characterized in that the number of substituted amino acids is between 1 and about fifteen. 9. The method of claim 8 wherein the number of substituted amino acids is between 1 and about 5. Process according to one of Claims 1 to 9, characterized in that the thrombolytic protein also contains a modification of one of the aminoglycosylation consensus binding sites, to a different binding consensus site. Process according to one of claims 1 to 9, characterized in that the thrombolytic protein also contains the modification of two consensus aminoglycosylation binding sites, for sites other than these. 12. Process according to one of Claims 1 to 9, characterized in that the thrombolytic protein also contains the modification of three consensus aminoglycosylation binding sites to other different binding sites. 13. Process according to one of Claims 2 to 12, characterized in that the thrombolytic protein is replaced by Arg-275 with a peptide bond with an amino acid other than Arg.