Neurite-promoting factor and process for the manufacture thereof

Claim 2

A process for the preparation of essentially pure human glia-derived neurite-promoting factor (GdNPF), related polypeptides or fragments thereof retaining neurite-promoting activity, characterized in that the desired compound is isolated from cells producing it or is synthesized by condensation reactions.

Claim 3

A process according to claim 1, comprising the steps of a) isolating a DNA coding for GdNPF or a fragment thereof from a cDNA library of glial cells or a genomic DNA library and optionally mutating it, or chemically synthesizing such a DNA, b) incorporating the DNA into an appropriate expression vector, c) transferring the obtained hybrid vector into a recipient host, d) selecting the transformed host from untransformed hosts by culturing under conditions under which only the transformed host survives, e) culturing the transformed host under conditions which allow expression of the heterologous polypeptide, and f) isolating the human GdNPF, related peptide, or fragment thereof.

Claim 4

A process according to claim 1, characterized in that a N-protected amino acid is coupled to a suitable resin, the protect­ing group is removed, a sencon N-protected amino acid is condensed with the amino group of the first amino acid, the cycle of depro­tection/condensation with the next N-protected amino acid is repeated until the peptide residue of the desired composition is complete, and finally this peptide residue is cleaved from the resin and deprotected.

Claim 5

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare human GdNPF of the formula X₁-Ser-His-Phe-Asn-Pro-Leu-Ser-Leu-Glu-Glu-Leu-Gly-Ser-Asn-Thr-Gly- 16 Ile-Gln-Val-Phe-Asn-Gln-Ile-Val-Lys-Ser-Arg-Pro-His-Asp-Asn-Ile- 32 Val-Ile-Ser-Pro-His-Gly-Ile-Ala-Ser-Val-Leu-Gly-Met-Leu-Gln-Leu- 48 Gly-Ala-Asp-Gly-Arg-Thr-Lys-Lys-Gln-Leu-Ala-Met-Val-Met-Arg-Tyr- 64 Gly-Val-Asn-Gly-Val-Gly-Lys-Ile-Leu-Lys-Lys-Ile-Asn-Lys-Ala-Ile- 80 Val-Ser-Lys-Lys-Asn-Lys-Asp-Ile-Val-Thr-Val-Ala-Asn-Ala-Val-Phe- 96 Val-Lys-Asn-Ala-Ser-Glu-Ile-Glu-Val-Pro-Phe-Val-Thr-Arg-Asn-Lys- 112 Asp-Val-Phe-Gln-Cys-Glu-Val-Arg-Asn-Val-Asn-Phe-Glu-Asp-Pro-Ala- 128 Ser-Ala-Cys-Asp-Ser-Ile-Asn-Ala-Trp-Val-Lys-Asn-Glu-Thr-Arg-Asp- 144 Met-Ile-Asp-Asn-Leu-Leu-Ser-Pro-Asp-Leu-Ile-Asp-Gly-Val-Leu-Thr- 160 Arg-Leu-Val-Leu-Val-Asn-Ala-Val-Tyr-Phe-Lys-Gly-Leu-Trp-Lys-Ser- 176 Arg-Phe-Gln-Pro-Glu-Asn-Thr-Lys-Lys-Arg-Thr-Phe-Val-Ala-Ala-Asp- 192 Gly-Lys-Ser-Tyr-Gln-Val-Pro-Met-Leu-Ala-Gln-Leu-Ser-Val-Phe-Arg- 208 Cys-Gly-Ser-Thr-Ser-Ala-Pro-Asn-Asp-Leu-Trp-Tyr-Asn-Phe-Ile-Glu- 224 Leu-Pro-Tyr-His-Gly-Glu-Ser-Ile-Ser-Met-Leu-Ile-Ala-Leu-Pro-Thr- 240 Glu-Ser-Ser-Thr-Pro-Leu-Ser-Ala-Ile-Ile-Pro-His-Ile-Ser-Thr-Lys- 256 Thr-Ile-Asp-Ser-Trp-Met-Ser-Ile-Met-Val-Pro-Lys-Arg-Val-Gln-Val- 272 Ile-Leu-Pro-Lys-Phe-Thr-Ala-Val-Ala-Gln-Thr-Asp-Leu-Lys-Glu-Pro- 288 Leu-Lys-Val-Leu-Gly-Ile-Thr-Asp-Met-Phe-Asp-Ser-Ser-Lys-Ala-Asn- 304 Phe-Ala-Lys-Ile-Thr-X₂-Ser-Glu-Asn-Leu-His-Val-Ser-His-Ile-Leu- 320 Gln-Lys-Ala-Lys-Ile-Glu-Val-Ser-Glu-Asp-Gly-Thr-Lys-Ala-Ser-Ala- 336 Ala-Thr-Thr-Ala-Ile-Leu-Ile-Ala-Arg-Ser-Ser-Pro-Pro-Trp-Phe-Ile- 352 Val-Asp-Arg-Pro-Phe-Leu-Phe-Phe-Ile-Arg-His-Asn-Pro-Thr-Gly-Ala- 368 Val-Leu-Phe-Met-Gly-Gln-Ile-Asn-Lys-Pro 378 (I), optionally glycosylated, wherein Cys is optionally in the disulfide form, X₁ is hydrogen, acyl, the peptide residue of the formula or a fragment of the residue of formula II comprising between 1 and 18 amino acids from the carboxyl end, optionally in acylated form, and X₂ is Arg or Thr-Gly, related polypeptides retaining neurite-­promoting activity, wherein one or more single amino acids in the compound of the formula I are replaced by other amino acids, and fragments of the compound of the formula I comprising at least 10 consecutive amino acids selected from the amino acid chain between the amino acid 29 and amino acid 378 and optionally one or more other amino acids.

Claim 6

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare human GdNPF of the formula I, wherein Cys is optionally in the disulfide form and X₁ is hydrogen, acetyl, the peptide residue of the formula II or a fragment of the residue of formula II comprising between 1 and 18 amino acids from the carboxyl end.

Claim 7

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare human GdNPF of the formula I, wherein Cys is optionally in the disulfide form and X₁ is hydrogen, the peptide residue of the formula II, the fragment comprising the amino acids -18 to -1 of the formula II, Pro-Ser-Ile-Cys-, Ser-Ile-Cys-, Ile-Cys-, or Cys-.

Claim 8

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare human GdNPF of formula I, wherein X₂ is Arg.

Claim 9

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare human GdNPF of formula I, wherein X₂ is Thr-Gly.

Claim 10

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a GdNPF-related polypeptide of formula I, wherein X₁ and X₂ have the meaning given in claim 4 and wherein one, two, three or four single amino acids are replaced by other amino acids.

Claim 11

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment comprising at least 10 consecutive amino acids of the compound of the formula I, wherein X₂ is Arg or Thr-Gly, selected from the amino acid chain between the amino acid 29 and amino acid 378, and optionally one or more other amino acids.

Claim 12

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment comprising amino acids 2 to 378, 3 to 378, 4 to 378, 5 to 378, 6 to 378, or 7 to 378 of the compound of the formula I, wherein X₂ is Arg or Thr-Gly.

Claim 13

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment comprising 10 to 50 consecutive amino acids of formula I, wherein X₂ is Arg, including ³¹⁰ Arg and ³¹¹Ser and/or ³⁴⁵Arg and ³⁴⁶Ser and optionally one, two or three other amino acids.

Claim 14

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment comprising 10 to 50 consecutive amino acids of formula I including amino acids selected from the amino acid chain 72 to 96, 134 to 146, 159 to 195 or 314 to 378, and optionally one, two or three other amino acids.

Claim 15

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment of the formula Gln-Lys-Ala-Lys-Ile-Glu-Val-Ser-Glu-Asp-Gly-Thr-Lys-Ala.

Claim 16

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment of the formula His-Lys-Ala-Lys-Ile-Glu-Val-Ser-Glu-Asp-Gly-Thr-Lys-Ala.

Claim 17

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment of the formula Arg-Ser-Ser-Pro-Pro-Trp-Phe-Ile-Val-Asp-Ser-Phe.

Claim 18

A process according to claim 1, 2 or 3, characterized in that the starting material is so chosen as to prepare a fragment of the formula Lys-Ser-Arg-Phe-Gln-Pro-Glu-Asn-Thr-Lys-Lys-Arg-Thr-Phe.

Claim 19

A process for the preparation of DNAs coding for GdNPF, mutants thereof, DNAs coding for related polypeptides retaining neurite-­promoting activity and fragments of such DNA comprising culturing a transformed host and isolating the desired DNA therefrom or synthe­sizing it by nucleotide condensation.

Claim 20

A process for the preparation of DNAs coding for GdNPF, mutants thereof, DNAs coding for related polypeptides retaining neurite-­promoting activity and fragments of such DNA comprising culturing a transformed host and isolating the desired DNA therefrom or synthe­sizing it by nucleotide condensation.

Claim 21

A process according to claim 18, comprising the steps of a) isolating poly(A) messenger RNA (mRNA) from glial cells, optionally enriching mRNA coding for GdNPF or fragments thereof and preparing single-stranded DNA complementary to that mRNA and therefrom double-stranded complementary DNA (ds cDNA), or b) isolating genomic DNA from suitable cells and selecting the desired DNA using a DNA probe, and c) incorporating ds cDNA of step a) or ds DNA of step b) into an appropriate expression vector, d) transforming an appropriate host with the obtained hybrid vector, e) selecting the transformed hosts which contain GdNPF DNA or fragments thereof from hosts containing no GdNPF DNA or fragments thereof, and f) isolating the desired DNA.

Claim 22

A process according to claim 18, characterized in that an oligonucleotide is condensed with a mono- or oligonucleotide by chemical or enzymatic condensation.

Claim 23

A process according to claim 19, characterized in that in step a) the mRNA is isolated from the human glioma cell line LN-340, which has been deposited at the "Collection Nationale de Cultures de Microorganismes", Institut Pasteur, Paris, on February 5, 1986 under the number I-518.

Claim 24

A process accordding to claim 18, 19 or 20, characterized in that the starting material is so chosen as to prepare a DNA of the formula Z₁-Y₁₃-Y₃-Y₁₈-Y₉-Y₁₁-Y₁₅-Y₁₁-Y₁₄-Y₁₁-Y₁₁-Y₁-Y₁₆-Y₂₀-Y₁₇-Y₁₁-Y₁₅-­Y₁₆-Y₁₀-Y₅-Y₁₆-Y₉-Y₁₄-Y₃-Y₁₅-Y₁₁-Y₁₆-Y₁₁-Y₇-Y₇-Y₁₁-Y₈-Y₁₆-Y₃-­Y₁₇-Y₈-Y₁₀-Y₆-Y₂₀-Y₁₄-Y₃-Y₆-Y₁₀-Y₂₀-Y₁₂-Y₁₆-Y₂-Y₁₅-Y₉-Y₄-Y₃-Y₁₀-­Y₂₀-Y₁₀-Y₁₆-Y₁₅-Y₉-Y₈-Y₁₀-Y₁-Y₁₆-Y₂₀-Y₁₁-Y₈-Y₁₃-Y₁₁-Y₆-Y₁₁-­Y₈-Y₁-Y₄-Y₈-Y₂-Y₁₇-Y₁₂-Y₁₂-Y₆-Y₁₁-Y₁-Y₁₃-Y₂₀-Y₁₃-Y₂-Y₁₉-Y₈-Y₂₀-­Y₃-Y₈-Y₂₀-Y₈-Y₁₂-Y₁₀-Y₁₁-Y₁₂-Y₁₂-Y₁₀-Y₃-Y₁₂-Y₁-Y₁₀-Y₂₀-Y₁₆-Y₁₂-­Y₁₂-Y₃-Y₁₂-Y₄-Y₁₀-Y₂₀-Y₁₇-Y₂₀-Y₁-Y₃-Y₁-Y₂₀-Y₁₄-Y₂₀-Y₁₂-Y₃-Y₁-­Y₁₆-Y₇-Y₁₀-Y₇-Y₂₀-Y₁₅-Y₁₄-Y₂₀-Y₁₇-Y₂-Y₃-Y₁₂-Y₄-Y₂₀-Y₁₄-Y₆-Y₅-Y₇-­Y₂₀-Y₂-Y₃-Y₂₀-Y₃-Y₁₄-Y₇-Y₄-Y₁₅-Y₁-Y₁₆-Y₁-Y₅-Y₄-Y₁₆-Y₁₀-Y₃-Y₁-Y₁₈-­Y₂₀-Y₁₂-Y₃-Y₇-Y₁₇-Y₂-Y₄-Y₁₃-Y₁₀-Y₄-Y₃-Y₁₁-Y₁₁-Y₁₆-Y₁₅-Y₄-Y₁₁-Y₁₀-­Y₄-Y₈-Y₂₀-Y₁₁-Y₁₇-Y₂-Y₁₁-Y₂₀-Y₁₁-Y₂₀-Y₃-Y₁-Y₂₀-Y₁₉-Y₁₄-Y₁₂-Y₈-­Y₁₁-Y₁₈-Y₁₂-Y₁₆-Y₂-Y₁₄-Y₆-Y₁₅-Y₇-Y₃-Y₁₇-Y₁₂-Y₁₂-Y₂-Y₁₇-Y₁₄-Y₂₀-­Y₁-Y₁-Y₄-Y₈-Y₁₂-Y₁₆-Y₁₉-Y₆-Y₂₀-Y₁₅-Y₁₃-Y₁₁-Y₁-Y₆-Y₁₁-Y₁₆-Y₂₀-Y₁₄-­Y₂-Y₅-Y₈-Y₁₆-Y₁₇-Y₁₆-Y₁-Y₁₅-Y₃-Y₄-Y₁₁-Y₁₈-Y₁₉-Y₃-Y₁₄-Y₁₀-Y₇Y₁₁-Y₁₅-­Y₁₉-Y₉-Y₈-Y₇-Y₁₆-Y₁₀-Y₁₆-Y₁₃-Y₁₁-Y₁₀-Y₁-Y₁₁-Y₁₅-Y₁₇Y₇-Y₁₆-Y₁₆-Y₁₇-­ Y₁₅-Y₁₁-Y₁₆-Y₁-Y₁₀-Y₁₀-Y₁₅-Y₉-Y₁₀-Y₁₆-Y₁₇-Y₁₂-Y₁₇-Y₁₀-Y₄-Y₁₆-Y₁₈-­Y₁₃-Y₁₆-Y₁₀-Y₁₃-Y₂₀-Y₁₅-Y₁₂-Y₂-Y₂₀-Y₆-Y₂₀-Y₁₀-Y₁₁-Y₁₅-Y₁₂-Y₁₄-Y₁₇-­Y₁-Y₂₀-Y₁-Y₆-Y₁₇-Y₄-Y₁₁-Y₁₂-Y₇-Y₁₅Y₁₁-Y₁₂-Y₂₀-Y₁₁-Y₈-Y₁₀-Y₁₇-Y₄-­Y₁₃-Y₁₄-Y₄-Y₁₆-Y₁₆-Y₁₂-Y₁-Y₃Y₁₄-Y₁-Y₁₂-Y₁₀-Y₁₇-Y₂₂-Y₁₆-Y₇-Y₃-Y₁₁-­Y₉-Y₂₀-Y₁₆-Y₉-Y₁₀-Y₁₁Y₆-Y₁₂-Y₁-Y₁₂-Y₁₀-Y₇-Y₂₀-Y₁₆-Y₇-Y₄-Y₈-Y₁₇-­Y₁₂-Y₁-Y₁₆-Y₁-Y₁-Y₁₇-Y₁₇-Y₁-Y₁₀-Y₁₁-Y₁₀-Y₁-Y₂-Y₁₆-Y₁₆-Y₁₅-Y₁₅-Y₁₈-­Y₁₄-Y₁₀-Y₂₀-Y₄-Y₂-Y₁₅-Y₁₄-Y₁₁-Y₁₄-Y₁₄-Y₁₀-Y₂-Y₉-Y₃-Y₁₅-Y₁₇-Y₈-Y₁-­Y₂₀-Y₁₁-Y₁₄-Y₁₃-Y₈-Y₆-Y₁₀-Y₃-Y₁₂-Y₁₅-Y₂₁-Z₂ (III), wherein Y₁ codes for alanine (Ala) and is GCT, GCC, GCA or GCG, Y₂ codes for arginine (Arg) and is CGT, CGC, CGA, CGG, AGA or AGG, Y₃ codes for asparagine (Asn) and is AAT or AAC, Y₄ codes for aspartic acid (Asp) and is GAT or GAC, Y₅ codes for cysteine (Cys) and is TGT or TGC, Y₆ codes for glutamine (Gln) and is CAA or CAG, Y₇ codes for glutamic acid (Glu) and is GAA or GAG, Y₈ codes for glycine (Gly) and is GGT, GGC, GGA or GGG, Y₉ codes for histidine (His) and is CAT or CAC, Y₁₀ codes for isoleucine (Ile) and is ATT, ATC or ATA, Y₁₁ codes for leucine (Leu) and is TTA, TTG, CTT, CTC, CTA or CTG, Y₁₂ codes for lysine (Lys) and is AAA or AAG, Y₁₃ codes for methionine (Met) and is ATG, Y₁₄ codes for phenylalanine (Phe) and is TTT or TTC, Y₁₅ codes for proline (Pro) and is CCT, CCC, CCA or CCG, Y₁₆ codes for serine (Ser) and is TCT, TCC, TCA, TCG, AGT or AGC, Y₁₇ codes for threonine (Thr) and is ACT, ACC, ACA or ACG, Y₁₈ codes for tryptophan (Trp) and is TGG, Y₁₉ codes for tyrosine (Tyr) and is TAT or TAC, Y₂₀ codes for valine (Val) and is GTT, GTC, GTA or GTG, Y₂₁ is a stop codon TAA, TAG or TGA, Y₂₂ is Y₂ or Y₁₇-Y₈, Z₁ is a flanking DNA residue of 12 nucleotides or more containing a promoter sequence, Z₂ is absent or a flanking DNA residue of one or more nucleotides, and Z₁ and Z₂ are optionally linked, a double-stranded DNA consisting of a DNA of formula III and of a complementary DNA thereto, that complementary DNA itself, a mutant thereof, wherein one or more nucleotides are mutated, and fragments of such DNA comprising at least 15 nucleotides.

Claim 25

A process accordding to claim 18, 19 or 20, characterized in that the starting material is so chosen as to prepare a DNA of the formula wherein W is A or ACAG, X₂ is Arg or Thr-Gly and Z₃ and Z₄, inde­pendently, are absent or a flanking DNA residue of one or more nucleotides optionally linked, a double-stranded DNA consisting of a DNA of formula IV and of a complementary DNA thereto, that complementary DNA itself, a mutant thereof, wherein one or more nucleotides are mutated, and fragments of such DNA comprising at least 15 nucleotides.

Claim 26

A process according to claim 18, 19 or 21, characterized in that the starting material is so chosen as to prepare a DNA of the formula IV, wherein W is A and X₂ is Arg.

Claim 27

A process according to claim 18, 19 or 21, characterized in that the starting material is so chosen as to prepare a DNA of the formula IV, wherein W is ACAG and X₂ is Thr-Gly.

Claim 28

A process accordding to claim 18, 19 or 20, characterized in that the starting material is so chosen as to prepare a DNA, which hybridizes with a DNA of formula IV or with a DNA complementary to the DNA of formula IV.

Claim 29

A process accordding to claim 18, 19 or 20, characterized in that the starting material is so chosen as to prepare a DNA of the formula wherein Z₅ and Z₆, independently, are absent or a flanking DNA residue of one or more nucleotides optionally linked, a double-­stranded DNA consisting of a DNA of formula V and of a complementary DNA thereto, that complementary DNA itself, a mutant thereof, wherein one or more nucleotides are mutated, and fragments of such DNA comprising at least 15 nucleotides.

Claim 30

A process for the preparation of a RNA coding for a human GdNPF of formula I or for rat GdNPF, a mutant thereof, wherein one or more nucleotides are mutated, or a fragment of such RNA comprising at least 15 nucleotides, characterized in that it is isolated from glial cells.

Claim 31

A process for the preparation of a RNA coding for a human GdNPF of formula I or for rat GdNPF, a mutant thereof, wherein one or more nucleotides are mutated, or a fragment of such RNA comprising at least 15 nucleotides, characterized in that it is isolated from glial cells.

Claim 32

A host cell transformed with a hybrid vector comprising a DNA coding for GdNPF, related peptides or fragments thereof operatively linked to an expression control sequence.

Claim 33

A host cell transformed with a hybrid vector comprising a DNA coding for GdNPF, related peptides or fragments thereof operatively linked to an expression control sequence.

Claim 34

A host cell according to claim 29 which is a cell of an Escherichia coli strain.

Claim 35

A host cell according to claim 29 or 30 which is a cell of the strain E. coli HB 101.

Claim 36

A process for the preparation of a pharmaceutical composition, characterized in that human GdNPF, related polypeptides or fragments thereof retaining neurite-promoting activity are mixed with a pharmaceutically acceptable carrier.

Claim 37

A process for the preparation of a pharmaceutical composition, characterized in that human GdNPF, related polypeptides or fragments thereof retaining neurite-promoting activity are mixed with a pharmaceutically acceptable carrier.