Hybrid human leukocyte interferons, process for their microbial production, intermediates therefor and compositions containing them

Claim 2

An antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons.

Claim 3

A polypeptide according to claim 1 the N-terminal portion thereof consisting essentially of amino acids 1-92 of LeIF-D and the carboxy terminal portion thereof consisting essentially of amino acids 92-165 of LeIF-A.

Claim 4

A polypeptide according to claim 1 the N-terminal portion thereof consisting essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consisting essentially of amino acids 93-166 of LeIF-D.

Claim 5

A polypeptide according to claim 1 the N-terminal portion thereof consisting essentially of amino acids 1-63 of LeIF-D and the carboxy terminal portion thereof consisting essentially of amino acids 63-165 of LeIF-A.

Claim 6

A polypeptide according to claim 1 the N-terminal portion thereof consisting essentially of amino acids 1-62 of LeIF-A and the carboxy terminal portion thereof consisting essentially of amino acids 64-166 of LeIF-D.

Claim 7

A polypeptide according to claim 1 the N-terminal portion thereof consisting essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consisting essentially of amino acids 93-166 of LeIF-B.

Claim 8

A polypeptide according to claim 1 the N-terminal portion thereof consisting essentially of amino acids 1-91 of LeIF-A, the carboxy terminal portion thereof consisting essentially of amino acids 93-166 of LeIF-F.

Claim 9

Double stranded DNA comprising a strand encoding a polypeptide as claimed in any one of claims 1-5.

Claim 10

Double stranded DNA comprising a strand encoding a polypeptide as claimed in claims 6 and 7.

Claim 11

A replicable plasmidic expression vehicle capable, in a transformant microorganism, of expressing a polypeptide as claimed in any one of claims 1-5.

Claim 12

A replicable plasmidic expression vehicle capable, in a transformant microorganism, of expressing a polypeptide as claimed in claims 6 and 7.

Claim 13

A transformant microorganism containing the expression vehicle of claim 10.

Claim 14

A transformant microorganism containing the expression vehicle of claim 11.

Claim 15

A plasmid selected from the group consisting of pLeIF AD trp (Bgl II), pLeIF AD trp (Pvu II), pLeIF DA trp (Bgl II) and pLeIF DA trp (Pvu II).

Claim 16

A plasmid selected from the group consisting of pLeIF AD trp (Bgl II), pLeIF AD trp (Pvu II), pLeIF DA trp (Bgl II) and pLeIF DA trp (Pvu II).

Claim 17

A plasmid selected from the group consisting of pLeIF AB (Pvu II) and pLeIF AF (Pvu II).

Claim 18

A plasmid selected from the group consisting of pLeIF AB (Pvu II) and pLeIF AF (Pvu II).

Claim 19

A method of forming a double stranded DNA as claimed in claim 8 or 9 which comprises: a. selecting a first, double stranded DNA fragment comprising codons encoding all or a portion of the amino acid sequence of a first, naturally occurring leukocyte interferon, said fragment including codons for the amino terminal portion of said interferon and, spaced in the 3' direction therefrom, a first restriction endonuclease cleavage site; b. cleaving said fragment with said restriction endonuclease; c. selecting a second double stranded DNA fragment encoding all or a portion of the amino acid sequence of a second, different, naturally occurring leuko-. cyte interferon, the second fragment comprising an identical restriction endonuclease aleavage site essentially identically positioned from the standpoint of the amino acid numbering of the two interferons and additionally comprising, beyond said site, codons for the carboxyl terminal portion of the second interferon; d. cleaving the second fragment with said restriction endonuclease; and e. ligating restriction endonuclease digestion products of steps b and d to reconstitute said restriction endonuclease site and form a hybrid fragment comprising, in sequence, codons for the amino terminal portion of the first interferon and the carboxy .terminal portion of the second.

Claim 20

A method of forming an antiviral polypeptide as claimed in any one of claims 1-7 which comprises: a. deploying the hybrid DNA fragment resulting from the method claimed in claim 16 within a replicable plasmidic expression vehicle, transforming a-microorganism with the same, culturing the microorganism and occasioning the expression of the polypeptide encoded by the hybrid DNA fragment; and b. lysing the resulting microorganism and recovering therefrom the polypeptide.

Claim 21

A pharmaceutical composition containing a therapeutically effective amount of a polypeptide as claimed in any one of claims 1-7 and a pharmaceutically acceptable carrier vehicle.

Claim 22

The use of a polypeptide as claimed in any one of claims 1-7 in the treatment of viral or neoplastic diseases or in the preparation of pharmaceutical compositions useful in such treatment.

Claim 23

The products and processes for their preparation as hereinbefore described.

Claim 24

The products and processes for their preparation as hereinbefore described.

Claim 25

A process for producing an antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different,' naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons, which process comprises deploying a hybrid double stranded DNA fragment comprising a strand encoding the polypeptide within a replicable plasmidic expression vehicle, transforming a microorganism with the same, culturing the microorganism and occasion the expression of the encoded polypeptide, lysing the resulting microorganism and recovering therefrom the polypeptide.

Claim 26

A process for producing an antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different,' naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons, which process comprises deploying a hybrid double stranded DNA fragment comprising a strand encoding the polypeptide within a replicable plasmidic expression vehicle, transforming a microorganism with the same, culturing the microorganism and occasion the expression of the encoded polypeptide, lysing the resulting microorganism and recovering therefrom the polypeptide.

Claim 27

A process as claimed in claim 1 wherein the N-terminal portion of the antiviral polypeptide consists essentially of amino acids 1-92 of LeIF-D and the carboxy terminal portion thereof consists essentially of amino acids 92-165 of LeIF-A.

Claim 28

A process as claimed in claim 1 wherein the N-terminal portion of the antiviral polypeptide consists essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 93-166 of LeIF-D.

Claim 29

A process as claimed in claim 1 wherein the N-terminal portion of the antiviral polypeptide consists essentially of amino acids 1-63 of LeIF-D and the carboxy terminal portion thereof consists essentially of amino acids 63-165 of LeIF-A.

Claim 30

A process as claimed in claim 1 wherein the N - terminal portion of the antiviral polypeptide consists essentially of amino acids 1-62 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 64-166 of LeIF-D.

Claim 31

A process as claimed in claim 1 wherein the N-terminal portion of the antiviral polypeptide consists essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 93-166 of LeIF-B.

Claim 32

A process as claimed in claim 1 wherein the N-terminal portion of the antiviral polypeptide consists essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 93-166 of LeIF-F.

Claim 33

A process of forming a double stranded DNA coding for a hybrid human leukocyte interferon of approximately 165-166 amino acids which process comprises (a) selecting a first, double stranded DNA fragment comprising codons encoding all or a portion of the amino acid sequence of a first, naturally occurring leukocyte interferon, said fragment including codons for the amino terminal portion of said interferon and, spaced in the 3' direction therefrom, a first restriction endonuclease cleavage site; (b) cleaving said fragment with said restriction endonuclease; (c) selecting a second double stranded DNA fragment encoding all or a portion of the amino acid sequence of a second, different, naturally occurring leukocyte interferon, the second fragment comprising an identical restriction endonuclease cleavage site essentially identically positioned from the standpoint of the amino acid numbering of the two interferons and additionally comprising, beyond said site, codons for the carboxyl terminal portion of the second interferon; (d) cleaving the second fragment with said restriction endonuclease; and (e) ligating restriction endonuclease digestion products of steps b and d to reconstitute said restriction endonuclease site and form a hybrid fragment comprising, in sequence, codons for the amino terminal portion of the first interferon and the carboxy terminal portion of the second.

Claim 34

A process of forming a double stranded DNA coding for a hybrid human leukocyte interferon of approximately 165-166 amino acids which process comprises (a) selecting a first, double stranded DNA fragment comprising codons encoding all or a portion of the amino acid sequence of a first, naturally occurring leukocyte interferon, said fragment including codons for the amino terminal portion of said interferon and, spaced in the 3' direction therefrom, a first restriction endonuclease cleavage site; (b) cleaving said fragment with said restriction endonuclease; (c) selecting a second double stranded DNA fragment encoding all or a portion of the amino acid sequence of a second, different, naturally occurring leukocyte interferon, the second fragment comprising an identical restriction endonuclease cleavage site essentially identically positioned from the standpoint of the amino acid numbering of the two interferons and additionally comprising, beyond said site, codons for the carboxyl terminal portion of the second interferon; (d) cleaving the second fragment with said restriction endonuclease; and (e) ligating restriction endonuclease digestion products of steps b and d to reconstitute said restriction endonuclease site and form a hybrid fragment comprising, in sequence, codons for the amino terminal portion of the first interferon and the carboxy terminal portion of the second.

Claim 35

A process as claimed in claim 8 wherein the first, naturally occurring leukocyte interferon is LeIF-D and the second, naturally occurring leukocyte interferon is LeIF-A.

Claim 36

A process as claimed in claim 8 wherein the first, naturally occurring leukocyte interferon is LeIF-A and the second, naturally occurring leukocyte interferon is LeIF-D.

Claim 37

A process as claimed in claim 8 wherein the first, naturally occurring leukocyte interferon is LeIF-A and the second, naturally occurring leukocyte interferon is LeIF-B.

Claim 38

A process as claimed in claim 8 wherein the first, naturally occurring leukocyte interferon is LeIF-A and the second, naturally occurring leukocyte interferon is LeIF-F.

Claim 39

A process for the preparation of pharmaceutical compositions containing an antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences- corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons which process comprises mixing said polypeptide with non-toxic, inert, therapeutically compatible carriers and bringing the resulting mixture into a suitable pharmaceutical dosage form.

Claim 40

A process for the preparation of pharmaceutical compositions containing an antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences- corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons which process comprises mixing said polypeptide with non-toxic, inert, therapeutically compatible carriers and bringing the resulting mixture into a suitable pharmaceutical dosage form.

Claim 41

A pharmaceutical composition containing a therapeutically effective amount of an antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons and a non-toxic, inert, therapeutically compatible carrier.

Claim 42

A pharmaceutical composition containing a therapeutically effective amount of an antiviral polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons and a non-toxic, inert, therapeutically compatible carrier.

Claim 43

Double stranded DNA comprising a strand encoding a polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons.

Claim 44

Double stranded DNA comprising a strand encoding a polypeptide of approximately 165-166 amino acids, the amino acid sequence of said polypeptide comprising, in sequence, discrete sub-sequences corresponding in amino acid identity and number to sub-sequences of different, naturally occurring leukocyte interferons, the amino acid sequence of said polypeptide differing in overall composition from the amino acid sequence of naturally occurring leukocyte interferons.

Claim 45

Double stranded DNA as claimed in claim 15 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-92 of LeIF-D and the carboxy terminal portion thereof consists essentially of amino acids 92-165 of LeIF-A.

Claim 46

Double stranded DNA as claimed in claim 15 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consists, essentially of amino acids 93-166 of LeIF-D.

Claim 47

Double stranded DNA as claimed in claim 15 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-63 of LeIF-D and the carboxy terminal portion thereof consists essentially of amino acids 63-165 of LeIF-A.

Claim 48

Double stranded DNA as claimed in claim 15 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-62 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 64-166 of LeIF-D.

Claim 49

Double stranded DNA as claimed in claim 15 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 93-166 of LeIF-B.

Claim 50

Double stranded DNA as claimed in claim 15 wherein the N-terminal portion of the encoded polypeptide consists essentially of amino acids 1-91 of LeIF-A and the carboxy terminal portion thereof consists essentially of amino acids 93-166 of LeIF-F.

Claim 51

A replicable plasmidic expression vehicle capable in a transformant microorganism of expressing a polypeptide coded by a DNA sequence according to any one of claims 15-21.

Claim 52

A plasmid selected from the group consisting of pLeIF AD trp (Bgl II), pLeIF AD trp (Pvu II), pLeIF DA trp (Bgl II) and pLeIF DA trp (Pvu II).

Claim 53

A plasmid selected from the group consisting of pLeIF AD trp (Bgl II), pLeIF AD trp (Pvu II), pLeIF DA trp (Bgl II) and pLeIF DA trp (Pvu II).

Claim 54

A plasmid selected from the group consisting of pLeIF AB (Pvu II) and pLeIF AF (Pvu II).

Claim 55

A plasmid selected from the group consisting of pLeIF AB (Pvu II) and pLeIF AF (Pvu II).

Claim 56

A microorganism transformed with an expression vehicle of claim 23 or claim 24.