Abstract:
Monomeric analogues of human insulin have a single substitution of the amino acid in 12 th , 16 th  or the 26 th  position of the B chain of human insulin and may also have a terminal deletion in the B chain.

Description:
This application is a National Stage Application of International Application Number PCT/GB00/03460, published, pursuant to PCT Article 21(2), in English. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to novel monomeric analogues of human insulin (HI) obtainable by recombinant DNA technology. 
     BACKGROUND OF THE INVENTION 
     Insulin is highly effective in treating insulin-dependent diabetes, and has been used clinically for nearly 80 years. With advances in DNA technology and the development of biotechnology industries, insulin extracted from animal pancreas is gradually being replaced by recombinant forms of human insulin, produced in microbial systems. This trend is encouraged by two observations, the number suffering from diabetes mellitus is on the increase globally and the clinical dose required to treat them is in milligram (mg) quantities. 
     Currently, the organisms employed for the commercial production of recombinant human insulin are  E. coli  and  S. cerevisiae . The expression levels in  E. coli  are high but difficulties associated with downstream purification often lead to loss of yield. These difficulties are not encountered with  S. cerevisiae , because the insulin produced is secreted into the culture medium, facilitating purification. However, the level of expression observed in this organism is low and difficult to increase. 
     Until recently, introduction of Lispro®, clinical preparations of human insulin contained polymeric forms of insulin which are slow acting. Monomeric forms of insulin, as described in U.S Pat. No. 5618913, by contrast, are relatively fast-acting and mimic more closely the natural situation. They therefore demonstrate a great potential for clinical application. A commercial monomeric insulin, available as Lispro®, comprises inversion of amino acids 28 and 29 of the B chain of human insulin, and may be abbreviated as B28Lys,B29Pro. 
     Kristensen et al, J. Biol. Chem. 272(20):12978-83 (1997), discloses alanine substitution at various positions on the insulin molecule, including B12, B16 and B26. A single substitution with Ala affected the binding activity of the resultant insulin analogue in certain cases. 
     Wang et al, Biochem. Mol. Biol. Int. 39(6):1245-54 (1996), discloses B12Thr, i.e. an insulin analogue in which the 12 th  amino acid of the B-chain of human insulin (Val) is substituted by Thr. Again, an effect on binding activity was observed. 
     EP-A-0046979 discloses des-B30 derivatives of human insulin. 
     EP-A-0291863 discloses des-B1 derivatives of human insulin. 
     SUMMARY OF THE INVENTION 
     According to the present invention, novel human insulin analogues are monomeric variants of B12Thr, B16Ala and B26Ala; the latter have not previously been recognised as monomeric. In addition to replacement of any or all of the 12 th , 16 th  and 26 th  amino acids on the B-chain, such that the analogue is monomeric, the B-1 and/or B-30 terminal amino acids may be absent. The term “insulin analogue” as used herein means a compound having a molecular structure similar to that of human insulin, including disulphide bridges between A7Cys and B7Cys and between A20Cys and B19Cys, and an internal disulphide bridge between A6Cys and A11Cys, and having insulin activity. 
     Without wishing to be bound by theory, it appears that, in the primary structure of the insulin molecule, a number of the amino acids in the B-chain are responsible for the polymerisation of insulin in clinical preparations. These include those in positions B12, B16 and B26. In particular, the replacement of Val by Thr in position B12 or Tyr by Ala in position B16 or B26 significantly reduces the tendency of the insulin analogues to polymerise even at high concentrations (see Example 9). This enhanced tendency to exist as a monomeric structure is not affected by deletion of either one or both of the terminal amino acids of the B-chain. 
     DESCRIPTION OF THE INVENTION 
     The Scheme, below, shows the construction of the expression plasmids pNHI-2/AOX1, pNHI-3/AOX1, pNHI4/AOX1 and the engineering of recombinant cells YP99/NHI-2, YP99/NHI-3 and YP99/NHI-4. It sets out a representative procedure for the preparation of compounds of the invention, by analogy with the use of the human insulin target gene (HI) housed in the shuttle plasmid pHI/PGK. This shuttle vector is constructed from the plasmid pVT102-U (acquired from Canadian Research Institute) and subsequently multiplied by PCR (Maniatis et al (1989), Molecular Cloning A Laboratory Manual, 2 nd  ed. New York: Cold Spring Harbour Laboratory), to obtain multiple copies of human insulin target gene (HI) and flanking alpha mating factor leader (MFL) sequence. The target gene is then cloned into plasmid pPIC9 which is subsequently linearised with BglII prior to being employed to transform  P. pastoris  cell GS115 by the spheroplast method. Once plasmid pPIC9 containing the target gene is intemalised, it integrates into the chromosomal DNA of the host cell [1]. Transformed cells bearing a high copy number of the HI gene are selected using the antibiotic G418 by the method described by Scover et al [2]. The presence of multiple copies of the HI are ascertained by the dot blotting method [3]. Cells bearing a high copy number of the HI gene are utilised to generate the human insulin precursor by fermentation, and after purification converted to human insulin by tryptic transpeptidation. 
     In order to obtain recombinant forms of human insulin analogues according to this invention, target genes were produced. This was accomplished by the “gap double-stranded DNA” method described by Li Yiping et al. (1987 Biotech . J. 3:90) which permits site-directed mutations in the HI target gene. Primers specifically designed to give B12Thr, B16Ala and B26Ala were as follows; For B12Thr (NHI-2): refer to Wang et al.,  supra  For B16Ala (NHI-3): 5′ TGA GGC TTT GNN STT GGT TTG CG 3′ (SEQ ID No.1) in which N can be any nucleotide (G,A,T or C), and S is C or G. For B26Ala (NHI-4): 5′ GAA AGA GGTT TTC NNS ACT CCT AGG GC 3′ (SEQ ID No.2) in which N and S are as defined above. 
     Novel human insulin analogues may be obtained by removing B30Thr and/or B1Phe, e.g. yielding a des-B1 and/or des-B30 analogue. Deletion may be achieved by known methodology. Rather than tryptic transpeptidation, to produce des-B30 human insulin, limited hydrolysis has been adopted, using trypsin in the preferred method, which further simplifies the process and increases the yield of insulin. 
     The methylotrophic yeast,  Pichia pastoris  is the preferred host for use In this invention for the preparation of insulin analogues because, as the Examples show, it has the advantages of high expression, simple processing, low production cost and high density culture. Furthermore it offers the advantages of a eukaryotic cell system; the correct folding and post-translational processing of secreted protein These advantages greatly enhance the possibility of utilizing  P. pastoris  as the expression host in the scale-up of human insulin production. Its use in the expression of proteins of commercial importance has been documented elsewhere [3-5]. 
     Human insulin analogues of the invention may be used in therapy. Their application and utility will be readily evident to those of ordinary skill in the art, e.g. in the treatment of diabetes mellitus. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows the construction of pNHI2/AOX1 plasmid of  Pichia pastoris.    
     The following Examples illustrate the invention. 
    
    
     EXAMPLE 1 
     Cloning of Mutated HI Gene 
     The plasmid pVT102-U from Canadian Biotechnology Research Institute was used to construct the plasmid pHI/PGK according to the standard method described in Maniatis et al (1989). The construct pHI/PGK is a shuttle plasmid with phosphoglycerate kinase (PGK) promoter, followed by alpha mating factor leader sequence (MFL) to direct secretion of the product of the human insulin target gene (HI) flanked by a BamHI site at MFL 5′ end and a HindIII site at HI 3′ end. Using pHI/PGK as template, together with TCCGGATCCATGAGATTT (SEQ ID NO. 3) as the 5′ primer and TGAATTCTTCTAGTTGCAGTAGTTT (SEQ ID NO. 4) as the 3′ primer, DNA fragments containing MFL and HI with the BamHI site GGATCC at 5′ end and the EcoR1 site GAATTC at the 3′ end were obtained by PCR. To obtain DNA fragments containing MFL and the target gene NHI-2 (B12Thr), NHI-3 (B16Ala) and NHI-4 (B26Ala) the HI target gene in pHI/PGK plasmid was first mutated by site-directed mutagenesis then replicated by PCR. By inserting these fragments behind the AOX1 promoter of the plasmid pPIC9 (Invitrogen), expression plasmids pNHI-2/AOX1, pNHI-3/AOX1 and pNHI-4/AOX1 were obtained (see the Scheme and the accompany drawing; the latter shows the first plasmid, and the others may be prepared by the same procedure). The primers used to obtain the mutated genes in this invention have SEQ ID NOS. 1, 2 and 3. 
     EXAMPLE 2 
     Construction and Screening of Expression Cell 
     The expression plasmids were linearised by BglII and used to transform  P. pastoris  cell GS115 (Invitrogen) using the spheroplast method. The linearised plasmids, once internalized, integrate into the chromosomal DNA of the host cell [1]. The recombinant cells, designated YP99/NHI-2, YP99/NHI-3 and YP99/HNI-4 with high copy number of the target gene, were selected by antibiotic G418 [2] and identified by the dot blotting method [3]. 
     EXAMPLE 3 
     Preparation of Precursors of HI analogues 
     High density fermentation was carried out in a 15 litre fermenter [7]. The following salt solutions were used in the fermentation: BSM-H 3 PO 4  26.7 ml/l, CaSO 4 .H 2 O 0.93 g/l, K 2 SO 4  18.2 g/l, MgSO 4 .7H 2 O 14.9 g/l, KOH 4.13 g/l; PTM1 -CuSO 4 . 5H 2 O 6 g/l, Kl 0.08 g/l, MnSO 4 H 2 O 3.0 g/l, NaMoO 4 .H 2 O 0.2 g/l, H 3 BO 3  0.02 g/l, CoCl 2 6H 2 O 0.5 g/l, ZnSO 4  20.0 g/l, H 2 SO 4  5 ml/l, FeSO 4 .7H 2 O 65.0 g/l. 
     Fermentation medium containing 6 L of salt solution BSM and 300 ml of glycerol is sterilised in the fermenter. Its pH is adjusted to 5.5 with 50% ammonium hydroxide. A 5 ml aliquot of salt solution PTM1 containing 1 mg of biotin is added per 1 litre of culture medium. The expression cell is inoculated to 50 ml YPG and grown in a shake flask at 30° C. for 24 hr. The broth is added to 600 ml of YPG, shaken in 3 flasks for 24 hr, added to the culture medium and fermented for 24 hr to deplete glycerol. Methanol solution containing PTM1 (5 ml/l) and biotin (1 mg/l) is added to induce the expression. The inductive fermentation is continued for 84 hr by feeding the above methanol solution. During the fermentation, the pH is maintained at 5.5 by adding 50% ammonium hydroxide. The expression level is measured by radioimmunoassay, SDS-polyacrylamide gel electrophoresis [8] and HPLC. 
     EXAMPLE 4 
     Separation and Purification of the Precursors 
     The fermentation broth is centrifuged to remove the cell bodies. The supematant is applied to a C8 column and purified by HPLC. After a single step of purification, a product can be obtained that is homogeneous in native polyacrylamide gel electrophoresis. 
     EXAMPLE 5 
     Transpeptidation of the Precursors 
     Purified precursors of HI analogues from Example 4 are dissolved in DMSO/1,4-butanediol/H 2 O (15:70:15, v/v) to a concentration of 30 mg/ml. Thr(Bu t )-OBu t  is added in excess, and the pH is adjusted to 6.5 by ammonium hydroxide. TPCK-trypsin is added (substrate:enzyme=5:1) and the reaction mixture is incubated at 25° C. for 6 hr. The reaction is stopped by acidification. The product is precipitated using acetone, and purified by HPLC using C8 column. 
     EXAMPLE 6 
     Preparation of des-B30 analogues 
     Purified precursors of HI analogues are dissolved in pH 8, 0.1M ammonium bicarbonate to a concentration of 10 mg/ml. TPCK-trypsin is added (substrate:enzyme=200:1) and the reaction mixture is incubated at 25° C. overnight. The product is analysed by native polyacrylamide gel electrophoresis 
     EXAMPLE 7 
     Preparation of Des-B1 analogues 
     HI analogues are reacted with phenylisocyanate in a molar ratio of 1:2, prior to treatment with trifluoroacetic acid as described by Bradenburg &amp; Hoppe-Seyler, Physiol. Chem. 350:471. The products of this reaction are separated and analysed by electrophoresis and found to be almost exclusively des-B1 forms of insulin analogues. 
     EXAMPLE 8 
     Preparation of Des-B1, Des-B30 analogues 
     Prepared by processing precursors of HI analogues as described in Example 6 followed sequentially by that described in Example 7. 
     EXAMPLE 9 
     Determination of Structural Forms 
     The structural form of the recombinant human insulin analogues prior to deletion of the one or both terminal amino acids of the B-chain is determined electrophoretically. A preparation of each analogue is passed through Superdex G-75 column (HR 10/30). HI and [B28Lys, B29Pro] insulin (Lispro) are used as negative and positive controls respectively. Phosphate buffered saline pH 7.4 is used as an elution buffer and the flow rate fixed at 0.4 ml/min. The concentration of the sample preparation is 1.2 mg/ml. The retention times and the peak profiles of human insulin analogues are shown in the following Table. 
     
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample 
                 Retention Time, min 
                 Peak profile 
               
               
                   
                   
               
             
             
               
                   
                 Hl 
                 36.4 
                 Unsymmetrical 
               
               
                   
                 [B28Lys, B29Pro]Hl 
                 39.4 
                 Symmetrical 
               
               
                   
                 [B12Thr]Hl 
                 39.4 
                 Symmetrical 
               
               
                   
                 [B16Ala]Hl 
                 38.3 
                 Symmetrical 
               
               
                   
                 [B26Ala]Hl 
                 38.9 
                 Symmetrical 
               
               
                   
                   
               
             
          
         
       
     
     These results demonstrate that HI analogues B12Thr, B16Ala and B26Ala are all monomeric in form. They have a similar retention time and peak profile as the known positive control [B28Lys, B29Pro] human insulin. 
     References 
     1. Cregg et al (1985), Mol. Cell. Biol. 5:3376 
     2. Scover et a/ (1994), Bio/Technology 12:181 
     4. Hagenson et al (1989), Enzy. Micro. Tech. 11:650 
     5. Steinlein et al (1995), Prot. Exp. Pur. 6:619 
     3. Clare et al(1991), Gene 105:205 
     6. Li YiPing et al (1987). Biotech. J. 3:90 
     7. Laroche et al (1994), Bio/Technology 12:1119 
     8. Schagger et al (1987), Anal. Biochem. 166:368                           
     
       
         
           
             4 
           
           
             1 
             23 
             DNA 
             Artificial Sequence 
             
               Oligonucleotide NHI-3 
             
           
            1
tgaggctttg nnsttggttt gcg                                             23
 
           
             2 
             27 
             DNA 
             Artificial Sequence 
             
               Oligonucleotide NHI-4 
             
           
            2
gaaagaggtt ttcnnsactc ctagggc                                         27
 
           
             3 
             18 
             DNA 
             Artificial Sequence 
             
               Oligonucleotide MFL 5′ primer 
             
           
            3
tccggatcca tgagattt                                                   18
 
           
             4 
             25 
             DNA 
             Artificial Sequence 
             
               Oligonucleotide HI 3′ primer 
             
           
            4
tgaattcttc tagttgcagt agttt                                           25