Abstract:
The present invention is a composition for treating cancer composed of a novel oligonucleotide enclosed in a liposome together with Pokeweed Mitogen (PWM), which has been treated with activated polyethylene glycol PEG 2  to form PWM-PEG. The composition is given in conjunction with recombinant human interleukin-2, (rIL-2).

Description:
DETAILED DESCRIPTION OF INVENTION  
         [0001]    I am seeking a patent and trademark for an injectable drug called Macrimmune V. This drug is prepared as a mixture of two elements, Mitogen A:  
           [0002]    Polyethylene glycol coated Pokeweed Mitogen (PWM-PEG) 0.05 μg PWM protein/kg  
           [0003]    Recombinant Interleukin II (rIL-2) 100,00U./kg  
           [0004]    The Pokeweed mitogen is prepared by the method of Borjeson 1 . The root of the Pokeweed plant (phytolacca americana) is blenderized in a standard food processor/blender. The blenderized material is then extracted in PBS (phosphate buffered saline—0.01 M). The extraction is performed overnight @ 4 C., pH 7.3, using 1L of PBS per 1 lb. of pokeweed root. The supernatant is then filtered, the filtrate centrifuged @ 27,000 g for 15 min., and the precipitate removed.  
           [0005]    Now, TCA (trichloracetic acid 10%) is added to the solution. TCA is added volume for volume i.e. 1:1 at 0 C. The mixture is again centrifuged @ 27,000 g for 15 min. and applied to a hydroxylapatite column. The chromatographic peak is determined by O.D. (optical density) measure and the peak is eluted off in 0.005M phosphate @ pH 7.5.  
           [0006]    Purified Pokeweed mitogen, in its final form, is composed of 5 proteins: P a1-a5 . Purified PWM, as a lyophilized powder, is available from several biochemical companies such as GIBCO/BRL in the U.S.A. and Seikagaku Kogyo Co. in Japan.  
           [0007]    The PWM is complexed with the activated PEG2 ester PEG2-NHS(1-3), where NHS is N-hydroxysuccinimide. The protocol for the synthesis of PWM-PEG is as follows:  
           [0008]    To 0.4 mg of PWM (2.5 mg/ml) dissolved in 0.5 M borate buffer (pH 10.0), 50 mg of activated PEG2 is added. The PEG2 is added slowly over a ten minute period @ 37 C. with constant stirring. The mixture is allowed to complete reaction over 1 h @ 37 C. with continued stirring. The resulting PWM-PEG is purified by dialysis against PBS, and ultrafiltration/concentration in an Amicon system with a PM 10 membrane (cut-off 10 kd) to eliminate N-hydroxysuccinimide and reduce the PEG concentration. The PWM-PEG is further purified from the unreacted PEG by gel filtration on a Pharmacia Superose 12 column, operated by an FPLC instrument, using 10 mM. phosphate buffer of pH 7.2, with 0.15M NaCl as eluent.  
           [0009]    The activated PEG2-NHS can be obtained from Shearwater Polymers Inc., Huntsville, Ala. The above synthesis will put PEG on 52% of the free lysine molecules of 1 mg of PWM.  
           [0010]    Recombinant human interleukin (rIL-2) is produced in  E. Coli  transfected with the gene from the Jurkit cell line 2 . The protein is also available as a lyophilized powder from Chiron Pharmaceuticals (Proleuken).  
           [0011]    The use of interleukin 2 as immunotherapy for advanced cancer is well established 3 . It is believed that the principal effect of IL-2is the activation of a subset of Natural Killer (NK) lymphocytes, thereby generating lymphokine activated killer cells (LAK).  
           [0012]    Subsequently, it was found that LAK cells could be further stimulated by certain plant lectins. This generates lectin dependant cell-mediated cytotoxicity, termed LDCC. The most potent lectin stimulation of human T and B-lymphocytes can be obtained with the use of pokeweed mitogen, (PWM).  
           [0013]    The problem with PWM is that it is highly immunogenic. The Japanese have solved the problem of pokeweed&#39;s immunogenicity by conjugating it with activated polyethylene glycol (PEG) 4 . The resulting PWM-PEG conjugate is not as immunogenic as the pure PWM but it is, also, less active. The use of the NHS ester to activate the PEG rather than the 6-chloro-s-triazine used by the Japanese has improved the mitogenic activity of the PWM-PEG.  
           [0014]    The second element of the invention is Mitogen B:  
           [0015]    This is the oligonucleotide which is illustrated in SEQ No I.  
           [0016]    The immunostimulatory activity of bacterial DNA is well known. Recently the exact nature of this stimulatory motif has been determined. A CG dinucleotide is essential, flanked by two, 5′ purines and two, 5′ pyrimidines. The four most stimulating heximers have been found to be GACGTC, GACGTT, AACGTC, and AACGTT. 7    
           [0017]    Immunostimulatory activity of cationic lipid-DNA complexes have been noted when bacterial DNA was employed (in the form of plasmids). 8  We have noted that liposomal-CG-oligonucleotides also cause cytokine and cellular stimulation. This also appears to be the case when Poly(I:C) is used as the DNA source.  
           [0018]    We have prepared an oligonucleotide from the four most stimulating hexamers. DNA synthesis involves protection of the 5′ end of the first nucleotide by dimethoxytrityl(DMT) while the OH end is attached by a linker to silica. Afterwards DMT is removed by washing and the next nucleotide is activated and attached. Using iodine, 5′, 3′ linkage is oxidized to generate a phosphotriester bond, and one of the phosphate oxygen&#39;s is methylated. The reaction has been automated to 80 groups. 9    
           [0019]    After automated synthesis, the nucleotides are purified using polyacrylamide gel electrophoresis (PAGE). They are then incorporated in cationic lipids. Briefly: DOTAP(1, 2 dioleoyl-3-trimethylammonium-propane) Avanti Polar Lipids, Alabaster, Ala. and cholesterol(Sigma) are mixed in a 1:1 molar ratio, and dried in the bottom of round flasks. They are then rehydrated with 5% dextrose at 50 C. for 6 h.  10    
           [0020]    DNA is added at a ratio of 30 nmol lipid to 1 μgDNA to a final concentration of 100 μg DNA per 0.1 ml. dextrose. The dose of DNA necessary for maximal immunostimulation is about 50-100 μg/kg.  
       
    
    
     EXPERIMENT 1  
       [0021]    Eighty C57BL x DBA mice were divided into groups of 5. They were given either Meth A or EL-4 tumors at a dose of 5×10 5  cell, injected s.c. in the suprascapular region. They were Meth then treated with either mitogen-A, mitogen-B, both or neither at 1 or at 3 days post tumor implant. Animals were 6-8 weeks old and weight matched to 20 g +/−3 g (SD). They were given mitogen-A at a dose of 50 μg i.p. and mitogen-B at 10 μg (0.1 ml soln) i.v. Mitogen-A was given biweekly for up to three weeks. Mitogen-B was given weekly for up to 3 weeks. Tumor volumes are estimated in mm 3 +/−SD.  
                                                                                                                               TABLE 1                           Effect of single agent or combination therapy given on day 1 post tumor implant            Tumor   Agent   Day 7       Day 10       Day 14       Day 21       Cures       Deaths                    MethA   Saline       0   153(22)       450(60)       5000(3500           0       5       MethA   Mit A       0       0       0       0       5       0       MethA   Mit B       0       0       0       0       5       0       MethA   Mit A &amp; B       0       0       0       0       5       0       EL-4   Saline   75(25)       250(100)       500(150)       TL           0       5       EL-4   Mit A   P4           0       0       0       5       0       EL-4   Mit B       0       0       0       0       5       0       EL-4   Mit A &amp; B       0       0       0       0       5       0                                  
 
         [0022]    [0022]                                                                                                                               TABLE 2                           Effect of single agent or combination therapy given on day 3 post tumor            Tumor   Agent   day 7       day 10       day 14       day 21       Cures       Deaths                    Meth A   Saline   P3   0   100(25)       500(50)       5500(3500           0       5       Meth A   Mit A   P5           0        0        0       5       0       Meth A   Mit B   P5       P3            0        0       5       0       Meth A   Mit A &amp; B       0       0        0        0       5       0       EL-4   Saline   80(20)       250(100)       750(175)       LT           0       5       EL-4   Mit A   P5       P1            0        0       5       0       EL-4   Mit B   P1       P1           100       250       4       1       EL-4   Mit A &amp; B   P4       P1            0        0       5       0                                    
       EXPERIMENT 2  
       [0023]    Forty C57BL x DBA were given an intraperitoneal injection of 10 5  cells from EL-4 or Meth A, followed by i.p. injections of Mitogens A, B or both on day 1 after tumor implantation. Dosages of mitogens is the same as in Experiment 1.  
                                                                                               TABLE 3                           Effect of single or combined agent therapy on i.p. tumors            Tumor   Agent   # animals       deaths       days till death       cures                    Meth A   Saline       5       5   25-33(27)           0       Meth A   Mit A       5       5   21-43(38)       Meth A   Mit B       5       5   29-45(42)       Meth A   Mit A &amp; B       5       0               5       EL-4   Saline       5       5    7-9(8)           0       EL-4   Mit A       5       5   20-47(40)           0       EL-4   Mit B       5       3   40-47(43)           2       EL-4   Mit A &amp; B       5       1       57       4                  
 
       Mitogenic Properties of Compounds A and B  
     Test Articles and Reagents  
       [0024]    A and B were prepared as indicated. Concavalin A (Con A), lipopolysaccharide (LPS), and PHA-L were purchased from Sigma Chemical Co. (St. Louis, Mo.). All diluted samples and controls were filtered through a 0.2 μm filter to sterilize the stock solutions prior to assay.  
       Cells  
       [0025]    Female C57BL/6 mice were obtained from Charles River Laboratories (Raleigh, N.C.). Mice were approximately 6-8 weeks of age when used. Mice were sacrificed by CO 2  inhalation and spleens were removed aseptically. Single cell suspensions were prepared by disaggregating the cells with frosted glass slides. Cells were washed twice and resuspended in complete medium. Complete medium sonsisted of RPMI-1640 medium containing 25 mM HEPES buffer (Mediatech, Herndon Va.) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, 100 μg/ml penicillin, 10 μml gentamicin (GIBCO-BRL, Gaithersburg, Md.), 2 mM L-glutamine (Mediatech), and 2×10 −5  M 2-mercaptoethanol (Sigma).  
       Proliferation Assays  
       [0026]    Spleen cells (2×10 5 /well) were placed in 96-well flat micrometer plates Costar/Corning, Corning, N.Y.) and cultured in triplicate with either medium (no stimulus or background) or various concentrations of PHA-L, Con A, LPS, and compounds A and B. Cultures were incubated at 37° C. in humidified 5% CO 2  for three days, pulsed with 1 μCi 3 H-thymidine ( 3 H-TdR)/well for the final 6-16 hours of incubation, and harvested using a Skatron (Sterling, Va.) semi-automated harvester. Proliferation was measured by  3 H-TdR incorporation after counting samples in a Beckman LS 60001C (Fullerton, Calif.) liquid scintillation counter. Data were processed using Microsoft Excel software. The first experiment compares Mitogens/Compounds A and B with PHA-L.  
                                                                                                 TABLE 1                           Experiment 1 - Compound A (Mar. 21, 2001) and Compound B (Feb. 20, 2001)                Proliferative Response   Proliferative Response (minus Background)            Agent   Dosage   Raw CPM   Mean CPM ± SD   Raw CPM   Mean CPM ± SD                    Media   —   1654   1765   1572    1735 ± 294                               1305   2101   2015               2236   2063   3008    2407 ± 556   501   328   1273     671 ± 556               3207   1998   1927       1472   263   192       Compound A   1.000 μg/mL   109961   128564   110816   116447 ± 10502   108226   126829   109081   114712 ± 10502       (Mar. 21,   0.500 μg/mL   156931   163253   157019   159068 ± 3625   155196   161518   155284   157333 ± 3625       2001)   0.250 μg/mL   195449   176014   *   185732 ± 13743   193714   174279   *   183996 ± 13743           0.200 μg/mL   179208   175642   176595   177149 ± 1846   177473   173907   174860   175413 ± 1846           0.100 μg/mL   177943   166293   191647   178627 ± 12691   176207   164557   189911   176892 ± 12691           0.050 μg/mL   209626   173578   232072   205092 ± 29510   207890   171842   230338   203356 ± 29510           0.025 μg/mL   203103   191088   177898   190696 ± 12607   201367   189352   176162   188961 ± 12607           0.010 μg/mL   159906   153294   187558   166919 ± 18177   158170   151558   185822   165184 ± 18177       Compound B   1.000 μg/mL   35639   35395   36498    35844 ± 579   33904   33660   34763    34109 ± 579       (Feb. 20, 2001)   0.500 μg/mL   22485   29819   23886    25397 ± 3893   20750   28084   22151    23661 ± 3893           0.250 μg/mL   17321   17373   14986    16560 ± 1363   15586   15838   13251    14825 ± 1363           0.200 μg/mL   15263   16211   18462    15979 ± 632   13528   14476   14727    14243 ± 632           0.100 μg/mL   11496   13790   9560    11615 ± 2118   9761   12055   7825    9880 ± 2118           0.050 μg/mL   9258   7905   9959    9041 ± 1044   7523   6170   8224    7305 ± 1044           0.025 μg/mL   8028   7520   19522    11690 ± 6788   6293   5785   17787    9955 ± 6788           0.010 μg/mL   4122   4311   3534    3989 ± 405   2387   2576   1799    2254 ± 405       PHA-L   20.00 μg/mL   30457   31747   22272    28159 ± 5139   28722   30012   20537    28424 ± 5139           10.00 μg/mL   81240   83980   95301    86840 ± 7454   79504   82244   93565    85105 ± 7454            5.00 μg/mL   42263   54563   33665    43497 ± 10504   40528   52828   31930    41762 ± 10504            2.50 μg/mL   7492   7648   9452    8197 ± 1089   5757   5913   7717    6462 ± 108            1.00 μg/mL   5408   2417   4238    4021 ± 1507   3673   682   2503    2286 ± 150            0.50 μg/mL   2876   2238   3844    2986 ± 809   1141   503   2109    1251 ± 809            0.25 μg/mL   2198   3859   3306    3121 ± 848   483   2124   1571    1386 ± 846            0.10 μg/mL   2045   1880   1902    1942 ± 90   310   145   167     207 ± 90       Con A   3.000 μg/mL   156233   151182   174547   160654 ± 12294   154497   149446   172812   158919 ± 12294           1.500 μg/mL   147906   151367   *   149636 ± 2447   148170   149631   *   147901 ± 2447           0.750 μg/mL   150280   159314   127738   145777 ± 16263   148544   157579   126002   144042 ± 16263           0.375 μg/mL   130588   103471   122789   118949 ± 13960   128852   101736   121053   117214 ± 13960       LPS    25.0 μg/mL   88172   81155   *    84664 ± 4962   86437   79420   *    82928 ± 4962            10.0 μg/mL   46551   48269   39792    44870 ± 4481   44815   46533   38056    43135 ± 4481            5.0 μg/mL   46215   50369   43859    46814 ± 3296   44479   48633   42123    45079 ± 3296            2.5 μg/mL   40418   60794   54771    51994 ± 10468   38682   59059   53036    50259 ± 10468                          
 
         [0027]    [0027] 
     
       
       
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             24  
             DNA  
             Artificial Sequence  
             
               Synthetic sequence of 24 nucleotides related 
      to four immunostimulatory heximers of bacterial DNA  
             
           
            1 

gacgtcgacg ttaacgtcaa cgtt                                            24