Macrimmune V, a composition for treating cancer

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 PEG2 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 Borjeson1. 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. 1Borjeson J. Reisfield R. and Chessin L. N. (1966) Journal of Experimental Medicine 124:859-872.

[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: Pa1-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 line2. The protein is also available as a lyophilized powder from Chiron Pharmaceuticals (Proleuken). B 2Rosenberg S A, Grimm E A, McGrogan M, et al. Biological activity of recombinant human interleukin-2 produced in Escherichia coli. Science 1984; 4:1380-91.

[0011] The use of interleukin 2 as immunotherapy for advanced cancer is well established3. 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). 3Rosenberg S A, Grimm E A, Muul L M, et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Eng J Med 1987; 316:889-97.

[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'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. 4Ueno T et al. (1996) Journal of Biomaterial Science, Polymer Edition 7(9):753-8.

[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 7Krieg, A M 1996. Lymphocyte activation by CpG dinucleotide motifs in prokaryotic DNA Trends Microbiol, 4:73

[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. 8Freimark, B D, et al. 1998. Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid:cationic lipid complexes. J. Immunol. 160:4580

[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's is methylated. The reaction has been automated to 80 groups.9 9Ellington, A and Green, R 1990. Synthesis of Oligonucleotides. IN Current Protocols in Molecular Biology (F. M. Ausubel et al. eds.)pp.2.11.1-2.11.18, Green Publishing and Wiley-Interscience, New York.

[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 10Liu, Y D et al. 1995, Cationic liposome-mediated i.v. gene delivery in mice. J. Biol. Chem. 27:24864.

[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 5×105 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 mm3+/−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 & 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 & B		0		0		0		0		5		0
TL = too large &/or death
P = palpable # animals

[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 & 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 & B	P4		P1			0		0		5		0
P = palpable #
LT = tumor too large &/or death

EXPERIMENT 2

[0023] Forty C57BL x DBA were given an intraperitoneal injection of 105 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 & 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 & 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 CO2 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 (2×105/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% CO2 for three days, pulsed with 1 μCi3H-thymidine (3H-TdR)/well for the final 6-16 hours of incubation, and harvested using a Skatron (Sterling, Va.) semi-automated harvester. Proliferation was measured by 3H-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
*Outlier deleted.

[0027]

Claims

1. A composition (Mitogen A) that includes a plant mitogenic lectin (PWM), that is combined with an immunogenicity modifier(PEG and recombinant Human Interleukin II), so that the lectin does not cause an undesired reaction between T and B cells in the acquired immune system when administered systemically.

2. The oligonucleotide comprising sequence No. 1

3. A composition (Mitogen B) comprising the oligonucleotide of claim 2, enclosed in a liposome.

4. A composition (Macrimmune V) comprising Mitogen B of claim 3 and Mitogen A, of claim 1.

5. The composition of claim 4 in which where the oligonucleotide is replaced by an equal weight of Poly(I:C).

6. The composition of claim 1, in which the PWM has been replaced by the isolectin Lsub4 of phytohemagglutinin (PHA-Lsub4).