Bis-Met histones

Abstract

The present invention provides a nucleic acid molecule which encodes a polypeptide consisting of two methionine residues as the first and second N-terminal amino acid residues linked via a peptide bond to a mature eukaryotic histone. The present invention furthermore relates to a vector containing said nucleic acid molecule, a host transformed with said vector, polypeptides encoded by the nucleic acid molecule and pharmaceutical and diagnostic compositions. The present invention also relates to the use of the nucleic acid molecule, vectors, hosts and the polypeptide of the invention for the preparation of a composition for the treatment of diseases. Furthermore, the present invention relates to a method of testing for the presence of the nucleic acid molecule or the polypeptide in a sample and to a kit.

Claims

1. A bis-met histone polypeptide comprising two methionine residues as first and second N-terminal amino acid residues linked via a peptide bond to a mature eukaryotic H1 histone.

2. The bis-met histone polypeptide of claim 1, wherein the mature eukaryotic H1 histone is a histone H1.0, a histone H1.1, a histone H1.2, a histone H1.3, a histone H1.4, a histone H1.5 or a histone H1t.

3. The bis-met histone polypeptide of claim 1, wherein an amino acid sequence including the second N-terminal amino acid residue, the peptide bond and the mature eukaryotic H1 histone is SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

4. The bis-met histone polypeptide of claim 1, wherein an amino acid sequence including the second N-terminal amino acid residue, the peptide bond and the mature eukaryotic H1 histone is one having at least 85% sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 and retaining at least 20% of its biological activity.

5. The bis-met histone polypeptide of claim 4, wherein the amino acid sequence retains at least 50% of its biological activity.

6. The bis-met histone polypeptide of claim 5, wherein the amino acid sequence retains at least 75% of its biological activity.

7. The bis-met histone polypeptide of claim 6, wherein the amino acid sequence retains at least 80% of its biological activity.

8. The bis-met histone polypeptide of claim 7, wherein the amino acid sequence retains at least 85% of its biological activity.

9. The bis-met histone polypeptide of claim 8, wherein the amino acid sequence retains at least 90% of its biological activity.

10. The bis-met histone polypeptide of claim 9, wherein the amino acid sequence retains at least 95% of its biological activity.

11. The bis-met histone polypeptide of claim 4, wherein the amino acid sequence is one having at least 90% sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

12. The bis-met histone polypeptide of claim 11, wherein the amino acid sequence is one having at least 95% sequence identity to SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13.

13. The bis-met histone polypeptide of claim 1 which is SEQ ID NO: 3.

14. A composition comprising the bis-met histone polypeptide of claim 1 and optionally comprising a pharmaceutically acceptable carrier and/or a diluent.

15. The composition of claim 14 further comprising a mature eukaryotic histone.

16. The composition of claim 15, wherein the mature eukaryotic histone is a histone H1 or a histone H2A.

17. The composition of claim 14, wherein the bis-met histone polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

18. The composition of claim 17, wherein the bis-met histone polypeptide consists essentially of the amino acid sequence of SEQ ID NO: 3.

19. The composition of claim 18, wherein the bis-met histone polypeptide has the amino acid sequence of SEQ ID NO: 3.

20. A method of treating a leukemia, the method comprising administering a composition of claim 14 to an individual in need thereof, wherein the disease is a leukemia.

21. The method of claim 20, wherein the leukemia is acute myeloic leukemia.

Description

(1) The figures show:

(2) FIG. 1: Schematic overview of the purification procedure

(3) FIG. 2. QTOF Mass spectrum of the B1, M-H1A-P02 clinical batch

(4) FIG. 3: QTOF Mass spectrum of the B2, M-H1A-Pool01 clinical batch

(5) The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.

EXAMPLE 1

Cloning of hH1.3 Constructs

(6) Construction of Plasmid Vector pEGT1-rH1.3S1

(7) As shown in SEQ ID NO: 1, histones displaying a strong positive charge resulting from a very high content of lysine residues. As the codon usage for lysine differs strongly between Escherichia coli and human, a codon optimization was performed in order to adapt the human histone H1.3 sequence to the codon usage of E. coli.

(8) A synthetic gene was produced whose sequence is provided as SEQ ID NO: 2. The artificial sequence was flanked with two restriction sites, namely BspH1 and BamH1, to allow the subsequent introduction into pEGT1 expression vector. The translation initiation codon ATG was incorporated into a Nco1 restriction site CCATGG. The initial ATG was doubled which provides a BspH1 site TCATGA whose cohesive end CATG is compatible with Nco1. Thus, a second methionine residue was incorporated after the first one. An additional BamH1 site GGATCC was introduced after the termination codon TAA. The amino acid sequence encoded by this artificial gene is given in SEQ ID NO: 3.

(9) The optimised gene was excised from its plasmid by digestion with BspH1 and BamH1 and inserted into pEGT1 expression vector linearized by NCO1 and BamH1 according to standard protocols to yield plasmid pEGT1-rH13S1.

(10) The ligated vector pEGT1-rH13S1 was introduced into electrocompetent E. coli strain BL21[DE3] by electroporation using standard protocol and the transformed cells were selected on LB plate supplemented with kanamycine. One clone was selected and the sequence of the insert encoding for the histone was verified for its perfect match with SEQ ID NO: 2.

(11) Construction of Plasmid Vector pEGT1-rH1.3S2

(12) In order to suppress the insertion of a second methionine in the rH13S1 construct, a second synthetic gene was used. The second original codon TCC encoding for a Serine was changed into AGC also encoding for Serine to ensure compatibility with the BspH1 site. The DNA sequence is provided as SEQ ID NO: 4 and the recombinant protein encoded by this artificial gene is provided as SEQ ID NO: 5. The same cloning strategy as outlined above for pEGT1-rH13S1 was employed since the artificial gene of SEQ ID NO: 4 was flanked with the BspH1 restriction site TCATGA at the initiator codon and BamH1 CCATGG one base pair post terminator codon.

(13) The optimised gene was excised from its plasmid by digestion with BspH1 and BamH1 and inserted into pEGT1 expression vector linearised by NCO1 and BamH1 according to standard protocols to yield plasmid pEGT1-rH13S2.

(14) The ligated vector pEGT1-rH13S2 was introduced into electrocompetent E. coli strain BL21[DE3] by electroporation using standard protocol and the transformed cells were selected on LB plate supplemented with kanamycine. One clone was selected and the sequence of the insert encoding for the histone was verified for its perfect match with SEQ ID NO: 4.

EXAMPLE 2

Recombinant Production of Histone H1.3

(15) The Strain

(16) The bacterium used in the preparation of rh1.3S is a recombinant strain of Escherichia coli BL21 (DE3)/pEGT1/H1.3S. The constructions were used to transform BL21 (DE3) strain of E. coli. Three clones were selected to perform an expression screening and one clone was selected to do a pre-Master Seed (Pre-MS-05L23-H1B).

(17) A Master Seed (MS-06D05-H1B) has been produced using the Pre-MS-05L23-H1B and a Working Seed (WS-06D06-H1B) has been produced using the Master Seed.

(18) Seed Culture

(19) Two 2-liters shake-flasks containing each 500 ml of YES medium (30 g/l yeast extract, 5 g/l NaCl) are inoculated each with 100 μl of the Working Seed (WS-06D06-H1B). The culture is incubated at 37° C. with an agitation of 270 rpm for 5 h00 (+/−0.5 hour) to reach an O.D (600 nm) of more than 1.5.

(20) Fermentation

(21) A 100-liters fermentor is prepared with 100 liters of NRJ18 medium. The fermentor is sterilised for 30 minutes at 123° C. After sterilisation and before inoculation, 50 ml of SAG 471 (antifoam) are added aseptically. The fermenter is inoculated with the seed culture in order to reach a theoretical initial optical density at 600 nm of 8.75×10.sup.−7.

(22) The calculated inoculum volume is added to a transfer bottle containing 500 ml of YES medium.

(23) Fermentation is performed over-night at 37° C. During the fermentation process, pH is maintained at pH 7.0±0.2 by periodic addition of NaOH 4 M and HNO.sub.32.24M. Dissolved oxygen is feedback regulated on the agitation at 30%.

(24) When the culture reaches an OD.sub.600 between 15 and 20, the culture is induced with a solution of 1 mM IPTG (23.8 g dissolved in 500 ml of highly purified water).

(25) After 1 h30 of induction, OD.sub.600 is more than 24 and the fermentor is cooled down below 16° C. pH regulation is maintained at 7.0±0.2. Other parameters are kept constant during cooling except the pressure which is decreased down to 300 mbars and the agitation down to 200 rpm.

(26) When medium temperature is below 16° C., the culture volume is estimated. The complete culture is centrifuged with 2 Beckman Centrifuges JA10 equipped with JLA 8.1000 rotor (±6 L/centrifuge): 5200 RPM-4° C.-20 minutes.

(27) Cell pellets are harvested and stored progressively at −20° C. during the centrifugation step.

(28) Cell Disruption in a High Pressure Homogenizer

(29) The day before the cell disruption, the concentrated cells corresponding to 100 liters of culture are thawed at room temperature.

(30) The day of the disruption, the cell pellets are diluted at 250 g/l in 20 mM Na.sub.2HPO.sub.4.12H.sub.2O pH 7.0 and the temperature of the suspension is increased to 30° C.

(31) The suspension is then homogenised in a Heidolph R2R2100 propeller. The cells are then lysed in a high pressure homogenizer PONY (800 bars). The cell suspension is treated twice through the cell homogenizer.

EXAMPLE 3

Protein Purification

(32) All steps of purification are performed on total volume of fermentor (i.e. 100 l).

(33) 1. Precipitation with 2.5% Perchloric Acid and 8 M Urea Extraction

(34) To the harvested cell mass 1/7 of volume of HClO.sub.4 20% (final concentration: 2.5%) is added. The suspension is homogenised prior to a third cycle in the Pony homogeniser at 250 bars. The solution is kept under gentle agitation for one hour at room temperature. Then, the suspension is centrifuged for 15 minutes (12,200 g-7,000 rpm, 4° C.). The supernatant is collected, the pH adjusted at 4.0±0.1 with 10M NaOH and filtered through a 0.45/0.22 μm Sartopore 2 membrane (2000 cm.sup.2) into a sterile bag.

(35) Urea is added to obtain an 8 M concentration in a double final volume, the volume being adjusted with 20 mM Na.sub.2HPO.sub.4 pH 7.0 buffer. The solution is kept overnight under gentle agitation at room temperature. Then, the pH is adjusted at 4.0±0.1 with 37% HCl or 10M NaOH.

(36) 2. Q Sepharose Fast Flow Anion Exchange Chromatography (QSFF)—Negative Mode

(37) The aim of this step is a reduction of the Endotoxin and DNA content. Anion exchange chromatography is conducted with Q Sepharose Fast Flow (Amersham Biosciences cat. n.sup.o 17-0510-05) packed in a Moduline 350/500 column (Millipore BioProcess Division cat n.sup.o 86351211).

(38) The column is packed in highly purified water at an eluent flow rate of 120 cm/h (115.4 l/h). The dimensions of the packed column bed are: diameter 25 cm, cross-sectional area=961 cm.sup.2, bed=18 cm, packed volume=17.314±0.962 l. The column is sanitised with 1.5 to 2.5 column volume (CV) of 1M NaOH+2M NaCl with a contact time of 2 hours at a flow rate of 96.2 l/h (100 cm/h-1603 ml/min).

(39) All the chromatographic steps are performed at a linear flow rate of approximately 100 cm/h (±96.2 l/h). The pH is stabilised with 1 to 2 CV of 50 mM Ammonium acetate+1M NaCl pH 4.0. The column is then equilibrated with 3.5 to 5 CV of 50 mM Ammonium acetate+8M urea pH 4.0.

(40) The solution from urea extraction (see section 1) is diluted about 1.5 times with 50 mM Ammonium acetate+8M urea pH 4.0 in order to obtain a conductivity lower than 10 mS/cm. Only 8 CV of urea extraction solution (before dilution) are loaded at the same time. The H1 protein is collected in the flow through, the equilibration-elution being performed with 1.5 to 2.5 CV of 50 mM Ammonium acetate+8 M urea pH 4.0.

(41) After elution, the column is cleaned with 1.5 to 2.5 CV of 50 mM Ammonium acetate+1M NaCl pH 4.0. This elution in 1M NaCl allows to eliminate DNA and endotoxins. Then, the column is sanitised with 1.5 to 2.5 CV 1M NaOH 1M+2M NaCl (2 h) and stored at room temperature in 20 mM NaOH.

(42) 3. Macroprep High S Cation Exchange Chromatography (MHS-E)—Positive Mode:

(43) Cation exchange chromatography is conducted with Macroprep High S (Bio-Rad Laboratories cat. n.sup.o 156-0033) packed in a Vantage 180/500 column (Millipore BioProcess Division cat n.sup.o 87018001). The column is packed in highly purified water at an eluent flow rate of 260 cm/h (66.1 l/h). The dimensions of the packed column bed are: diameter 18 cm, cross-sectional area=254.4 cm.sup.2, bed=36 cm, packed volume=9.16±0.25 l.

(44) The column is sanitised with 1.5 to 2.5 CV of 1M NaOH+2M NaCl with a contact time of 2 hours at a flow rate of 40 l/h (157 cm/h). Maximum flow rate can be 250 cm/h. The pH is stabilised with 1.5 to 2.5 CV of 50 mM Ammonium acetate+2 M NaCl pH 2.0. The column is then equilibrated with 4 to 5.5 CV of 50 mM Ammonium acetate pH 2.0.

(45) The pH of the QSFF-FT fraction (see section 2) is adjusted to 2.0 with 37% HCl. This solution is loaded without prior dilution at a flow rate of 125 cm/h (±31.8 l/h). The binding capacity of the gel is 5 to 15 mg/ml matrix. After loading, the column is equilibrated with 2 to 3 CV of 50 mM Ammonium acetate pH 2.0 at a flow rate of 157 cm/h (40 l/h). Maximum flow rate is 200 cm/h.

(46) Elution is performed with a conductivity linear gradient on 10 CV between 25% (0.5M NaCl) and 75% (1.5M NaCl) with 50 mM Ammonium acetate pH 2.0 and 50 mM Ammonium acetate+2M NaCl pH 2.0. Elution is performed at a flow rate of 157 cm/h (40 l/h). Maximum flow rate is 200 cm/h. The eluted peak is collected in 2 liters fractions which are analysed by SDS-PAGE before pooling. After pooling, the MHS-E pool is stored at −20° C. until the next purification step or at 2-8° C. if used within 24 hours.

(47) Then, the column is sanitised with 1.5 to 2.5 CV 1M NaOH 1M+2M NaCl (2 h) and stored at room temperature in 20 mM NaOH.

(48) 4. Concentration—Diafiltration

(49) The concentration is conducted with two Sartocon cassette (0.6 m.sup.2, cut-off 5 kDa) Hydrosart Sartorius membranes (Sartopore cat n.sup.o 302 144 2906 E-SG). The membranes are mounted in a holder connected to a Proflux M12 system (Millipore Bioprocess Division). The membrane is rinsed with water for injection (WFI). The sanitisation is performed by continuous recirculation of 0.5 M NaOH for 60 minutes. Then, the membrane is rinsed with Na.sub.2HPO.sub.4.12H.sub.2O 20 mM pH 7.0, until permeate pH=7.0±0.1. Then, the membrane is equilibrated with PBS pH 7.4 (NaCl 8 g/l, KH.sub.2PO.sub.4 0.19 g/l, Na.sub.2HPO.sub.4 2.38 g/l) until permeate pH=7.4±0.1.

(50) The inlet pressure and outlet pressures are set to 1.5±0.1 bar and 1.2±0.1 bar respectively.

(51) The several Macroprep High S eluates are pooled together and, according to the total amount of protein, are concentrated to a volume corresponding to a theoretical concentration of 30 mg/ml. After concentration, the retentate is diafiltrated against 10 volumes of PBS pH 7.4 (NaCl 8 g/l, KH.sub.2PO.sub.4 0.19 g/l, Na.sub.2HPO.sub.4 2.38 g/l). The retentate is collected and 7 washes of the membrane are carried out, each one with 150 ml of PBS pH 7.4 (NaCl 8 g/l, KH.sub.2PO.sub.4 0.19 g/l, Na.sub.2HPO.sub.4 2.38 g/l) during 3 minutes with the same process parameters. The permeate line is closed during the washes.

(52) A BCA protein assay is carried out on the retentate and each separate wash fractions. The retentate is pooled with selected wash fractions to obtain a total concentration superior to 12 mg/ml with a yield superior to 90%, if possible.

(53) The membrane is rinsed with WFI. The sanitisation is performed by continuous recirculation of 0.5 M NaOH for 60 minutes. The membrane is then stored in NaOH 0.1 M.

(54) 5. Sterile Filtration

(55) Sterile filtration of the retentate+the selected wash fractions is performed on a 1000 cm.sup.2 0.45/0.22 Sartopore 2 filter (Sartorius cat n.sup.o 544-1307-H8-00) at room temperature. The membrane is rinsed with about 500 ml of PBS pH 7.4 (NaCl 8 g/l, KH.sub.2PO.sub.4 0.19 g/l, Na.sub.2HPO.sub.4 2.38 g/l) before use.

(56) The filtration is performed with a peristaltic pump at a flow rate of about 100 ml/min and the filtrate is collected in sterile and pyrogen free 5 L or 10 L single-use bottle.

(57) According to a BCA assay performed on the filtrated bulk, the concentration is adjusted to 10 mg/ml with PBS pH 7.4 (NaCl 8 g/l, KH.sub.2PO.sub.4 0.19 g/l, Na.sub.2HPO.sub.4 2.38 g/l) added by filtration. After sampling, the sterile bulk is aliquoted in PETG 2000 ml Nalgene bottles (±1500 ml to 1700 ml/bottles). The sterile bulk is stored at −20° C. A schematic summary of production steps is provided in FIG. 1.

EXAMPLE 4

hH1.3 and Bis-Met hH1.3 Purification Efficiency

(58) Culture of BL21[DE3]-bis-met rH1.3 in 50 L fermenter resulted in a yield at harvest time of at least 600 mg/L of culture, as assessed by SDS page analysis on serial dilution of total lysed cells. Final yield after the complete purification process was in excess of 500 mg/L of purified bis-met rH13.

(59) Culture of BL21[DE3]-hH1.3 in 50 L fermenter resulted in a yield at harvest time of at least 600 mg/L of culture, as assessed by SDS page analysis on serial dilution of total lysed cells. The cells were processed through homogenisation and precipitation with HClO.sub.4 according to the standard protocol. The results obtained were according to expectation. Loading on the MacroPrep High-S was also performed as usual, however, the rhH1.3 protein could not be eluted from the column using the conductivity linear gradient on 10 CV between 30% (0.6M NaCl) and 75% (1.5M NaCl) with 10 mM NaCH.sub.3COO pH 2.0 and 10 mM NaCH.sub.3COO+2 M NaCl pH 2.0. Although the rhH1.3 protein was eluted at 2 M NaCl, this step did not allow for sufficient purification and the protein could not be further processed. Thus, the purification has to be considered as failed. This failure was confirmed on two independent purification trials made from two different fermentations.

EXAMPLE 5

Effect of Bis-Met Histone H1.3 In Vitro

(60) Inhibition Zone Assay

(61) To measure the effect of recombinant histone as an antimicrobial and antiviral agent an inhibition zone assay was performed according to standard methods. Furthermore, the effect of recombinant histone as an antifungal agent was tested. Bacteria and fungi were grown in the presence of the bis-met histone H1.3 produced according to the methods outlined above and the average zone diameter was determined (see table 1).

(62) Both gram-positive and gram-negative bacteria and fungi are efficiently eliminated as shown in table 1.

(63) TABLE-US-00001 TABLE 1 Inhibition Zone Assay Drug Concentration Average Zone Target Organism [μg/μl] Diameter [mm] Bacillus megaterium 5 11.4 2.50 10.5 1.25 9.7 0.625 8.6 0.31 7.9 Positive control LL-37 9.8 Escherichia coli D21 20.00 7.4 10.00 6.3 5.00 4.8 2.50 0 1.25 0 Positive control LL-37 5 Candida albicans 20.00 11.8 5.00 8.1 2.50 6.1 1.25 4.4 Positive control Nystain 20.9

(64) Cytotoxicity Assay:

(65) This cell test is detecting the toxic effect of the histone on a histone-sensitive leukemia cell line (e.g. U-937). The vitality of leukemia cancer cells after incubation at different histone concentrations is monitored by means of the AlamarBlue assay, based on the observation of the fluorescence of the redox indicator, which is changing in response to the vitality of the cell. The histone anticancer activity is characterized by IC.sub.50, which corresponds to the histone concentration by which the 50% cancer cell viability is observed. The batches used for the cytotoxicity assay as well as the clinical trials are summarised in table 2. As shown in Table 1, all tested samples show similar, high cytotoxicity against the tumor cell line U-937, irrespective of different contents of H1.3 and bis-Met (cf. Table 3).

(66) TABLE-US-00002 TABLE 2 Cytotoxicity of Batches used in the Clinical Trial Sample (drug product) IC.sub.50 [μM] Batch 1: M-H1A-P02 3.2 ± 0.5 Batch 2: M-H1A-Pool01 3.1 ± 0.5 Batch 3: M-H1A-Pool02 3.1 ± 0.5 Batch 4: M-H1A-Pool03 2.2 ± 0.5

(67) In addition to the bacteria and fungi shown in table 1, further bacteria, fungi and viruses may conveniently be tested by methods known in the art, such as any of the methods outlined herein. Non-limiting examples include Epstein-Barr-Virus, Staphylococcus aureus, Aspergillus niger, Enterococcus, Pseudomonas, Haemophilus influenzae and Salmonella.

EXAMPLE 6

Clinical Data

(68) A phase I/II dose-escalation-trial to evaluate the maximum tolerable dose (MTD) of recombinant human histone H1.3 (rhH1.3) in patients with relapsed or refractory AML and in patients that refused chemotherapy or that are not eligible for chemotherapy was carried out. Inclusion criteria of patients were: signed informed consent, any race, both gender, at least 18 years of age, cytologically proven AML with at least 20% blasts in bone marrow, failure after, unfit for or refusal of standard chemotherapy, adequate performance state (Karnowsky index>60%) and a life expectancy of at least 30 days. Criteria that lead to exclusion of patients were significant organ deficiency, known HIV infection, known hepatitis C virus or hepatitis B virus infection, gravidity or nursing, other malignancies, circulating anti-H1 antibodies, heparin treatment during the two weeks before Visit 1 or during study participation, active medical conditions known to potentially interfere with rhH1.3 treatment such as rheumatoid arthritis or systemic lupus erythematodes (SLE) as well as alcohol and/or drug abuse.

(69) Design of the Study

(70) Patients received 3 infusions per week in three consecutive weeks. The initial dose was 38 mg/m.sup.2. The dose escalation scheme used is shown in table 3.

(71) TABLE-US-00003 TABLE 3 Dose Escalation Scheme Dose Dose Number of Level in mg/m.sup.2 Patients Planned Tested Treatment 1 38 3 7 Complete 2 60 3 7 Complete 3 96 3 3 Complete 4 153  3 3 Complete New Dose Escalation 5, 5, 6 245, 245, 392 1 (cycle 1) 1 Complete 6, 6, 7 392, 392, 628 (cycle 2, the same Complete patient as above) 6, 6, 7 392, 392, 628 1 1 Complete

(72) The clinical trial Phase I/II was done in the Saarland University Hospital in Homburg with AML (acute myeloic leukemia) patients. The drug product batches used in this trial were: B1, B2, B3 and B4. The characteristics of these four batches and of one GLP batch, used in the toxicology study are presented in the table 4 below:

(73) TABLE-US-00004 TABLE 4 Characteristics of Batches of Polypeptide used in the Study Evaluation- Anticancer Endotoxin Endotoxin Rh H1.3 MS activity in cell Batches Description [EU/mg] Level peaks [Da] Test IC.sub.50 [μM] L-H1A-03B07* 5 below AL H1.3 + bis Met 1.7-2.7 M-H1A-P02 B1 12 above AL H1.3 + bis Met 3.2 M-H1A-Pool01 B2 95 above AL mainly bis Met 3.1 M-H1A-Pool02 B3 0.8 below AL mainly bis Met 3.1 M-H1A-Pool03 B4 1.7 below AL mainly bis Met 2.2 * Used in tox study, not clinical study AL: acceptable limit MS peak: 22221 Da H1.3 no Met 22481 Da bis Met N-terminal Met-Met
6.1 Preliminary Evaluation

(74) The table 5 summarizes the preliminary clinical results of the first 22 AML patients treated with recombinant human histone H1.3 (rh H1.3, “Oncohist”) at increasing dose levels (the so-called Fibonacci scheme).

(75) Patients WW13 and WW27 received two treatment cycles each (one cycle comprised 3 infusions a week over three weeks, altogether 9). WW27 had a dose escalation in each cycle i.e. 5-5-6: two weeks dose level 5, third week dose level 6 and similarly in the second cycle dose levels: 6-6-7.

(76) TABLE-US-00005 TABLE 5 Clinical Results of AML Patients Treated with Recombinant Human Histone H1.3 (“Oncohist”) Obtained in the Preliminary Evaluation Patient Drug Composition Endotoxin Initials Dose Product According to MS Contamination & No. Level Remarks Batch (H1.3, bis Met)* [EU/mg] Side Effects AS 01 1 TTI B1 H1.3 + bis Met 12 moderately tolerated NM 02 1 TTI B1 H1.3 + bis Met badly tolerated HS 03 1 TLI B1 H1.3 + bis Met well tolerated RH 04 1 TTI B1 H1.3 + bis Met well tolerated MF 05 1 TTI, TLI B1 H1.3 + bis Met badly tolerated RS 07 1 TTI B1 H1.3 + bis Met well tolerated PS 10 1 TTI B1-21 vials H1.3 + bis Met well tolerated B2-4 vials MT 11 2 B2 Mainly bis Met 95 badly tolerated MG 12 2 TLI B2 Mainly bis Met badly tolerated WW 13 2 PR, TN B2 Mainly bis Met badly tolerated AH 15 2 TTI, TLI B2 Mainly bis Met badly tolerated GB 16 2 TN, TLI B2 Mainly bis Met badly tolerated HF 18 2 TTI, TLI B2 Mainly bis Met badly tolerated Start of Histone Drug without Endotoxin ES 19 2 PR, TTI, TLI B3 Mainly bis Met 0.8 NSE IG 20 3 B3 Mainly bis Met NSE BG 21 3 TTI B3 Mainly bis Met NSE GR 22 3 B3 Mainly bis Met NSE EL 23 4 TTI, TLI B3 Mainly bis Met NSE EW 24 4 B3 Mainly bis Met NSE BH 26 4 B3 Mainly bis Met NSE WW 27 C1: 5, 5, 6 PR, TTI B3/B4 Mainly bis Met B4: 1.7 NSE C2: 6, 6, 7 PF 28 6, 6, 7 B3 Mainly bis Met NSE *preliminary analysis PR: partial remission TTI: temporary thrombocyte increase TN: thrombocytes with normal levels TLI: temporary leukocyte increase NSE: no side effects H1.3: mature recombinant human histone H1.3; bis Met: N-Met-Met-H1.3

(77) As is evident from Table 5, drug side effects occur only as a consequence of endotoxin contamination (see also Table 3). Both the naturally occurring histone H1.3 and the “bis Met” derivative show similar properties as far as clinical signs of efficacy are concerned.

(78) Immunogenicity

(79) All patients were screened for the existence of anti-histone H1.3 autoantibodies before, during and after treatment. None of the treated patients developed autoantibodies during the treatment, neither those with one treatment cycle nor those who received two cycles. Histone H1 is an evolutionary very conservative protein and is neither expected nor proven to be immunogenic. The clinical data confirm that no immunogenic activity can be observed using either the naturally occurring histone H1.3 or the “bis Met” derivative.

(80) Therapeutic Effects

(81) About 50% of the patients showed an increase of thrombocytes and part of them also increase of leukocytes, both very critical biomarkers for AML. Three patients had a partial remission (decrease of tumor cells to less than 50% the initial value. Patient WW13 showed an increase of thrombocytes to normal level (210×10.sup.9/l) which lasted 18 months. His thrombocyte count before the treatment with Oncohist was equal to 47×10.sup.9/l.

(82) 6.2 Final Evaluation of Clinical Results

(83) Table 6 summarizes the clinical results obtained after more detailed analysis of the 22 AML patients treated with recombinant human histone H1.3 (rh H1.3, “Oncohist”) at increasing dose levels (Fibonacci scheme).

(84) As described above, patients WW13 and WW27 had received two treatment cycles each (one cycle comprised 3 infusions a week over three weeks, altogether 9). WW27 had a dose escalation in each cycle i.e. 5-5-6: two weeks dose level 5, third week dose level 6 and similarly in the second cycle dose levels: 6-6-7.

(85) TABLE-US-00006 TABLE 6 Clinical Results of AML Patients Treated with Recombinant Human Histone H1.3 (“Oncohist”) Obtained in the Final Evaluation Patient Drug Composition Endotoxin Initials & Dose Product According to MS Contamination No. Level Remarks Batch (H1.3, bis Met)* [EU/mg] Side Effects AS 01 1 B1 H1.3 + bis Met 12 moderately tolerated NM 02 1 TI, LI B1 H1.3 + bis Met poorly tolerated HS 03 1 TLI B1 H1.3 + bis Met moderately tolerated RH 04 1 B1 H1.3 + bis Met well tolerated MF 05 1 B1 H1.3 + bis Met poorly tolerated RS 07 1 B1 H1.3 + bis Met well tolerated PS 10 1 B1-21 vials H1.3 + bis Met well tolerated B2-4 vials MT 11 2 B2 Mainly bis Met 95 poorly tolerated MG 12 2 LI B2 Mainly bis Met poorly tolerated WW 13 2 PR, TN B2 Mainly bis Met poorly tolerated AH 15 2 LI B2 Mainly bis Met poorly tolerated GB 16 2 B2 Mainly bis Met poorly tolerated HF 18 2 B2 Mainly bis Met poorly tolerated Start of Histone Drug without Endotoxin ES 19 2 PR, TI B3 Mainly bis Met 0.8 well tolerated IG 20 3 B3 Mainly bis Met well tolerated BG 21 3 TI B3 Mainly bis Met well tolerated GR 22 3 B3 Mainly bis Met well tolerated EL 23 4 TI.sup.b, LI B3 Mainly bis Met well tolerated EW 24 4 B3 Mainly bis Met well tolerated BH 26 4 B3 Mainly bis Met well tolerated WW 27 C1: 5, 5, 6 PR, TI, LI B3/B4 Mainly bis Met B4: 1.7 well tolerated C2: 6, 6, 7 PF 28 6, 6, 7 B3 Mainly bis Met Day 8**, day 19 SAE*** *preliminary analysis **after one of 9 infusions AE, recovered ***during last infusion SAE, atrial fibrillation in 74 years patient, relation questionable PR: partial remission TI: thrombocyte increase TN: thrombocytes with normal levels LI: leukocyte increase AE: adverse events; SAE: serious adverse events H1.3: mature recombinant human histone H1.3; bis Met: N-Met-Met-H1.3

(86) Therapeutic Effects

(87) According to the final evaluation, seven of 22 patients showed an increase of thrombocytes and part of them also increase of leukocytes, both very critical biomarkers for AML. Three patients had a partial remission (decrease of tumor cells to less than 6-25% with concomitant improvement of other blood values). Patient WW13 showed an increase of thrombocytes to normal level (210×10.sup.9/l) which lasted 18 months. His thrombocyte count before the treatment with Oncohist was equal to 47×10.sup.9/l.

(88) Safety Evaluation

(89) A Clinical Study Report showed that rhH1.3 (Oncohist) is safe at doses treated so far. No serious side-effects were observed except for one atrial fibrillation under infusion of rhH1.3, which was considered to be possibly related to the study drug. Seventeen (17) patients completed one course of therapy (8-9 administrations), and two responding patients received a second course without side effects. No dose-limiting toxicities were observed and the maximal tolerated dose has not been reached at 628 mg/m.sup.2.

(90) Most importantly, the pure, endotoxin-free study drug was tolerated well by patients, i.e. without side effects, contrary to cytostatics. This result is in accordance with preclinical studies, showing that the recombinant human histone H1.3 derivative does not damage healthy blood cells and does not cause resistance.

EXAMPLE 7

Evaluation of the Presence of Bis-Met hH1.3 in a Sample

(91) The “bis-Met” histone hH1.3 is easily distinguished by MS from endogenous histone H1. This can be analysed directly with ESI-QTOF detection of original unprocessed rhH1.3 drug product solution or in an RP-HPLC-ESI-MS process with RP-HPLC chromatographic separation and subsequent ESI-MS detection. As can be seen in FIG. 2, batch B1 contains both histone H1.3 and the N-Met-Met-derivative. FIG. 3 shows one of three batches (B2) which consist mainly of N-Met-Met-H1.3. Independent of composition, the different batches show comparable cytotoxic activity against leukemia cells (Table 3).

(92) The following spectra were obtained by tandem mass spectrometry (QTOF, a combination of quadrupole and time-of-flight spectrometry in a single instrument). As can be seen in FIG. 2, batch B1 contains both histone H1.3 and the N-Met-Met-derivative. FIG. 3 shows one of three batches (B2) which consist mainly of N-Met-Met-H1.3. Independent of composition, the different batches show comparable cytotoxic activity against leukemia cells (Table 3).