Patent Application
20190151427
A computer readable text file, entitled “SequenceListing.txt,” created on or about Jun. 7, 2016 with a file size of about 1 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
The present invention pertains to the field of anti-cancer vaccines. More particularly, the invention relates to a method for preparing a vaccine formulation with an optimized polypeptide comprising three epitopes and MONTANIDE as adjuvant, for use in anti-cancer immunotherapy.
WO2007073768 (A1) discloses a polypeptide having the sequence Vx006 (YLQVNSLQTVYLEYRQVPVYLEEITGYL, SEQ ID NO: 1), which comprises three universal tumor-antigen-derived optimized cryptic peptides (TERT988Y, MAGE-A248V9 and HER-2/neu402Y). Contrary to a simple mixture of the three peptides and to other polypeptides comprising the same epitopes but in a different order, the polypeptide of SEQ ID NO: 1 elicits a polyspecific CTL response against the native epitopes TERT988, HER-2/neu402, MAGE-A248G9 and MAGE-A248D9, expressed by tumor cells. Since almost 100% of tumors express at least one of the three antigens, Vx006 thereby constitutes an excellent candidate for anticancer immunotherapy.
Immunological adjuvants are substances which are added to vaccine formulations to increase their efficacy. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination. IFA (Incomplete Freund's adjuvant) is one of the most commonly used adjuvants in research. It is prepared from non-metabolizable oils. Vaccine formulations with IFA are water-in-oil (W/O) emulsions. IFA induces an immune response through the formation of a depot at the injection site and the stimulation of antibody producing plasma cells. However, adverse reactions, including sterile abscesses requiring surgical intervention in a number of cases, and some preclinical findings suggesting that IFA could be carcinogenic in laboratory animals, prevented further clinical development of IFA-adjuvanted vaccines. IFA-type detoxified adjuvants with improved specifications and quality control steps have then become available. For example, the mineral oil-based MONTANIDE ISA 51 and squalene-based MONTANIDE ISA 720 (SEPPIC, France) emulsions are currently undergoing clinical developments in vaccine studies against cancer, malaria and HIV. However, because W/O emulsions persist at the site of injection where they tend to cause local reactions, the application of these W/O emulsion adjuvants may remain limited to the field of therapeutic and cancer vaccines in humans. A vaccine against non-small-cell lung cancer, the CIMAVAX EGF vaccine using MONTANIDE ISA 51 VG, is undergoing Phase III studies and has already been licensed at least in Cuba.
MONTANIDE ISA 51 is a mixture of a highly purified mineral oil (Drakeol 6VR) and a surfactant (Mannide monooleate). In 2006, owing to concerns of prion contamination, the formulation of MONTANIDE ISA 51 was changed from using oleic acid isolated from beef tallow to that isolated from olives (MONTANIDE ISA 51 VG).
Process used to perform water in oil (W/O) emulsified vaccines can be obtained by a variety of protocols, using different devices such as high shear mixers, vortex, syringes, or syringes and connectors (T- or I-connectors). The methods used to obtain the emulsion are not all equivalent regarding vaccines physico-chemical parameters and the efficacy of vaccination.
Classically, to obtain an emulsified peptide with MONTANIDE ISA 51, two silicone-free syringes connected by a connector (I or T-connector) are used to bring a high shear in order to entrap droplets of water into surrounding oil phase. Typically, the peptide is dissolved in a saline solution (most often PBS or NaCl 0.9% saline buffer) and one volume of this solution is mixed with one volume of MONTANIDE ISA 51. The mix is then loaded into the device consisting of two silicone-free syringes connected for example by an I-connector, which is used to perform a pre-emulsification step of 20 low speed cycles (i.e., cycles lasting approximately 4 seconds), followed by a 40 rapid cycles emulsification step. A cycle is defined as the transfer of the whole solution (aqueous phase and adjuvant) from a first syringe to the other, followed by a transfer of the whole solution back to its syringe of origin. Recently, Ascarateil and Koziol presented the results obtained with a new equipment optimized for automatic W/O emulsion process (Journal for ImmunoTherapy of Cancer 2013). This device, disclosed in the patent application EP 2 322 132 A1, is an automated closed system allowing to control the emulsification process.
The inventors have tried to obtain W/O emulsions with MONTANIDE ISA 51 and an aqueous phase comprising the polypeptide of SEQ ID NO: 1, by the classical protocol described above. When doing so, they encountered a number of unexplained difficulties which are illustrated in the experimental part below. Since the standard protocol for obtaining an emulsion with a peptide solution and MONTANIDE failed, the inventors tried a number of parameters variations in the emulsifying process, without success. Finally, they found that surprisingly, the polypeptide of SEQ ID NO: 1 could be emulsified with MONTANIDE ISA 51 VG only if the two following conditions are fulfilled:
the polypeptide of SEQ ID NO: 1 must be dissolved in pure water instead of saline solution; and
the pre-emulsification step must comprise at least 2 very slow cycles, each lasting at least 6 seconds.
The present invention hence pertains to a method for obtaining a water in oil emulsion for administration of a polypeptide of sequence YLQVNSLQTVYLEYRQVPVYLEEITGYL (SEQ ID NO: 1) to a human subject, comprising the following steps:
(i) obtaining an aqueous antigenic phase by dissolving lyophilized polypeptide of SEQ ID NO: 1 in pure water (water for injection);
(ii) loading one volume of said aqueous antigenic phase and one volume of MONTANIDE ISA 51 into a device comprising two syringes linked by a connector;
(iii) pre-emulsifying the formulation by performing at least three cycles of transfer of the whole formulation from one syringe to the other and then back to the first syringe, wherein each cycle lasts at least 6 seconds; and
(iv) emulsifying the formulation by performing at least 15 additional transfer cycles, wherein each cycle lasts at most 2 seconds.
In the present text, “MONTANIDE ISA 51” designates the presently sold MONTANIDE ISA 51 VG, as well as any bioequivalent adjuvant derived therefrom (for example, by replacing the oleic acid isolated olives by that isolated from another source or a synthetic one).
It should also be kept in mind that in step (ii), the 50/50 ratio of water and oil phases can vary. Indeed, the adjuvant can represent up to 60 percents of the whole solution. Hence, unless otherwise specified, the phrase “mixing one volume of aqueous phase and one volume of MONTANIDE ISA 51” means “mixing aqueous phase and MONTANIDE ISA 51 in a W/O ratio comprised between 50/50 and 40/60”.
Importantly, the syringes used to perform the above process must be appropriate for MONTANIDE, i.e., they must be silicon-free and rubber free (i.e., without any rubber tip free on the plunger), and preferably also latex-free. Examples of syringes which can be used to perform the invention are:
OR 2 ml INKJET (Ref: 4606701V from B-Braun, Germany),
OR 5 ml INKJET (Ref: 4606710V from B-Braun, Germany),
OR 2 ml Norm-Ject (Ref: 4020.000V0 from Henke Sass Wolf GMBH, Germany),
OR 5 ml Norm-Ject (Ref: 4050.000V0 from Henke Sass Wolf GMBH, Germany).
In the method according to the present invention, step (i) can be performed by hydrating a lyocake of polypeptide of SEQ ID NO: 1 with pure sterile water and waiting until the dissolution is completed. When this step is performed at a temperature between 10 and 40° C., a few seconds to 2 minutes are necessary to obtain a complete dissolution of the lyocake; a gentle agitation of one to a few seconds, by taking the vial into two fingers, can be applied if necessary. The dissolution step can also be performed at a lower temperature, for example in the fridge (1-4° C.).
According to a particular embodiment, step (i) is performed so that a volume of 0.5 to 6 ml, preferably around 1 ml or 5 ml of aqueous antigenic phase is obtained.
According to a particular embodiment, step (i) is performed so that the final concentration of peptide of SEQ ID NO: 1 in the aqueous antigenic phase is between 2 and 20 mg/ml, preferably in the range of 2 to 10 mg/ml.
In a particular embodiment, step (ii) is performed by loading one volume of the aqueous antigenic phase into a first syringe and at least one volume of MONTANIDE ISA 51 into a second syringe, and then bridging the two syringes by the connector, so that the final aqueous phase/MONTANIDE ISA 51 ratio is comprised between 50/50 and 40/60. The simplest way to transfer liquids from vials is to use a syringe and a needle. However, this method has a risk of sting. Another safer solution is to use a vial adapter such as a 13 mm or a 20 mm vial adapters from West Pharmaceutical Services, Inc. or from Promepla (Monaco). For example, step (ii) can be performed by following the protocol described below:
Remove the cover from the vial adapter package while handling the vial adapter through the blister package;
Attach the adapter to the vial; use the blister package to handle the adapter. Seat the adapter on the vial by pushing it down until the spike penetrates the elastomeric stopper and the adapter snaps in place. Remove and discard the blister package;
Twist the syringe onto the adapter;
Withdraw 1 volume (typically, 1 ml) of sterile MONTANIDE ISA 51 VG into the syringe No 1 and remove air. Keep the syringe plugged on the vial;
Repeat the same operation with a new vial adapter and the second syringe to withdraw 1 volume of the aqueous antigenic solution from the vial into syringe No 2;
Remove syringe No 2 from the adapter and connect it onto the connector;
Push the plunger very slowly in order to drain the maximum of air from the system (this step can also be performed with syringe No 1 instead of syringe No 2);
Remove syringe No 1 from the adapter and twist it onto the connector; so that the system is ready for emulsification.
According to a preferred embodiment, the connector used in step (ii) is an I-connector. Non-limitative examples of such connectors are:
the I-connector developed by Green Peptide (Japan) for use with the emulsifying disclosed in the patent application EP 2 322 132 A1,
the connector of reference DIDRACDLLFT from Didanorm (France),
the I-connector (ref: ODG0015ST) from Promepla (Monaco), and
the I-connector (ref: MX494) from Smiths medical (US), which is a non-DEHP formulation, latex-free, lipid resistant, non-PVC connector with and approximate priming volume of 0.2 ml and an approximate overall length of 5 cm.
As already mentioned, the pre-emulsification step (step (iii)) of the claimed method comprises at least two slow cycles, wherein each cycle corresponds to the transfer of the whole formulation (antigenic phase plus adjuvant), from one syringe to the other and then back to the first syringe and lasts at least 6 seconds; in a preferred embodiment, this step comprises 3 to 20 cycles, for example 3, 4, 5, 6, 7, 8, 9, 10 or 20 cycles. Each of these slow cycles preferably lasts between 6 and 30 seconds, more preferably between 8 and 15 seconds and even more preferably between 8 and 12 seconds, for example 8, 9, 10, 11 or 12 seconds. Remarkably, when the slow cycles are performed as indicated above, 5 slow cycles are sufficient for the pre-emulsion step, contrary to the typical protocol which comprises 20 slow cycles. Hence, at least when the pre-emulsification step is performed manually, the number of cycles of step (ii) is preferably between 3 and 8, for preferably between 4 and 6, and even more preferably 5.
The inventors have observed that once the pre-emulsification is correctly performed, a classical emulsification step produces a satisfying emulsion, even if no more than 15-20 fast transfer cycles are performed. Of course, an increased number of rapid cycles can be performed, such as at least 20 cycles, at least 30 cycles, at least 40 cycles and up to 60 cycles or even more. In what precedes, a “rapid” or “fast” cycle lasts between 0.5 and 2 s at most 2 seconds, preferably less than 1.5 s, more preferably less than 1 s and even more preferably less than 0.7 s (as fast as possible).
The present invention also pertains to a kit for obtaining a water in oil emulsion containing the Vx006 polypeptide of SEQ ID NO: 1 and MONTANIDE ISA 51, wherein said kit comprises at least 1 mg of lyophilized polypeptide of SEQ ID NO: 1 (for example 2 mg or 10 mg), at least 1 ml of MONTANIDE ISA 51 (for example, 3 ml of MONTANIDE ISA 51), two silicon- and rubber-free syringes, an I-connector and a notice of use describing the process according to any of the preceding claims. Optionally, the kit can also comprise a vial of water for injection and/or a vial adapter. A sterile needle can also be comprised, for vaccinating the patient after obtaining the emulsion. The syringes in the kit can be, for example, 2 ml or 5 ml syringes. Optionally, the lyophilized Vx006 polypeptide can be replaced by a solution of Vx006 in sterile water.
Another aspect of the present invention is a water in oil emulsion containing the polypeptide of SEQ ID NO: 1 and MONTANIDE ISA 51, wherein the peptide concentration in said emulsion is in the range 1 to 10 mg/ml.
Emulsions can be characterized by different parameters, including DvX, wherein X is comprised between 0 and 100, and which is the maximum particle size (diameter) for X % volume the sample. For example, Dv50 is the maximum particle diameter below which 50% of the sample volume exists, and Dv90 is the maximum particle diameter below which 90% of the sample volume exists. In a preferred embodiment of an emulsion according to the invention Dv 50 is below 3 μm, preferably below 2 μm and more preferably about 1.2 μm. For such emulsions, Dv90 is usually below 5 μm, preferably below 3 μm, and more preferably below 2 μm.
The invention is further illustrated by the following figure and examples.
The examples have been performed using the following materials and methods:
Peptides. Peptides were synthesized by BACHEM (Basel, Switzerland). Vx006 (SEQ ID NO: 1) has been prepared in ammonium solution prior to lyophilisation.
MONTANIDE ISA51VG. MONTANIDE ISA51VG was provided by SEPPIC (Castres, France), Batch number T 134201.
Micelle kun and emulsification devices. A prototype of the Micelle kun (described in EP 2 322 132 A1), was used at SEPPIC (Castres, France). Silicone free syringes of 2 ml are produced and marketed by BBraun (reference 4606701V). I-connector are produced and marketed by didactic GROUP (Etainhus, France) (reference RACDLLFTF).
Emulsion characteristisation. To characterize the emulsion, two tests are commonly used; the drop test and the measure of the drops size.
Drop test: The drop test allows checking the type of emulsion: W/O (aqueous phase drops in oily base) or O/W (drops of oil (adjuvant) in aqueous phase). In a beaker with clear water, lay 1 drop of emulsion on the surface of water and mix gently.
If the emulsion drop stays on the surface, it is a W/O emulsion.
If the emulsion disperses in water, it is an O/W emulsion.
Size of drops: The size of drops in the dispersed phase was checked by laser scattering granulometer. Particle size of emulsions was measured with a Malvern Mastersizer S.
The average particle size was represented by:
Dv 50 means: 50% of the volumes of particles have a size lower than the Dv50,
DV 90 means: 90% of the volumes of particles have a size lower than the Dv90.
Emulsifying MONTANIDE ISA 51 VG requires a saline aqueous phase. It is known that the use of saline buffer (such as PBS and NaCl 0.9%) allows producing thinner emulsions than pure water. To exemplify this, investors have compared emulsions prepared by five independent manipulators with MONTANIDE ISA 51 VG with either pure water or NaCl 0.9% saline solution. 0.7 ml of placebo solution was loaded into a silicone free syringe and 0.7 ml of MONTANIDE ISA 51 VG into a second syringe. After connection of both syringes onto an I-connector, the classical protocol was applied, i.e., 20 slow cycles followed by 40 rapid cycles. The size of drops in the dispersed phase was checked by laser scattering granulometer. Particle size of emulsions was measured with a Malvern Mastersizer S.
Table 1 shows that Dv50 and Dv90 are significantly lower with NaCl 0.9% than with water. Dv50 and Dv90 measured with emulsion of NaCl 0.9% with MONTANIDE were 0.5 and 1.1 μm in average, compared to 0.8 and 1.7 μm in emulsions with water. More importantly, emulsions performed with NaCl 0.9% saline buffer were much more stable than the emulsion with sterile water, since after 24 hours at 4° C., Dv50 and Dv90 measured with emulsions of NaCl 0.9% with MONTANIDE were respectively 1.1 and 1.5 μm in average, compared to 3.6 and 8.3 μm in emulsions with water. These results are in accordance with the recommendation of SEPPIC, MONTANIDE ISA51VG producer.
Vx006 was produced by BACHEM (Basel, Switzerland). For clinical trial, Vx006 was produced with a GMP grade, as vials containing 10 mg of lyophilized peptide.
For vaccinating a patient, 1 ml of solvent is added on the lyocake to resuspend the peptide, 0.7 ml of peptide in solution is then loaded in a silicone free syringe and 0.7 ml of MONTANIDE ISA51VG in a second syringe. Both syringes are then connected to the I-connector for the emulsification protocol.
In practice, solubilizing a peptide can be quite a challenge, although the following chemical rules exist to predict peptide solubility (disclosed, for example, in Sigma-Aldricht's web site):
1. Peptides which are shorter than 5 residues are generally soluble in aqueous media, except in extreme cases where all the residues are very hydrophobic (W, I,
8
2. Hydrophilic peptides, containing >25% charged residues (E, D, K, R and H) and <25% hydrophobic residues also generally dissolve in aqueous media, provided that the charged residues are fairly distributed throughout the sequence. Peptides are generally purified with 0.1% TFA/water and 0.1% TFA/ACN solvent system. Therefore, if the peptide is dissolved in aqueous solution which is unbuffered or insufficiently buffered, the resulting peptide solution can be acidic. The pH of the solution must be around neutrality before trying other means of solubilization. Both acidic peptides (E+D residues>K+R+H residues) and basic peptides (R+K+H residues>E+D residues) are more soluble at neutral pH than at acidic pH.
3. Hydrophobic peptides containing 50% to 75% hydrophobic residues may be insoluble or only partially soluble in aqueous solutions, even if the sequence contains 25% charged residues. It is best to first dissolve these peptides in a minimal amount of stronger solvents such as DMF, acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4-8M GdnHCl or urea, DMSO (if the sequence does not contain C, W or M), and other similar organic solvents, and then slowly add (drop wise) the solution to a stirred aqueous buffer solution. If the resulting peptide solution begins to show turbidity, the solubility limit might have been reached and it will be futile to proceed.
4. Very hydrophobic peptides containing >75% hydrophobic residues will generally not dissolve in aqueous solutions. These peptides generally require initial solubilization in very strong solvents such as TFA and formic acid and may precipitate when added into an aqueous buffered solution. The final peptide solution may require a higher concentration of organic solvent or denaturant, which may not be applicable in biological studies involving live cells.
5. Peptide sequences containing a very high (>75%) proportion of S, T, E, D, K, R, H, N, Q or Y are capable of forming extensive intermolecular hydrogen bonding network and have a tendency to form gels in concentrated aqueous solutions. These peptides may have to be treated similarly to step #3.
Since the Vx006 peptide of SEQ ID NO: 1 comprises 15 hydrophobic residues (between 50 and 75%), 4 charged residues (<25%) and 16 (<75%) residues of the list cited in point 5 above, it theoretically needs a treatment as described in the above point 3, but may be partially soluble in water.
According to SEPPIC recommendations, to the fact that the emulsion is performed for human administration (preventing the use of solvents as DMSO etc.), results presented in Example 1 above and solubility prediction, Vx006 was initially diluted in saline buffer. Two saline solvents were tested, NaCl 0.9% and PBS. In both cases, the peptide in solution was cloudy, the solubilisation was not complete. More importantly and surprisingly, after a few minutes, the solution became viscous and finally a gel was formed, preventing any emulsification of the peptide with MONTANIDE. Moreover, the addition of several solvents developed by Seppic and compatible with injection in humans and emulsion with MONTANIDE were tested during the Vx006 manufacturing development. None of these solvents had any impact on the Vx006 solubilisation.
After a series of unsuccessful tries, inventors discovered that the peptide was perfectly soluble in water for injection, and that the pH of the obtained solubilised peptide was compatible with injection into humans (pH around 7). The lyocake was hence hydrated with a small volume of pure water (1 to 5 ml), and left at room temperature (15-25° C.) until full dissolution (10 seconds to a few minutes). The vial can be gently agitated to accelerate the dissolution, if necessary. The dissolution can also be performed at cold temperature (0-10° C.). The peptide in solution was totally clear and no gel formation was observed (
Vx006 in solution in pure water at a concentration of 10 mg/ml was then emulsified with MONTANIDE ISA51VG. First of all, 0.7 ml of peptide in solution was loaded in a silicone free syringe and 0.7 ml of MONTANIDE ISA51VG in a second syringe. After connection of both syringes onto an I-connector, the typical protocol recommended by SEPPIC was applied, i.e., 20 slow cycles (of 4 seconds) followed by 40 rapid cycles. Four manipulators trained for performing emulsion have applied the same protocol.
The drop test was then performed to evaluate the emulsions. As shown in table 2, in approximately 25% of the emulsifications, the emulsions were oil in water (O/W) emulsions, instead of water in oil (W/O). Moreover, even if the peptide was diluted 5 times (final concentration 2mg/ml), the result remained the same, showing that the emulsification capabilities of the peptide are not due to peptide concentration but to intrinsic characteristics of the Vx006 peptide. Such variability is not acceptable in the context of clinical trials, since oil in water emulsion do not play the role of adjuvant in the vaccination. Inventors then modified several parameters to define a robust protocol for emulsifying Vx006.
To avoid the manipulator bias, next tests were performed using a “Micelle kun”, which is an automatized apparatus conceived to perform controlled emulsion processes (EP 2 322 132 A1). A prototype of the device was provided by SEPPIC. Three slow-cycle speeds were tested, “0” which corresponds to 12 s for one cycle, “5” which corresponds to 6 s for one cycle and “10”, which is 4 s per cycle. 20 slow cycles and 40 rapid cycles were then applied (“Micelle kun” performs the rapid cycles at the constant speed of 0.7 s per cycle). Finally, both peptide concentrations 10 mg/ml and 2 mg/ml were tested. Results in table 3 show that “Micelle kun” was always able to produce W/O emulsions at speeds “0” and “5” but not at “10”, showing the importance of applying very slow cycles during the pre-emulsifying step. It is important to note that the supplier of the “Micelle kun” initially envisaged to produce the “Micelle kun” with a unique “low” speed, but SEPPIC requested that the “low” speed can vary and reach cycles as long as 12 seconds, in order to ensure that it could be used to emulsify all peptides, including particular peptides such as Vx006. Indeed, the specialists from SEPPIC never worked with any peptide as sensitive to the parameters of the pre-emulsification step as the peptide of SEQ ID NO: 1.
To simplify the preparation of the vaccine in the hospital, the inventors decided to determine the minimum number of slow cycles necessary to obtain a W/O emulsion. Using the “Micelle kun”, the number of slow cycles was decreased. The protocol that allows to obtain each time W/O emulsions of Vx006 and MONTANIDE ISA 51 VG with the “Micelle kun” is: 5 cycles at very low speed (speed “0”) followed by 40 cycles at high speed (table 4). It is to be noted that only 3 slow cycles can be sufficient, when these cycles are performed manually at a very low speed (at least 10-15 seconds per cycle).
Finally, to test the robustness of the defined protocol, three manipulators applied 5 very slow cycles and 40 rapid cycles. Table 5 shows that 100% of the emulsions were W/O, thin and stable at 24 hours. Interestingly, the emulsions with Vx006 are more stable than those without the peptide (compare the data of Table 5 below with those of Table 1).
Ascarateil and Koziol: New equipment optimized for emulsified vaccine preparation in the hospital. Journal for ImmunoTherapy of Cancer 2013 1(Suppl 1):P196.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13306769.4 | Dec 2013 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15104858 | Jun 2016 | US |
| Child | 16263023 | US |
