The invention relates to a vaccination procedure and to products for use in such procedures. More precisely, the invention relates to a method of eliciting enhanced titres of antibodies specific for a selected target hapten molecule by using at least two different hapten carrier conjugate vaccines wherein the haptens are the same selected target hapten and the carriers are different and/or the carriers are bound at different positions to the hapten directly or via a linker and/or the linker structures are different. Further, the invention relates to hapten carrier conjugate vaccine combinations and to commercial packages containing them.

Background

A common method to create a potent vaccine that elicits antibodies against low molecular weight non-immunogenic molecules, so-called haptens, and other substances with low immunogenicity is to conjugate such non-immunogenic molecule or low-immunogenic substance to a suitable high molecular weight immunogenic carrier. Several such conjugate vaccines have been tested in clinical trials for potential therapies, such as treatments against drug dependences, e.g. nicotine or cocaine dependence. Many of such conjugate vaccines have proven to be safe, and have also demonstrated some efficacy in both human clinical studies as well as in animal testing.

However, vaccination of animals and humans by using these conjugate vaccines elicits specific antibodies against the hapten as well as against both the carrier and against the chemical linker structure between the hapten and the carrier ¹ . In addition, the total amount of antibodies against the desired free antigen (free hapten) is rather low, and the clinical effect is often regarded to be insufficient ^2,3,4,5 .

The main goal when immunizing an individual with a drug vaccine (e.g. a nicotine vaccine) is to generate antibodies that bind to the inhaled, ingested or injected drug, thereby preventing the drug in question from reaching the brain where it exerts its rewarding effects. By binding the drug, the generated antibodies will alter the distribution of the drug in the body, resulting in elevated serum drug levels and decreased brain drug levels. If the antibodies bind strongly to the drug, the drug-antibody complex remains in the blood stream and thus there will be less free drug available for distribution to the brain ⁶ .

In general, a low-molecular weight substance, e.g. the nicotine molecule, is not immunogenic by itself. In order to be immunogenic the low molecular weight substance, e.g. nicotine, must be conjugated to a suitable immunogenic carrier in order to elicit antibodies in the body. The hapten, e.g. nicotine molecule, is conjugated to the carrier with a suitable linker. The structure of the linker will influence the properties of the nicotine molecule and, therefore, the choice of the linker is of great importance. The position of the linker on the hapten structure is also very important for the quality of the antibodies.

A nicotine vaccine is in general comprised of a low molecular weight nicotine molecule (the hapten) which is conjugated to a high molecular weight carrier protein via a linker. The carrier is normally an immunogenic active protein that will elicit antibodies to itself as well. The nicotine part of the conjugate vaccine presents its three dimensional structure as well as its physical-chemical properties to the immunological system in the vaccinated body.

One undesirable effect of the published nicotine vaccines used so far is that the vaccines elicit considerable amounts of antibodies against both the linker structure as well as against the carrier, especially after repeated immunizations using the same vaccine. The main goal for the vaccination is to generate as large amounts as possible of high affinity antibodies that bind strongly to the nicotine molecule. Additionally, they should be selective and not recognize nicotine's metabolites or other structures, such as the carrier and linker structure. It is important to avoid generation of antibodies against the carrier as much as possible because of the risk of a suppression effect at repeated immunization.

Several nicotine conjugated vaccines have been developed and tested in clinical trials ^2,3 . The position of the linker on the nicotine molecule and the used carriers are described in the Table below for four different vaccines that can be used in the invention described below. The structure of the linker is also different in the vaccines (not shown in the Table).

The abbreviations in the Table of the company names indicate the following companies: Independent Pharmaceutica AB [SE], Xenova Group pic [UK], Nabi Biopharmaceuticals [US], and Cytos Biotechnology AG [CH], repectively. The structure of Nicotine and one of the vaccines mentioned in the Table, namely NIC-VAX, are shown in Figure 1. TABLE

Active immunization with the above mentioned nicotine vaccines generated nicotine antibodies as well as antibodies against the carrier protein. However, competition

experiments (ELISA) show that the generated antibodies have high affinity also to the linker structure plus the hapten. Booster immunizations, i.e. repeated immunizations, using the same vaccine may results in suppression of the amount of antibody produced. All the human trials reported so far using nicotine vaccines use a repeated booster scheme with the same vaccine. The booster immunizations are given to increase the antibody titer further than obtained by the primary immunization. However, it is known that injection with an immunogenic dose of a carrier followed by immunization with hapten-carrier conjugate may selectively suppress the anti- hapten antibody response. It is reported that in a phase I clinical trial of a birth control vaccine using gonadotrophin subunits linked to tetanus toxoid, some of the subjects failed to evoke a booster antibody response to human chorionic gonadotrophin (beta hCG). Changing the tetanus toxoid to diphtheria toxoid in subsequent immunizations restored the anti-hCG response ⁷ .

Recently, DE Keyler et al ⁸ investigated the combined administration of two distinct nicotine immunogens in rats as a means of enhancing the total serum nicotine antibody response, and also reducing individual variability. The two immunogens utilized different linker positions, linker composition, and carrier proteins in order to provide distinct hapten presentations. It was hypothesized that immune responses to the two immunogens would be independent so that combining the two immunogens into a bivalent vaccine would not compromise the immune response to each individual immunogen, and would produce additive serum antibody levels. The effects of these vaccines on the distribution and serum protein binding of a single nicotine dose were also studied. The main findings of their study were that 1 ) the use of nicotine haptens differing in the location and structure of the linker, as well as the carrier protein to which they are conjugated, resulted in distinct immunogens which elicited non cross-reacting antibodies, 2) the concurrent administration of these two nicotine immunogens in a bivalent vaccine did not compromise their immunogenicity, and 3) the resulting total serum antibody concentration correlated with the magnitude of vaccine effects on nicotine pharmacokinetics. The authors concluded that the total nicotine antibody concentration in the bivalent vaccine group was additive compared to that of the monovalent vaccines alone.

However, a bivalent vaccine, which may be initially more effective than a certain monovalent vaccine, will, when administered as a booster dose, result in the same type of immunological drawbacks such as suppressed response, linker effects as the repeated administration of the monovalent vaccine described above.

DESCRIPTION OF THE INVENTION

The current invention is based on a sequential vaccination technique in order to optimize the concentration of antibodies against the hapten (e.g. nicotine) and suppress the formation of antibodies against the linkers as well as antibodies against the carriers or both. This is accomplished by sequential vaccination with at least two different vaccines that have different carriers and/or different linker structures but the same hapten. It is also important to vary the position of the linker on different sites of the hapten molecule to increase the efficacy of the vaccine therapy thus exposing different parts of the hapten molecule to the immune recognition system. To enhance the formation of antibodies against the free hapten molecule e.g. the nicotine molecule and suppress the formation of antibodies against the linkers and carriers the individual vaccines (Vaccine A and Vaccine B) are administered sequentially at different points of times, e.g. with a few weeks intervals as booster doses. The booster dose of a different vaccine ("Vaccine B) after the primary vaccination with "Vaccine A" will result in an enhanced activation of the plasma cells expressing high affinity antibodies against the pure hapten molecule e.g. the nicotine and suppression of the formation of antibodies against the linker structure and/or the carrier of vaccine A, which was used in the primary immunization. This means that if it is desirable to increase the amount of antibody against a hapten, e.g. nicotine, there is a clear advantage to vaccinate the individual at different points of time with a series of different vaccines having different carriers, different linkers and the linkers on different positions on the hapten structure.

Description of the drawings

Fig. 1 illustrates the structure of nicotine and of a typical nicotine vaccine, NIC-VAX, which consists of a conjugate between the nicotine molecule and Pseudomonas aeruginosa protein A mixed with alum as adjuvant.

Fig. 2 shows two examples according to invention of schematic vaccination schedules. The schemes are examples and can be different both regarding types of vaccines and times of the different vaccinations. In the schemes the Vaccine A, Vaccine B, Vaccine C and Vaccine D are different hapten carrier conjugate vaccines.

Short description of the invention

The present invention provides an improved vaccination procedure for eliciting enhanced titers of antibodies against a selected target hapten in a subject as well as a combination of at least two different hapten carrier conjugate vaccines.

In addition to vaccination of a subject, the invention can be used to provide suitable B-cells to be used in appropriate cell systems, e.g. hybridomas, for production of monoclonal antibodies against a selected hapten that can be used in immunotherapies

In short, the invention is based on at least two different hapten carrier conjugates that are administered to a subject at different points of time as a primary vaccine and a boost vaccine, both containing the same hapten, to elicit enhanced titers of antibodies against the hapten molecule, e.g. nicotine or ketamine. After the first boost vaccine, one to several further booster doses may be administered using the primary vaccine, the first boost vaccine or e.g. a second, third or fourth different boost vaccine. Thus, in a first aspect of the invention there is provided a hapten carrier conjugate for use in a method for eliciting antibodies specific for a target hapten molecule in a subject, the method comprising the steps of

a) immunizing the subject with a first hapten carrier conjugate bearing the target hapten molecule, and subsequently

b) immunizing the subject with a second hapten carrier conjugate bearing the target hapten molecule,

wherein the first and second hapten carrier conjugates have a structural difference in relation to each other, and wherein the target hapten has a molecular weight of less than 1 kDa.

In a second aspect of the invention, there is provided a method for eliciting antibodies specific for a target hapten molecule in a subject, comprising the steps of

a) immunizing the subject with a first hapten carrier conjugate bearing the target hapten molecule, and subsequently

b) immunizing the subject with a second hapten carrier conjugate bearing the target hapten molecule,

wherein the first and second hapten carrier conjugates have a structural difference in relation to each other, and wherein the target hapten has a molecular weight of less than 1 kDa.

In a third aspect, of the invention, there is provided a use of a hapten carrier conjugate in the manufacture of a medicament for use in a method for eliciting antibodies specific for a target hapten molecule in a subject, the method comprising the steps of

a) immunizing the subject with a first hapten carrier conjugate bearing the target hapten molecule, and subsequently

b) immunizing the subject with a second hapten carrier conjugate bearing the target hapten molecule,

wherein the first and second hapten carrier conjugates have a structural difference in relation to each other, and wherein the target hapten has a molecular weight of less than 1 kDa.

The method of the first, second or third aspects may further comprise the step of

subsequently immunizing the subject with a third hapten carrier conjugate bearing the target hapten molecule, wherein the first, second and third hapten carrier conjugates have a structural difference in relation to each other. Said method may further comprise the step of re-immunizing the subject with the first hapten carrier conjugate bearing the target hapten molecule subsequent to step (b).

Said method may also comprise timing the immunizations such that 7-90 days elapse between the immunizations. More preferably, 7-60 days elapse between the immunizations. Most preferably, 7-30 days elapse between the immunizations.

The hapten carrier conjugate(s) of the first, second or third aspects may comprise a linker connecting the hapten to the carrier.

The structural difference mentioned in the first, second and third aspects may comprise a difference in a feature selected from the group consisting of: carrier structure, linker structure, position of attachment of a linker to the carrier, position of attachment of the hapten to the carrier and position of attachment of the hapten to a linker.

Said structural difference may preferably comprise a feature selected from the group consisting of: linker structure and position of attachment of the linker to the hapten.

The carrier moieties of the conjugates mentioned in the first, second and third aspects may comprise T-cell epitope-containing proteins or particles. The T-cell epitope-containing proteins or particles may be selected from proteins such as lysozymes, bovine serum albumin, ovalbumin and keyhole limpet hemocyanin; particles such as virus particles and virus-like particles (VLP); and bacterial toxoids and bacterial toxins such as tetanus toxoid, diphtheria toxoids, cholera toxin, cholera toxin subunit B (CTB) and recombinant CTB. Said carrier moieties are preferably selected from the group consisting of tetanus toxoids, viruslike particles, recombinant cholera toxin B and Pseudomonas aeruginosa exoprotein.

The target hapten of the first, second or third aspects may be selected from the group consisting of nicotine, cocaine, ketamine, benzodiazepines, amphetamines,

tetrahydrocannabinols, opiates, cholesterol and low molecular weight hormones such as steroid hormones such as estradiol, testosterone and anabolic steroids. Preferably, the target hapten is nicotine.

The hapten carrier conjugates of the first, second or third aspects may be selected from the group consisting of Niccine™ , TA-Nic™, NicVax™ and Νίοθβ™.

The method of the first, second or third aspects may further comprise the step of identifying a subject in need of antibodies to the target hapten molecule. The subject of the first, second or third aspects is preferably human.

In a fourth aspect of the invention, there is provided a package comprising:

a) A primary vaccine comprising a first hapten carrier conjugate bearing the target hapten molecule and separately b) A booster vaccine comprising a second hapten carrier conjugate bearing the target hapten molecule,

wherein the first and second hapten carrier conjugates are structurally different, and wherein the hapten has a molecular weight of less than 1 kDa.

The package of the fourth aspect may further comprise a third hapten carrier conjugate bearing the target hapten molecule, wherein the first, second and third hapten carrier conjugates have a structural difference in relation to each other.

At least one of the hapten carrier conjugates of the fourth aspect may comprise a linker connecting the hapten to the carrier.

The structural difference of the fourth aspect may comprise a difference in a feature selected from the group consisting of: carrier structure, linker structure, position of attachment of a linker to the carrier, position of attachment of the hapten to the carrier and position of attachment of the hapten to a linker. Said structural difference may preferably comprise a feature selected from the group consisting of: linker structure and position of attachment of the linker to the hapten.

The carrier moieties of the conjugates of the fourth aspect may comprise T-cell epitope- containing proteins or particles. Said T-cell epitope-containing proteins or particles may be selected from proteins such as lysozymes, bovine serum albumin, ovalbumin and keyhole limpet hemocyanin; particles such as virus particles and virus-like particles (VLP); and bacterial toxoids or toxins such as tetanus toxoid, diphtheria toxoids, cholera toxin, cholera toxin subunit B (CTB) and recombinant CTB. Said carrier moieties are preferably selected from the group consisting of tetanus toxoids, virus-like particles, recombinant cholera toxin B and Pseudomonas aeruginosa exoprotein.

The target hapten of the fourth aspect may be selected from the group consisting of nicotine, cocaine, ketamine, benzodiazepines, amphetamines, tetrahydrocannabinols, opiates, cholesterol and low molecular weight hormones such as steroid hormones such as estradiol, testosterone and anabolic steroids. Preferably, the target hapten is nicotine.

The hapten carrier conjugates of the fourth aspect may be selected from the group consisting of Niccine™, TA-Nic™, NicVax™ and NicQp™.

The package of the fourth aspect may further comprise a set of instructions for performing the method of the second aspect.

The package of the fourth aspect may be for use in a method of the second aspect. Detailed description of the invention Depending on the size of the hapten antibodies induced by hapten-carrier conjugates will to varying degrees be selected to react against not only the hapten but also against linker specific-determinants created by the hapten when linked to the carrier. For example, nicotine can be regarded as a comparatively small hapten in this regard, thus the relative proportion of antibodies that have their specificity for nicotine+linker will be high when using a fixed hapten-carrier complex. Boosting with the same same nicotine-carrier complex will normally result in antibodies with an increase in avidity/affinity in parallel for the nicotine-linker complex epitopes. However, an optimal selection and production of high affinity antibodies for the free hapten (e.g. nicotine), will not be achievable using such a protocol.

In order to obtain the highest possible concentration of high affinity antibodies against the free target hapten (e.g. nicotine), that is antibodies with the most efficient capacity to neutralize the target hapten, a protocol encompassing the usage of a series of hapten-carrier complexes is provided herein.

Ideally, the various hapten carrier complexes should only have one common denominator, the presence of the nicotine hapten, whereas especially the linker but also the carrier should be different. After a first immunization with hapten-linker X-carrier A, a second immunization using hapten-linker Y-carrier B will have a select capacity to preferentially induce those relatively few memory B cells induced by hapten-linker X-carrier A with very high affinity for the hapten (that is not also reactive with linker determinants). A similar outcome would also take place if the first and second carrier proteins used are the same but the linkers used for coupling the hapten were distinctly different and not generating crossreacting antibodies against the linker parts.

Thus, an aspect of the current invention is directed to a method of eliciting, in a subject, enhanced titers of antibodies specific for a selected target hapten molecule, which hapten molecule is included in at least two different hapten carrier conjugates. The method comprises the steps of initially immunizing the subject with one hapten carrier conjugate, wherein the hapten is the selected hapten molecule, and subsequently booster immunizing the subject with at least one other hapten carrier conjugate, wherein the hapten is the selected hapten molecule. The first two immunizations are optionally and probably most commonly, followed by at least one further booster immunization of the subject with a hapten carrier conjugate selected from the group consisting of the initial one hapten carrier conjugate and other hapten carrier conjugates wherein the hapten is the selected hapten molecule.

The subject, that will be immunized according to the method of the invention, can be selected from various kinds of animals, preferably mammals, such as humans and laboratory animals, e.g. rats and mice, or domestic animals, e.g. rabbits and dogs.

The enhanced titers of selective antibodies against the free hapten that will result from the method of the invention is not only enhanced compared to the use of only one hapten carrier conjugate but also to the use of two different hapten carrier conjugates in a bivalent vaccine. Such a bivalent vaccine has been reported in the prior art ⁸ to give additive titers compared to a monovalent vaccine.

The subsequent booster immunization(s) will be made after a period of time that is commonly used for booster immunizations, i.e. after a few weeks, such as after 4, 5 and/or 10 weeks.

The immunizations are performed in commonly known ways and are suitably made with appropriate doses of the conjugates by oral, intradermal, subcutaneous, nasal, sublingual, intramuscular, intraperitoneal and/or transdermal administration.

In the invention, the different hapten carrier conjugates, that include the same selected target hapten molecule, are preferably selected from hapten carrier conjugates that have and at least one differing feature selected from the group consisting of different carriers, carriers that are bound at different positions to the hapten directly or via a linker, and different linker structures.

Examples of a selected hapten molecule is a low molecular weight molecule selected from the group consisting of nicotine, cocaine, ketamine, benzodiazepines, amphetamines, terahydrocannabinols, opiates, low molecular weight hormones and cholesterol. Examples of the carriers in the different hapten carrier conjugates are carriers selected from the group consisting of different T-cell epitope-containing large molecules, preferably proteins, virus particles, virus-like particles (VLP), bacterial toxoids, such as tetanus toxoid, diphtheria toxoids, cholera toxoids, and derivatives of toxins and toxoids.

In an embodiment of the invention the one hapten carrier conjugate and the one other hapten carrier conjugate are nicotine carrier conjugates, wherein the carriers are selected from the group consisting of tetanus toxoids, virus-like particles, recombinant cholera toxin B and Pseudomonas aeruginosa exoprotein.

Another aspect of the invention is directed to a hapten carrier conjugate vaccine combination comprising at least two different hapten carrier conjugate vaccines,

one hapten carrier conjugate primary vaccine and

at least one hapten carrier conjugate boost vaccine,

wherein the hapten of the one hapten carrier conjugate primary vaccine as well as the hapten of the at least one hapten carrier conjugate boost vaccine are the same selected hapten molecules, and

wherein the hapten carrier conjugates have at least one differing feature selected from the group consisting of different carriers, carriers that are bound at different positions to the hapten directly or via a linker, and different linker structures. The recommended immunization doses of the different hapten carrier conjugate vaccines and final pharmaceutically acceptable composition of the vaccines will be decided by the vaccine producers based on the contemplated the kind of subject and route of administration. Common vaccine components are, in addition to the immunizing component, adjuvants, diluents, surfactants, lubricants, preservatives and stabilizers.

Examples of a selected hapten molecule is a low molecular weight molecule selected from the group consisting of nicotine, cocaine, ketamine, benzodiazepines, amphetamines, terahydrocannabinols, opiates, low molecular weight hormones and cholesterol; examples of the carriers in the different hapten carrier conjugates are carriers selected from the group consisting of different T-cell epitope-containing large molecules, preferably proteins, virus particles, virus-like particles (VLP), bacterial toxoids, such as tetanus toxoid, diphtheria toxoids, cholera toxins, and derivatives of toxins and toxoids; and in an embodiment of the invention the one hapten carrier conjugate and the one other hapten carrier conjugate are nicotine carrier conjugates, wherein the carriers are selected from the group consisting of tetanus toxoids, virus-like particles, recombinant cholera toxin B and Pseudomonas aeruginosa exoprotein.

A yet further aspect of the invention is directed to a commercial package that comprises the vaccine combination according to the invention. The commercial package can be designated a kit. In addition to the at least two different hapten carrier conjugate vaccines of the invention in separate containers, vials or other units, the commercial package or kit will contain instructions for use and optionally aids useful for the contemplated administration of the vaccines, such as syringes and tags. In case one of the two different hapten carrier conjugate vaccines is formulated as a sustained release preparation, then both of the two vaccines can be contained in the same administration unit for simultaneous administration so that the primary vaccine will be released first in the body and the boost vaccine is released subsequently after an appropriate time.

The invention will in the following be further illustrated by non-limiting examples.

EXPERIMENT

Development of murine hybridoma cell lines producing IgG antibodies against ketamine

The aim of the experiment was to develop specific monoclonal antibodies (mAbs) for the detection of the free form of the low molecular weight drug molecule ketamine. Earlier experiments showed that when mice were immunized with only one type of a conjugated ketamine with a linker, the mice showed a good titer of antibodies against the ketamine- conjugate but no or very low titer of antibodies against free ketamine molecules.

To investigate the possibility to increase the specificity of antibodies against the free ketamine, a sequential vaccination protocol is tested with the aim to find hybridoma clones that are produce antibodies which are specific for the free ketamine. In the experiments described below two different vaccines (ketamine-conjugated proteins) were used sequentially in the immunization protocol. Neither vaccine gave any clones that produce antibodies which are specific for the free ketamine if when used alone.

Materials

Mice

BALB/c mice (female, about 8 weeks old), were used.

Myeloma cell line

Spleen cells were immortalized by cell fusion using myeloma cell line SP2/0-Ag14.

Antigen for immunization and screening

Two different ketamine conjugates ("conjugate A" and "conjugate B", proprietary structures) were used in the immunization protocols.

Methods

Immunization

Five mice were intraperitoneally immunized over a period of 39 days. For immunization a water-in-oil emulsion of equal volumes of antigen and Freund ^' s complete or incomplete adjuvant was prepared. Preparation of the antiserum

Antiserum was prepared during the immunization in order to check the immune response in an ELISA assay.

Fusion

The mice were killed and spleens were aseptically removed, pooled and homogenized. The spleen cells and the myeloma cells were fused in the presence of PEG 3350, re-suspended in complete growth medium, plated out onto eight 96-well tissue culture flat-bottom plates fed twice with HAT medium, before testing the culture supernatants by ELISA. ELISA Screening assay

An ELISA was used for screening of IgG antibodies in cell culture supernatants. Polystyrene microtiter plates were coated with the respective ketamine conjugates. For detection of bound antibodies, plates were incubated with anti-mouse IgG conjugated to alkaline phosphatase 1 :5000) followed by several washes and addition of 150 μΙ/well substrate buffer (2 mM 4-nitrophenyl phosphate in 5% diethanolamine + 0.5 mM MgCI2, pH 9.8). The optical density (OD) was estimated in a microplate reader at 405 nm.

Wells with OD405nm values that were twofold higher than the OD405nm value of the average plate were selected as positive. Competitive ELISA

The procedure of the competitive ELISA was the same as the procedure of the screening ELISA. The ELISA plates were coated and blocked as described above. The cell culture supernatants were mixed with blocking buffer containing various concentrations of free ketamine. As control the same concentration of cell culture supernatant in blocking buffer was used. The mixtures were pre-incubated for 1 h at room temperature and subsequently transferred to the coated and blocked ELISA plates with ketamine-conjugate. ELISA procedures were then continued as described above.

Selection of antibody producers

Cells from positive IgG producing wells were transferred into wells of 48-well plates and cultivated for 2 - 4 days (depending on growth characteristic of cells). During this time a competitive ELISA with free ketamine was carried out in order to detect the cultures that were producing antibodies specific for ketamine.

Results

The polyclonal antiserum from mice (dilution 1 :1000) was pre-incubated with 10 pg/ml of free ketamineand subsequently transferred to ELISA plates with immobilized ketamine-conjugate. All mice that have been immunized with only "conjugate A" or only "conjugate B" showed high titre against the conjugate, but almost no competition with the free Ketamine. However, when the mice in one of the vaccinated groups (Conjugate A immunized) were boosted with the second conjugate ("Conjugate B") a high degree of competition with the free Ketamine was shown and we were able to detect 7 positive clones that showed good competition against the free ketamine after the fusion and screening procedure.

The results from this experiment indicate that the specificity of the produced antibody against free ketamine increased.

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