HK1119065A - A METHOD FOR PREPARING A STABLE IMMUNOGENIC PRODUCT COMPRISING ANTIGENIC HETEROCOMPLEXES OF TNFα AND A CARRIER PROTEIN - Google Patents

Description

Method for preparing a stable immunogenic product comprising antigenic heterocomplexes of TNF alpha and a carrier protein

Technical Field

The present invention relates to a stable immunogenic product comprising antigenic heterocomplexes of TNF α and a carrier protein for obtaining a humoral immune response in a mammal, producing antibodies that neutralize the biological activity of TNF α, and methods of making the same.

The stabilized immunogen product may also be referred to as an anti-TNF α immunogen product or may also be referred to as an immunogen product for inducing anti-TNF α antibodies.

The invention also relates to a vaccine composition comprising said stable immunogen product, said vaccine composition comprising antigenic heterocomplexes of TNF α and a carrier protein, and to a process for the preparation thereof.

Background

A general method for preparing a stable immunogenic product comprising antigenic heterocomplexes comprising one or more antigenic proteins of interest and at least one carrier protein as already described in the PCT application published under application number WO2004/024189 in the name of neoacs. Notably, the PCT application discloses a method for preparing such heterocomplexes using TNF α as the antigen of interest and KLH as the carrier protein. When carrying out the preparation of a heterocomplex comprising TNF α and KLH, the general process of this prior art comprises the following steps:

a) obtaining a mixture of KLH and TNF α;

b) adding glutaraldehyde to the mixture to a final concentration of 0.026M;

c) removing excess glutaraldehyde by performing dialysis;

d) adding formaldehyde to the dialyzed solution and maintaining the presence of formaldehyde for 48 hours;

e) adding glycine to the solution obtained at the end of step d; and

f) dialysis is performed with the solution obtained at the end of step e.

The examples in PCT application No. WO2004/024189 show that the synthetic TNF α/KLH immunogenic product is stable and can increase the production of antibodies with good neutralizing activity against native TNF α.

However, over time, considering the high level requirements of many health authorities, including in the us and europe, in order to prepare suitable vaccine compositions comprising said heterocomplexes as the main active ingredient, it appears that more effective heterocomplexes are needed.

Detailed Description

Applicants have found that the general process disclosed in PCT application No. WO2004/024189, when applied to a heterocomplex comprising TNF α and a carrier protein, results in a final immunogenic product in which the deactivation and immunogenicity of TNF α must be improved in order for the anti-TNF α vaccine composition prepared to be approved by multiple health authorities worldwide. Applicants have also found that the stability of the whole immunogenic product should be improved, in particular during storage.

Applicants have now discovered a novel method for preparing a stable immunogenic product comprising antigenic heterocomplexes of TNF α and a carrier protein, which product achieves the various objectives described above.

The object of the present invention comprises a method for the preparation of a stable immunogenic product comprising antigenic heterocomplexes of TNF α and a carrier protein. Which comprises the following steps:

a) providing a solution comprising TNF α;

b) adding EDTA to a solution comprising TNF α of step a) above;

c) adding a carrier protein to the solution finally obtained in step b in order to obtain a mixture of TNF α and said carrier protein;

d) adding glutaraldehyde to the mixture obtained at the end of step c, so that the molecular fraction of TNF α is covalently conjugated to the above-mentioned carrier protein and a heterocomplex between TNF α and the carrier protein is obtained;

e) removing glutaraldehyde, as well as TNF α and free molecules of the carrier protein, from the mixture obtained at the end of step d, so as to obtain a solution containing purified heterocomplexes between TNF α and the carrier protein;

f) adding formaldehyde to the solution obtained at the end of step e and maintaining the presence of formaldehyde for a period of time ranging from 96 hours to 192 hours;

g) adding a reagent for blocking the reaction with formaldehyde to the solution containing the heterocomplexes between TNF α and the carrier protein obtained at the end of step f; and

h) in order to obtain a solution comprising the above-mentioned stable immunogenic product comprising heterocomplexes between TNF α and the carrier protein, formaldehyde and blocking reagent are removed from the solution obtained at the end of step h).

By carrying out the above method, the resulting stable immunogenic product is endowed with an enhanced ability to induce the production of antibodies that neutralize native TNF α when administered to a mammal in combination with one or more immunoadjuvant compounds.

A "stable immunogenic product comprising antigenic heterocomplexes of TNF α and a carrier protein", as used herein, consists of an immunogenic product of antibodies that induce the biological activity of neutralizing TNF α, the immunogenic product comprising protein interactions between (i) TNF α molecules and (ii) carrier molecules, wherein less than 40% of the TNF α molecules are bound to the carrier molecules by covalent chemical bonds.

The stable immunogenic product prepared by the above method is "immunogenic" in that it induces antibodies against native TNF α in a subject, which, upon injection into the subject, induces the production of antibodies that more specifically neutralize the biological activity of TNF α.

The immunogenic product prepared using the above method is "stable" in that it has its own isoelectric point which is distinguishable from at least the isoelectric points of either (i) TNF α or (ii) the carrier molecule, and since the immunogenic product migrates as a protein band in an isoelectric focusing assay, the protein band is distinct from at least one of the two protein bands corresponding to (i) TNF α and (ii) the carrier molecule, respectively. This means that the immunogenic product according to the invention comprises neither free unbound TNF α molecules nor free unbound carrier protein molecules.

Because the immunogenic product comprises (i) TNF α of the antigen and (ii) carrier protein molecules of the antigen that are bound together (a) in part by covalent bonds (less than 40% covalent bonds) and (b) in part by weak non-covalent bonds (more than 60% non-covalent bonds), including ionic interactions, hydrogen bonds, van der waals interactions, and the like, the immunogenic product prepared using the above method comprises "antigenic heterocomplexes" or "heterocomplexes" of TNF α and carrier protein.

The stable immunogenic product, which comprises antigenic heterocomplexes of TNF α and a carrier protein as defined above, can be prepared according to the method of the invention, and can be defined herein more simply as an immunogenic product for use in induction in humans or animals, or for the induction of antibodies to TNF α; or alternatively an anti-TNF α immunogenic product.

The stable immunogenic product finally obtained according to the invention, thanks to the combined steps of the method, has a higher reproducibility than the heterocomplexes comprising TNF α and a carrier protein obtained according to the above cited prior art method disclosed in PCT application No. WO 2004/024189.

In particular, it has been found that the immunogenic activity of the stabilized immunogenic product prepared by carrying out the method according to the invention is highly reproducible from one preparation to another.

As shown in the examples herein, the stable immunogenic product prepared by carrying out the process according to the invention, due to its ED₅₀At doses greater than 50ng/ml, and even greater than 400ng/ml (when expressed as the final concentration of TNF α), there was no detectable TNF α biological activity in vivo and in vitro.

Also, it is shown herein that, in addition to inducing high titers of anti-TNF α antibodies, the stable immunogenic products prepared by practicing the present methods according to the present invention induce the production of high neutralizing anti-TNF α antibodies with NC₅₀Exceeding 1/1000 neutralization capacity.

Furthermore, the stable immunogenic products prepared by carrying out the present method according to the present invention have also been shown to be able to be safely administered to mammals, since these products do not induce any adverse side effects and, in particular, do not have inflammation or alteration of any organs such as heart, lung, liver and spleen even when administered in amounts 4000 times higher than the Single Human Therapeutic Dose (SHTD).

Furthermore, the stable immunogenic products prepared by implementing the method according to the invention induce the production of large amounts of neutralizing antibodies against TNF α, in particular IgG isotypes, in rhesus monkeys (rhesussmasque), which show that these stable immunogenic products are able to break the immunological tolerance to endogenously produced proteins and induce an effective vaccination leading to the neutralization of TNF α, the vaccine of which is able to prevent the toxic effects of TNF α overdoses in all pathological situations in which they are involved.

The stable immunogenic product prepared by carrying out the method according to the invention has also been shown to be effective in vaccinating mammals against TNF α -induced arthritis. Furthermore, it was shown that these stable immunogenic products when used alone are capable of preventing and/or treating TNF α -induced arthritic inflammatory synovitis and joint damage without the need for adjuvant prevention or curative treatment with any additional anti-arthritic active ingredient such as methotrexate.

The stable immunogenic product prepared according to the method of the present invention has also been shown to prevent and/or treat lethal shock caused by overproduction of TNF α.

Thus, the stable immunogenic product prepared according to the invention induces an autoimmune protective effect against TNF α, preventing acute and chronic pathological conditions of TNF α dependence, with no detectable side effects.

Without wishing to be bound by any particular theory, applicants believe that the particular combination of steps a) through h) allows the steric conformation of the initial TNF α product to be preserved, while facilitating exposure of the primary epitope of TNF α to solvents, which, when administered to a mammal, allows for efficient presentation of TNF α antigens to immunocompetent cells.

Without wishing to be bound by any particular theory, the applicant also believes that the particular combination of steps a) to h) gives the final immunogenic product better structural and chemical stability when TNF α and KLH are used, compared to the method disclosed in PCT application No. WO 2004/024189. These particular properties of the final stable immunogen product obtained by this method should make it possible to prepare vaccine compositions which are also very stable over long term storage.

Moreover, it has been found that the process of the invention allows for the preparation of stableThe final immunogen has no residual biological activity of the initial product of TNF alpha. Notably, the ED of the TNF α initial product has been found in the present invention₅₀Is about 10 pg/ml. The stable immunogenic end product obtained by the process of the invention has an ED₅₀Values are higher than 50ng/ml and even in most cases higher than 400 ng/ml. Meanwhile, the stable immunogen final product has high immunogenicity.

Furthermore, it has been shown that the immunogenic product obtained according to the method of the invention retains the main conformation of native TNF α, since it binds to the receptors R1 and R2 of TNF α despite the loss of biological activity of the immunogenic product.

In step a), the native TNF α constituting TNF α is selected from the group consisting of mouse TNF α, rat TNF α, rabbit TNF α and human TNF α. Preferably, the TNF α consists of human TNF α.

According to the process of the present invention, step b) comprises in certain embodiments the steps of:

b1) adding EDTA to the solution comprising TNF α of step a); and

b2) adding DMSO to the solution obtained at the end of step b 1).

It has been found that the addition of DMSO in step b2) according to the invention enhances the immunogenicity of the stable immunogen end product.

In certain embodiments of the present method, step d) comprises the steps of:

d1) adding glutaraldehyde to the mixture obtained at the end of step c), so as to covalently conjugate the TNF α molecules to the carrier protein and obtain a heterocomplex between TNF α and the carrier protein; and

d2) EDTA is added to the heterocomplex between TNF alpha and the carrier protein obtained at the end of step d 1).

Thus, the solution obtained at the end of step d1), comprising a product containing (i) a part of the TNF α molecules bound to the carrier molecule by covalent bonds and (ii) a part of the TNF α molecules bound to the carrier molecule by non-covalent bonds.

The method according to the invention comprises a step h) for removing formaldehyde and blocking reagent from the solution obtained at the end of step g) to obtain a purified stable immunogenic product comprising a heterocomplex between TNF α and said carrier protein.

At the end of step h), a stable immunogen solution is obtained, in a colorless translucent state, practically without significant particulate matter.

In certain embodiments, the method may further comprise the additional step of i) freezing the solution obtained at the end of step h).

In certain embodiments, the method may further comprise an additional step i) of lyophilizing the solution obtained at the end of step h) in order to obtain a white powder that can be preserved for a long period of time before use.

In a preferred embodiment of step a), the TNF α concentration ranges from 0.1mg/ml to 50mg/ml, and even more preferably from 0.5mg/ml to 10 mg/ml.

In a preferred embodiment of step b), the final concentration of EDTA ranges from 1mM to 500mM, and most preferably from 1mM to 10 mM.

Preferably, EDTA is added as a buffer solution at a pH ranging from 7 to 8.5, more preferably from 7.5 to 8.1.

In a preferred embodiment of step b2), the final concentration of DMSO ranges from 0.5% v/v (by volume) to 20% v/v, most preferably from 5% v/v to 20% v/v.

Preferably, step b2) is carried out for a period of time of from 10 minutes to 50 minutes, more preferably from 20 minutes to 40 minutes.

In a preferred embodiment of step c), the molar ratio of TNF α to the carrier protein is in the range of from 5: 1 to 100: 1, and more preferably in the range of from 20: 1 to 80: 1. In the most preferred embodiments of step c), the molar ratio of TNF α to carrier protein is in the range of from 30: 1 to 70: 1, alternatively from 40: 1 to 60: 1.

In a preferred embodiment of step d), the final concentration of glutaraldehyde ranges from 0.05% w/w (by weight) to 0.5% w/w. A final concentration of glutaraldehyde in the range of from 0.02M to 0.03M is suitable, with a final concentration of 0.026M being most preferred.

Preferably, step d2) is carried out for a period of time of from 30 minutes to 60 minutes, more preferably from 40 minutes to 50 minutes.

In a preferred embodiment of step d2), the final concentration of EDTA ranges from 1mM to 10 mM.

In a preferred embodiment of step e), glutaraldehyde is removed by performing dialysis, ultrafiltration using diafiltration, or performing Tangential Flow Filtration (TFF).

When dialysis is performed, it is preferred to use a dialysis membrane with a cut-off value of 6-8 kDa.

The dialysis step preferably comprises three steps including (i) the first two steps using a working buffer (100mM phosphate, 150mM NaCl pH7.8, EDTA 5mM) and (ii) the last step using PBS.

Typically, the dialysis step is performed with a conventional buffer solution, such as Phosphate Buffered Saline (PBS), using a buffer in a volume of 200 to 400 times that of a solution comprising a heterocomplex between TNF α and the carrier protein in which the TNF α moiety is covalently bound to the carrier protein used.

When ultrafiltration using diafiltration is performed, a filter with a cut-off of 10000kDa is preferably used. It is understood that the heterocomplex between TNF α and the carrier protein is found to remain in the ultrafiltration retentate. Typically, the liquid used for diafiltration consists of a buffer, for example Phosphate Buffered Saline (PBS). Three diafiltration cycles are generally performed with equal volumes of buffer.

In a preferred embodiment of step f), the final concentration of formaldehyde ranges from 1% w/w to 10% w/w, and even more preferred ranges from 2% w/w to 5% w/w.

In step f), the presence of formaldehyde is maintained for a time ranging from 96 hours to 192 hours.

It has been found that maintaining formaldehyde and heterocomplexes for less than 96 hours in accordance with the present invention results in a significant decrease in the stability of the final immunogenic compound over time when compared to the stabilized immunogenic compound obtained according to a preferred embodiment of the method of the present invention.

On the other hand, it has been found that maintaining formaldehyde in coexistence with the heterocomplexes for more than 192 hours according to the present invention results in a final immunogenic product with high stability, but with a significantly reduced ability to induce antibodies with high neutralizing activity against native TNF α.

More preferably, the formaldehyde is maintained in step f) for a period of time of from 120 hours to 168 hours.

Most preferably, the formaldehyde is maintained in step f) for a period of time from 130 hours to 150 hours.

Advantageously, step f) is carried out at a temperature ranging from 30 ℃ to 42 ℃, more preferably from 35 ℃ to 39 ℃.

The reagent that blocks the reaction of the protein molecule with formaldehyde in step g) may consist of any suitable compound comprising at least one amino group that will terminate the chemical reaction of the protein molecule with formaldehyde.

In certain preferred embodiments, the blocking reagent consists of glycine.

In a preferred embodiment of step g), the final concentration of glycine ranges from 0.01M to 10M, and even more preferred ranges from 0.05M to 2M.

In certain other preferred embodiments of step g), the blocking reagent consists of lysine.

In a preferred embodiment of step g), the final concentration of lysine ranges from 0.01M to 10M, preferably from 0.05M to 2M, and most preferably from 0.05M to 0.5M.

In a preferred embodiment, the protein molecule is contacted with lysine for an incubation time of from 1 hour to 10 days, and most preferably from 5 days to 10 days.

The incubation with lysine for more than 3 days, and most preferably at least 5 days, results in optimal irreversibility of the final product chemical structure and contributes to its good immunogenicity and stability.

When step h) is carried out, it is advantageous to adjust the pH of the solution to a value in the range from 6.8 to 7.8, more preferably in the range from 7.0 to 7.6, for example by using a base such as NaOH.

In a preferred embodiment of step h), the formaldehyde and blocking agent are removed by dialysis, ultrafiltration using diafiltration or Tangential Flow Filtration (TFF).

The conditions of dialysis, ultrafiltration using diafiltration or Tangential Flow Filtration (TFF) used in step h) are generally the same as those previously determined for step e) above, or correspond to the conditions disclosed in the examples herein.

The term "carrier protein" or "carrier protein molecule" is used herein according to its conventional meaning to a person skilled in the art, i.e. a protein which, when coupled to an antigenic molecule, including a hapten molecule, is capable of inducing an immune response against the antigenic molecule in a host organism, particularly in a mammal, including a human. As used herein, the immune response includes the production of antibodies directed against the antigenic molecule.

According to the method of the invention, a wide variety of carrier proteins known to the person skilled in the art can be used in step c). The carrier protein should carry sufficient helper T-cell epitopes to activate T-helper and B cells and induce these cells to release sufficient IL-1 and IL-2 to induce B cell clonal expansion that will produce neutralizing anti-TNF α antibodies.

The "carrier protein molecule" included in the stabilized immunogen products of the present invention means any protein or peptide (regardless of its amino acid sequence) of at least 15 amino acids in length and which, when covalently bound to a TNF α molecule moiety to form a protein heterocomplex, constitutes the immunogen product of the present invention, such that a large number of TNF α molecules are presented to B lymphocytes.

The carrier protein molecule preferably consists of a protein or a peptide of at least 15 amino acids in length, or alternatively an oligomer of such a peptide, comprising one or more helper T epitopes ("helper cells") capable of activating helper T lymphocytes ("T helper cells") of the host organism to produce cytokines, including interleukin 2, such cytokines which, in turn, activate and induce B lymphocytes to proliferate, which upon maturation will produce antibodies against TNF α.

The carrier protein molecules may also be present in, derived from, homooligomers or homopolymers of native proteins, as well as being derived from homooligomers or homopolymers of peptide fragments of native proteins. The antigenic protein of interest may also be present in hetero-oligomers or hetero-polymers comprising a combination of several different peptide fragments originally contained in the native protein from which it was derived.

Examples of carrier proteins that may be used when carrying out the method according to the invention include diphtheria tetanus toxoid (including DT, DT CRM197, respectively, other DT mutants, e.g. position Glu-148 etc. [ see, e.g., U.S. patent application nos. 4,709,017, WO93/25210, WO95/33481 etc. ] and TT (and TT fragment C)), maple Keyhole Limpet Hemocyanin (KLH), OMPC from neisseria meningitidis (n.meningidis) and purified tuberculin protein derivatives (PDD).

The function of the carrier is to provide cytokine help to enhance the immune response to TNF α. An incomplete list of vectors that may be used in the present invention includes: keyhole Limpet Hemocyanin (KLH), serum albumins such as Bovine Serum Albumin (BSA), inactivated bacterial toxins such as tetanus or diphtheria toxins (TT and DT), or recombinant fragments thereof (e.g., domain 1 of fragment C of TT, or the translocation domain of DT), or purified tuberculin protein derivatives (PDD).

In an embodiment of the method, the carrier is protein D from haemophilus influenzae (EP 0594610B 1). Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted patent EP 0594610B 1). In some cases, for example in recombinant immunogen expression systems, it may be desirable to use fragments of protein D, such as protein D1/3. sup.rd (comprising the N-terminal 100-110 amino acids of protein D (WO 99/10375; WO 00/50077)).

Thus, in a preferred embodiment of the method, the carrier protein is selected from the group consisting of Diphtheria Toxin (DT) and mutants thereof, Tetanus Toxin (TT), Keyhole Limpet Hemocyanin (KLH), and purified tuberculin protein derivatives (PDD), Bovine Serum Albumin (BSA) and protein D from haemophilus influenzae.

Most preferably, the carrier protein consists of Keyhole Limpet Hemocyanin (KLH).

The invention also relates to a method for preparing a vaccine composition comprising the steps of:

a) preparing a stable immunogenic product comprising antigenic heterocomplexes of TNF α by the above method; and

b) mixing said stable immunogenic product comprising antigenic heterocomplexes of TNF α prepared in step a) with one or more immunoadjuvants.

As shown in the examples herein, the stable immunogenic product, which may also be referred to herein as an anti-TNF α immunogenic product, consists of the final product obtained according to the method of the invention, possessing specific physicochemical and biological properties.

In general, the anti-TNF α immunogenic products according to the invention have a molecular weight range from 50kDa to 8000kDa (8MDa), with the proteins representing about 30% of the total protein mass having a molecular weight below 1000kDa (1 MDa).

Furthermore, the anti-TNF α immunogenic products according to the invention exhibit a molar ratio of TNF α to carrier protein in the range of from 40: 1 to 60: 1.

Furthermore, the anti-TNF α immunogenic product according to the present invention, when incubated under denaturing conditions, in which the non-covalent bonds are removed, such as in SDS-containing buffers, produces a product of more than one protein, indicating that the TNF α molecules and carrier protein molecules contained therein are at least partially bound together by non-covalent bonds, such as weak bonds (including ionic interactions, hydrogen bonds, and van der waals bonds).

Furthermore, it has been found that in an anti-TNF α immunogenic product according to the invention the covalent bonds between the TNF α molecules and the carrier protein molecules always show less than 40% of the total number of bonds between these molecules, since more than 60% of the TNF α molecules in said anti-TNF α immunogenic product are always released as molecules which are not bound to the carrier protein under denaturing conditions.

As shown by way of example herein, including example 8, an anti-TNF α immunogen product according to the present invention includes at least the following protein classes:

-a TNF α molecule covalently bound to a carrier protein molecule; and

-a molecule bound by non-covalent bonds comprising:

-a monomer of a TNF α molecule;

-dimers of TNF α molecules;

-a trimer of TNF α molecules;

-polymers of dimers and/or trimers of TNF α molecules;

-a monomer of a carrier protein molecule; and

-a polymer of carrier protein molecules.

Importantly, the anti-TNF α immunogenic product according to the invention induces anti-TNF α antibodies with TNF α Neutralizing Capacity (NC)₅₀) Exceeding 1/1000.

The TNF α neutralizing capacity consists of a serum dilution that neutralizes 50% of the TNF α cytotoxic activity. The biological properties of the anti-TNF α immunogen products according to the present invention can be readily assessed directly and reliably by those skilled in the art using conventional techniques as disclosed in the examples and as further elaborated in the present specification.

NC of anti-TNF alpha immunogenic product of the invention₅₀The values are significantly different from those found for NC of stable immunogenic products prepared according to the method disclosed in the previous PCT application No. WO2004/024189₅₀The value is obtained.

NC of specific anti-TNF alpha immunogenic product in which the carrier protein consists of KLH₅₀The comparison between the values is detailed in the examples herein, in particular in table 3.

The TNF α -KLH immunogen product according to the present invention consists of a batch of product designated "K7", whereas the corresponding stabilized immunogen product prepared according to the method disclosed in the previous PCT application No. WO2004/024189 consists of a batch of product designated "K10", as seen in table 3.

As shown in Table 3, a stable immunogenic product was prepared according to the method previously disclosed in PCT application No. WO2004/024189, and its NC was found₅₀The value is 1/700. NC of anti-TNF alpha immunogenic product according to the invention₅₀Value 1/1200, and is therefore approximately the NC of the previous existing product₅₀Half the value.

By the combination of specific properties of the method according to the invention, comprising the use of EDTA in step b) and the maintenance of the presence of formaldehyde from 96 hours to 192 hours in step f), the final product of the anti-TNF α immunogenic product according to the invention is provided with a higher TNF α neutralizing capacity.

In particular, as shown in table 3 herein, maintaining the presence of formaldehyde in step f) from 96 hours to 192 hours is such as to obtain a product having an optimally low NC₅₀The final product of the anti-TNF α immunogen.

In addition, as shown in Table 3 herein, maintaining the presence of formaldehyde from 96 hours to 192 hours in step f) also makes it possible to obtain an anti-TNF α immunogen end product in which the cytotoxic activity of TNF α is almost completely blocked, the ED of which₅₀(effective dose)₅₀) Values greater than 50ng/ml, preferably greater than 400ng/ml, and sometimes at least as high as 10 pg/ml.

TNF alpha neutralizing capacity NC for anti-TNF alpha immunogenic products according to the invention₅₀The evaluation of the values is disclosed in detail in example 3 and summarized in the following.

One skilled in the art can directly and reliably determine the TNF α Neutralizing Capacity (NC) of an anti-TNF α immunogenic product₅₀) The general method of values comprises the steps of:

a) administering to the mouse by intramuscular route a mixture of an anti-TNF α immunogenic product to be tested and Complete Freund's Adjuvant (CFA);

b) on day 21 after step a), the same mice were administered intramuscularly with a mixture of the anti-TNF α immunogenic product to be tested and Incomplete Freund's Adjuvant (IFA);

c) on day 28 after step a), a blood sample was taken from each mouse treated in steps a) and b) above.

d) Determining at which dilution of the serum of the blood sample collected in step c) the cytotoxic activity of a standard amount of TNF α is 50% inhibited.

To implement the NC₅₀Value evaluation method, the cytotoxic activity of TNF α was determined according to a conventional cytotoxicity assay using the L929 cell line. Preferably, the standard amount of TNF α in the culture of L929 cells is a final concentration of 20 ng/ml.

As in the examples hereinIn particular as disclosed in Table 3 herein, the ED₅₀Values consisted of the final concentration of the anti-TNF α immunogenic product according to the invention, which induced 50% cytotoxicity in the conventional L929 cytotoxicity assay.

It has been found that another property of the anti-TNF α immunogenic product according to the invention which differs from the stabilized immunogenic product disclosed in PCT application No. WO2004/024189 is due to ED₅₀Constitution, which is greater than 50ng/ml, even greater than 400ng/ml, and sometimes up to 10pg/ml, whereas ED was found for prior art products₅₀Approximately 15 ng/ml.

Without wishing to be bound by any particular theory, applicants believe that the anti-TNF immunogenic product obtained according to the method of the invention has better biological properties, including a lack of TNF α activity and high immunogenicity, suggesting that the anti-TNF immunogenic product according to the invention is structurally different from the stable immunogenic product disclosed in PCT application No. WO2004/024189, although specific structural changes may not be readily detectable.

The invention also relates to a stable immunogenic product comprising a heterocomplex of an antigen of TNF α and a carrier protein, said product having one or more of the following technical features:

(i) comprising (1) a TNF α molecule and (2) a carrier protein molecule, (a) joined together by less than 40% covalent bonds and (b) by more than 60% non-covalent bonds;

(ii) it displays a molar ratio of TNF alpha to the carrier protein ranging from 40: 1 to 60: 1;

(iii) its induced production has TNF alpha Neutralizing Capacity (NC)₅₀) An anti-TNF α antibody less than 1/1000; and

(iV) it has an ED of more than 50ng/ml, and even more than 400ng/ml in the L929 cytotoxicity assay₅₀The value is obtained.

(V) it possesses a molecular weight range from 50kDa to 8000kDa (8M Da), the molecular weight of the carrier protein being below 1000kDa (1MDa) accounting for about 30% of the total protein mass.

It is apparent that the anti-TNF α immunogenic product of the present invention has the characteristics (i) to (iv) described above.

In certain embodiments, the carrier protein consists of KLH.

The percentage of TNF α proteins of interest and the percentage of carrier protein molecules covalently linked to each other in the immunogenic product of the invention can be readily detected by one skilled in the art.

For example, determining the percentage of TNF α molecules covalently linked to a carrier protein molecule in an immunogenic product of the invention can be accomplished by:

(i) submitting the immunogenic product in solution to denaturing and reducing conditions;

(ii) (iii) performing size exclusion chromatography with the product obtained at the end of step (ii) during which a plurality of protein components of progressively decreasing molecular weight are successively eluted from the size exclusion chromatography carrier;

(iii) determining the number of TNF α molecules covalently linked to the carrier molecule in the fraction of the eluate comprising the highest molecular weight protein component;

(iv) (iv) comparing the amount of TNF α measured in step (iii) with the total amount of TNF α initially contained in the starting immunogenic product.

In step (i) of the method for determining the percentage of covalent bonds described above, an amount (in moles or weight) of the immunogenic product of the invention is incubated under denaturing and reducing conditions, resulting in the dissociation of weak bonds between the various protein components which are not linked to each other by covalent bonds.

In the preferred denaturing conditions urea is present, e.g., at a final concentration of 8M; or SDS may be present, for example, at a final concentration of 1% by weight of the total solution containing the immunogenic product. In preferred reducing conditions beta-mercaptoethanol is present, e.g., at a final concentration of 5% of the total volume of the solution containing the immunogenic product.

In step (ii) of the method for determining the percentage of TNF α molecules and carrier protein molecules covalently linked to each other, the person skilled in the art selects a size exclusion chromatography carrier according to his technical general knowledge. For example, Superdex 75 from Pharmacia may be used by those skilled in the art^TM、Superdex 200^TMAnd Superdex 400^TMCommercially available chromatographic supports are available under the trademark Perkin Elmer (R).

In step (ii), the fraction of molecules corresponding to the carrier molecules covalently linked to the TNF α molecules is first eluted before the fraction of the eluate containing the antigen of interest in free form. The amount of TNF α eluted in free form corresponds to the fraction of the antigen of interest that is non-covalently linked to the carrier molecule in the starting immunogen product. The amount of TNF α covalently linked to the carrier protein molecule is determined in the high molecular weight protein fraction, for example, in an immunoenzyme assay, in a radioimmunoassay or in an immunofluorescence assay, directly or indirectly ("sandwich method"), using an antibody specific for TNF α and which does not have any immunological cross-reactivity with the carrier protein molecule.

In step (iii), the amount of TNF α covalently linked to the carrier protein molecule, determined as described above, is compared with the initial amount of TNF α contained in a given amount (in moles or weight) of the starting immunological product, and from this the percentage of TNF α, which is covalently linked to the carrier protein molecule in the immunogenic product of the invention, is calculated.

The percentage of carrier protein molecules and the percentage of TNF α covalently linked to each other in the immunogenic product of the invention can be readily detected by one skilled in the art using a less preferred method comprising the following steps.

a) Immobilization on a support of specific antibodies against the carrier protein;

b) contacting an antibody directed against the carrier protein, immobilized on the support in step a), with a known molecular mass of an immunogenic product to be tested comprising the carrier protein and TNF α;

c) removing the molecules of the immunogenic product not linked to the immobilized anti-carrier protein antibody in step a) by means of a buffer solution comprising one or more protein denaturants.

d) d1) contacting (i) the immunogenic complex formed in step c) between the immobilized anti-carrier protein antibody and the molecule of the immunogen product with (ii) a specific anti-carrier protein antibody;

d2) separately from step d1), contacting the immunogenic complexes formed in step c) between the immobilized anti-carrier protein antibodies and the molecules of the immunogenic product with (ii) specific anti-TNF α antibodies;

e) e1) quantifying the antibody added in step d1) that has been linked to the carrier protein;

e2) quantifying the antibody that has been linked to TNF α added in step d 2);

f) the ratio between the following two is calculated:

(i) the amount of bound anti-carrier protein antibody determined in step e 1); and

(ii) the amount of bound anti-TNF α antibody determined in step e 2).

This ratio is the ratio of carrier protein molecules and TNF α molecules covalently bound to each other in the starting immunogenic product.

In step c) of the above method, the use of an aqueous elution solution comprising one or more protein denaturants causes the denaturation of the immunogen product bound to the anti-carrier protein antibody, so that TNF α molecules which are not covalently bound to the carrier protein molecules are released into the elution solution. Thus, in step d2) of the method, only the TNF α molecules covalently bound to the carrier protein are quantified.

The denaturing buffer used in step c) preferably contains a surfactant, such as Tween ® 20, at a final concentration of 0.1% v/v.

In steps d1) and d2), the amount of bound antibody is preferably determined by incubating the antigen-antibody complex formed at the end of each of the above steps with a detectable molecular marker of the new antibody, respectively.

(i) In step d1), the novel antibody is directed against an anti-carrier protein antibody and labeled with a detectable molecule;

(ii) in step d2), the novel antibody is directed against an anti-TNF α antibody and labeled with a detectable molecule.

Radioactive molecules, fluorescent molecules or enzymes, these detectable molecules being indiscriminate. As an enzyme, peroxidase may be more commonly used, and its presence is shown by colorimetry after incubation with an o-Phenylenediamine (POD) substrate.

The details of the above mentioned process are described in the examples.

By way of illustration, it has been shown, according to the invention, that the above-mentioned first or second quantitative method is applied: less than 40% of the TNF α molecules in the immunogenic product comprising heterocomplexes between the KLH carrier molecule and the human TNF α molecules are covalently bound to the KLH carrier protein molecules.

To prepare the immunogenic or vaccine compositions of the invention, the stabilized immunogenic product obtained according to the process of the invention is adjusted to the appropriate concentration, optionally mixed with suitable vaccine adjuvants and packaged for use.

The present invention also relates to immunogenic compositions comprising (i) a stabilized immunogen product comprising TNF α and an antigenic heterocomplex of a carrier protein prepared by the methods disclosed herein, or (ii) a stabilized immunogen product comprising TNF α and an antigenic heterocomplex of the above-described carrier protein in combination with one or more pharmaceutically acceptable excipients.

The invention also relates to vaccine compositions comprising (i) a stable immunogenic product comprising TNF α and antigenic heterocomplexes of a carrier protein prepared by the methods disclosed herein, or (ii) a stable immunogenic product comprising TNF α and antigenic heterocomplexes of the above carrier protein in combination with one or more immunological adjuvants.

As used herein, the term "adjuvant" is intended to be used in its ordinary sense, i.e., any substance mixed therewith that enhances an immune response to an antigen. Adjuvants useful in the present invention include, but are not intended to be limiting, Freund's, mineral gums such as aluminum hydroxide and surface active substances such as lysolecithin, polyether polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacille Calmette-Guerin) and Corynebacterium parvum are potentially useful adjuvants.

Any adjuvant known in the art may be used in the above vaccine compositions, including oily adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial Lipopolysaccharides (LPS), peptidoglycans (e.g., murein, mucopeptide or glycoproteins such as N-Opaca, muramyl dipeptide [ MDP ], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), Sibirans (e.g., 01K2), immunostimulatory complexes of EP 109942, EP180564, and EP231039, aluminium hydroxide, saponin, DEAE-dextran, neutral oils (e.g. miglyol), vegetable oils (e.g. arachid oil), liposomes, pluronic.rtm. polyols, Ribi adjuvant systems (see, e.g. GB-a-2189141), or interleukins, particularly those which stimulate cell-mediated immunity. An alternative adjuvant consists of an extract of Amycolata of the order Actinomycetales of the genus Bacteria, which has been described in U.S. Pat. No. 4,877,612. Furthermore, all adjuvant mixtures are commercially available. The adjuvant used depends to some extent on the recipient organism. The amount of adjuvant administered will depend on the type and size of the animal. The optimum dosage can be readily determined by conventional means.

Suitable adjuvants include, but are not limited to, surfactants such as hexadecylamine (hexadecylamine), octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N-dioctadecyl-N' -N-methylidene (2-hydroxyethyl-propanediamine), methoxyhexadecyl-glycerol, and pluronic polyol; polanions, such as pyrans, dextran sulfate, multimeric immune complexes, polyacrylic acid, carboxyvinyl polymers, peptides, such as muramyl dipeptide, MPL, aimethylglycine, phagocytic stimulatory peptides, oil emulsions, alum, and mixtures thereof. Other potential adjuvants include the B peptide subunit of e.coli (e.coli), the heat labile toxin of cholera toxin. McGhee, j.r., et al, "On vaccatendevelop," seq.hematol, 30: 3-15(1993).

The adjuvant properties of saponins have long been known because of their ability to increase antibody titers against immunogens. As used herein, the term "saponin" refers to a group of plant-derived surface-active glycosides, consisting of a hydrophilic region (usually several sugar chains) linked to a hydrophobic region of a steroid or triterpene structure. While saponins are available from different sources, saponins with useful adjuvant activity have been obtained from Quillaja saponaria (Molina). Saponins from these sources are used to isolate a "homogeneous" fraction denoted "Quil a" (Dalsgaard, k., (1974), arch. gesamtevirusforsch.44: 243).

A major concern for veterinary and human use of Quil a in vaccine formulations is dose-site reactivity. One way to avoid such toxicity of Quil a is to use an immune stimulating complex (well known as iscom. tm., abbreviation for immune stimulating complex). This is mainly because Quil a is less reactive when incorporated into an immunostimulatory complex, reducing its cell lysis effects by association with cholesterol in the complex, reducing its binding to cholesterol in the cell membrane. In addition, a lower amount of Quil a is required to produce similar levels of adjuvant effect.

The immunomodulatory properties of Quil a saponins and the additional benefits derived from these saponins when incorporated into immunostimulatory complexes have been described in various publications, such as Cox and Cox, j.c. and Coulter, a.r. advances in advanced Technology and application Animal park Control Biotechnology, chapter 4, Yong, edit w.k., CRC Press (1992); cox, j.c. and Coulter, A.R. (1997) Vaccine, 15 (3): 248-256; cox, j.c. and Coulter, A.R. (1999) BioDrugs 12 (6): 439 — 453); dalsgaard, (1974) (supra); morein et al (1989) "Immunostilatingcomplete (ISCOM)", In "Vaccines: recent Trends and Progress ″.g.gregoriadis, a.c.allison and g.poster (editors). plenum Press, New York, p.153; australian Patent Specifications Nos.558258, 589915, 590904 and 632067.

Classical ISCOMs, which are immunostimulatory complexes, are formed by the combination of cholesterol, saponins, phospholipids and immunogens, such as viral packaging proteins. An immunostimulatory complex matrix composition (called iscoma trix. tm.) was formed identically, but without the viral proteins. The immunostimulatory complex appears to stimulate both humoral and cellular immune responses. Immunostimulatory complexes have been made with proteins of various viral origins, including HSV-1, CMV, EBV, Hepatitis B Virus (HBV), rabies and influenza. See, e.g., i.g. barr et al adv. drug Delivery Reviews, 32: 247-271(1998). It has been observed that naked DNA or polypeptides derived from infectious agents are poorly immunogenic when administered by themselves and that inclusion within immunostimulatory complexes has enhanced their immunogenicity. A number of proteins formulated with immunostimulatory complexes have been shown to induce CTL, primarily in rodent models. Ber-zofsky (1991), Biotechnol. Ther.2: 123-135; hsu et al, (1996), Vaccine 14: 1159-1166; lipford et al, (1994), Vaccine 12: 73-80; mowat et al, (1991), Immunology 72: 317 and 322; osterhaus et al, (1998), Dev.biol.Stand.92: 49-58; rimmelzwaan et al, (1997), J.Gen.Virol.78 (pt.4): 757 and 765; sambhara et al, (1998), j.infect.dis.177: 1266-; sambhara et al, (1997), mech. aging dev.96: 157-169; sjolander et al, (1997), Vaccine 15: 1030-; sjolander et al, (1998), J.Leukoc.biol.64: 713-723; takahashi et al, (1990), Nature 344: 873-875; tarpey et al, (1996), Vaccine 14: 230-; trudel et al, (1987), Vaccine 10: 107-112; verschoor et al, (1999), J.Virol.73: 3292-; Villacres-Eriksson (1995), clin. exp. immunol.102: 46-52; zugel et al (1995), eur.j.immunoll.25: 451-458.

It is generally believed that the combination of the stable immunogenic product obtained by the method of the invention and the adjuvant is important for inducing an optimal immune response. A number of studies have confirmed this hypothesis, including studies with virosomes and iscoms. (Ennis, F.A., Crux, J., Jameson, J., Klein, M., Burt, D., and Thippawong, J.1999.virology 259: 256. 261. nurbriggen, R., Novak-Hofer, I., Seelig, A. and Gluck, R. (2000), Progress in lipid Research 39: 3-18. Voeten, J.T.M., Nieuwkoop, N.J., Lovgren-Bengtsson, K., Osteraus, D.M.E., and Rimmelzwan, G.F.2000. Eueuro J Imm (Submitt-ed)). Usually the binding between iscom.tm. and antigen has been successful by incorporating the ampholytic antigen into the iscom.tm. structure during its formation. (Morein, B., B.Sundquist, S.Hoglund, K.Dalsgaard, and A.Osterhaus.1984.Nature 308: 457). This incorporation is accomplished by hydrophobic interactions. In contrast, a recent approach has been to use chelation and electrostatic interactions to bind antigens to preformed protein-free immunostimulatory complexes (ISCOMATRIX. TM.) (International patent application Nos. PCT/AU98/00080-WO 98/36772 and PCT/AU 00/00110).

In certain embodiments of the vaccine composition according to the invention, the vaccine composition comprises, e.g., a pharmaceutical excipient, one or more charged inorganic carriers. Examples of charged organic carriers, which are adjuvants suitable for use in the present invention, include, but are not limited to, saponins, saponin complexes, any one or more components of an immunostimulatory complex of saponins, cholesterol and lipids (e.g., a saponin component and/or a phospholipid component), liposomes, or oil-in-water emulsions, known as iscomatrix. (the components and preparation of ISCOMATRIX. TM. are described in detail in International patent application No. PCT/SE86/00480, Australian patent Nos.558258 and632067, and European patent publication No. 0180564, the disclosures of which are incorporated herein by reference). Additional adjuvant examples include, but are not limited to, those detailed in publications by Cox and Coulter, 1992, 1997 and 1999. It is understood that the organic vector being tested may be naturally occurring or may be synthetic or recombinantly derived.

The vaccine composition may also include additional adjuvants to enhance the effectiveness of the composition. Suitable adjuvants include, but are not limited to: (1) aluminum salts (aluminum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations (with or without other specific immunostimulants such as muramyl peptides (see below) or bacterial cell wall components), such as (a) MF59(PCT publication No. WO 90/14837) comprising 5% squalene, 0.5% tween 80 and 0.5% span 85 (optionally comprising varying amounts of the muramyl tripeptide phosphatidylethanolamine (see below), although not necessarily) formulated as submicron particles, (b) safhe comprising 10% squalene, 0.4% tween 80, 5% pluronic blocking polymer and thr-MDP (see below), or microfluidized into submicron emulsion or vortexed to produce large-particle emulsion, and (c) a rubimil. tm. adjuvant system (riras immunom, hatton, Mont.) comprising 2% squalene, 0.2% tween 80 and one or more components of bacterial cell wall, it is derived from the group consisting of monophosphoryl lipid a (MPL), Trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS), preferably MPL + CWS (detox. tm.); (3) saponin adjuvants, such as Stimuon. TM. (Cambridge Bioscience, Worcester, Mass.); (4) complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), and the like; (6) detoxified mutants of bacterial ADP-ribosylating toxins such as Cholera Toxin (CT), Pertussis Toxin (PT) or E.coli (E.coli) thermolabile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PT-K9/G129; see, e.g., WO93/13302 and WO 92/19265; (7) other substances that act as immunostimulants to enhance the efficacy of the composition; and (8) microparticles having adsorbed macromolecules as described in International patent application No. PCT/US 99/17308. Aluminum and MF59 are preferred.

As described above, suitable muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 '-2' -dipalmitoyl-s-N-glycero-3-hydroxyphosphoryloxy) -aminoethane (MTP-PE), and the like.

Additional adjuvants for inducing a systemic response of mucosal or anti-immunogenic compounds according to the invention may be those described in Vogel et al (Vogel F.R., Powell M.F. and Alving C.R., A complex of vaccine additions and amplifications; 2 nd edition; Vogel F.R. and Powell MF, 1995; A summary complex of vaccine additions and amplifications. in Powell MF, edited by Newman MJ,. Vaccidin: the vaccine and antigen approach.N. New York: environmental publication, 1995: Ple 228))).

In general, adjuvants that may be included in vaccine compositions according to the present invention include, but are not limited to:

(i) gel-type adjuvants, such as aluminum hydroxide or aluminum phosphate (Glenny AT et al, 1926; JPathol Bacteriol; Vol 29: 38-45; Gupta RK and Siber GR, 1994; Biologicals; Vol 22: 53-63);

(ii) adjuvants for microorganisms, for example:

DNA CpG motifs (Chu RS et al 199; J Exp Med; volume 186: 1623-; monophosphoryl lipid A (Schnerson R et al, 1991; J Immunol; Vol. 147: 2136-2140); cholera toxin (Holmgren J et al, 1993; Vaccine; Vol. 11: 1179-1184; Okahashi N et al, 1996; infection Immun; Vol. 64: 1516-1525);

escherichia coli heat-labile toxins (Lycke N et al, 1992; Eur J Immunol; Vol.22: 2277-;

pertussis toxin (Roberts M et al, 1995; infection Immun; volume 63: 2100-;

muramyl dipeptide (Ellouz F et al; 1974; Biochem Biophys Res Commun; Vol 59: 1317-; cohen LY et al, 1996; cell Immunol; volume 169: 75-84);

(iii) oil emulsions and emulsifier-based adjuvants, such as:

freund's incomplete adjuvant (Dhiman N et al, 1997; Med Microbiol Immunol (Berlin); Vol.186: 45-51; Putkonen P et al, 1994; J Med Primatol; Vol.23: 89-94);

MF59(Dupuis M et al, 1998; Cell Immunol; Vol.186: 18-27; KahnJO et al, 1994; J infection Dis; Vol.170: 1288-1291; Ott G et al, 1995; vaccine. Vol.13: 1557-1562);

SAF (Allison AC; 1998; Dev Biol Stand; Vol.92: 3-11; Gupta RK et al, 1993; Vaccine; Vol.11: 293-;

(iv) particulate adjuvants, for example:

immune Stimulating Complexes (ISCOMs) (Putkonen P et al, 1994; J Med Primatol; Vol.23: 89-94; Gupta RK et al, 1993; Vaccine; Vol.11: 293-;

liposomes (Richards RL et al, 1998; infection Immun; Vol.66: 2859-;

biodegradable microspheres (Men Y et al, 1996; Vaccine; Vol.14: 1442-;

saponins (QS-21) (Newman MJ et al, 1992; J Immunol; Vol.148: 2357-;

(v) synthetic adjuvants, for example:

nonionic blocking copolymers (Hunter RL et al, 1994; AIDS Res hum retroviruses; Vol.10(Supl 2); S95-S98; Newman MJ et al, 1997; MechAgeing dev; Vol.93: 189-);

muramyl peptide analogs (Cohen LY et al, 1996; Cell Immunol; Vol.169: 75-84; Fast DJ and Vosika GJ, 1997; Vaccine; Vol.15: 1748-;

polyphosphazenes (Payne LG et al, 1998; Vaccine; Vol.16: 92-98);

synthetic polynucleotides (Johnson AG, 1994; Clin Microbiol Rev; Vol.7: 277-289; Harrington DG et al, 1979; infection Immun; Vol.24: 160-166); and

(vi) cytokines, for example:

immunoreactive fibronectin-gamma (IFN-. gamma.) (Odean MJ et al, 1990; InfectImmun; Vol.58: 427-432);

interleukin-2 (Nunberg J et al, 1989; Proc Natl Acad Sci USA; Vol.86: 4240-;

interleukin-12 (Luis C et al, 1994; Science; Vol.263: 235-237; Bliss J et al, 1996; J Immunol; Vol.156: 887-894; Jankovic D et al, 1997, J Immunol; Vol.159: 2409-2417).

Another aspect of the invention therefore relates to the use of a stable immunogenic product of the vaccine composition as defined above for inducing an immune response, including a humoral immune response, in a mammal, wherein the antibody neutralizes immunosuppressive, apoptotic or pro-angiogenic properties of the native cytokine.

Another object of the invention consists of a method for inducing the production of antibodies that neutralize the activity of native TNF α in a mammal, comprising the steps of administering to the mammal (i) a vaccine composition as disclosed above or (ii) a stable immunogenic product comprising antigenic heterocomplexes of TNF α and a carrier protein as described above together with one or more immunological adjuvants.

The vaccine composition may optionally include a liquid, semi-solid or solid vaccine acceptable (i.e., sterile and non-toxic) diluent for use as a pharmaceutical vehicle, excipient or vehicle. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl and propyl hydroxybenzoates, talc, alginates, starch, lactose, sucrose, dextrose, sorbitol, mannitol, acacia, calcium phosphate, mineral oil, cocoa butter, and cocoa butter.

The vaccine composition according to the present invention may further comprise one or more pharmaceutically acceptable carriers and/or diluents, such carriers including any carrier which does not itself induce an adverse response to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamines, amino acid copolymers, lipid aggregates (e.g. oil droplets or liposomes) and inactivated virus particles. Such vectors are well known to those of ordinary skill in the art.

The vaccine composition according to the present invention may typically further comprise a diluent such as water, salt, glycerol, ethanol and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in the composition.

Suitable formulations of the vaccines of the present invention include injectable liquid solutions or injectable suspensions. Solid forms suitable for dissolution or suspension in a liquid pharmaceutically acceptable carrier prior to injection may also be prepared. The vaccine formulation may be emulsified. Additional substances that may be included in the products useful in the present methods include, but are not limited to, one or more preservatives, such as disodium or tetrasodium salts of ethylenediaminetetraacetic acid (EDTA), thimerosal, and the like.

The vaccine composition optionally includes a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semi-solid, or solid diluent suitable for use as a pharmaceutical vehicle, excipient, or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl and propyl hydroxybenzoates, talc, alginates, starch, lactose, sucrose, dextrose, sorbitol, mannitol, acacia, calcium phosphate, mineral oil, cocoa butter, and cocoa butter.

The vaccine composition may be packaged in a form that facilitates delivery. The compositions may be packaged in capsules, caplets, sachets, cachets, gelatin, paper or other containers. These delivery forms are preferred when compatible with the immunogenic composition entering the recipient organism, and, in particular, when the immunogenic composition is delivered in a unit dose. The dosage units may be packaged, for example, in tablets, capsules, suppositories, or cachets.

Forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. They must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of microbial activity can be achieved by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many instances, it will be preferable to include isotonic agents, for example, sucrose or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of pharmaceutical compositions which delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the necessary other ingredients from those enumerated above. As to sterile powders for the preparation of sterile injectable solvents, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When the active immunogenic compound according to the invention should be protected, it may be administered orally, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the diet. For oral therapeutic administration, the active immunogenic compounds according to the invention may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such vaccine compositions and formulations should contain at least 1% by weight of active immunogenic compounds. The percentages of the composition and formulation may of course vary and may conveniently be between about 5% to about 80% of the weight of the unit. In such therapeutically useful vaccine compositions, the amount of active immunogenic compound is the appropriate dose obtained. Preferred vaccine compositions or formulations are prepared according to the invention so that the unit form contains between about 0.1g and 2000mg of the active immunogenic compound.

Vaccine compositions suitable for oral administration according to the invention may also be prepared in the form of liquid solutions, including liquid aerosol formulations.

Liquid aerosol formulations contain an immunogenic product and a dispersing agent in a physiologically acceptable diluent. The dry powder aerosol formulations of the present invention consist of finely divided immunogenic product in solid form and a dispersing agent. Using liquid or dry aerosol formulations, the formulation must be aerosolized. That is, it must be broken down into liquid or solid particles to ensure that the aerosolized dose actually reaches the mucosa of the nasal passages or lungs. The term "aerosol particles" is used herein to describe particles suitable for nasal or pulmonary administration, i.e. liquid or solid that can reach the mucosa. Other considerations, such as the construction of the delivery device, additional ingredients in the formulation, and the nature of the particles are important. These aspects of pulmonary drug delivery are well known in the art, and formulation procedures, aerosolization methods, and construction of delivery devices require, at most, routine experimentation by one of ordinary skill in the art. In a specific embodiment, the mass median kinetic (the mass median kinetic) parameter will be 5 microns or less in order to ensure that the drug particles reach the alveoli [ week, l.l., crit.rev.in the ther.drug Carrier Systems 8: 333(1991)].

Aerosol delivery systems, such as metered dose inhalers and dry powder inhalers, are disclosed in Newman, s.p. (Aerosols and the Lung, Clarke, s.w. and Davia, d. eds., pages 197-22) and may be used in conjunction with the present invention.

The vaccine composition according to the present invention may be administered to a subject to be immunized by any conventional method, including, for example, by oral, intravenous, intradermal, intramuscular, intramammary, intraperitoneal, or subcutaneous injection; by oral, transdermal, sublingual, intranasal, anal or vaginal delivery. The treatment may consist of a single dose or multiple doses administered over a period of time.

The invention is further illustrated by the following figures and examples.

Drawings

Figure 1 consists of a scheme illustrating the general procedure for making a stable immunogenic product according to the invention.

Figure 2 illustrates the humoral response in mice immunized with (a) KLH alone or (B) the stable immunogenic product made as disclosed in example 1. The abscissa: (ii) an individual; ordinate: IgG anti-TNF α antibody titers.

FIG. 3 illustrates the anti-TNF α neutralizing activity of serum antibodies generated by immunizing mice with (A) KLH alone or (B) a stable immunogen product made as disclosed in example 1. Each curve corresponds to serum from one mouse. The abscissa: percent neutralization of TNF α activity; ordinate: serum dilution.

FIG. 4 illustrates the humoral response of rhesus monkeys immunized with (A) KLH alone or (B) 20 μ g of the stabilized immunogenic product manufactured as disclosed in example 1, and (C) 80 μ g of the stabilized immunogenic product manufactured as disclosed in example 1. The abscissa: days after the first injection; ordinate: IgG anti-TNF α antibody titer.

FIG. 5 illustrates the anti-TNF α neutralizing activity of serum antibodies raised against rhesus monkeys immunized with (A) KLH alone or (B) 20 μ g of the stabilized immunogenic product manufactured as disclosed in example 1, and (C) 80 μ g of the stabilized immunogenic product manufactured as disclosed in example 1. The abscissa: percent neutralization of TNF α activity; ordinate: serum dilution.

Figure 6 illustrates the humoral response of transgenic huTNF α B6.sjl-tg (tnf) N2 mice immunized with (a) control PBS buffer, (B) KLH alone, (C) the stabilized immunogenic product manufactured as disclosed in example 1 and (D) the stabilized immunogenic product manufactured as disclosed in example 1 in combination with methotrexate. The abscissa: (ii) an individual; ordinate: IgG anti-TNF antibody titers.

FIG. 7 illustrates the clinical assessment of arthritis in transgenic huTNF α B6.SJL-Tg (TNF) N2 mice injected with

Sodium chloride [. diamond-solid ];

-KLH alone [ ■ ];

stable immunogenic product [. tangle-solidup ] manufactured as disclosed in example 1; and

stable immunogenic product in combination with methotrexate [ ■ ] produced as described in example 1

The abscissa: the number of days after the first injection; ordinate: clinical assessment of arthritis.

FIG. 8 illustrates the properties of hTNF α -Kinoid.

FIG. 8-a) 1: RPN800 molecular weight standard reference, 2: KLH (0.8. mu.g), 3: hTNF α (0.8 μ g), 4: mu.g of Kinoid, loaded on a 12% SDS-polyacrylamide gel. Visualization was by silver staining or western blotting.

FIG. 8-b) autoradiogram: 1: a molecular weight standard reference; 2: 125I-hTNF alpha; 3: kinoid labeled on hTNF α; 4: kinoid labeled on KLH; 5: 125I-KLH.

FIG. 8-c) Kinoid (50. mu.g) was filtered on a superose 6 column (10X 300). Eluted with 0.2ml DBPS. The column was first calibrated with a series of molecular weight standard references.

FIG. 8-d) comparative evaluation of TNF α (. diamond-solid.) and kinoid (. diamond-diamond.) biological activities in L929 cells. e) The binding of TNF α to receptors I (. diamond.) and II (■) was compared, as was the binding of kinoid to receptors I (. diamond.) and II (□).

FIG. 9 illustrates the antibody response in TTg and Balb/c mice.

FIG. 9-a) serum anti-TNF α antibody titers in kinoid, inactivated TNF α (iTNF α), or KLH-immunized TTg, compared to Balb/c mice.

FIGS. 9-b) to 9-e): evaluated in kinoid (. diamond.) and KLH (. diamond-solid.) immunized mice

FIG. 9-b) neutralizing power according to serum dilution,

FIG. 9-c) serum neutralization capacity as a function of time,

FIG. 9-d) inhibition of receptor I binding by serum hTNF α,

FIG. 9-e) IgG neutralizing ability,

FIG. 9-f) inhibition of TNF α receptor I binding by IgG.

1-immunization protocol: 2 intramuscular injections, 20 μ g at 3 week intervals and booster immunizations (5 μ g at 6 weeks); 2: 2 intramuscular injections, 10. mu.g and 5. mu.g booster.

FIG. 10 shows cell-mediated immunity of mouse splenocytes to hTNF α.

Splenocytes purified from KLH (n-3) (. diamond-solid.) and kinoid (n-2) (. diamond-solid.) immunized mice. Balb/c and TTg mice were immunized on days 1, 8, and 29. Boosts were performed on day 90 and spleens were sacrificed (day 120). Splenocytes were stimulated in vitro with a) hTNF α, b) murine TNF α (mutTNF α) and c) KLH antigen for 96 hours. Specific cell proliferation was determined by 3H-thymidine incorporation and expressed as the stimulation index (FIG. 10-A) or measurement of IL2 (FIG. 10-B) and IFN- γ (FIG. 10-C) products in the culture supernatant after 44 hours and 72 hours, respectively (see materials and methods).

FIG. 11 shows that vaccination with hTNF α kinoid improves clinical arthritis in huTNF α transgenic mice.

Seven-week-old mice were immunized with KLH-TNF α (open loop) (n ═ 8) or KLH (closed loop) (control, n ═ 7) on days 1, 7, and 28. Differences between any of the parameter groups were statistically significant:

FIG. 11-a): the progress of weight gain began from the start of the test. P < 0.05 (analysis of variance);

FIG. 11-b): the number of affected limbs. P < 0.0001 (analysis of variance);

FIG. 11-c): arthritis clinical score. P < 0.0001 (analysis of variance);

FIG. 11-d): the daily prevalence (percentage) of arthritis showed a reversal of the disease in the vaccinated groups (P < 0.01, ANOVA).

This experiment was repeated twice with similar results.

Examples

Examples 1 to 7

Example 1: a method of making a stable immunogenic product according to the invention.

A. Material

Table 1: compounds and reagents

Reagent	Grade	Suppliers of goods	Details of	Applications of
					Glutaraldehyde 25%	Class 1	Sigma	G5882 10×1mL	PD&GMP
Formaldehyde 37%	-	Sigma	F-1635 25mL	PD
					Formaldehyde 37%	EP	Sigma	15513	GMP
Disodium hydrogen phosphate (Anhydrous)	Analar	BDH	10249	PD
					Disodium hydrogen phosphate (Anhydrous)	USP	Merck	1.06585 5kg	GMP
Ethylenediaminetetraacetic acid (EDTA)	Analar	BDH	10093	PD
					Ethylenediaminetetraacetic acid (EDTA)	USP	Merck	108421 1kg	GMP
Glycine	Analar	BDH	10119	PD
					Glycine	USP	Merck	500190 1kg	GMP
Sodium chloride	Analar	BDH	10241	PD
					Sodium chloride	BP	Merck	116224	GMP
Dimethyl sulfoxide (DMSO)	99.5％	Sigma	D5879	PD
					Dimethyl sulfoxide (DMSO)	USP	Sigma	D2438 10mL	GMP
Dulbecco's Phosphate Buffer (DPBS)	-	Sigma	D8537	PD
					Dulbecco's Phosphate Buffer (DPBS)	tbc	tbc	tbc	GMP

Table 2: consumable product

Description of the invention	Suppliers of goods	Applications of
			Slide-a-Lyzer ® expression cassette 7kDa MWCO (0.5-3mL)	Perbio；66370	Dialysis
Pellicon XL PES Membrane (5 or 10kDa MWCO)	Micropores; PXB010A50	Tangential flow filtration
			50mL test tube	Nunc；362696	Sample container
Polypropylene wide-mouth bottle (150mL)	VWR；215-0520	Sample container
			Polypropylene wide-mouth bottle (250mL)	VWR；215-5683	Sample container
Freezing pipe (3.5 mL)& 2mL)	Sigma；V1138；V9637	Sample container
			Laboratory-sized TFF unit with 500mL acrylic resin container	Micropores; XX42LSS13	Tangential flow filtration

B. Detailed description of the method

The procedures disclosed in this example illustrate embodiments of the method of the invention for preparing a stable immunogenic product comprising antigenic heterocomplexes of TNF and KLH.

In addition, the process detailed below was designed to produce a batch of 120mg of stable immunogen product.

Step a)

30.05ml of TNF α (concentration 4.2mg/ml) were taken and allowed to thaw overnight at 4 ℃ (note: 126.21mg of TNF α were required).

Step b) (or step b1) in certain embodiments of the method)

90.15ml of "dilution buffer" were added to obtain a "working buffer" solution with TNF α of 1mg/ml (. + -. 10%).

130mM disodium hydrogen phosphate, 133mM NaCl, 6.6mM EDTA, ph7.8, dilution buffer

Working buffer 100mM disodium hydrogen phosphate, 150mM NaCl, 5mM EDTA, ph7.8

Step b2)

To the remaining 120ml of 1mg/ml TNF α (+ -10%) 1.2ml DMSO was added. The mixture was left at room temperature for 30 minutes. Mix well every 10 minutes and try to avoid foaming.

Add 61.34ml of "working buffer" to the mixture. Mix gently by inverting the container and try to avoid foaming.

Step c)

Addition of 51.6mg of KLH. Note that: 5.26ml of KLH solution at 9.81mg/ml were added. Mix gently by inverting the container. The total volume was 187.8ml at this time.

Step d)

Dilute 25% glutaraldehyde stock to 2.5% with "working buffer". It is finished immediately before use.

20.86ml of a diluted 2.5% glutaraldehyde solution are added. Note that the total volume at this point was 208.56 ml. Mix gently by inverting the container.

The bottles were closed and incubated for 45 minutes at room temperature. The bottle was gently inverted every 15 minutes.

Step e)The solution was diafiltered against the working buffer using Tangential Flow Filtration (TFF).

Dialysis 3 times against 20 volumes of pH7.6 in 10mM phosphate buffer containing 150mM NaCl.

Dialysis for 2 hours at volume 1

Dialysis for 2 hours at volume 2

Volume 3 ═ dialysis overnight

Step f)

The formaldehyde stock solution (37%) was diluted 10-fold with "working buffer" to obtain a 3.7% solution. It is finished immediately before use.

-adding diluted 3.7% formaldehyde to the TNF α -KLH solution to obtain a final concentration of 0.2%.

For example, if the volume recovered is 200ml, then the amount of 3.7% formaldehyde added will be 11.43 ml.

The bottles were closed and incubated at 37 ℃ for 6 days. The bottle was inverted once a day.

Step g)

After-6 days, 2M glycine (formulated in WFI) was added to a final concentration of 0.1M. Incubate at room temperature for 1 hour, during which the bottle is gently inverted.

Step h)

Check the pH of dpbs (sigma) for diafiltration and, if necessary, adjust to a final pH of 7.3 ± 0.2 with 0.1M NaOH. No pH adjustment should generally be required.

Diafiltration of the solution against DPBS using Tangential Flow Filtration (TFF).

A2X 5kDa MWCO TFF membrane was used.

A500 ml (acrylic resin) container on a Labscale TFF system was used.

The starting material was recovered from the membrane by draining the concentrate (ultrafiltration retention) to a pre-weighed polypropylene bottle.

Example 2: through the rootExperimental procedures for obtaining Stable immunogenic products according to the methods of the invention

The experimental procedures disclosed in example 2 can be referred to by those skilled in the art. However, one skilled in the art may also perform the tests according to any of the test procedures disclosed in the various embodiments herein.

2.1. Determination of the total protein content by colorimeter: examples of Bradford assays

Protein content was determined using the Bradford technique (Bradford, M.anal.biochem.1976.72, 248-254).

Briefly, a calibration curve was established with Bovine Serum Albumin (BSA) by pipetting 0, 10, 20, 30, 40. mu.l of a 0.2mg/ml BSA solution in PBS into a series of tubes. Subsequently, each tube volume was finally brought to 500. mu.l by adding the corresponding volume of DI water. Then 500. mu.l Bradford reagent was added to each tube. Two blanks were made with 200. mu.l PBS. After 5 minutes at room temperature, the contents of each tube were vortexed and read at 595 nm.

200 μ l of an appropriately diluted solution of the protein to be tested was reacted with Bradford reagent as described above.

TNF α protein content in KLH-TNF α: ELISA (enzyme Linked immunosorbent assay)

Samples of 4 KLH-TNF α taken from 4 vials were diluted to 5 μ g/ml in Phosphate Buffered Saline (PBS) at pH 7.2. These dilutions were used to coat microtiter plates (Costar 3590) at 100. mu.l per well. After allowing to coat at 4 ℃ overnight, the plate was washed with PBS containing 0.1% Tween 20, and the wells were saturated with 2% Fetal Calf Serum (FCS) solution (in 100. mu.l PBS-Tween) for 2 hours at 37 ℃. After washing the plate with PBS-tween, 100 μ l of serially diluted anti-TNF α serum was pipetted into each well and the plate was incubated at 37 ℃ for 2 hours, after which it was thoroughly washed clean and incubated again at 37 ℃ for 2 hours after adding 100 μ l of horseradish peroxidase-labeled anti-IgG antiserum to each well.

After incubation, the plates were rinsed and 100 μ l of an o-phenylenediamine (OPD) solution was added to each well. After 3 minutes, 100. mu.l of 2N sulfuric acid was added to each well and the plate was read at 490nm using a multiscan spectrophotometer.

Purity grade of KLH-TNF α immunogen: isoelectric focusing (IEF) + Western blot

The purity level of the immunogen was assessed by migration of KLH-TNF α immunogen alongside KLH alone and TNF α alone by isoelectric focusing (IEF) on 1% agarose plates, within a pH gradient of 3 to 10. After each sample was appropriately diluted to 250. mu.g/ml, electrophoresis was performed using a rapid gel electrophoresis system apparatus (Amersham Pharmacia) under the following conditions.

First step (premigration): 500V, 2.5mA, 2.5W, 15 ℃, 5aVh

The second step (application): 200V, 2.5mA, 2.5W, 15 ℃, 5aVh

Third step (migration): 1500V, 2.5mA, 2.5W, 15 ℃, 450aVh

After migration, the proteins were transferred to PVDF membranes by micro-capillary action and visualized by western blotting.

Western blot detection

The nitrocellulose membrane was saturated by soaking in 5% milk-TBS tween 20 overnight at room temperature, after which it was incubated with either the polyclonal anti-TNF α (human) primary antibody or the anti-KLH primary antibody diluted in 10% milk-TBS tween 20 for 1 hour at room temperature. Subsequently, the membrane was washed 4 times with TBS-Tween 20 over 5 minutes, after which it was incubated with horseradish peroxidase-labeled secondary antibody diluted with 10% milk-TBS Tween 20 for 1 hour at room temperature. Again, the membrane was washed 4 times with TBS-Tween 20 and the spots were visualized by chemiluminescence using the ECL enhancement kit (Amersham Pharmacia).

3. Percentage of TNF alpha-KLH covalent bonds in a stabilized immunogenic product

3.1. method 1: size exclusion chromatography under denaturing and reducing conditions

The kinoid solution to be tested was placed under denaturing (urea at 8M final concentration) and reducing (5% final concentration of β -mercaptoethanol) conditions leading to dissociation of the non-covalent bonds. The resulting solution was loaded with Superdex200^TM(Pharmacia) eluted from a size exclusion column, Superdex200 was selected because its 600kDa cut limit was much lower than the molecular weight of KLH and the covalent KLH-TNF α construct. Only those cytokine molecules covalently bound to KLH remain in the exclusion volume. The latter TNF α (specific TNF α antigen) was determined by sandwich elisa using anti-TNF α antibodies that did not cross-immunoresponse with KLH. The results are expressed as a percentage of TNF α titer in the starting solution, as determined by the same enzyme-linked immunosorbent assay. This percentage is equal to% TNF α -KLH covalent bonds in TNF α kinoid.

3.2. method 2: dual-sandwich enzyme-linked immunoassay using tween wash

A microtiter plates (Costar, high binding) were coated with 1mg/ml of anti-KLH polyclonal antibody in PBS (10mM pH7.3, NaCl 150mM) at 100. mu.l per well for 2 hours at 37 ℃. After washing 3 times with PBST (PBS containing 0.1% v/v Tween 20), each well was saturated with PBS containing 2% fetal bovine serum for 1.5 hours. The wells were washed 3 more times with PBST.

Two identical serial dilutions of the kinoid to be tested (10, 5, 0.156. mu.g/ml) were then added to the wells and incubated for 2 hours. The wells were washed 3 times with PBST, which dissociation (via tween 20) cleared all molecules non-covalently bound to KLH, which was immobilized to the capture antibody of the coated support. The first series of test dilutions was incubated with anti-KLH antibody and the second series with anti-TNF α antibody. After incubation at 37 ℃ for 1.5 hours, wells were washed as above and then incubated with a specific peroxidase-conjugated secondary antibody.

Addition of OPD (peroxidase substrate) enables quantitative colorimetric detection of immobilized anti-TNF α antibodies in the first series and immobilized anti-KLH antibodies in the second series. The fixed antibody ratio between the two series gives the percentage of TNF α covalently bound to KLH in kinoid.

Immunological Activity of KLH-TNF alpha KINOID

And (3) testing antigenicity: enzyme-linked immunosorbent assay (ELISA)

The purpose of this test is to determine the binding capacity of an immunogen (or antigen derivative, or antigen conjugated to a carrier protein) to specific antibodies associated with the native antigen. The test is essentially based on reverse ELISA.

A series of dilutions of increasing immunogen in the assay were dispensed into polystyrene microtiter plate wells. A specific polyclonal antibody directed against the protein to be tested is allowed to react with the immunogen immobilized in the well. After the excess unreacted antibody is removed by washing the plate, quantitative display is performed by reacting the antibody immobilized by the antigen in the well with an antibody against the primary antibody labeled with horseradish peroxidase. The yellow color developed by addition of the appropriate substrate is directly proportional to the amount of protein immobilized. The Optical Density (OD) per well was measured with a microplate photometer. And measuring the protein content of the sample to be measured from the calibration curve.

Immunogenicity of KINOID

The immunogenicity of the immunogen preparation is:

1-its level of ability to induce the formation of specific anti-TNF α polyclonal antibodies (in vivo assay); and

-2-its neutralizing capacity. The latter is measured in vitro by quantifying the ability of antisera sampled from immunized mice to inhibit the specific biological activity of TNF α.

-1-for this purpose, 20 groups of 7 week old Balb/c mice weighing 18-20g, housed in a single cage per 5 animals, were fed on standard pellets and without restriction of drinking water and immunized on days 0, 7, 14 and 21 by intramuscular injection of 0.1ml (10. mu.g) of the immunogen to be tested in ISA51 (1: 1v/v emulsion in ISA 51). On day 60, booster immunizations were given by injection of 5 μ g of immunogen (0.1ml, 1: 1 emulsion in ISA 51). Blood samples were taken by retroorbital puncture 2 days before the start of immunization and again 12 days after the last injection for detection of antibody levels by enzyme linked immunosorbent assay. Three control mice received a 1: 1v/v emulsion of PBS in IFA.

Serum antibody titers were expressed as the reciprocal of the dilution that produced OD > 0.3.

6. Immunotoxicity-in vitro

Testing of proliferation of T cells (by 3H-thymidine) treated with various kinoid doses and stimulated by PPD/TT antigen.

Freshly isolated peripheral blood mononuclear cells were obtained from healthy donors by separating blood by ficoll gradient centrifugation. Cells were seeded into round bottom 96-well plates of RPMIc medium containing 10% fetal bovine serum, 15,000 cells per well. Kinoid was added to a concentration of 1. mu.g/ml to 100ng/ml, followed by PPD or TT to 0.16%. Place the plate at 37 ℃ 5% CO₂And (4) incubating for 6 days. Tritiated thymidine was added 18 hours before the end of incubation, 0.5. mu. Ci/well. Cell proliferation was analyzed using a beta scintillation counter.

Biological characterization of KLH-TNF α KINOID

TNF- α bioassay:

cell lysis of murine L929 cells in the Presence of Actinomycin D

Material

-L929 mouse fibroblast cell line (ATCC commercial number CCL-1); KLH-TNF alpha (mouse or human) kinoid in PBS (Standard), NEOVACS

-recombinant murine TNF- α Peprotech (315-01A) or human TNF- α Peprotech (300-01A); medium (RPMI supplemented with 10% fetal bovine serum, 2mM glutamine, 100U/ml penicillin-streptomycin)

Assay medium (RPMI supplemented with 2% fetal bovine serum, 2mM glutamine, 100U/ml penicillin-streptomycin)

-pre-incubation medium: HL1 supplemented with 2mM glutamine, 100U/ml penicillin-streptomycin

-96 well flat bottom plates (Costar, 3595)

Actinomycin D, 1000. mu.g/ml, stored at 4 ℃ (protected from light)

MTT solution (Sigma, M5655), 5mg/ml aliquot stock in PBS, stored at-20 ℃ (protected from light)

-DMSO(SIGMA，471267)

Duration of the test

24 hours incubation

1 hour assay preparation

Method

1. KLH-TNF α mouse or human kinoid and standards were diluted in 50 μ l of assay medium per well of 96-well plates, leaving row 1 as blank, with a series of two-fold dilutions starting at the appropriate dilution, from row 2 to row 11.

2. L929 cell suspensions were prepared at a density of 7.5105/ml in assay medium supplemented with 1. mu.g/ml actinomycin D (protected from light).

3. Placing the plate in a humidified incubator at 37 deg.C and 5% CO₂Incubate for 24 hours.

4. With no Ca content²⁺、Mg²⁺Was rinsed twice with PBS.

5. Add 50. mu.l MTT solution to assay medium to 40% per well and 5% CO at 37 ℃ in humidified incubator₂Incubate for 4 hours.

6. The plates were emptied and 50. mu.l DMSO was added to each well.

7. Reading plate at 550-630nm

8. The data is analyzed.

Examples3：

Comparative study of various KLH-TNF alpha (human) kinoid formulations

Various preparations of human TNF α kinoids consisting of human TNF α complexed with a specific carrier protein KLH have been accomplished.

More precisely, various formulations of KLH-TNF α (human) have been produced by the same general procedure disclosed in example 1 but varying (i) the percentage of DMSO, (ii) the glutaraldehyde concentration (2.5 or 22.5mM) and (iii) the duration of incubation of the intermediate with formaldehyde (duration of incubation time from 0.2 to 6 days).

Then, loss of biological activity of human TNF α has been determined. Furthermore, the preservation of conformational B-epitopes in the final kinoid product has been examined by (i) cytotoxicity assays of human TNF α in the L929 cell line, and (ii) by immunogenicity assays in the present examples published herein.

A.Materials and methods

Determination of the absence of TNF alpha biological Activity in KLH-TNF alpha (human) kinoid

Human TNF α, cytotoxic to murine fibroblasts of the L929 cell line in the presence of actinomycin D, was assessed for cytotoxicity by the cell survival assay using MTT (3- [4, 5-dimethylthiazol-2-yl ] -2, 5-biphenyl-tetrazolium bromide). The assay is based on the reduction of MTT by the mitochondrial NAPDH reductase of living cells to the formazan, a desirable product, which has a bluish-purple color.

In this assay, the metabolic activity of the cell enables the viability to be assessed.

The KLH TNF-alphaThe evaluation of the biological activity consists in a gradual reduction (from 50ng/ml to 0ng/ml) of the quantity of saidL929 cells were incubated in microplate wells in the presence of product mixed with D actinomycin (1. mu.g/ml).

In the presence of 5% CO₂In a humidified atmosphere at 37 ℃ for a period of 24 hours.

After 24 hours of incubation, the cell culture supernatant was discarded and replaced with MTT test solution.

After incubation with MTT for 4 hours at 37 ℃, MTT solution was discarded and DMSO was added.

After mixing well, the optical density of the cell culture supernatant was measured with a spectrophotometer at a wavelength of 570 nm.

The results are expressed as optical density at 570nm (o.d.). In parallel, cell cultures were incubated with TNF α ranging from 0 to 10ng/ml as a control.

The final result is expressed as half the lethal dose, also known as LD₅₀It is the dose that induces 50% lysis of the L929 cultured cells.

A.2 after chemical treatment with glutaraldehyde and formaldehyde, in the final KLH-TNF α (human) Determination of human TNF α conformation B-epitope preservation in kinoid: immunogenicity assay

The immunogenic activity was tested in C57Bl/6 weighing 18-20g and the preservation of the B epitope conformation of human TNF α in the final KLH-TNF α (human) kinoid after chemical treatment was studied.

On day 0, three mice in the same group were injected intramuscularly with 0.2ml (50 μ g) of an emulsion in Complete Freund's Adjuvant (CFA).

On day 21, a second injection was given with 25 μ g of kinoid product in incomplete Freund's adjuvant.

Retro-orbital blood samples were taken from each mouse prior to the first injection and at day 28. Serum was collected from each group of mice.

The humoral response is determined by detecting antibodies of the IgG isotype directed against human-TNF alpha in the sera of the immunized mice. Humoral responses were measured by ELISA and expressed as antibody titers (dilutions)^-1Giving an optical density greater than 0.3).

As described later, the assay for TNF α cytotoxicity on L929 cells has been carried out using KLH-TNF α (human)The neutralizing capacity of the serum was determined in the immunized mice.

L929 cells were treated with various dilutions from serum pools sampled on day-2 and day 28 (dilutions from 1/100 to 1/12800), and then incubated at room temperature for 40 min, followed by 20ng/ml human TNF α for 20 min at 4 ℃.

In the presence of 5% CO₂The cells were cultured in the humidified air at 37 ℃ for further 24 hours.

After 24 hours of cell culture, the cell culture supernatant was discarded and replaced with MTT solution.

After 4 hours incubation at 37 ℃, MTT was discarded and DMSO was added.

After mixing well, the optical density of the cell culture supernatant was measured with a spectrophotometer at a wavelength of 570 nm.

The results are expressed in terms of optical density (o.d.) of the cell culture supernatant. Cell cultures were incubated in parallel with TNF α ranging from 0 to 10ng/ml as a control.

Serum that neutralizes TNF α cytotoxicity avoids its cytotoxicity to L929 cells.

The results are expressed as 50% neutralization capacity or NC₅₀It corresponds to the serum dilution that neutralizes 50% of human TNF α cytotoxicity.

B: results

TABLE 3 Final KLH TNF α (human) prepared with various chemical treatmentsThe results obtained are shown below.

TABLE 3

Code	Glutaraldehyde (mM)	Formaldehyde (Tian)	EDTA	DMSO(％)	ED50(ng/ml)	Antibody titer	NC50
								K1	2.5	2	5mM	0	12.5	＞64000	1/650
K2	2.5	2	5mM	2	20	＞64000	1/600
								K3	22.5	0	5mM	1	0.15	＞64000	1/600
K4	22.5	2	5mM	1	200	＞64000	1/750
								K5	22.5	2	5mM	2	225	＞64000	1/400
K6	22.5	2	5mM	5	250	＞64000	1/700
								K7	22.5	6	5mM	1	＞400	＞64000	1/1200
K8	22.5	6	5mM	2	＞400	＞64000	1/300
								K9	22.5	6	5mM	5	＞400	＞64000	1/350
K10	22.5	2	0mM	0	15	＞64000	1/700

Production of KLH TNF α (human) by chemical treatment of (i) DMSO at a concentration of 1% and (ii) glutaraldehyde at a concentration of 22.5mM, followed by (iii) chemical treatment with formaldehyde for 6 daysThe best results were obtained with the method of (1), as shown in table 3 above. The resulting final KLH-TNF α (human) product detoxifies human TNF α and induces the production of polyclonal antibodies that neutralize the biological activity of native human TNF α.

Thus, human TNF α cytokines contained therein that retain the conformationally B-epitope were inactivated according to the manufacturing procedure disclosed in the K7 condition in table 3 above.

The KLH-TNF α (human) end product prepared according to the conditions referred to as "K7" in table 3 above is the same as that prepared according to example 1 above.

The best TNF alphaThe product has been studied in the following examples, in particular in transgenic mouse models, considering its ability to induce polyclonal antibodies that neutralize the native TNF α in autologous systems.

Example 4: study of acute toxicity of KLH-TNF alpha (human) kinoid in mice

The aim of this study is to define the characteristics of the vaccine composition comprising KLH-TNF α (human) kinid according to the invention, as a broad-spectrum therapeutic vaccine for the treatment of cancer cachexia and/or various autoimmune diseases (e.g. arthritis, crohn's disease and psoriasis).

In particular, vaccine compositions have been investigated with respect to their tolerability and non-risk properties.

Therefore, primary toxicity studies have been performed in SWISS mice.

A.Materials and methods

Forty-six (46) female SWISS mice were assigned to (i) a group of 4 untreated animals and (ii) three groups of fourteen animals, which received:

a) control group (adjuvant vehicle): n-14

Intramuscular Injection of (IM) Phosphate Buffered Saline (PBS) into the thigh;

the control group was used to evaluate the local and systemic reactivity of the adjuvants used in the formulations.

b) Treatment group (first dose: 2.000 times the single therapeutic dose in humans, STHD °/N +14)

KLH-TNF α (human) kinase in Phosphate Buffered Saline (PBS) was injected Intramuscularly (IM) in the thigh as 50 μ g/mouse.

c) Treatment group (second dose: 4000-fold of single treatment dose in humans, STHD): n-14

KLH-TNF α (human) kinase in Phosphate Buffered Saline (PBS) was injected Intramuscularly (IM) in the thigh as 100 μ g/mouse.

d) Untreated group of mice first used in the experiment included in the study (n ═ 4)

Dose calculations were performed for single doses related to human use, which were 80 μ g/injection/individual: 1SHTD (single therapeutic dose in humans), i.e., 1.15. mu.g/kg.

Injection of KLH-TNF α (human) kinase had been completed on the fifth day of the experiment (D5).

Animals have been tested for response to the above treatments according to the following parameters:

a-number of immediate, short-term and long-term final deaths (observation period of 10 days after injection)

b-local or systemic reactivity to the treatment.

The c-curve shows the overall state and body weight of the animal up to day 10 after injection of KLH-TNF α (human) kinase (D15).

Five (5) days after the observation period (D20), the surviving animals were sacrificed and subjected to treatment for the following controls.

d-microscopic pathodissection examination of the muscle at the injection site to assess local tolerance.

e-determining the weight of lung, heart, liver and spleen as index values of the organ response to the administration of the immunostimulating substance.

B. Results

When administered to mice at greatly elevated doses (approximately 4000 times the single therapeutic dose for humans in each mouse), the KLH-TNF α (human) kinid did not cause any adverse reactions throughout the 10 day observation period and for each test group, as illustrated by:

1) no immediate or intermediate death;

2) there was no local reaction at the injection site, nor any systemic reaction;

3) in any of the test groups, there was no effect on the growth curve of the mice.

Furthermore, macroscopic examination of the organs of the sacrificed animals at the end of the experiment (D20) showed no changes in the organs or any increase in spleen and liver volume.

Moreover, the weights of the heart, lungs, liver and spleen varied in a similar manner for the entire group of test animals.

Example 5 study of the KLH-TNF α (human) in a transgenic mouse model adapted for human TNF Immunogenicity of kinoid.

The immunogenicity (humoral) of KLH-TNF α (human) kinoid formulations, compared to that of KLH, has been studied in mice B6.SJL-Tg (TNF) N2 at 5 weeks of age (a group of 10 mice). These mice were supplied by Taconic corporation (usa) and included transgenic (hemizygous) mice for the human TNF α gene.

A. Materials and methods

On days 0 and 7, mice (a group of 10 mice) had received an injection of 0.2ml (30 μ g) of the emulsion in ISA51 by intramuscular root. A second injection of 25 μ g of the emulsion in ISA51 was given on day 28. Each mouse was then subjected to a retroorbital blood sample on day 35.

B. Results

1. Humoral response

The humoral response is determined by the presence in the serum of the immunized mice of antibodies of the IgG type directed against human TNF α; humoral responses were determined by ELISA assay and antibody titers (dilutions)^-1Giving an optical density greater than 0.3). FIG. 2 illustrates the antibody titers obtained.

Mice immunized with the KLH-TNF α (human) end product obtained according to example 1, the sera derived from them having high levels of IgG-type antibody titers, whereas the sera derived from mice immunized with KLH are devoid of these antibodies.

The neutralizing activity of these antibodies was measured in L929 cells using a TNF α cytotoxicity assay. The results are presented in fig. 3.

The KLH-TNF alpha (human) kinoid preparation induced antibodies with high levels of neutralizing activity.

Example 6 investigation of the immunogenicity of KLH-TNF alpha (human) kinoid in rhesus monkeys

The humoral immunogenic activity of KLH-TNF α (human) kinoid, compared to that of KLH alone, has been studied in rhesus monkeys supplied by MDS Pharma (Lyon-France). It is noteworthy that the naturally occurring TNF α from rhesus monkey has 98.1% amino acid homology with human TNF α.

A. Materials and methods

On days 0, 21 and 49, rhesus monkeys had received 0.5ml of an emulsion of kinoid dissolved in ISA51 for injection by the intramuscular route, including (i)80 or 20 μ g of KLH-TNF α (human) kinoid formulation, or (ii) KLH alone. Blood samples were taken from each animal on days 28, 56, and 68.

B. Results

1-humoral response:

the humoral response was measured by detecting the presence of antibodies of the IgG isotype directed against human TNF α in the sera of immunized rhesus monkeys. The humoral response has been determined using an ELISA assay and expressed as antibody titer (dilution)^-1Giving an optical density greater than 0.3). The results obtained for the antibody titers are reported in figure 4.

Sera derived from rhesus monkeys immunized with KLH-TNF α (human) preparations had anti-TNF α IgG isotype antibody titers, while sera derived from rhesus monkeys immunized with KLH alone lacked these antibodies.

The anti-TNF α antibody titers were higher in rhesus monkey sera that received 80 μ g of KLH-TNF α (human) kinase.

The neutralizing activity of this antibody has been determined in L929 cells using a TNF α cytotoxicity assay. The results of these assays are reported in figure 5.

The KLH-TNF alpha (human) kinase-induced antibody has high neutralizing activity.

Example 7 therapeutic Effect of anti-TNF α Autoimmunization in huTNF α transgenic mice Evaluation of (2)

In 5-week-old transgenic mouse huTNF α B6.SJL-Tg (TNF) N2 supplied by Taconic corporation (USA), the therapeutic efficacy of an anti-TNF α autoimmunization strategy using KLH-TNF α (human) kinoid formulation as an active ingredient was evaluated. These TNF α transgenic mice develop spontaneous polyarthritis at 4 to 5 weeks of age.

A-materials and methods

1-immunization

Mice received injections of 0.2ml of emulsion in ISA intramuscularly on days 0, 7 and 28. The treatments that have been performed on four groups of 10 mice are detailed below:

group A: PBS: 200 μ l PBS

-group B: KLH: 200 μ l KLH

-group C: KLH-TNF: 200 μ l KLH-TNF

-group D: KLH-TNF + MTX: 200. mu.l KLH: TNF and methotrexate (1mg/kg), starting from immunization N.sup.1 and up to sacrifice three times a week (intraperitoneal injections, 200. mu.l each).

The mice have received (i) at day 0 and day 7: 30 μ g of KLH-TNF α (human) kinase formulation and (ii) on day 28: treatment of 15. mu.g of the same formulation.

On day 35, retroorbital blood samples were taken from each mouse. Blood samples were also taken at the time of sacrifice on day 57.

Clinical examination and quantitative assessment of 2-arthritis

Clinical examinations were performed at the beginning of the trial and then twice a week.

Evaluation was performed by an observer with no knowledge of the treatment applied. The clinical severity of arthritis at each joint (finger, tarsal, ankle, carp) was quantified by a score attribute varying from 0 to 4, where: 0 is normal; 1 ═ erythema; 2 ═ swelling; deformation 3 ═ deformation; and 4 ═ most deformation or necrosis. To obtain the arthritis score for each animal, these scores were summed up daily. The average value for each group was calculated for each day of treatment.

Histological examination and quantitative assessment of 3-arthritis

All animals were sacrificed on day 57 after the start of the experiment. The hind paws were removed, fixed in formalin, decalcified, then dehydrated and subsequently embedded in paraffin blocks. Then, a5 μm thick tissue section was prepared with a microtome. To ensure a correct stereoscopic assessment of the joint pathology, at least a serial section is made for each paw. The slide specimen was then stained with hematoxylin and eosin and then observed under an optical microscope. Each section was assessed quantitatively for lesions on a scale of 3 data points (0 normal; 3 severe). This histological score can be divided into two parameters: on the one hand, destruction of cartilage and destruction of bone (thickness of cartilage and presence of thickness, irregularities and erosion of bone); on the other hand, inflammation (increased synovial fluid, inflammatory infiltration of cells).

4, counting:

the resulting values are given as mean and Standard Deviation (SDM). student's test and analysis of variance (ANOVA) have been completed.

B. Results

1-humoral response:

the humoral response was measured by detecting the presence of antibodies of the IgG isotype directed against human TNF α in the sera of immunized rhesus monkeys. The humoral response was determined by ELISA assay and expressed as antibody titer (dilution)^-1Giving an optical density greater than 0.3).

The results obtained for the antibody titers are reported in figure 6.

Clinical examination and quantitative assessment of 2-arthritis

Change in clinical score over time

The change in clinical score over time is reported in figure 7.

Treatment of rhesus monkeys (i) induced a significant statistically significant reduction in arthritis scores assessed by clinical examination with KLH-TNF α (human) kinase or (ii) the combined use of KLH-TNF α (human) kinase and Methotrexate (MTX). By comparison, control animals treated with KLH or PBS buffer had much lower arthritis scores determined.

Assessment of disease occurrence and disease progression parameters

The assessment of disease development and disease progression is reported in table 4 below.

The number of days of disease development per animal has been determined by clinical examination. Animals not suffering from the disease were not considered.

The score "amax" corresponds to the highest score obtained for each animal during the test. The score "amax" represents a parameter of disease severity.

Incidence refers to the number of animals that develop arthritis before the end of the trial, taking into account the total number of animals per group.

TABLE 4

Treatment of	Days of disease onset (sick animals only)	Amax score. + -. standard deviation	Incidence of disease
				PBS	24.4±2.5	11.5±4.2	10/10
KLH	25.9±2.5	9.0±1.4	10/10
				KLH/TNF	45±2.3**/##	0.9±0.5**/#	3/10
KLH/TNF	43.5±3.3*/##	1.0±0.32**/#	6/10

^*p < 0.01 vs. KLH^**p < 0.001 vs KLH # p < 0.02 vs PBS # # p < 0.001 vs PBS (Student t-test)

The results show that treatment by KLH-TNF α and KLH-TNF α + Methotrexate (MTX) induced in a statistically significant manner:

-delaying the onset of arthritis compared to control animals treated with KLH or PBS alone.

-a decrease in the severity of arthritis; and

-reduction of the number of diseased animals.

Histological examination and quantitative assessment of 3-arthritis

The results are reported in table 5 below.

TABLE 5

Treatment of	Inflammation score. + -.sem	Disruption score ± sem
			PBS	1.30±0.09	0.71±0.11
KLH	1.53±0.21	1.10±0.23
			KLH/TNF	0.16±0.09**	0.08±0.03**
KLH/TNF	0.24±0.09**	0.21±0.12*

^*p < 0.01 vs KLH and PBS^**: p < 0.001 vs KLH and PBS (Student's t test)

The reduction of histological changes (destruction and joint inflammation parameters) was induced in a statistically significant manner by treatment with KLH-TNF α and KLH-TNF α + Methotrexate (MTX).

C-conclusion

Immunization of huTNF α transgenic mice with KLH-TNF α (human) kinoid formulations according to example 1 significantly protected the animals from inflammation and joint damage as shown by clinical and histological analysis results.

The group of animals treated with the KLH-TNF α (human) kinase preparation cannot be distinguished from the group of animals treated with the KLH-TNF α (human) kinase preparation and Methotrexate (MTX) in terms of joint protection; these results can be explained by the fact that under these experimental conditions the main efficacy of the KLH-TNF α (human) kinoid formulation alone masks the final beneficial effects of methotrexate.

The group of animals treated with KLH was not distinguishable from the group of animals treated with PBS buffer.

Additional results, which relate to the biological properties of the stabilized immunogen products according to the present invention, are disclosed in examples 8 to 12 below.

Examples 8 to 12

A.Materials and methods of examples 8 to 12

A.1. Reagent

KLH was purchased from Intracel (san diego, canada), hTNF α was purchased from boehringer ingelheim (mannheim, germany), murine TNF α was purchased from cytopab (Rehovot, israel), hTNF α receptors RI and RII, hTNF α, murine IL2 and murine IFN γ elisa assay kits were purchased from R & D systems (rel, france), ISA51 adjuvant was purchased from Seppic (paris, france).

A.2. Animal(s) production

Heterozygous hTNF transgenic mice (1006-T) of 4-5 week old females and males were purchased from Taconic, 6-8 week old female C57Bl/6 and Balb/C mice from Charles River (L' arms, France). Mice were bred in germ-free conditions. Rabbits were bred in Charles River breeding (L 'Arbresles, France) and rhesus monkeys in MDSPhama (St-Germain-sur-L' Arbresles, France).

A.3. Preparation of immunogens

hTNF α -KLH immunogen was prepared by the method disclosed in example 1.

SDS/Polyacrylamide gel electrophoresis, silver staining and immunoblotting

SDS-polyacrylamide gel electrophoresis analysis was performed under non-reducing conditions according to the method of Laemmli (Laemmli, U.K. (1970), Nature 227: 680-85). Proteins were visualized by silver staining and autoradiography in 12% SDS-polyacrylamide gel electrophoresis (Le Roy F., Bisbal C., Silhol M., Martinand C., Lebleu B. & Salehzada T. (2001). The Journal of biological Chemistry 276: 48473-482.) or immunoblot analysis with appropriate antibodies.

A.5. Gel filtration chromatography

The heterocomplex was loaded onto a Superose 610/300 GL gel filtration column (Amersham bioscience, Orsay, France) pre-equilibrated with DPBS and eluted with 0.2ml/min using the same buffer as used at equilibration.

A.6. Cell culture

Mouse L929 cell line (ATCC, CCL 1) was cultured in RPMI-1640 containing 10% fetal bovine serum. Purified murine splenocytes were resuspended in RPMI medium containing 5% fetal bovine serum and 50. mu.M 2-. beta. -mercaptoethanol and incubated in medium alone or with KLH (30. mu.g/ml), hTNF α (3. mu.g/ml) and murine TNF α (1. mu.g/ml) antigen.

A.7.T cell proliferation assay

Antigen-activated splenocytes were cultured for 4 days and then irradiated with 3H-thymidine for 18 hours. After 18 hours the cells were collected in a filter and the incorporation of DNA into thymidine was quantified using a beta-ray counter. The stimulation index is expressed as: [ (mean of cpm from stimulated cells) - (mean of cpm from unstimulated cells) ]/(mean of cpm from unstimulated cells). A SI value greater than 2 is considered positive.

TNF alpha and receptor assays

A.8.1. Direct receptor binding assay:

plates were pre-coated with 50 ng/well of hTNF α RI or RII to determine the presence of this heterocomplex and hTNF α binding to its target receptor. Serially diluted samples were incubated with their receptors and detected with biotinylated goat polyclonal anti-hTNF α antibodies (R & D Systems). To assess the ability of serum or IgG to inhibit hTNF α binding to its receptor, hTNF α was first pretreated with serial dilutions of test serum or IgG prior to transfer to plates on which hTNF α RI was immobilized. Blocking titers were expressed as the reciprocal of the dilution of serum that neutralized 50% of hTNF α binding.

A.8.2. anti-hTNF alpha antibody dropAnd (3) measuring the degree:

the specific anti-hTNF α antibody titers in the sera of immunized and control mice were determined by direct enzyme-linked immunosorbent assay (Elisa). Elisa plates pre-coated with 50 ng/well hTNF α were incubated with serially diluted sera from immunized and control mice. Specific IgG was detected using peroxidase rabbit anti-mouse IgG (Zymed, Canada). The final titer is expressed as the reciprocal of the highest sample dilution giving an optical density value of 0.3.

A.8.3. And (3) cytokine quantification:

hTNF α, murine IL-2 and murine IFN γ were measured in serum and culture supernatant by enzyme-linked immunosorbent assay.

Bioassay of hTNF α

hTNF α activity was assessed using the L929 cytotoxicity assay (Blaqul 2004). Serial dilutions of hTNF α and heterocomplexes were incubated with L929 cells in the presence of actinomycin D (1 μ g/ml) for 18 hours and the number of viable cells was determined by MTT assay. Hyperimmune serum or IgG were similarly assayed for the ability to neutralize hTNF α activity after incubation of serum and IgG with hTNF α. Neutralization titers were expressed as the reciprocal of the serum dilution that neutralized 50% of hTNF α activity.

A.10. Immunization

Animals were immunized by 3 or 4 intramuscular injections with kinoid (seppic) or control formulation (5-30 μ g) adjuvanted with ISA 51. Sera were collected 8 to 12 days after 3 or 4 immunizations and at sacrifice.

A.11. Antibody purification

Immunoglobulins (IgG) and specific anti-hTNF α or anti-KLH antibodies were purified from sera of immunized or control mice using protein G IgG purification kit (Pierce) or affinity chromatography on hTNF or KLH-coupled sepharose 4B columns (Sigma chemical Co., St.Louis, Mo). Avidity was determined using BIAcore 3000 techniques (Lofas, s., johnson B. (1990) j.chem.soc.chem.comm.21: 1526; Karlsson, r., fall a., (1997) j.immunol.methods 200: 121).

A.12. Lethal shock

Peritoneal injections of hTNF α in 20mg D-galactosamine dissolved in PBS stimulated the response of control and hTNF α -kinoid immunized mice (C57Bl/6 or TTg) 10 days after the last immunization injection. Animals surviving 24 hours after injection were recorded with a lethal dose of hTNF α of 80-90% (Lehmann V J Exp Med 1997657). In these experiments, mice sacrificed 8 hours after TNF α injection were grouped and visual examination of the liver was performed.

A.13. Arthritis tracking

Human TNF α transgenic mouse 1006-T suffers from idiopathic arthritis from week 8, such as Tg197 strain (Keffer, J., Probert, l., Cazlaris, h., Georgopoulos, s., Kaslaris, e., kiusis, D. & Kollias, G. (1991) Embo J10, 4025-31). Mice were monitored blindly for signs of arthritis in the four paws. Clinical severity was scored from 0 (normal) to 3 (severe inflammation with deformation) for each limb (Williams, r.o., Feld-mann.m. and Maini, R.N. (1992) Proc Natl Acad Sci USA89, 9784-8; Thwin, m.m., Douni, e., Aidinis, v., Kollias, g., Kodama, k., to Sato, k., Satish, r.l., mahendan, R. & Gopalakri-shanakone, P. (2004) Arthritis rester 6, R282-94). The mean arthritis score was calculated for each treatment group for each day of clinical observation. The legs were sectioned separately and processed as described elsewhere for histological analysis. (Bessis, n., Guery, l., Mantovani, a., Vecchi, a., Sims, j.e., Fradelizi, D. & Boissier, M.C. (2000) Eur J Immunol 30, 867-75). A large number of slices were cut out per paw and at least four slices were examined. Injuries at each joint of the knee, ankle and foot were assessed blindly, as previously described with a 4-point partition (0-3, where 0 is normal and 3 is severe) or synovitis (synovial fluid increase, inflammatory cell infiltration) or joint injury (Bone and cartilage thickness, irregularity and appearance of erosion) (Saidenberg-kerrnac' h, N., corado, a., Lemeiter, d., dervernejoul, m.c., Boissier, M.C. & Cohen-Solal, M.E. (2004) Bone 35, 1200-7, Miellot, a., Zhu, r., Diem, s., Boissier, m.c., Herbelin, a. & besiss, N. (2005) Eur J35, 4-13).

A.14. And (5) carrying out statistical analysis. All statistical analyses were done with StatView version 5.0 software. Analysis of variance is used to analyze repeated measurements such as clinical score, number of affected limbs, weight gain, prevalence. For comparison of quantitative data, a nonparametric Mann Whitney test was used. Qualitative data were compared using the Yates modified chi-square test.

B. Results of examples 8 to 12

Example 8: biological Properties of Stable immunogenic product (hTNF. alpha. kinoid)

A. Immunological and biochemical characteristics

Human TNF α kinoid is a KLH-hTNF α heterocomplex that migrates as 3 bands in SDS-polyacrylamide gel electrophoresis, exhibiting Molecular Weights (MW) of approximately 250, 105 and 40kDa (FIG. 8-a). A significant proportion of kinoids do not migrate and are visible at the lamination glue level. Both hTNF α and KLH were identified using specific antibodies (FIG. 8-a).

By using¹²⁵Similar migration patterns were obtained for I-labeled kinoids on KLH or hTNF α, as shown in FIG. 8-b. 4 peaks were resolved on superose 6 gel filtration. One peak was in the exclusion volume and the other 3 were molecular weights 440, 158 and 13.7kDa (FIG. 8-c).

Deletion of TNF alpha biological Activity

Kinoid lacks any TNF α -induced cytotoxicity even at the highest concentrations (FIG. 8-d) as assessed by the standard L929 assay.

Despite its desirable lack of biological activity, human TNF α -kinoid binds to both hTNF α receptor I (p55) and receptor II (p75) (FIG. 8-e).

Example 9: induction of anti-TNF alpha antibodies in human TNF alpha transgenic mice (TTg mice)

Immunization with TNF α kinoid induced high titers of anti-TNF α antibodies in TTg mice, however none of the detectable anti-TNF α antibodies could be induced with inactivated TNF α or with control KLH. In contrast, anti-TNF α antibodies were induced in Balb/c mice with either kinoid or inactivated TNF α (FIG. 9-a). KLH and kinoid immunization resulted in the production of anti-KLH antibodies in TTg and Balb/c mice (not shown). The high dilution of hyperimmune serum from kinoid immunized TTg mice was able to neutralize the biological activity of TNF α even at high dilution, however the serum from KLH immunized mice did not have this effect as assessed by the standard L929 cytotoxicity assay (FIG. 9-b). Activity peaked 2-4 weeks after booster immunization, decreased significantly (> 50%) (fig. 9-c) within 3 months and blocked TNF α R1 and R2 receptor binding (fig. 9-d). anti-hTNF α antibodies are mainly IgG1 (52%) and IgG2a (48%). The amounts of IgG3, IgM and IgE were negligible when detected with the isotype Elisa kit. IgG finally purified from hyperimmune serum showed a range of KD from 5X 10 when evaluated using the Biacore technique^-8M to 10^-10M huTNF α, and blocks hTNF α binding to its receptors I and II (fig. 9-f). These results explain why circulating hTNF α could not be detected in immunized mice, instead it was present at 9pg/ml in unimmunized mice.

Example 10: safety of stable immunogenic products (hTNF. alpha. kinoids)

High doses of TNF α kinoid (up to 100 μ g) injected in mice of different strains (Balb/C; C57 Bl/6; Swiss), rabbits or rhesus monkeys produced no local or systemic clinical side effects. Furthermore, although administration of native hTNF α in D-galactosamine treated C57Bl/6 mice resulted in death within 24 hours (8 out of 8 mice), hTNF α kinoid did not have this effect. Furthermore, kinoid immunization does not elicit adverse effects, including an undesirable anti-TNF α cellular autoimmune response and impairment to long-term growth and survival of immunized TTg mice.

As detected by T-cell proliferation and cytokine (IL-2 and IFN γ) production in cell culture supernatants, splenocytes from kinoid-immunized TTg mice cultured in vitro did not elicit any cell-mediated immune response to autologous hTNF α (FIG. 10(A, B, C) -a). In contrast, kinoid-immunized Balb/c mice showed significant cell-mediated immunity to heterologous hTNF α, as compared to control non-immunized animals (not shown). No cellular response to murine TNF α was detected in the immunized mice compared to the control (FIG. 10(A, B, C) -B). Finally, in the immunized TTg mice, there was a comparable positive T cell response to KLH antigen, which was identical to that observed for the control animals receiving KLH, with no detectable clinical effect (fig. 10(A, B, C) -c).

Growth of kinoid immunized mice (n ═ 8) when measured by body weight, was comparable to non-immunized animals (n ═ 7) up to 50 days post-immunization, but became significantly better at a later stage (fig. 11-a). Mice sacrificed 120 days after immunization were used for histological comparison studies of joint tissues. Other groups of TTg mice were followed for longer periods of time in order to observe the clinical course of arthritis. Of these animals, control mice (n-3) were sacrificed on day 150 due to the severity of their arthritis. In contrast, TTg mice immunized with 3 hTNF α -kinoids remained unaffected by severe arthritis (now over 210 days).

Example 11: antibody pre-treatment induced by neutralization of stable immunogen product (hTNF. alpha. kinoid) Prevent TNF alpha galactosamine lethal shock.

Intraperitoneal administration of TNF α can induce lethal shock in mice in the presence of galactosamine. The effect is dose dependent. As shown in Table 1, this lethal shock occurred at a dose of 11 μ g in C57Bl/6 mice, and the recorded death dose was as low as 1 μ g in TTg mice.

All non-vaccinated controls died, but C57Bl/6 mice immunized with hTNF α -kinoid treated with 11 μ g hTNF α did not die (Table 6). It is noteworthy that the immunized animals are not only resistant to lethal shock, but they remain clinically healthy. Furthermore, repeated injections of hTNF α -galactosamine at one month intervals had no effect (not shown).

Importantly, kinoid immunized TTg mice also resisted hTNF α -dependent lethal shock, while control TTg mice died (table 6). Higher doses of hTNF α (2 μ g) were administered with no effect on TTg mice immunized with hTNF α kinase (Table 6). These animals survived 2 weeks after repeated hTNF α shock and remained completely healthy to date.

Protection from shock in immunized mice is due to neutralizing anti-TNF α antibodies. Whereas control C57Bl/6 mice receiving nonspecific IgG (1mg) died within 24 hours after 1 hour of TNF α (11 μ g) -D-galactosamine administration, mice given specific purified polyclonal IgG (which was derived from hyperimmune serum) survived (P < 0.001 vs control). In these experiments, additional control groups and immunized mice were sacrificed 8 hours after administration of hTNF α, and macroscopic organ examination showed liver atrophy in the control, but not in the immunized mice (p < 0.02 vs control) (table 7).

Example 12: TTg mouse protection immunized with a stable immunogen product (hTNF α kinoid) Protect it from arthritis

TTg mice, 7 weeks old, were immunized with KLH/TNF- α kinoid and monitored over a 120 day period. The control group consisted of mice treated with KLH and saline, both of which did not affect the development of arthritis. All control mice suffered from polyarthritis with inflammation and joint deformity (FIG. 11), whereas kinoid immunized mice only suffered from marginal joint disease (P < 0.0001) (FIG. 4-c), with a delay in the onset of clinical signs (P < 0.05), a low score for the highest clinical index (P < 0.01) (Table 8) and significantly reduced disseminated disease and much fewer affected limbs (P < 0.0001) (FIG. 11-b). Although all animals developed clinical arthritis, the disease was reversed only in the TNF- α kinoid immunized group (FIG. 11-d); all but one animal in the treated group was protected during 120 days (table 8) (P < 0.001) as opposed to the control group.

Histological evaluation showed a significant reduction in inflammation and joint damage in the treated animals, which showed a low histological score (p < 0.01) (table 8); in contrast, control mice showed clear signs of arthritis with proliferative synovitis and cellular infiltration of mononuclear and polymorphonuclear cells with cartilage and bone erosion. The incidence of histological synovitis or destruction was significantly reduced in TNF α -kinoid treated mice (table 8).

Table 6.TNF α -dependent lethal shock:

control and kinoid immunized C57Bl/6 and TTg mice were given 10 days after the last boost of immunization by intraperitoneal injection of hTNF α in 0.1ml PBS in the presence of D-galactosamine

Prevention of lethal shock assessed by animal survival was only observed in immunized mice after 24 hours.^*P < 0.01 vs control (chi fang test)

	hTNF alpha (mu g/mouse)	Mice surviving 24 hours (live mice/total mice)
						C57Bl/6		TTg
		Control	Of immunization	Control	Of immunization
						Test 1 test 2 "	11121	6/60/66/66/6	6/66/66/66/6	0/6 Do not do 0/6	6/6*To make 6/66/6*

TABLE 7 neutralization of TNF α -dependent shock by hyperimmune IgG

30 minutes before intravenous injection of protein G purified mouse IgG (derived from control and kinoid immunized C57Bl/6 mice), groups of mice received D-galactosamine and hTNF α in 0.1ml PBS by intraperitoneal injection.^*P < 0.001 vs. control (Chi Fang test) * P < 0.02 (Chi Fang test)

	hTNF alpha (mu g/mouse)	Mice surviving 24 hours (live mice/total mice)	Atrophy of liver
				TNF-kinoid immunized mice as control mice	1111	0/1010/10*	5/50/5*

TABLE 8 clinical and histological scores of arthritis at day 120 in TNF-alpha kinoid (KLH-TNF) immunized mice and in control (KLH) mice.

The incidence of inflammation/damage assessed histologically is the number of mice with an inflammation/damage score of 0.5 or more. Amax: arthritis clinical highest score. Results are given as mean ± SEM.^*p < 0.05 vs. KLH, * p < 0.01 vs. KLH (Mann Whitney); p < 0.001 vs KLH (chi-square test)

Immunization	Animal(s) production	Clinical evaluation			Histology
									KLHKLH-TNF	78	Arthritis (arthritis)			Inflammation(s)		Damage of
Start (sky)	Amax score	Prevalence rate	Score of	Prevalence rate	Score of	Prevalence rate
							36.1±4.549.5±3.2*	8.6±0.61.4±0.2*			71+	1.5±0.10.1±0.1*	70+	1.2±0.10.1±0.1*	71+

Claims (27)

1. A method for preparing an anti-TNF α immunogenic product comprising the steps of:

a) providing a solution containing TNF α;

b) adding EDTA to the TNF alpha-containing solution of step a);

c) adding a carrier protein to the solution obtained at the end of step b);

d) adding glutaraldehyde to the liquid mixture obtained at the end of step c);

e) removing glutaraldehyde and also TNF α and free molecules of the carrier protein from the solution obtained at the end of step d);

f) adding formaldehyde to the solution obtained at the end of step e) and maintaining the presence of formaldehyde for a time ranging from 96 hours to 192 hours;

g) adding to the solution obtained at the end of step f) a reagent that blocks the reaction with formaldehyde; and

h) removing formaldehyde and blocking reagent from the solution obtained at the end of step h), thereby obtaining a solution containing the anti-TNF α immunogenic product described above.

2. The method of claim 1, wherein step b) comprises the steps of:

b1) adding EDTA to the solution containing TNF α of step a); and

b2) DMSO was added to the solution obtained at the end of step b 1).

3. The method according to any one of claims 1 or 2, wherein step d) comprises the steps of:

d1) adding glutaraldehyde to the liquid mixture obtained at the end of step c); and

d2) EDTA is added to the heterocomplex between TNF alpha and the carrier protein obtained at the end of step d 1).

4. The method according to any one of claims 1 to 3, wherein the concentration of TNF α in step a) ranges from 0.1mg/ml to 50 mg/ml.

5. The method according to any one of claims 1 to 3, wherein the concentration of TNF α in step a) ranges from 0.5mg/ml to 10 mg/ml.

6. The method according to any one of claims 1 to 5, wherein the final concentration of EDTA in step b) ranges from 1mM to 500 mM.

7. The method according to any one of claims 2 to 6, wherein the final concentration of DMSO in step b2) ranges from 0.5% v/v to 20% v/v.

8. The method according to any one of claims 1 to 7, wherein the molar ratio of TNF α to the carrier protein in step c) ranges from 5: 1 to 100: 1.

9. The method according to any one of claims 1 to 8, wherein the final concentration of glutaraldehyde in step d) ranges from 0.05% w/w to 0.5% w/w.

10. The method according to any one of claims 3 to 9, wherein the final concentration of EDTA at step d2) ranges from 1mM to 10 mM.

11. The method according to any one of claims 1 to 10, wherein glutaraldehyde is removed in step e) by performing dialysis, by ultrafiltration using diafiltration or by Tangential Flow Filtration (TFF).

12. The process according to any one of claims 1 to 11, wherein the final concentration of formaldehyde in step f) ranges from 1% w/w to 10% w/w.

13. The method according to any one of claims 1 to 12, wherein the final concentration of formaldehyde in step f) ranges from 2% w/w to 5% w/w.

14. The process according to any one of claims 1 to 13, wherein in step f) the presence of formaldehyde is maintained for a time ranging from 120 hours to 168 hours.

15. The method according to any one of claims 1 to 14, wherein in step g) the blocking reagent consists of glycine.

16. The method according to claim 15, wherein the final concentration of glycine in step g) ranges from 0.01M to 10M.

17. The method according to claim 15, wherein at step g) the final concentration of glycine ranges from 0.05M to 2M.

18. The method according to any one of claims 1 to 14, wherein in step g) the blocking reagent consists of lysine.

19. The method of claim 18, wherein the final concentration of lysine in step g) ranges from 0.01M to 10M.

20. The method of claim 18, wherein the final concentration of lysine in step g) ranges from 0.05M to 0.5M.

21. The method according to any one of claims 1 to 20, wherein in step h) the formaldehyde and blocking reagent are removed by performing dialysis, by ultrafiltration using diafiltration or by Tangential Flow Filtration (TFF).

22. The method according to any one of claims 1 to 21, wherein the carrier protein is selected from the group consisting of Diphtheria Toxoid (DT) and mutants thereof, Tetanus Toxin (TT), acer Keyhole Limpet Hemocyanin (KLH), and purified protein derivatives of tuberculin (PPD), OMPC from neisseria meningitidis, purified protein derivatives of tuberculin (PPD), Bovine Serum Albumin (BSA), and protein D from haemophilus influenzae.

23. A method according to any one of claims 1 to 22, wherein the carrier protein consists of Keyhole Limpet Hemocyanin (KLH).

24. A method for preparing a vaccine composition comprising the steps of:

a) preparing an anti-TNF α immunogenic product by performing the method of any one of claims 1 to 23; and

b) combining the anti-TNF α immunogen product prepared in step a) with one or more immunoadjuvants.

25. An anti-TNF α immunogenic product having one or more of the following technical characteristics:

(i) comprising (a) a TNF-alpha molecule and (2) a carrier protein molecule joined together by less than 40% covalent bonds and (b) by more than 60% non-covalent bonds;

(ii) it shows a molar ratio of TNF-alpha to the carrier protein ranging from 40: 1 to 60: 1;

(iii) its ability to induce TNF alpha Neutralization (NC)₅₀) An anti-TNF α antibody less than 1/1000; and

(iv) it has an ED of more than 50ng/ml and even more than 400ng/ml in the L929 cytotoxicity assay₅₀The value is obtained.

26. An immunogenic composition comprising (i) an anti-TNF α immunogenic product prepared according to the method of any one of claims 1 to 23, or (ii) an anti-TNF α immunogenic product according to claim 25, in combination with one or more pharmaceutically acceptable excipients.

27. A vaccine composition comprising (i) an anti-TNF α immunogenic product prepared according to the method of any one of claims 1 to 23, or (ii) an anti-TNF α immunogenic product according to claim 25, in combination with one or more immunoadjuvants.