WO2001045745A2

WO2001045745A2 - A reversible linkage technology for controlled conjugation

Info

Publication number: WO2001045745A2
Application number: PCT/GB2000/004935
Authority: WO
Inventors: Nicholas Flinn; Tony Johnson
Original assignee: Acambis Research Limited; Smithkline Beecham Biologicals S.A.
Priority date: 1999-12-21
Filing date: 2000-12-21
Publication date: 2001-06-28
Also published as: AU2206701A; WO2001045745A3

Abstract

The present invention describes a linkage for use in the conjugation of compounds (e.g. peptides) to carrier vehicles (e.g. macromolecules, polymers, dendrimers, proteins etc.), producing constructs of biological and immunological relevance. The ability to link an Epitope (such as a peptide) to a carrier (such as a protein) in a controlled and specific manner is of a paramount importance in the development of a potent, pharmaceutically relevant, immunogenic hapten-carrier construct, such as a vaccine. The invention provides a reversible linkage technology for controlled conjugation of compounds such as peptides or peptidic Epitopes to carriers such as proteins. It utilises an aryl aldehyde moiety to introduce an aldehyde functionality on to a carrier molecule (e.g. on to the surface of a protein carrier molecule). It uses a 2-hydroxy-4-alkoxy linker based on an aryl aldehyde to provide protection in the conjugation of a peptide to a carrier, by virtue of imine formation.

Description

A reversible linkage technology for controlled conjugation

The present invention describes a linkage for use in the conjugation of compounds (e.g. peptides) to carrier vehicles (e.g. macromolecules, polymers, dendrimers , proteins etc.), producing constructs of biological and immunological relevance .

Introduction

The ability to link an Epitope (such as a peptide) to a carrier (such as a protein) in a controlled and specific manner is of paramount importance in the development of a potent, pharmaceutically relevant, immunogenic hapten-carrier construct. The linkage should:

• enable covalent bonding between specific functionalities present in the Epitope and carrier, utilising mild, selective, non-denaturing conditions,

• be of a simple design to limit introduction of neodeterminants, which may reduce the specificity of the immune response to the Epitope,

• be reversible, allowing the Epitope to be examined for chemical changes that may occur during the conjugation process and to assess the level of carrier 'loading' and • allow control over the level of loading of the Epitope to the carrier, to maintain a soluble construct.

Current Status and Problems

Current methods of conjugation of peptidic Epitopes to carrier molecules (including synthetic macromolecules) rely mainly upon activation with gluteraldehyde or carbodiimide, to form a covalent linkage. The linkage is often formed non- specifically and haphazardly between Epitope and antigen due to the involvement of numerous reactive groups within these multifunctional molecules. More recently, favoured methods of conjugation utilise the more selective maleimide based reagents. Here, an Epitope containing a free thiol moiety is selectively reacted with a maleimide derivatised carrier to form an irreversible covalent bond. However, many useful Epitopes contain disulphide bridges to maintain their immunogenic structure, without which they are useless. The presence of an additional free thiol group (for conjugation purposes) would, potentially, lead to 'scrambling' of the disulphide bridge resulting in structurally altered Epitopes . The irreversible nature of the thiol-maleimide construct does not allow the degree to which this 'scrambling' has occurred, to be assessed.

An additional problem arising from the use of uncontrolled conjugation methods, is the inability to reliably control the level of attachment of the Epitope to the carrier. This may have repercussions for both the immunogenic-potential of the construct and its aqueous solubility. A low Epitope-to- carrier ratio may result in a poor immunogenic response.

Attachment of too many Epitopes to the carrier may lead to inter-Epitope (intra-construct) interactions and the formation of new, detrimental, discontinuous Epitope (s) that will give rise to an unwanted response. Alternatively, inter-Epitope interaction may prevent recognition by components of the immune system, through steric congestion. In addition, replacement of charged or polar surface residues on the carrier may dramatically reduce the aqueous solubility of the construct .

The techniques currently available are not sufficiently developed to enable controlled, directed, selective conjugation of diverse Epitopes to numerous types of carriers.

The design of a vaccine construct requires a number of criteria to be met (see above) .

Surprisingly, with the importance afforded to vaccine research programmes, little progress has been made towards a universal linker technology. This issue has become more important as more chemically complex Epitopes are discovered through such techniques as phage display libraries. For the rapid preparation of peptides and their derivatives, solid phase synthesis is the method of choice. This technique may be used to incorporate, selectively, a linker moiety at any position in a peptide chain. This can be achieved, by those skilled in the art, through the appropriate choice of orthogonally protected amino acids [e.g. Lys (Dde) ] . Further modifications of the peptide, particularly disulphide bridge formation, are performed in solution after release of peptide from the solid support. It is crucial that any linker should not interfere with this or any other post cleavage peptide

(Epitope) modifications. Further, the linker should contain a non-proteinogenic functionality to engender a specific, but mild, chemical reaction with a protein partner.

Derivatisation (if necessary) of the protein should introduce, in a non-denaturing manner, an unnatural species whose reactivity is complimentary to that introduced into the peptide Epitope. Ideally, covalent linking of peptide and carrier should be formed in a facile manner without the need for further reagents. Once formed, the linkage should be amenable to cleavage in a selective fashion to yield the intact, disulphide-bridged Epitope for process verification. Chemis try

Chemistries have been described utilising hydroxylamine or hydrazine substituted peptides and aldehyde derivatised macromolecular carriers (to form oxime and hydrazones respectively) . The aldehyde functionality is introduced onto the synthetic macromolecular carriers using either nonspecific oxidative transformations of amino acids (serine and threonine) or via chemical introduction of an alkyl aldehyde. When formed, such oxime and hydrazone constructs are reported to be largely stable for many hours over a large pH range. See for example Shao, J. and Tarn, J.P. (1995) Unprotected peptides as building blocks for the synthesis of peptide dendrimers with oxime, hydrazone and thiazolidine linkages. J. Am. Chem. Soc, 117(14), 3893-3899. Rose, K. et al . (1996) Natural peptides as building blocks for the synthesis of large protein-like molecules with hydrazone and oxime linkages. Bioconj . Chem. 7, 552-556.

Protein modification (aldehyde formation) by uncontrolled oxidation or currently practised chemical reactions is unacceptable. In addition, literature described oxime and hydrazone bonds are too stable, they do not readily allow for quantitative cleavage of carrier-attached Epitope. Cleavage of the peptide Epitope must be achieved in a selective manner to yield intact Epitope i.e. unmodified by the cleavage process. A general methodology must take into account the presence of sensitive functionalities contained within peptide Epitopes, especially the presence of disulphide bridges. The sensitivity of certain amino acids to oxidative, reductive, nucleophilic and basic conditions implies that acidolysis is an acceptable, mild procedure.

As stated above, oximes and hydrazones derived from alkyl aldehydes are too stable. Aryl aldehydes would also allow the formation of oxime and hydrazone derivatives when reacted with a suitable partner. An advantage of aryl aldehydes is the potential to modify their acid lability through the introduction of electron donating or withdrawing groups onto the aryl moiety. This system thereby provides a mechanism by which to fine-tune the acid lability of the peptide-carrier linkage. Our previous work (Johnson, Quibell and Sheppard; J. Peptide Science, Vol. 1, 11-25 (1995) and also WO 98/17628) has shown that the 2-hydroxy-4-alkoxy linker could be used to anchor amide bonds to a support and be cleaved using acidolysis. As reported previously, the acid functionality of the backbone amide linker (BAL) is attached to an amine derivatised support by activation with known coupling reagents, in organic media. Excess reagents are used to force reactions to completion. These uncontrolled, non-aqueous conditions are totally unsuitable for use with multifunctional sensitive protein carriers.

The Invention Consequently, despite the efforts of the prior art to improve on peptide linker chemistry, there is still a need for a reversible linkage technology for controlled conjugation of compounds such as peptides or peptidic Epitopes to carriers such as proteins. The problems encountered and the functional requirements in, for example, vaccine production go far beyond those of simple peptide-peptide or peptide-resin linkages.

In its broadest aspect, the present invention utilises an aryl aldehyde moiety to introduce an aldehyde functionality on to a carrier molecule (e.g. on to the surface of a protein carrier molecule) .

In a further aspect the invention uses a 2 -hydroxy-4 -alkoxy linker based on an aryl aldehyde to provide protection in the conjugation of a peptide to a carrier, by virtue of imine formation.

The present invention provides in a first aspect an intermediate of general formula (A) :

Epitope-R,-X-NH₂ (A)

In a second aspect the invention provides an intermediate of general formula (B) : ? — Carrier (B)

Both of the above are used in a method of preparation of compounds of general formula (C) :

H

Epitope-R_rX-N-_- _^ <^c) R₉- Carrier wherein; Epitope = Ep-F and Ep = a drug or other organic molecule or peptide (linear or constrained) comprising or consisting of amino acids, the amino acids being proteinogenic or synthetic non-proteinogenic and constrained being a peptide containing a disulphide bridge, lactam (amide) bridge or other chemical entity, any of which linking two side chains together or side-chain to backbone or backbone to backbone:

F = G-NH-, G-N(R)- (where R is a C5 alkyl group), G-CO-, G-S- or G-CH= , -G- S0₂-, the attachment point to R_x (defined below) that is an integral part of the Epitope and may be derived from proteinogenic or non-proteinogenic amino acid derivatives positioned within a peptide chain, at the N-terminus or at the C-terminus : G = and n = 1 to 6 and m = 0 to 6

R_x = I-J-V-, a bifunctional molecule:

V = -CO-, -NH-, -S0₂- and -S- ; preferably -CO-

J =

and n = 2 to 12, preferably 2 , m = 0 to 6 and R₃ = -N0₂, -C0₂H, -S0₃ :

I is attached to J as shown to provide: -NH-J, -CO-J, -S-J, - N(R)-J (where R is a C5 alkyl group), -S0₂-J and -(CH₂)_n-J (where n = 1 to 6)

-CH₂ - , -O- -NH- or -CO-

R2 is general formula (K)

where R₅ contains any of the following;

"(CH₂)_n- where n = 2 to 12, preferably 4 and m = 0 to 6, preferably 1:

R₄ = H or CH₃-(CH₂)_n- where n = 0-5, preferably 0: R₆ = -NH-, -N(R)- (where R is a C5 alkyl group), -N=, -CO-, - S-, -S0₂-, -CH=, -(CH₂)- -N-CO-or -O-CO- ; preferably -CO- or - NH- : and

Carrier is any macromolecular species containing at least four sites for attachment of an Epitope through the following surface groups attached as shown to the carrier; carrier-NH-, carrier-CO-, carrier-S-, carrier-S0₂- , carrier-N(R) - , carrier- CH= or carrier- (CH₂) _n- (where n = 1 to 6) .

It is a particular advantage of the invention that the carrier may be a protein molecule. Carriers that can be used with the invention will be readily known to the man skilled in the art.

A non-exhaustive list of carriers which may be used in the present invention include:

Keyhole Limpet Haemocyanin (KLH) , serum albumins such as bovine serum albumin (BSA) , inactivated bacterial toxins such as tetanus or diptheria toxins (TT and DT) , or recominant fragments thereof (for example, Domain 1 of Fragment C of TT, or the translocation domain of DT) , or the purified protein derivative of tuberculin (PPD) . Alternatively the mimotopes or Epitopes may be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing T-cell help. Preferably the ratio of peptides to carrier is in the order of 1:1 to 20:1, and preferably each carrier should carry between 3-15 peptides. A preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 Bl) . Protein D in an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926m granted EP 0 594 610 Bl) . Fragments of Protein D, for example Protein D l/3^rd (comprising the N-terminal 100-110 amino acids of Protein D (GB 9717953.5)), can also be used.

A further advantage of the invention is that it enables the conjugation of constrained peptides and proteins onto a carrier molecule without the loss of the constraining disulphide bond within the peptide. The linker moiety of the invention is independent of the

Epitope and carrier that are to be conjugated. By this it is meant that a special choice of sequences or of residues to be present in the body of the Epitope is not required. Many prior art conjugation methods require certain residues to effect the conjugation. For example, to conjugate peptides to a carrier using certain techniques, it is necessary to modify the peptide so that no lysine residues are present within the peptide other than the lysine residue which is specifically used in the conjugation procedure. The presence of additional lysine residues would cause non-specific and uncontrolled conjugation. The linker moiety described herein is independent of the Epitope and therefore affords greater flexibility to the Epitope structure/sequence.

The invention allows for the controlled conjugation of a peptide Epitope (antigen) to a protein, so as to form immunogenic conjugate which may be able to raise a protective antibody response in an animal or human patient.

The immunogenic conjugation of the invention may be formulated with a suitable adjuvant and used in active immunisation. The compounds or compositions of the invention can be used for the treatment of IgE mediated disease, or use in the manufacture of medicaments that can be used for treatment of IgE mediated disease .

Furthermore, anitbodies raised by immunisation with a conjugate or vaccine of the invention may be isolated and purified (ie separated from contaminating lost material) and used as such in passive immunisation.

Description of Drawings

Figure 1: Solid Phase Peptide Synthesis: wherein X' is optionally a terminal group or a protecting group; AA_X is an optional amino acid residue; AA_n is an optional amino acid residue; C(Trt) is a cysteine residue with trityl protection;

CO(-SH) is a cysteine residue with a free thiol group;

Lys (Dde) is a lysine residue with 1- (4 , 4-dimethyl-2 , 6-dioxy- cyclohexylidine) ethyl protection; HBTU is 2,-(lH benzotriazole-1-yl) -1,1,3,3,- tetramethyluroniumhexaflurophosphate; HOBt is N- hydroxybenzotriazole; and NMM is methylmorpholine .

Figure 2 : Modified Carrier Preparation

Figure 3: Peptide/Carrier Conjugation: wherein AA_X is an optional amino acid residue; AA_n is an optional amino acid residue; and C...C is a peptide containing a disulphide link between two cysteine residues .

Figure 4: C67-8 Anti-IgE Data

Figure 5: Competition assay with soluble IgE and IgE C67-8 peptide

Figure 6: PT1079 Anti-IgE Data

Figure 7: Competition assay with soluble IgE and PT1079 peptide Figure 8: PT1078 Anti-IgE Data

Figure 9: Competition assay with soluble IgE and PT1078 peptide

Figure 10: Inhibitory activity of mouse BSA-C67-8 induced antisera

Figure 11: Inhibitory activity of mouse antisera induced by

BSA 1078 and BSA 1079

Figure 12: HPLC monitoring of acid hydrolysis of conjugate c:\gilson\_nipoint\nick\jan00\130100\bsaavπl.001\bsaavπl.gdt.WL_1 AV-1 (Nle)-BSA.inj.Number:1 c:\gilson\unipoint\nick\jan00\170100\hclav1 001\hclav1 gdt.WL_1 BSA-AV1 (Nle)'+HC! 15mιrππ_j.Number:2 c-\gιlson\uπιpoιπt\nιck\jan00\170100\hclav1 001\hclav1 gdt:WL_1 BSA-AVI (Nle) +HCI 1 houππj Number:3 cΛgιlsoπ\unιpoιπt\πιck\jaπ00\170100\hclav1 001\hclav1 gdt:WL_1 BSA-AVI (Nle) +HCI 2 houπn_j Number4

The present invention is illustrated by but not limited to the following examples .

Example 1

Methodology

Hydroxylamine and acylhydrazine peptide derivatives were prepared on the solid phase as shown in Scheme 1. Acid mediated cleavage afforded the linear, deprotected, modified peptide. This could be readily oxidised and purified to yield the disulphide-bridged modified Epitope.

Introduction of the aryl aldehyde functionality utilised the succinimido active ester shown in scheme 2. The BAL-linker was introduced in a mixed aqueous-organic system through the N- hydroxy succinimide active ester (Scheme 2), known to be compatible with use in aqueous conditions. Further, the use of an isolated, activated derivative allows control of surface substitution and releases innocuous chemical moieties during the course of the reaction. Substitution of the ammo functions of BSA (bovine serum albumin) to -90% unexpectedly led to insoluble, precipitated protein. Modification of reaction conditions led to a method for the controlled deπvatisation of BSA, which at -50% yielded modified protein with acceptable aqueous solubility (Scheme 3) .

Simple combination of modified peptide and derivatised BSA afforded peptide-BSA constructs readily isolated by dialysis (Figure 3 (Scheme 4) ) . SDS-PAGE was used to confirm an increase molecular weight. Attempted acidolytic cleavage of the oxime linker was unsuccessful even strong acid conditions. Surprisingly, however, the hydrazone-linked construct underwent rapid acid mediated hydrolysis to yield the original intact d sulphide-bridged Epitope.

Example 2. Production of mi otopes of PI, and immunogenicity/functional activity thereof

2.1 Production of immunogens

Mimotopes of PI (EDGQVMDVD Seq ID No . 1) were derived either by bacteriophage display techniques or by rational design by molecular modelling of the C-D loop of Cε2 domain of IgE. The following peptides (Table 1) were synthesised and formulated into BSA-peptide conjugates. It will be noted that the peptides in Table 1 contain two cysteine residues within the sequence that can be used to constrain the peptide by the formation of a disulphide bond. The conjugation method described herein enables such peptides to be conjugated to a suitable carrier without the loss of constraint of the disulphide bond.

Table 1: Peptide sequences

The peptides/protein carrier constructs were produced as follows. Acylhydrazine peptide derivatives were prepared on the solid phase as shown in Figure 1 (Scheme 1) . These peptide derivatives can be readily prepared using the well- known 'Fmoc' procedure, utilising either polyamide or polyethyleneglycol-polystyrene (PEG-PS) supports in a fully automated apparatus, through techniques well known in the art [techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical Approach' by E. Atherton and R.C. Sheppard, published by IRL at Oxford University Press (1989)]. Acid mediated cleavage afforded the linear, deprotected, modified peptide. This could be readily oxidised and purified to yield the disulphide-bridged modified Epitope using methodology outlined in 'Methods in Molecular Biology, Vol. 35: Peptide Synthesis

Protocols (ed. M. . Pennington and B.M. Dunn), chapter 7, pp91-171 by D. Andreau et al .

The peptides thus synthesised can then be conjugated to protein carriers (in this case Bovine Serum Albumin, BSA) using the following technique :

2.2 Modified Carrier Synthesis

Introduction of the aryl aldehyde functionality utilised the succinimido active ester (BAL-OSu) prepared as shown in Figure 2 (Scheme 2) (see WO 98/17628 for further details) . Substitution of the amino functions of BSA (bovine serum albumin) to -50% gave routinely soluble modified protein. Greater substitution of the BSA led to insoluble constructs . BSA and BAL-OSu were mixed in equimolar concentration in DMSO/buffer (see Figure 2; Scheme 3) for 2hrs . This experimentally derived protocol gave -50% substitution of BSA as judged by the Fluorescamine test for free amino groups. 2.3 Peptide-BSA construct

Simple combination of modified peptide and derivatised BSA afforded peptide-BSA constructs readily isolated by dialysis

(Figure 3: Scheme 4). SDΞ-PAGE was used to confirm an increase in molecular weight.

2.4 Immunogenicity studies

The mimotope/BSA constructs were purified and formulated into vaccines and adjuvanted with and oil in water emulsion containing QS21 and 3D-MPL described in WO 95/17210 the (25μg BSA conjugate dose) . These vaccines were administered into groups of 10 BalbC mice, and boosting was be performed on day 21 and on day 42 and sera can be harvested on day 42 and 56. The immune response to anti-plate bound IgE and receptor orientated IgE, was then followed using techniques known in the art. Also, the activity of the antiserum in the inhibition of histamine release from allergic basophils was measured using known techniques .

2.5 Results

All BSA constructs induced high titres of anti -IgE antibodies, when the IgE was bound directly to the ELISA plate, and when orientated on the high affinity receptor. Moreover, all of these responses were confirmed to be specific, in that they were competed by free IgE and the mimotope itself, and not by non-specific peptides. The anti-IgE induced by these immunogens were capable of inhibiting histamine release from human basophils derived from an allergic donor (rye grass, LOLP1) .

For the results for C67-8 see figures 4, 5 and 10. For the results for PT1078 see figures 8, 9 and 11. For the results for PT1079 see figures 6, 7 and 11.

Moreover, the immune responses generated by these peptide mimotopes were not anaphylactogenic .

Table 2, Anaphylactogenicity of the PI mimotope antisera

Footnote to table, Cells from a LolPl-sensitive donor were treated with diluted mouse serum for 30 mins . Released histamine was determined by a commercially available histamine specific EIA. Data are mean ± S.E.M. (n = 10) .

Example 3. Acid Hydrolysis of the Hydrazone Linkage

Using the disulphide constrained peptide Arginine Vasopressin (AVPCYFQNCPRG - Sequence ID Number 283) as an example, the reversible nature of the hydrazone linkage was demonstrated. The AVP sequence was assembled with a additional nor-leucine and lysine residues at the C-terminus, which was then modified (as previously described herein) with the succinic acid hydrazine linker, oxidised and purified to yield the disulphide constrained, modified AVP. Conjugation with BSA-BAL using conditions previously described herein, yielded the BSA-AVP conjugate.

A sample of the conjugate in ammonium carbonate buffer was treated with an equivalent volume of IN HC1 and the sample composition monitored by reverse phase-HPLC. Over a time of 1-2 hours, the intact, disulphide constrained, modified peptide was released from the conjugate (Figure 12.) and its identity confirmed by mass spectrometry . Thus, the linkage between Epitope and carrier may be cleaved to allow quality control of the conjugation process. This ensures that any chemical modification or side reaction of the Epitope, arising during the conjugation process, may be identified and rectified before any immunisations of the conjugates are carried out .

Example .

Other peptides derived from or mimotopes of the Ce2 region of IgE, have been conjugated to a carrier using the reversible linker of the invention. These peptides are listed below.

Peptide Sequence Location sequence and ΞEQ ID

Name IgE Domain; Identity NO.

PI EDGQVMDVD Cε2 (Glu270-Asp278) 1

P2 STTQEGEL Cε2 (Ser283-Leu290) 2

P3 SQKHWLSDRT Cε2 (Ser300-Thr309) 3

P4 GHTFEDSTK Cε2 (Gly318-Lys327)

P5 GGGHFPPT Cε2 (Gly245-Thr250) 5

P6 PGTINI Cε2 (Pro262-Ile267) 6

P7 FTPPT Cε2 (Phe231-Thr235) 7

P15 CLEDGQVMDVDLL-NHj PI mimotope 8

P15r LLDVDMVQGDELC-NH₂ PI retro mimotope 9

P15p LEDGQVMDVDLC PI mimotope 10

P15g CLEDGQV DVDLC PI mimotope 11

C67/8 CFINKQMADLELCPRE PI mimotope 12

C67 CFMNKQLADLELCPRE PI mimotope 13

PT1079 CLEDGQVMDVD CPREAAEGDK PI mimotope 14

PT1079GS CLEDGQVMDVDLCGGSSGGP PI mimotope 15

PT1078 CLEDGQVMDVDCPREAAEGDK PI mimotope 16

P15S QVMDVDL PI mimotope 17

EEC39-I KCREVWLGESETIMDCΞ PI mimotope 18

EEC39-J ACREVWLGESETIMDCD PI mimotope 19

EEC39-10 SCREVWLGESETVMDCG PI mimotope 20

EEC40-9 NCQDLMLREDAGCWSKM PI mimotope 21

EEC47-3 DCEEPMCSPVLLQQLKL PI mimotope 22

P15t LEDGQVMDVD PI mimotope 23

P16 CSTTQEGELA-NH₂ P2 mimotope 24

P2sh TTQEGΞ P2 mimotope 25

P17 CSQKH LSDRT-NH₂ P3 mimotope 26

P4ex TYQGHTFEDSTKKCADSNPRGV P4 mimotope 27

P5sh GGHFPP P5 mimotope 28

Sequence SEQ ID No.

CFINKQMAD ELC 29 CFMNKQLADLELC 30 KCREVWLGESETIMDC 31

HCQQVFFPQDYLWCQRG 32

SCREVWLGGSEMIMDCE 33

ECNQNLSGSLRHVDLNC 34

DCEEPMCSPVLLQKLKP 35

SCRΞVWLGGSEMIMDCE 36

RCDQQLPRDSYTFCMMS 37

SCPAFPREGDLCAPPTV 38

FCPEPICSPPLSRMTLS 39

VCDECVSRELAL 40

WCLEPECAPGLL 41

VCDECVSRELAL 42

DCLSKGQMADLC 43

SCQGREVRRECW 44

WCREVWLGEΞETIMDCE 45

ACREVWLGESETIMDCD 46

GCAEPKCWQALHQKLKP 47

ECRGPNMQMQDHCPTTD 48

QCNAV EGLQMVDHC 49

CCVADPETQMTPSSEMF 50

HCKNEFKKGQWTYSCΞD 51

QCRQFVMNQSEKEFGQC 52

NCFMNKQLADLELCPRE 53

SCAYTAQRQCSDVPNPG 54

GCFMNKQMADLELCPRTAA 55

ACFMNKQMADLELCPRVAA 56

GCFINKQLADLELCPRVAA 57

GCFMNKQLAD ELCPRAAA 58

ECFMNKQLADSELCPRVAA 59

GCFMNKQLADPELCPREAE 60

GCFMNKQLVDLELCPRGAA 61

GCFMNKQLADLELCPREAA 62

GCFMNKQQADLELCPRGAA 63

GCFINKQMADLELCPREAA 64

Code No. Sequence SΞQ ID No.

PT1078HBC CLEDGQVMDVDCPREAAEGD 65

PT1079HBC CLEDGQVMDVDLCPREAAEGD 66

IgEC26 QCNAVLEGLQMVDHCWN 67

IgEC29 CCVADPETQMTPSSEMF 68

IgEC42 ECLKIEQQCADIVEIPR 69

IgEC69 SCAYTAQRQCSDVPNPG 70

IgEC9 ECRGPNMQMQDHCPTTD 71

IgEC13 ECLVYGQMADCAAGGWP 72

IgEC56 QCRQFVMNQSEKEFGQC 73

IgEC43 HCKNEFKKGQWTYSCSD 74

IgEC81 CCVTDVQTTNMDVPAGQ 75

IgEC83 TCCVTDIPPPDYEQSLG 76

IgEC70 CCESDIPLNELHALADP 77

IgEC64 CCKΞDIPSPVTQFNTMK 78

IgEC73 CCQSDVPHQPGINDLHV 79

IgEC72 CCMSDTPDISRLPVPDS 80

IgEC66 CCMSDSPADPNRGLPIW 71

IgEC75 CCLSDDAPTLPVRR 82

ESC18 CCITDVPQGVMYKGΞPD 83

ESC45 ECKVDGQLSDSPLLRNN 84 ESC12 CCMTDDPMDPNΞTWAIR 85

ESC43 CCMTDDPMYTNSTWAIR 86

ESC1 CCVDDTPNSGLAMRVSK 87

ESC4 CCEVDDFPTHHPGWTLR 88

ESC46 SCNLNHQSCDIPPVKQI 89

ESC20 CCMADQELDLGHNAANA 90

ESD36 CCVMDLELASGF 91

ESD14 CCVMDIEVRGS 92

ESD38 CCQRDVELVFGS 93

ESD15 CCRADFEVGNGG 94

ESD6/10/40 CCVΞDEPAGVRD 95

ESB4/35 GAGWQEKDKELR 96

ESB25 GAMTAGQLΞDLP 97

ESB10/38 VAGGQWDRELK 98

ESB8 KAGEQAMDMELR 99

ESB29/36 RGRNQIMDLEI 100

ESB15 QIDRQITDTLL 101

ESB26 REQQISDVPRV 102

ESB12 CQAMDAEILNQV 103

ESBl/6etC GQMMDTELLNR 104

ESB7 SMEGQVRDIQV 105

ESB18 YQQRDLELLAΞ 106

ESB9 SMGQKVDRELV 107

ESB40 SMGQEVDRELV 108

SB21/33/31 AENDQMVD EI 109

ESB32 GGWQEΞDIPGR 110

ESB4/35 GGWQEKDKELR 111

ESB24 HCCRIDREVSGA 112

ESB13 CAPGMGCWESVK 113

EEC39/50/129 SCREVWLGGSEMIMDCE 114

EEC131 SCPAFPREGDLCAPPTV 115

EEC147 FCPEPICΞPPLSRMTLS 116

EEC40 ECNQNLSGSLRHVDLNC 117

EEC115/3/48 RCDQQLPRDSYTFCMMS 118

EEC36 HCQQVFFPQDYLWCQRG 119

ΞEC17/47/25 DCEEPMCSPVLLQKLKP 120

EEC40A NCQDQMLREDAGCWSKI 121

EEC51/48/53 HCΞEPEYSPATRVFCGR 122

EEC2/23/44/132 DCDWINPPDPPHFWKDT 123

EEC41 ACFSRNGQVTDVPHSCY 124

EEC135 KCPTYPKPNDRCLWPVP 125

EEC116 YCPKYPLEGDCLLDNDY 126

EEC21/19 RCEEWLCIPPAPAFAPP 127

EEC55 TCGQSELRCASLETHHV 128

EEC5 NCNDNPMLDCMPAWSS 129

EEB33 DALDERAWRARA 130

EED183 SCQGREVRRECW 131

EED35/53/164 VCDECVSRELAL 132

EED38 WCLEPECAPGLL 133

EED147/173 DCLSKGQMADLC 134

EED35/53/164 VCDECVSRELAL 135

EED36 GCPTWPRVGDHC 136

EED131/138/102 RCQSARWPECW 137

EED18/47/48 SCAPSGDCGYKG 138

EED132 GCPMWPQPDDEC 139

EED139 ECPRWPLMGDGC 140

EED134 GCQVGELVWCRE 141

EED33 QCVRDGTRKVCM 142 EED50 TCLVDRQESDVC 143

EED34/104 DCWDGDRLVCL 144

EED 1/56 RCEQGALRCVGE 145

EED51 VCPPGWKNLGCN 146

EED57 MCQGWEIVSECW 147

IgEC67-10 ADGAGCFMNKQMADLELCPREAAEA 148

IgEC67-l ADGAGCFMNKQMADLELCPRTAAEA 149

IgEC67-2 ADGAACFMNKQMADLELCPRVAAEA 150

IgEC67-3 ADGAGCFINKQLADLELCPRVAAEA 151

IgEC67-12 ADGAGCFINKQLADLELCPREAAEA 152

IgEC67-9 ADGAGCFMNKQLADLEMCPRDDAEA 153

IgEC67-4 ADGAGCFMNKQLADPELCPREAEEA 154

IgEC67-5 ADGAGCFMNKQLVDLELCPRGAAEA 155

IgEC67-6 ADGAGCFMNNQLADWELCPRAAAEA 156

IgEC67-ll ADGAGCFMNKQMADWEMCPRAAAEA 157

IgEC67-14 ADGAGCFMNKQQADLELCPRGAAEA 158

IgEC67-13 ADGAECFMNKQLADSELCPRVAAEA 159

IgEC67-7 ADGAGCFMNKQLADLELCPREAAEA 160

1-3 ADGAGCFINMQMADQELCPRAAAEA 161

2-13 ADGAGCFINKQMSDFELCPREAGEA 162

3-11 ADGAGCFINKQMADLELCTREAAEA 163

3-1, 3-9, 3-10 ADGAGCFINKQMADLELCPRQAAEA 164

1-11 ADGAGCFINNQMADLELCPRGGAEA 165

2-15 ADGAGCFINKQMADWELCPREGAEA 166

4-9 ADGAGCFINKQMADLELCPSQAAEA 167

1-4, 1-2, 1-12 ADGAGCFINKQMADLELCPREGAEA 168

5-16 ADGAGCFINKQMADSELCPREPAEA 169

4-1 ADGAGCFIKKQMADLELCPREAWEA 170

2-12 ADGAECFINKQMADRELCAREVAEA 171

1-9, 2-5 ADGAGCFIDKQMADLELCPRAAAEA 172

2-9, 2-6 ADGAGCFINKQMADLELCRREAGEA 173

1-16 ADGAGCFKNKQMVDSELCARQAAEA 174

1-5 ADGAGCFQNKQMADLELCPREAAEA 175

4-2, 4-3 ADGAECFINKQRADLELCPGEAAEA 176

1-10 ADGAGCFINKQMADSELCPAAAAEA 177

5-11 ADGAGCFINRQMADPELCPREAAEA 178

1-8 ADGAGCFIEKQMADMELCQARAAEA 179

5-10 ADGAGCFINKQMADWELCPREAAEA 180

5-2 ADGAGCFINNQMADLELCPREAAEA 181

1-1 ADGAGCFIEKQMADMELCQRETAEA 182

2-3 ADGAGCFINKQMADMELCPREAAEA 183

2-8, 1-13, 4-11, 1-14 ADGAGCFINKQMADLELCPREAAEA 184

1-6 ADGAGCFRNKQMADLELCPREAAEA 185

1-7 ADGAGCFINKQMADLELCPARAAEA 186

2-11, 2-4, 2-10, 2-7 ADGAGCFINRQLADMELCSRGAAEA 187

4-4 ADGAECFINRQMADLELCGREAAEA 188

6-9, 5-1, 6-2, 6-8,6-4 ADGAGCFISPQLADWKRCMREAAEA 189

6-12, 5-8 ADGAGCSIHTQMADWERCLREGAEA 190

6-10 ADGAGCSIHRQMADWERCLREGAEA 191

P5longl CSSCDGGGHFPPTIQC P5 mimotope 192 P51ong2 CLQSSCDGGGHFPPTIQLLC P5 mimotope 193 Exa ple 5 .

Other peptides derived from or mimotopes of the Ce3/4 region of IgE, have been conjugated to a carrier using the reversible linker of the invention. These peptides are listed below. Peptide Sequence Sequence SEQ ID

Location and NO.

IgE Domain

P5X RASGKPVNHSTRKEEKQRNGTL Cε3 197

P6X GTRDWIEGE Cε3 198

P7X PHLPRALMRSTTKTSGPRA Cε3/Cε4 199

P8 PEWPGSRDKRT Cε4 (Pro451-T r461) 200

P9 EQKDE Cε4 201

P200 LSRPSPFDLFIRKSPTITC Cε3 202

P210 WLHNEVQLPDARHSTTQPRKT Cε4 203

1-90N LFIRKS Cε3 204

2-90N PSKGTVN Cε3 205

3 - 9ON LHNEVQLPDARHSTTQPRKTKGS Cε4 206

4-90N SV-.PGK Cε4 207

Pll CRASGKPVNHSTRKEEKQRNGLL P5 mimotope 208

PIla (Ac) GKPVNHSTGGC P5 mimotope 209

Pllb (Ac) GKPVNHSTRKEEKQRNGC P5 mimotope 210

Pile CGKPVNHSTRKEEKQRNGLL (NH₂) P5 mimotope 211

PIId (Ac) RASGKPVNHSTGGC P5 mimotope 212

P12 CGTRDWIEGLL P6 mimotope 213

P12a CGTRDWIEGETL (NH₂) P6 mimotope 214

P12b (Ac) GTRDWIEGETGC P6 mimotope 215

P13 CHPHLPRALMLL P7 mimotope 216

P13a CGTHPHLPRALM (NH₂) P7 mimotope 217

P13b (Ac) THPHLPRALMRSC P7 mimotope 218

P13c (Ac) GPHLPRALMRSSSC P7 mimotope 219

P14 APEWPGSRDKRTC P8 mimotope 220

P14a (Ac) APEWPGSRDKRTLAGGC P8 mimotope 221

P14b CGGATPEWPGSRDKRTL (NH₂) P8 mimotope 222

P14c CTRKDRSGPWEPA (NH₂) P8 retro 223

P14d* (Ac) APCWPGSRDCRTLAG P8 mimotope 224

(cyclic)

P14d (Ac) ACPEWPGSRDRCTLAG P8 mimotope 225

(cyclic)

C-1C14 CATPEWPGSRDKRTLCG P8 mimotope 226

C-1C13 CATPEWPGSRDKRTCG P8 mimotope 227

C3C12 TPCWPGSRDKRCG P8 mimotope 228

P9a CGAEWEQKDEL (NH₂) P9 mimotope 229

P9b (Ac) AEWEQKDEFIC P9 mimotope 230

P9b* (Ac) GEQKDEFIC P9 mimotope 231

P9a* CAEGEQKDEL (NH₂) P9 mimotope 232

Carll CPEWPGCRDKRTG P8 mimotope 233

Carl2 TPEWPGCRDKRCG P8 mimotope 234 Peptide sequence Mimotope of SEQ ID NO.

CSRPSPFDLFIRKSPTITC A-B loop of Cε3 235 CSRPSPFDLFIRKSPTC A-B loop of Cε3 236 CPSPFDLFIRKSPTITC A-B loop of Cε3 237 CPSPFDLFIRKSPC A-B loop of Cε3 238

Peptii ie sequence

C S R P S P F D L F I R K S P T I C 239 c s R P S P F D L F I R K S P C 240

C R P s P F D L F I R K S P C 241

C R P s P F D L F I R K S P T C 242

C R P s P F D L F I R K S P T I C 243

C R P s P F D L F I R K S P T I T C 244

C P s P F D L F I R K S P T I C 245

C P S P F D L F I R K S P C 246

C Y A F A T P E W P G S R D K R T L A 247

C Y A F A T P E W P G S R D K R T L C 248

C Y A F A T P E W P G S R D K R T C 249

C Y A F A T P E W P G S R D K R C 250

C A F A T P E W P G S R D K R C 251

C A F A T P E W P G S R D K R T c 252

C A F A T P E W P G S R D K R T L c 253

C A F A T P E W P G S R D K R T L A C 254

C F A T P E W P G S R D K R T L A C 255

C F A T P E W P G S R D K R T L c 256

C F A T P E W P G S R D K R T C 257

C F A T P E W P G S R D K R C 258

C T W s R A S G K P V N H S T R C 259

C T W Ξ R A S G K P V N H S T C 260

C T W S R A S G K P V N H S c 261

C T W S R A S G K P V N H C 262

C W S R A S G K P V N H C 263

C W S R A S G K P V N H S C 264

C W S R A S G K P V N H S T c 265

C W S R A S G K P V N H S T R C 266

C s R A S G K P V N H S T R c 267

C s R A S G K P V N H Ξ T C 268

C s R A S G K P V N H S C 269 c s R A S G K P V N H C 270

C Q W L H N E V Q L P D A R H S C 271

C Q W L H N E V Q L P D A R H C 272

C Q W L H N E V Q L P D A R C 273

C Q W L H N E V Q L P D A C 274

C W L H N E V Q L P D A C 275

C W L H N E V Q L P D A R C 276

C W L H N E V Q L P D A R H C 277

C W L H N E V Q L P D A R H S C 278

C L H N E V Q L P D A R H S C 279

C L H N E V Q L P D A R H C 280

C L H N E V Q L P D A R C 281

C L H N E V Q L P D A C 282

Claims

1. A compound of general formula (A) : Epitope-R,-X-NH₂ (A) wherein Epitope = Ep-F;

Ep = a drug or other organic molecule or peptide (linear or constrained or cyclised) comprising or consisting of amino acids, the amino acids being proteinogenic or synthetic non- proteinogenic and constrained being a peptide containing a disulphide bridge, lactam (amide) bridge or other chemical entity, any of which linking two side chains together or side- chain to backbone or backbone to backbone;

F = G-NH-, G-N(R)- (where R is a C5 alkyl group), G-CO- , G-S- or G-CH= , -G- S0₂-, the attachment point to Rj. (defined below) that is an integral part of the Epitope and may be derived from proteinogenic or non-proteinogenic amino acid derivatives positioned within a peptide chain, at the N-terminus or at the C-terminus ; G =

-(^CH2)n- -(CH₂) -(CH₂), and n = 1 to 6 and m = 0 to 6 ; R = I-J-V-, a bifunctional molecule; wherein V = -CO-, -NH-, -S0₂- and -S- ; preferably -CO-; and n = 2 to 12, preferably 2 , m = 0 to 6 and R₃ = -N0₂, -C0₂H, -S0_3;

I = attached to J as shown to provide: -NH-J, -CO-J, -S-J, - N(R)-J (where R is a C5 alkyl group), -S0₂-J and -(CH₂)_n-J (where n = 1 to 6)

and X = -CH₂-, -O- , -NH- or -CO-.

2. A compound according to claim 1 wherein I of R_x = -CO-

3. A compound according to claim 1 wherein J of R_x = (CH₂)₂

4. A compound according to claim 1 wherein V of R_x = -CO-

5. A compound according to claim 1 where R_x = -CO- (CH₂) ₂-C0-

6. A compound according to claim 1 where X = -NH-

7. A compound of general formula (B) : :—Carrier (B) wherein

R2 = general formula (K) : where R₅ contains any of the following;

(CH₂)_π- -(CH₂)- V(CH₂) where n = 2 to 12, preferably 4 and m = 0 to 6, preferably 1;

R₄ = H or CH₃-(CH₂)_n- where n = 0-5, preferably 0;

R₆ = -NH-, -N(R)- (where R is a C5 alkyl group), -N=, -CO-, -

S-, -S0₂-, -CH=, -(CH₂)- -N-CO-or -0-CO-; preferably -CO- or -

NH- ; and

Carrier is any macromolecular species containing at least four sites for attachment of an Epitope through the following surface groups attached as shown to the carrier; carrier-NH-, carrier-CO-, carrier-S-, carrier-S0₂- , carrier-N(R) - , carrier-

CH= or carrier- (CH₂) _n- (where n = 1 to 6) .

8. A compound according to claim 7 wherein R₄ of K = -H

9. A compound according to claim 7 wherein R₅ of K = -(CH₂)₄-

10. A compound according to claim 7 wherein R₆ of K = -CO¬

IL A compound of general formula (C) : l_|

Epitope-R_rX-N _=< ^(c)

R₂- C a r r i e r which is a conjugate of a compound of formula A in claim 1 with a compound of formula B in claim 7 .

12. A compound according to claim 11 wherein the compound of formula C is

Carrier- CO-(CH₂)

13. A compound according to any of claims 1 to 12 wherein the moiety Ep is a constrained or cycl sed peptide sequence.

14. A compound according to any of claims 1 to 13 wherein the moiety Carrier is a protein.

15. A compound according to claims 11 - 14 which s an immunogenic conjugate of compounds of formula (A) and (B) as defined m claims 1 to 10.

16. A compound according to any of claims 1 to 6 wherein the moiety Ep is selected from the group consisting of peptides of SEQ ID Nos . 1-282.

17. A compound according to any of claims 7 to 15 wherein the carrier is selected from the group consisting of: Keyhole Limpet Haemocyanm, Serum albumin, Bovine serum albumin,

Inactivated bacterial toxins,

Tetanus toxin,

Diptheria toxin,

Recombinant fragments of tetanus toxins,

Recombinant fragments of diptheria toxins,

Domain 1 of Fragment C of tetanus toxin,

Translocation domain of diptheria toxin,

Purified protein derivative of tuberculin,

Protein D,

Fragments of Protein D,

Protein D l/3^rd

Lipsome carriers

18. An immunogenic composition comprising or consisting of a immunogenic conjugate of a compound according to claim 16 and a compound according to claim 17.

19. A vaccine composition comprising or consisting of an immunogenic conjugate according to claim 15 or 18, together with an adjuvant.

20. A vaccine composition as claimed in claim 19 wherein said adjuvant is selected from aluminium or calcium salts.

21. An antibody induced by a vaccine as claimed in any one of claims 19 and 20, or induced by an immunogenic conjugate as claimed in any one of claims 15 and 18.

22. An isolated and purified antibody according to claim 21.

23. A method of making an immunogenic conjugate as claimed in claim 15 or 18 comprising fusing an Epitope to a carrier by chemical conjugation of a compound of formula (A) to a compound of formula (B) .

24. A method of making a vaccine comprising manufacturing an immunogenic conjugate as claimed in claim 15 or 18 and formulating said immunogen with an adjuvant.

25. A pharmaceutical composition comprising a compound of claim 15 or a composition of claim 18 and a pharmaceutically acceptable excipient for the treatment of IgE mediated disease .

26. The use of a compound of claim 15 or a composition of claim 18 in the manufacture of a medicament for the treatment of IgE mediated disease.

27. A method for treatment for a patient suffering from an IgE mediated disease comprising the administration to the patient of an effective amount of: (i) A compound according to any of claims 11 to 17

(ii) An immunogenic composition according to claim 18

(iii) A vaccine according to claims 19 or 20

(iv) An antibody according to claims 21 or 22

(v) A pharmaceutical composition according to claim 5