AU744911B2 - Catalytic antibodies and a method of producing same - Google Patents

Description

WO 99/15563 PCT/AU98/00783 -1- CATALYTIC ANTIBODIES AND A METHOD OF PRODUCING SAME FIELD OF THE INVENTION The present invention relates generally to a growth factor precursor and its use to select production of antigen specific catalytic antibodies. Such catalytic antibodies are produced following B cell activation and proliferation induced by catalytic cleavage products of a target antigen portion of the growth factor precursor of the present invention. A particularly useful form of the growth factor precursor is as a nucleic acid vaccine. The nucleic acid vaccine of the present invention preferably further comprises a molecular adjuvant. Another aspect of the present invention comprises a growth factor precursor in multimeric form. The growth factor precursor of the present invention is useful for generating catalytic antibodies for both therapeutic, diagnostic and industrial purposes.

BACKGROUND OF THE INVENTION The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. A particularly important area of research is the use of recombinant antigens to stimulate immune response mechanisms and outcomes. However, recombinant techniques have not been fully effective in generating all components of the humoral response. One such important yet not fully exploited component is the catalytic antibody.

Catalytic antibodies are highly substrate specific catalysts which can be used, for example, to proteolytically activate or inactivate proteins. Catalytic antibodies have great potential as therapeutic agents in human diseases such as rheumatoid arthritis, AIDS and Alzheimer's disease amongst many others.

I! Antibody therapy has been used in patients. Antibodies have a half-life of about 23 days in the circulation of humans which-is a clear advantage over other drugs. Catalytic antibodies, however, are considered to be even more effective. They are recycled after their antigenic U I ri~ WO 99/15563 W099/5563PCT/AU98/00783 -2encounter and are not bound to the antigen as occurs with "classical" antibodies. Catalytic antibodies should, therefore, function at a much lower dose than classical antibodies and could be used at sub-immunogenic doses. Catalytic antibodies would be particularly useful in long term therapy.

Traditionally, catalytic antibodies have been generated by imimunising mice with transition state analogs. Such antibodies have been shown to catalyse several chemical reactions.

However, this approach has a severe limitation in that it is difficult to predict the structure of transition state analogs which effect proteolysis of specific proteins. Immunising a mouse with a transition state analog is by definition inefficient since it selects B cells on the ability of surface immunoglobulins to bind the analogs and not on the ability of a surface immunoglobulins to catalytically cleave the analogue. This is one of the reasons why catalytic antibodies have relatively low turn-over rates and cannot compete with the naturally occurring enzyme counterparts, in the case where they exist.

Another approach has been the mutation of conventional antibodies to alter their activity to be catalytical like in nature. However, to date, such an approach has not proved successful.

As a consequence, catalytic antibodies have not previously achieved prominence as therapeutic, diagnostic or industrial tools.

There is a need, therefore, to develop a more efficaciou s approach to generating catalytic antibodies having desired catalytic specificity.

International Patent Application No. PCT/AU97/00194 filed on 26 March 1997 and is herein incorporated by reference provided a means for selecting catalytic B cells. The method contemplated a growth factor comprising two Ig binding domains from protein L of Peptostreptococcus ma gnus as B cell surface molecule binding portions flanking a T cell surface molecule binding portion (designated from hen egg lysozyme (HEL). Thie specificity of the LHL growth factor for catalytic B cells was provided by an antigen masking or attached to a molecule masking one or more of the B cell surface molecule binding WO 99/15563 PCTIAU98/00783 -3portions. Catalytic cleavage of the antigen exposed the B cell surface molecule binding portions to permit catalytic antibody production.

In accordance with the present invention, there is provided an improved growth factor precursor.

SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Sequence Identity Numbers (SEQ ID NOs.) for nucleotide and amino acid sequences referred to herein are defined following the Examples.

One aspect of the present invention is directed to a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor, associate together by intra- and/or inter-domain bonding and substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule.

Another aspect of the present provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent WO 99/15563 PCT/AU98/00783 -4thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor associate together by intra- and/or interdomain bonding and substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule wherein if said growth factor precursor comprises a single B cell surface molecule binding portion, then the growth factor precursor further comprises a multimerising inducing element.

Yet another aspect of the present invention provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least two B cell surface molecule binding portions, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain such that in the growth factor precursor, these variable chain domains associate together by intra- and/or inter-domain bonding and, when associated together, substantially prevent at least one of the B cell surface molecule binding portions from interacting with a B cell surface molecule wherein upon cleavage of said antigen by a catalytic antibody, the at least two B cell surface molecule binding portions induce activation and proliferation of a B cell expressing said catalytic antibody.

p' WO 99/15563 PCT/AU98/00783 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation showing the structure of LgL comprising ompA and the hexa-his-Tag on the C terminus.

Figure 2 is a photographic representation showing production of OHLgL in E. coli using w/v PHAST-gels.

Figure 3 is a graphical representation of the 280 nm absorbance trace showing purification of LgL on a HPLC superose 12 column.

Figure 4 is a photographic representation of LgL fractions from a HPLC superose 12 column on a 20% w/v PHAST gel.

Figure 5 is a graphical representation showing biological potency of LgL as demonstrated by B7-1 and B7-2 expression after overnight stimulation.

Figure 6 is a diagrammatic representation showing structure of ccMTLgL comprising LgL with TEV cleavage signal and disulphide linked single chain Fv from McPc603.

Figure 7 is a photographic representation of ccMTLgL containing fractions from a FLAG M1 affinity column analysed on a PHAST-gel.

Figure 8 is a graphical representation of the 280 nm absorbance trace of fractions containing ccMTLgL from an HPLC superose 12 gel.

Figure 9 is a photographic representation of ccMTLgL fractions from HPLC superose 12 gel analysed on PHAST gel.

Figure 10 is a photographic representation showing presence of inter-domain disulphide bond in ccMTLgL on 20% w/v PHAST gel under reducing and non-reducing conditions, before and after cleavage with TEV.

f p WO 99/15563 PCT/AU98/00783 -6- Figure 11 is a graphical representation showing B7-1 expression after overnight stimulation of mesenteric lymph node cells with anti-g, LgL, ccMTLgL and ccMTLgL TEV.

Figure 12 is a graphical representation showing the results of repeating the experiment associated with Figure 11 except that TEV is also added in situ to the overnight B cell cultures.

Figure 13 is a schematic representation of ompL.

Figure 14 is a schematic representation of Fv-catAb.

Figure 15 is a photographic representation of a silver stained 20% w/v PAGE SDS PHASTgel analysis of scM603 purified from periplasmic fraction via an L-column.

V. WO 99/15563 PCT/AU98/00783 -7- The following abbreviations are used in the specification.

ccMTLgL Growth factor precursor comprising LgL linked to variable heavy and light chain domains from antibody McPc603 via TEV sensitive peptide FSC Forward light scatter g Glycine-serine linker having the structure (GGGGS) 4 H T cell surface molecule binding portion from hen egg lysosyme (HEL) hulgG Human immunoglobulin G L B cell surface molecule binding portion from protein L of Peptostreptococcus magnus LgL Two L molecules linked via glycine-serine peptide LHL Growth factor comprising H flanked by two L molecules McPc603 Antibody having anti-phosophorylcholine specificity TLHL LHL linked to kappa light chain via TEV sensitive peptide and g attached to N terminus region 01 t, WO 99/15563 PCT/AU98/00783 -8-

MOLECULE

LHL

CATAB-TEV

ThHL LHLseq FLAG epitope Kappa LHL-omnp Strep-tag ccMTLgL SUMMARY OF SEQ ID NOs.

SEQ ID NO.

Nucleotide Amino acid 1 2 3 4 5 6 7 8 V WO 99/15563 PCT/AU98/00783 -9- DETAILED DESCRIPTION OF THE INVENTION The present invention provides in part an improved growth factor precursor capable of selecting catalytic B cells. The selected catalytic B cells then undergo mitogenesis including activation and proliferation as a pre-requisite for the production of catalytic antibodies.

Accordingly, one aspect of the present invention is directed to a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor associate together by intra- and/or inter-domain bonding and, when associated together, substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule wherein if said growth factor precursor comprises a single B cell surface molecule binding portion, then the growth factor precursor further comprises a multimerising inducing element.

The present invention further provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a Svariable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor associate together by intra- and/of ifiterdomain bonding and, when associated together, substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that ar WO 99/15563 WO 9915563PCT/AU98/00783 upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permnits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule wherein if said growth factor precursor comprises a single B cell surface molecule binding portion, then the growth factor precursor further comprises a multimerising inducing element.

The growth factor precursor is deemed a "precursor" since it is substantially incapable of inducing B cell rnitogenesis activation and proliferation followed by antibody production) in the absence of catalytic cleavage of a portion of the growth factor precursor which masks at least one B cell surface molecule binding portion on the molecule. By masking the B cell surface molecule binding portion, the growth factor precursor is substantially incapable of inducing B cell mitogenesis such as by, but not limited to, cross-linking of B cell surface immunoglobulins. The term "masks" or "masking" includes the steric, conformational, electrostatic and/or physical interference at or proximal to at least one B cell surface molecule binding. portion on the growth factor precursor thus preventing interaction between the B cell surface molecule binding portion and a B cell surface molecule. One of the catalytic products of the growth factor precursor of the present invention is a growth factor capable of inducing B cell mitogenesis.

The growth factor precursor of the present invention may be synthesised as a single polypeptide chain. The polypeptide chain comprises various regions such as a component of the variable heavy chain and a component of a variable light chain of an immunoglobulin (referred to herein as variable light chain and variable heavy chain domains), a target antigen, a T cell surface molecule binding portion and at least one B cell surface molecule binding portion. Additional regions may also be included such as purification tags including FLAG and hexa-his and a molecular adjuvant such as but not limited to CMd, CThA4 and/or CD4OL. Such a polypeptide may be produced from fusing together a series of nucleotide sequences to produce a single nucleic acid molecule which, when expressed in an appropriate host cell, produces a single amino acid sequence in the form of the polypeptide.

WO 99/15563 WO 9915563PCT/AU98/00783 -11 I- Alternatively, the polypeptide chain may be made in modular form and the modules bound, ligated, linked or otherwise associated together. For example, the growth factor precursor may comprise a multimodular molecule having a module comprising a B cell surface molecule binding portion, a module comprising a T cell surface molecule binding portion, and one or more modules comprising the variable heavy chain domain and variable light chain domain.

The modular components may be bound, ligated or otherwise associated together by any convenient means such as but not limited to peptide bonding, electrostatic attraction, covalent bonding, di-suiphide bridges and/or hydrogen binding. A combination of covalent and peptide bonding and disulphide bridging are particularly preferred in forming a growth factor precursor from the modules.

The growth factor of the present invention functions after catalytic processing. Where the growth factor precursor comprises two B cell surface molecule binding portions, the masking effect of the variable heavy and light chains may be in respect of both B cell surface molecule binding portions or only one B cell surface molecule binding portion. Where the growth factor precursor molecule comprises only one B cell surface molecule binding portion then a multimerizing inducing unit or multimer forming portion may also be included in order to form multimers of the B cell surface molecule binding portion of the growth factor.

In a related aspect, the present invention provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor, associate together by intra- and/or inter-domain bonding and substantially prevent thfe at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said 0, WO 99/15563 PCT/AU98/00783 -12variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule.

The T cell surface molecule binding portion provides T cell dependent help for the B cell.

The T cell surface molecule binding portion is preferably part of the growth factor precursor but may alternatively be exogenously supplied. An example of an exogenously supplied portion having T cell dependent help from a B cell is anti-CD40L antibodies or functional equivalents thereof.

In a further aspect of the present invention, the multimizing inducing portion comprises a signal peptide such as from the outer membrane protein A (ompA) or a functional equivalent or derivative thereof linked preferably to the C-terminal portion of the growth factor.

In a particularly preferred embodiment, the B cell surface molecule binding portions comprises a B cell surface binding portion such as a B cell surface immunoglobulin although the present invention extends to a range of B cell surface molecules the binding, interaction and/or cross-linking of which leads to or facilitates B cell mitogenesis.

The present invention further contemplates a composition of matter capable of inducing B cell mitogenesis of a catalytic B cell after catalytic processing said composition of matter comprising components selected from: a recombinant or synthetic molecule capable of inducing a B cell surface molecule binding portion in multimeric form; (ii) a recombinant or synthetic molecule of comprising a further portion providing a T cell surface molecule binding portion; and (iii) separate compositions mixed prior to use or used sequentially or simultaneously comprising in a first composition a component having a B cell surface molecule binding portion and in a second composition a molecule capable of providing aT cell surface molecule binding portion; 1P6~-~if~T~-iP ill L ~_i WO 99/15563 PCT/AU98/00783 -13said composition of matter further comprising a recombinant or synthetic B cell surface molecule binding portion masked by components of a variable heavy chain domain and a variable light chain domain which variable heavy and light chains are associated together by intra- and/or inter-domain bonding.

In a related embodiment, the present invention is directed to a composition of matter capable of inducing B cell mitogenesis of catalytic B cells after catalytic processing said composition of matter comprising components selected from: a recombinant or synthetic molecule comprising a B cell surface molecule binding portion; (ii) a recombinant or synthetic molecule comprising a B cell surface molecule binding portion and a signal peptide linked to the C-terminal portion of the B cell surface molecule binding portion; (iii) a recombinant or synthetic molecule of or (ii) comprising a further portion providing a T cell surface molecule binding portion; and (iv) separate compositions mixed prior to use or used sequentially or simultaneously comprising in a first composition a component having a B cell surface molecule binding portion and in a second composition a molecule capable of providing a T cell surface molecule binding portion; said composition of matter further comprising a recombinant or synthetic B cell surface molecule binding portion masked by components of a variable heavy chain domain and a variable light chain domain which variable heavy and light chains are associated together by intra- and/or inter-domain bonding.

Preferably, for example to facilitate cross-linking of B cell surface molecules to induce mitogenesis activation and proliferation), the growth factor comprises at least two B cell surface molecule binding portions. Alternatively, where the growth factor is present in multimeric form or is capable of being presented in multimeric form, the molecule may comprise a single B cell surface molecule binding portion.

WO 99/15563 PCT/AU98/00783 14- The presentation of a T cell surface molecule binding portion on the surface of a B cell allows for B cell mitogenesis. The term "B cell mitogenesis" is used herein in its broadest context and includes B cell activation and proliferation, clonal expansion, affinity maturation and/or antibody secretion as well as growth and differentiation.

In accordance with the present invention, a multimer comprises two or more growth factor molecules or a precursor thereof. Examples of portions inducing multimerisation include but are not limited to an antibody, a region facilitating formation of cross-linked molecules or a signal peptide. Cross-linkage in this context includes any interaction that provides bonding adequate to lead to multimer formation including but not limited to covalent linkage, ionic linkage, lattice association, ionic bridges, salt bridges and non-specific molecular association.

A particularly preferred embodiment of the present invention is directed to the use of a signal peptide such as the signal peptide of ompA [Skerra, Gene, 151: 131-135, 1994] or a functional derivative thereof. A "functional derivative" in this context is a mutant or derivative of the ompA signal peptide (or its functional equivalent) which still permits multimer formation of the growth factor.

An example of a suitable B cell surface molecule binding portion is protein L from Peptostreptococcus magnus. Protein L has five immunoglobulin-binding domains. Other immunoglobulin binding molecules include protein A, protein G and protein H. The present invention, however, extends to any molecule capable of binding to a B cell surface component including, for example, a ligand of a B cell receptor.

The portion of the recombinant or synthetic molecule defining a T cell surface molecule binding portion is presented to a preferably already primed T cell to induce B cell proliferation and affinity maturation of an antibody in the germinal centre. This is generally accompanied by immunoglobulin class switching and antibody secretion into the serum.

Generally, the T cell surface molecule binding portion is interalised within the B cell and presented on major histocompatibility complex (MHC) class I.

1. WO 99/15563 PCT/AU98/00783 An example of a T cell surface molecule binding portion is from hen egg lysozyme (HEL) [Altuvia et al, Molecular Immunology, 31: 1-19, 1994] or is a derivative thereof such as a peptide comprising amino acids 42 to 62 from HEL or a homologue or analog thereof. This T cell surface molecule binding portion is recognised by the T cell receptor (TCR) of HEL specific T cells when presented by an antigen presenting cell (APC) on the MHC class II molecule H-2AK in mice or other MHC class II molecules or their equivalents in other mammals such as humans. Examples of other T cell surface molecule binding portions include but are not limited to tetanus toxoid, ovalbumin, malarial antigens as well as other regions of HEL. One skilled in the art would readily be able to select an appropriate T cell surface molecule binding portion.

In an alternative embodiment, the portion providing the T cell surface molecule binding portion functions like a T cell epitope. An example of such a portion is an antibody.

As stated above, the B cell surface molecule binding portions induce B cell activation and blast formation. The internalisation and processing of the growth factor leads to the presentation of the antigen on MHC II. T cell recognition of MHC I with the antigen signals the activated B cell to proliferate and undergo antibody class switching and secretion.

The mitogenic growth factor of the present invention is most useful in generating antibodies of desired catalytic specificity when, in a precursor form, it selects "catalytic" B cells. The precursor growth factor comprises a target antigen to which a catalytic antibody is sought and contains components which mask antigen-independent clonal expansion of B cells. Upon cleavage of the antigen by a selected B cell surface immunoglobulin, the growth factor can induce B cell mitogenesis.

In effect, then B cells are selected on the catalytic activity of their surface immunoglobulin rather than on their binding to a transition state analog. This allows for affinity maturation in the germinal centres and ensures "catalytic-maturation" to obtain the highest enzymatic turnover rate possible in vivo. This aspect of the present invention is achieved by designing r- 1, WO 99/15563 PCT/AU98/00783 -16growth factor precursor shielded and substantially inactive until released through cleavage by a catalytic antibody on a B cell surface. The term "cleavage" in this context is not limiting to the breaking of bonds but includes an interaction adequate to remove or reduce shielding of the B cell growth factor.

The liberated growth factor activates the catalytic B cell via the B cell surface molecule binding portion domains. The growth factor is then internalised and processed analogous to a normal antigen. Intracellular processing permits the T cell surface molecule binding portion being presented on the B cell surface and this leads to T cell dependent clonal expansion of the B cell as well as catalytic maturation and secretion of the catalytic antibody. The catalytic antibodies can then be detected in serum and "catalytic" B cells can be recovered by standard techniques.

The antigen according to this aspect of the present invention is any antigen to which a catalytic antibody is sought. Examples include cytokines such as but not limited to tumor necrosis factor (TNF), an interleukin (IL) such as IL-1 to IL-15, interferons (IFN) such as IFNa, IFNp or IFNy, colony-stimulating factors (CSF) such as granulocyte colonystimulating factor (G-CSF), granulocyte-macrophase colony-stimulation factor (GM-CSF), blood factors such as Factor VII, erythropoietin and haemopoietin, cancer antigens, docking receptors from pathogenic viruses such as HIV, influenza virus or a hepatitis virus (eg. HEP A, HEP B, HEP C or HEP E) and amyloid plaques such as in Alzheimer's disease patients or myeloma patients. More particularly, in the case of TNF, proteolytic inactivation of TNF would be useful in the treatment of rheumatoid arthritis and toxic shock syndrome. By targeting viral, docking receptors, pathogenic viruses such as HIV, hepatitis viruses and influenza viruses are rendered effectively inactive. Catalytic antibodies will also be useful in the clearance of amyloid plaques in Alzheimer's disease or myeloma disease patients.

Targeting IgE, for example, may provide a mechanism for treating inflammatory conditions such as asthma.

The catalytic antibodies of the present invention may also be useful in detoxifying drugs such as drugs consumed by an individual. For example, the effects of cannabis or heroin or other J, WO 99/15563 PCT/AU98/00783 17drugs could be treated in an individual by the administration of catalytic antibodies directed to the active components of those drugs (Mets et al. Proc. Natl. Acad. Sci. USA 95: 10176- 10181, 1998). Furthermore, catalytic antibodies may be useful in the treatment of autoimmune and inflammatory disease conditions such as by targeting autoimmune antibodies. Catalytic antibodies also have a use in environmental and other industrial situations and could be directed to environmental pollutants such as petroleum products and plastics. In all these situations, suitable antigens would be selected and incorporated into the growth factor precursor of the present invention.

In a related aspect of the present invention, the "antigen" portion of the growth factor precursor can be mimicked by a target site such as an amino acid linker sequence comprising a protease cleavage site. Examples include an amino acid linker sequence comprising the tabacco etch virus (TEV) protease cleavage site. More particularly, in the case of a TEV protease cleavage site, cleaving of the amino acid linker sequence by the TEV protease would be useful for producing characteristics similar to those of a catalytic antibody. This provides a useful model system for developing growth factor molecules.

The growth factor precursor enables an antigen to be recognised by a B cell via a growth factor capable of inducing B cell mitogenesis. The growth factor is in "precursor" form until cleavage of all or part of the antigen. It is important, however, that the B cell surface molecule binding portions be "masked" until catalytic B cells induce cleavage of the target antigen and exposure of the B cell surface molecule binding portions. Masking is provided by molecules capable of binding to or otherwise associating with the B cell surface molecule binding portion. In a particularly preferred embodiment, the masking molecules are all or a portion of the variable heavy chain domain and variable light chain domain of an immunoglobulin.

In a particularly preferred embodiment, a fragment comprising a variable heavy and light chain (Fv domains) is employed which is a single chain (sc) and/or disulphide stabilize-d (ds).

The scdsFV fragment is conveniently obtainable from plasmacytoma McPc603, described in (Freund et al. Biochemistry, 33: 3296-3303, 1994). The variable light and heavy chain i- WO 99/15563 PCT/AU98/00783 -18regions are preferably present as a single amino acid sequence. The regions fold and associate together by inter-domain attractive forces. Intra-domain attractive forces may also be involved. Preferably, the intra- and inter-domain attractive forces are disulphide bonds but the present invention extends to other forces capable of stabilising the domains such that they fold over or are in close proximity to at least one B cell surface molecule binding portion thus preventing B cell surface molecule binding portion interaction with a B cell surface molecule.

Reference to inter- and intra-domain bonding means bonding with the polypeptide chain of the growth factor precursor and not to bonding between different polypeptide chains.

Accordingly, another aspect of the present invention is directed to a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor, associate together by intra- and/or inter-domain bonding and substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule.

In a related embodiment, the present invention provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from b6tiha variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain domains in the growth factor precursor associate t. WO 99/15563 PCT/AU98/00783 -19together by intra- and/or inter-domain bonding and substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule wherein if said growth factor precursor comprises a single B cell surface molecule binding portion, then the growth factor precursor further comprises a multimerising inducing element.

Another aspect of the present invention provides a growth factor precursor comprising a recombinant polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least two B cell surface molecule binding portions, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain such that in the growth factor precursor, these variable chain components associate together by intra- and/or inter-domain disulphide bridges and, when associated together, substantially prevent at least one of the B cell surface molecule binding portions from interacting with a B cell surface ligand for said epitope wherein upon cleavage of said antigen by a catalytic antibody, the at least two B cell surface molecule binding portions induce activation and proliferation of a B cell expressing said catalytic antibody.

A particularly useful masking molecule is derived from the variable heavy and light chain of McPc603. The latter molecule is expressed in the periplasmic space of DH10B and can be purified on an L-column. The variable heavy and light chain components is preferably present on a single peptide chain.

In a particularly preferred embodiment, the recombinant or synthetic growth factor precursor substantially prevents binding of at least one B cell surface molecule binding portion to a cognate B cell surface immunoglobulin thereby preventing B cell activation by having immunoglobulin peptide(s) or chemical equivalents thereof linked, fused or otherwi-seassociated with the growth factor precursor to facilitate masking of the B cell activating effects of the growth factor. In a particularly preferred embodiment, the precursor comprises ''WO 99/15563 PCT/AU98/00783 an antigen to which a catalytic antibody is sought and portions capable or masking a B cell surface molecule binding portionon the growth factor precursor. The precursor preferably contains domains for variable heavy and light chain components which associate together and exhibit inter- and intra-domain disulphide bridges.

Generally, the immunoglobulin molecules which bind to the B cell surface molecule binding portion of the growth factor are linked to the N-terminal and/or C-terminal portions of the growth factor. For example, one particularly preferred embodiment of the present invention provides a growth factor precursor comprising the structure: I' A X, [X 2 ]d [X 3 ]a I" wherein: X, and X 3 are B cell surface molecule binding portions; CA i s Oo' 1 orI a is 0 or 1 or I' and I" are either both present or only one is present and they may be the same or different and each is a blocking reagent for X, and/or X 3 such as a variable heavy and light chain or a sc-ds-Fv molecule; A is the target antigen for which a catalytic antibody is sought;

X

2 is an entity providing T cell dependent help to a B cell; and r is 0, 1 or >1, wherein a catalytic antibody on the surface of said B cell is capable of cleaving all or part of A from said recombinant or synthetic molecule resulting in the molecule X [X 3 wherein A' is optionally present and is a portion of A after cleavage with the catalytic antibody wherein said resulting molecule is capable of inducing T cell dependent B cell mitogenesis of the B cell to which X, and X 3 bind.

The molecular components of I'A X, X 2

X

3 A I" may be in any sequence order.

In another embodiment, the I' A Xi X 2

X

3 A I" molecule or part thereof may be in multimeric form. This is particularly the case when all or part of the molecule includes a multimerisation component such as but not limited to the signal peptide of ompA. The monomeric units may be bound or otherwise associated together by any number of binding WO 99/15563 PCT/AU98/00783 -21 means such as contemplated above including covalent bonding, salt bridges, disulphide bridges and hydrophobic interactions amongst many others. Depending on the extent of multimerisation, this may impair the masking ability of B cell surface molecule binding domains of the growth factor and some antigen-independent clonal expansion may occur.

This may not be too disadvantageous where there is at least some catalytic antibody dependent B cell mitogenesis.

According to this embodiment, there is provided a growth factor precursor comprising the structure: A X 1

[X

2 ']o[X 2

X

3 [A]p wherein: I' and I" are both present or only one is present and each is a blocking reagent for X, and/or

X

3 such as a variable heavy or light component or an sc-ds-Fv; A is the target antigen for which a catalytic antibody is sought; X, and X 3 are B cell surface molecule binding portions;

X

2 and X 2 may be the same or different and each is an entity capable or providing T cell dependent help for a B cell; o may be 0 or 1; Smay be 0 or 1; n indicates the multimeric nature of the component in parentheses and may be 0, 1 or >1; m indicates the multimeric nature of the component in parenthesis and may be 1 or >1.

Preferably, n and m are each from about 1 to about 10,000 more preferably from about 1 to about 1,000 and still more preferably from about 1 to about 200.

Preferably, if n is 0, then o is 1.

In alternative embodiments, the growth factor precursor comprises the structure AX X 3 2 A I"]m or A X, [X2t]o]n[X, X 3 A I"]m] 0 WO 99/15563 PCT/AU98/00783 -22- The exact number ascribed to n and m may not be ascertainable but the multimeric nature identified functionally or physically by size (eg. determined using HPLC or PAGE).

The present invention is now described by way of example only with reference to a particular growth factor precursor analogue. This analogue is capable of mimicing a growth factor precursor but uses an enzyme sensitive molecule in place of the antigen. Such an analogue is a useful model for designing growth factor precursors.

The growth factor precursor analogue comprises modular components linked together by a glycine-serine bridge referred to as [ggggs] 4 The unit is present four times. It is abbreviated herein Two B cell surface molecule binding portions, L, are linked by a g bridge to form the core L-g-L. On the carboxy end of the B cell surface molecule binding portion, a hexahis Tag is linked to form: L-g-L-6xHis. The N terminal end of the molecule comprises a TEV protease cleavage site to provide the molecule: TEV-L-g-L-6xHis.

The blocking or masking region is provided by a single chain molecule comprising portion of a variable heavy chain and a variable light chain of McPc603. The variable portions associate together and are stabilised by inter- and intra-domain disulphide bridges. These mask at least one of the B cell surface molecule binding portions on L. The molecule may alternatively only comprise a single L.

In the formula: A X, [X 2 ']o[X 2

X

3 I' and I" may both be present or one or other is present and represent a single amino acid sequence comprising a portion of the variable heavy and variable light chain of McPc603.

Element A is the target antigen to which a catalytic antibody is sought. Element A may be present once or twice. Accordingly, p is 0 or 1. X, and X 3 are the B cell surface molecule binding portions. Two B cell surface molecule binding portions are preferred but one B cell surface molecule binding portion may surfice. In one embodiment, when the growth factor SWO 99/15563 PCT/AU98/00783 -23precursor carries a multimerizing component such as the ompA, signal peptide then the growth factor precursor may contain only a single epitope. In these cases, n is 0. X 2 and X 2 are T cell surface molecule binding portions providing T cell dependent help for a B cell. If a single T cell surface molecule binding portion is present, o is 0. Where the growth factor precursor is in multimeric form n and m are >1 and up to about 10,000 and 200, respectively.

The elements may be in any order.

The growth factor precursor of the present invention may also contain elements to assist in purification of the molecule. Examples include the hexa-His affinity tag and FLAG-tag.

The g bridge is preferred but the present invention extends to any linking mechanism and is most preferably a flexible linking peptide.

In the example referred to above, TEV is the target site further TEV protease which mimics the cleavage by a catalytic antibody.

Another aspect of the present invention contemplates a nucleic acid molecule encoding the growth factor precursor herein described. According to this aspect of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain components in the growth factor precursor, associate together by intra- and/or inter-domain bonding and, when associated together, substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain component permits the at least one B cell surface molecule binding portion to interact with a V WO 99/15563 PCT/AU98/00783 -24- B cell surface molecule.

The preferred nucleic acid molecule of the present invention encodes the growth factor precursor defined herein as ccMTLgL having the amino acid sequence substantially as set forth in SEQ ID NO: 16. The present invention further contemplates molecules having growth factor precursor activity with an amino acid sequence with at least about similarity to ccMTLgL. Alternative percentage similarities include at least about 70%, at least about 80% and at least about 90% or above similarity to SEQID NO: 16.

In a particularly preferred embodiment, the nucleic acid molecule comprising a nucleotide sequence substantially set forth in SEQ ID NO: 15 or a nucleotide sequence having at least similarity thereto or a nucleotide sequence capable of hybridising thereto under low stringency conditions of 42 'C.

Reference herein to a low stringency at 42°C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions.

Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions.

The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, II ill i k WO 99/15563 PCT/AU98/00783 biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. Any number of programs are available to compare nucleotide and amino acid sequences.

Preferred programs have regard to an appropriate alignment. One such program is Gap which considers all possible alignment and gap positions and creates an alignment with the largest number of matched bases and the fewest gaps. Gap uses the alignment method of Needleman and Wunsch Mol. Biol. 48: 443-453, 1970). Gap reads a scoring matrix that contains values for every possible GCG symbol match. GAP is available on ANGIS (Australian National Genomic Information Service) at website http://mel l.angis.org.au..

In a related embodiment, the present invention provides a nucleic acid molecule encoding the growth factor precursor herein described. According to this aspect of the present invention, there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain components in the growth factor precursor, associate together by intra- and/or inter-domain bonding and, when associated together, substantially prevent the at least one B ceii surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain component permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule wherein if said growth factor precursor comprises a single B cell surface molecule binding portion, then the growth factor precursor further comprises a multimerising inducing element.

In another embodiment, the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a I-WO 99/15563 PCT/AU98/00783 -26polypeptide chain or a molecule having modular peptide components or a synthetic equivalent thereof wherein said polypeptide chain or modular peptide molecule comprises at least one B cell surface molecule binding portion, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable heavy chain and variable light chain components in the growth factor precursor, associate together by intra- and/or inter-domain bonding and, when associated together, substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain component permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule.

Preferably, the nucleic acid molecule is in form of a genetic "vaccine". In this regard, a genetic vaccine conveniently comprises the nucleic acid molecule in, for example, a viral vector or other suitable nucleic acid transferring medium. Generally, one or more pharmaceutically acceptable carriers and/or diluents are also included. The genetic vaccine is introduced to cells either directly intramuscularly), or systemically or cells are removed from an individual, the genetic vaccine introduced into the cells and then the cells are returned to the individual or a genetically related individual. The nucleic acid in the genetic vaccine after introduction to cells is expressed to produce the growth factor precursor of the present invention.

In a particularly preferred embodiment, the nucleic acid molecule in the genetic vaccine further comprises a nucleotide sequence encoding a molecular adjuvant. Examples of suitable molecular adjuvants include CTLA4 (Boyle et al. Nature 392: 408-411, 1998), CD40L (Lane et al. J. Exp. Med. 177:1209-1213, 1993) and C3d (Dempsey et al. Science 27: 348-350, 1996; Lou and Kohler, Naurve Biotechnology 16: 458-462, 1998).

The present invention extends to recombinant polypeptides defining the growth factor precursor and further comprising a molecular adjuvant attached thereto.

i. t. WO 99/15563 PCT/AU98/00783 -27- Upon cleavage of the growth factor precursor by a catalytic antibody recognising the antigen (for example, a TNF peptide portion), the covalent linkage between the B cell surface molecule binding portion and the variable heavy and light domains is broken. The blocking variable chains will dissociate from the B cell surface molecule binding portion due to the relatively low affinity (-10 7 M) of individual domains for each other. This will release the mature growth factor which can bind to and crosslink the surface immunoglobulin.

Catalytic antibodies can be detected in the serum using any number of procedures such as ELISA based assays and catalytic B cells may be recovered with standard hybridoma technology. Where the catalytic antibodies are from non-human animals, these can be humanised by recombinant DNA technology and produced for therapeutical applications in humans. Alternatively, the antibodies may be generated in a "humanized" animal such as a humanized mouse which is transgenic for the human Ig loci.

The present invention contemplates derivatives of the growth factor and/or its precursor. A derivative includes a mutant, part, fragment, portion, homologue or analogue of the growth factor and/or precursor or any components thereof. Derivatives to amino acid sequences include single or multiple amino acid substitutions, deletions and/or additions.

Particularly useful derivatives include chemical analogues of the growth factor precursor and/or its components. Such chemical analogues may be useful in stabilizing the molecule for therapeutic, diagnostic and industrial use.

Analogues of the growth factor precursor contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde WO 99/15563 PCT/AU98/00783 -28followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.

Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hiydiroxy-5phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or Dj. WO 99/15563 PCT/AU98/00783 -29isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

Crosslinkers can be used, for example, to stabilise 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and N,-methylamino acids, introduction of double bonds between Ca and Cp atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention further contemplates chemical analogues of the growth factor precursor capable of acting as antagonists or agonists of same. These may be useful in controlling the immunological response. Chemical analogues may not necessarily be derived from the growth factor precursor but may share certain conformational similarities.

Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of the growth factor precursor. Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening of, for example, coral, soil, plants, microorganisms, marine invertebrates or seabeds.

Screening of synthetic libraries is also contemplated by the present invention.

t. WO 99/15563 WO 99/ 5563PCT/AU98/00783 30 TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid a-aniinobutyric acid a-am-ino-a-methylbutyrate amninocyclopropanecarboxylate amninoisobutyric acid aminonorbomylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamnine D-glutamnic acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-omnithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan Abu Mgabu Cpro Aib Norb Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dile Dleu Dlys Dmet Domn Dphe Dpro Dser Dthr Dtrp L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid Chexa L-N-methylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylomithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltiyptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmhis Nmile Nmleu Nm-lys Nmmet Nmnle Nmnva Nmom Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmnval Nmetg Nmtbug NMe Nva WO 99/15563 WO 9915563PCT/AU98/00783 -31 D-tyrosine D-valine D-a-methylalanine D-a-methylarginine D-.a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-a-methylglutamine D-a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-a-methyllysine D-a-methylmethionine D-a-methylornithine D-a-methylphenylalanine D-a-methylproline D-a-methylserine D-a-methylthreonine D-a-methyltryptophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutaxnine D-N-methylglutamate D-N-.methylhistidine D-N-methylisoleucine D-N-methylleucine Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dihis Dmile Dmleu Dmlys Dmmet Dmom Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnaia Dnmarg Dnmasn Dnmasp Dncys Dnrngln Dnmglu Dnnmbis Dnmile Dnmfleu a-methyl-aminoisobutyrate ix-methyl-y -aminobutyrate a-methylcyclohexylalanine a-methylcylcopentylalanine a-methyl-a-napthylalanine ct-methylpenicillamine N-(4-arninobutyl)glycine N-(2-amninoethyl)glycine N-(3-am-inopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamyhnethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl)glycine N-.(3,3-diphenylpropyl)glycine N-(3-guanidinopropyl)glycine N-(1-hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(imidazolylethyl))glycine N-(3-indolylyethyl)glycine Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Norn Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr -N~er Nhtrp WO 99/15563 WO 99/ 5563PCT/AU98/00783 32 D-N-methyllysine N-methylcyclohexylalanine D-N-methylornithine N-methylglycine, N-methylam-inoisobutyrate 1 -methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-mnethyltyrosine D-N-methylvaline y-amninobutyric acid L-t-butylglycine L-ethylglycine L-homophenylalanine L-a-methylarginine L-ct-methylaspartate L-a-methylcysteine L-a-methylglutamine L-cx-methylhistidine L-a-methylisoleucine L-a-methylleucine L-a-methylmethionine L-a-methylnorvaline L-a-methylphenylalanine L-a-methylserine L-a-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl) carbamyhnethyl)glycine Dnn-lys Nmchexa Dnmom Nala Nmaib Nile Nleu Dnmtrp Dintyr Dnval Gabu Thug Etg Hphe Marg Masp Mcys Mgln Mhis Mile Mlieu Mmet Mnva Mphe Mser Mtrp Mval Nnbhm N-methyl-y -aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine 1-methylethyl)glycine N-methyla-napthylalanine N-methylpenidilamnine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamnine L-a-methylalanine L-a-methylasparagine L-a-methyl-t-butylglycine L-methylethylglycine L-a-methylglutamate L-a-methylhomophenylalanine N-(2-methylthioethyl)glycine L-a-methyllysine L-a-methylnorleucine L-a-methylomithine L-a-methylproline L-a-methylthreonine L-a-.methyltyrosine L-N-methylhomophenylalanine ,3-diphenylpropyl) carbamylmethyl)glycine 1-carboxy-l1-(2,2-diphenyl-Nmbc ethylaniino)cyclopropane Nmgabu Dnminet Nmcpen Dnmphe Dnmpro Drnser Dnmthr NvaI Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Ivlhphe Nmet Mlys MNle Mom Mpro Mthr Mtyr Nmhphe Nnbhe ''WO 99/15563 PCT/AU98/00783 -33- Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule.

Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

Still a further aspect of the present invention extends to a method for producing catalytic antibodies to a specific antigen, said method comprising administering to an animal an effective amount of a growth factor precursor comprising an antigen capable of interacting with a B cell bound catalytic antibody said antigen linked to or otherwise associate with a B cell surface molecule binding portion and a portion capable of providing T cell dependent help to a B cell. The growth factor precursor further comprises a B cell surface molecule binding portion masking entity such as a portion of a variable heavy and light chain linked to the antigen.

Alternatively, the growth factor precursor may comprise a B cell surface molecule binding portion in multimeric form linked to an antigen for which a target antibody is sought. The portion providing T cell dependent help is preferably a T cell surface molecule binding portion and is preferably part of the precursor. However, it may be a separate entity administered simultaneously or sequentially to an animal. Again, the B cell surface molecule binding portion is masked as above.

The present invention also provides catalytic antibodies produced by the above method.

Such catalytic antibodies may be directed to any antigen such as but not limited to a cytokine, for example, tumor necrosis factor (TNF), an interleukin (IL) such as IL-1 to ILinterferons (IFN) such as IFNa, IFN3 or IFNy, colony-stimulating factors (CSF) such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colonystimulation factor (GM-CSF), blood factors such as Factor VIII, erythropoietin and haemopoietin, cancer antigens, docking receptors from pathogenic viruses such as HIV, influenza virus or a hepatitis virus (eg. HEP A, HEP B, HEP C or HEP E) and amyloid plaques such as in Alzheimer's disease patients or myeloma patients.

VWO 99/15563 PCT/AU98/00783 -34- The catalytic antibodies of the present invention have particular therapeutic and diagnostic uses especially in relation to mammalian and more particularly human disease conditions.

Accordingly, the present invention contemplates a pharmaceutical composition comprising a growth factor precursor or a derivative thereof and optionally a modulator of growth factor precursor activity and one or more pharmaceutically acceptable carriers and/or diluents. More particularly, the pharmaceutical composition comprises catalytic antibodies generated by the growth factor precursor of the present invention. These components are hereinafter referred to as the "active ingredients".

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or sterile powders for the extemporaneous preparation of sterile injectable solutions. It 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.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization such as by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

PWO 99/15563 PCT/AU98/00783 When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound 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 compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of active compound, preferably between about 0.1 pg and 1500 mg and more preferably between about 1 gg and 100 mg.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry, orange or mango. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations. WO 99/15563 WO 99/ 5563PCT/AU98/00783 36 Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated.

Supplementary active ingredients can also be incorporated into the compositions. These may include immune potentiating molecules, multimer facilitating molecules and pharmaceutically active molecules chosen on the disease conditions being treated.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0. 1 ng to about 2000 mg, more preferably ranging from 0. 1 g.g and 1500 mg and even more preferably ranging between I gtg and 1000 mg.

Expressed in proportions, the active compound is generally present in from about 0.5 pig to about 2000 mg/mi of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Still another aspect of the present invention is directed to antibodies to the growth factor precursor and its derivatives. Such antibodies may be monoclonal or polyclonal and are independent to the catalytic antibodies selected by the precursor. The (non-catalytic) antibodies to recombinant or synthetic the growth factor precursor or its derivatives of the present invention may be useful as therapeutic agents but are particularly useful as diagnostic agents. Antibodies may also be generated to the catalytic antibodies generated by the growth factor precursors. All these antibodies have particular application in diagnostic assays for the growth factor or catalytic antibody inducer thereof.

For example, specific antibodies can be used to screen for catalytic antibodies. The laiter would be important, for example, as a means for screening for levels of these antibodies in a biological fluid or for purifying the catalytic antibodies. Techniques for the assays k.WO 99/15563 PCT/AU98/00783 -37contemplated herein are known in the art and include, for example, sandwich assays and

ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of antigen, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting an antigen in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for said antigen or its derivatives or homologues for a time and under conditions sufficient for an antibody-antigen complex to form, and then detecting said complex. In this context, the "antigen" may be a growth factor, its precursor, a component thereof or a catalytic antibody induced thereby.

i'WO 99/15563 PCT/AU98/00783 -38- The presence of antigen may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain an antigen including cell extract, supernatant fluid, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supematant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody having specificity for the antigen or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid r WO 99/15563 PCT/AU98/00783 -39surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g.

2-40 minutes, or overnight if more convenient) and under suitable conditions from room tempterature to about 40°C such as 25-37 to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, p WO 99/15563 PCT/AU98/00783 beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigenantibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.

As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibodyhapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method.

However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention may use any number of means to clone genetic sequences encoding catalytic antibodies. For example, a phage display library potentially capable of expressing a catalytic antibody on the phage surface may be used to screen for catalysis of defined antigens.

j. WO 99/15563 PCT/AU98/00783 -41 The present invention further contemplates the use of the products of catalysis of a growth factor precursor to induce B cell mitogenesis to generate catalytic antibodies to a specific antigen.

More particularly, the present invention contemplates the use of a growth factor precursor comprising an antigen to which a catalytic antibody is sought linked, fused or otherwise associated to a B cell surface molecule binding portion in the induction of B cell mitogenesis following catalytic cleavage of all or part of said antigen.

Still another embodiment of the present invention contemplates the use of an antigen linked, fused or otherwise associate to a B cell surface molecule binding portion in the manufacture of a growth factor precursor to induce B cell mitogenesis following catalytic cleavage of all or part of said antigen.

The present invention is further described by the following non-limiting examples.

EXAMPLE 1 GENERATION OF LHL FROM SYNTHETIC OLIGONUCLEOTIDES LHL was generated from three overlapping synthetic oligos, a 115mer, a 116mer and a 105mer, using the proofreading DNA polymerase Pfu in two 20 cycle PCR reactions. The two PCR products (290bp and 200bp) were purified and blunt end cloned into the expression vector pASK75. The sequence was verified by automated sequencing. All subsequent PCRs were done in a similar fashion as described in the literature. The nucleotide and corresponding amino acid sequence for LHL is shown in SEQ ID NO: 1 and SEQ ID NO:2 respectively.

WO 99/15563 PCT/AU98/00783 -42- EXAMPLE 2 EXPRESSION OF LHL IN E. COLI AND PURIFICATION OVER A HUMAN IgG (hulgG) AFFINITY COLUMN The expression vector pASK75 directs protein expression via the ompA signal peptide into the periplasm of E. coli. Protein expression was induced with 200ng/ml anhydrotetracycline for 16 hrs in midlog E. coli DH10B cultures. Cells were lysed and soluble LHL purified over a hulgG affinity column. Extensive washes with 0.5% v/v Triton X-100 were performed on the affinity column in order to eliminate endotoxins from the preparations.

Expression levels were estimated at 20mg per litre of culture.

EXAMPLE 3 GENERATION OF AN LHL PROTEIN CARRYING THE N-TERMINAL FLAG EPITOPE AND THE C-TERMINAL STREP-TAG A form of LHL (referred to herein as "LHL.seq") was generated by PCR containing the FLAG epitope at its N-terminus and the so called strep-tag at its C-terminus. The nucleotide and corresponding amino acid sequence for LHL.seq is shown in SEQ ID NO:7 and SEQ ID NO:8, respectively. The FLAG epitope comprises the amino acids DYKDDDDK (SEQ ID NO:9) and the strep-tag the amino acids AWRHPQFGG (SEQ ID NO: 14). The FLAG epitope is recognised by several anti-FLAG monoclonal antibodies and the strep-tag by streptavidin. The strep-tag was used for purification of LHL.seq over a streptavidin column. LHL.seq was washed with 0.5% v/v Triton X-100, Tween20 and PBS while bound to the column in order to minimise endotoxin levels. LHL.seq was eluted with either 100mM glycine pH2.0 or with 1mg/ml diaminobiotin in PBS. In this method LHL.seq was not purified on the basis of binding immunoglobulin, thereby eliminating potential contamination of other unknown bacterial proteins which also bind immunoglobulins. The biological activity of LHL.seq, however, remained identical to that of LHL. The FLAGepitope was added to the N-terminus in order to facilitate the secretion of LHL.seq into the periplasmic space. As in previous expression studies, this was unsuccessful and LHL.seq 11 WO 99/15563 PCT/AU98/00783 -43needed to be purified from total bacterial lysate. As a result of this, the ompA signal peptide is not removed, which in turn led to formation of LHL.seq multimers.

EXAMPLE 4 MITOGENIC ACTIVITY OF LHL ON B CELLS Mitogenic activity of LHL on B cells was tested in overnight cultures of splenocytes and mesenteric lymphocytes as well as on purified B cells. The activation status of B cells was analysed by FACS, examining B cell size and induction of B7-2 surface expression. LHL's activation potency is similar to LPS (10 gg/ml), a bacterial mitogenic lipopolysaccharide and anti-IgM antibody (25 which crosslinks surface IgM. The results have been independently obtained in several different mouse strain e.g. B 10.A(4R), CBA, C3H/HeJ and BALB/c. B cells showed a clear dose response to LHL when titrated in 5-fold dilutions .g/ml to 1.6 ng/ml) in the activation assay. Parallel experiments analysing the T cell activation status within the same cultures demonstrated that LHL has no effect on T cells. T cells did not show any blast formation nor did they upregulate activation markers, e.g. IL-2 receptor alpha chain EXAMPLE BLOCKING OF LHL MITOGENICITY BY HuIgG In the same experiments, soluble hulgG (500 gg/ml) which binds to the L domains was used to specifically block the activity of LHL. These results rule out that B cell activation was due to a contamination of the bacterially produced LHL with endotoxins.

EXAMPLE 6 PROCESSING OF LHL BY B CELLS AND PRESENTATION OF THE H EPITOPE TO THE HEL-SPECIFIC HYBRIDOMA 3A9 Splenocytes or mesenteric lymphocytes were cocultured with the T cell hybridoma 3A9 in the presence of LHL. 3A9 is specific for the HEL peptide 52-61aa presented on MHC II H- WO 99/15563 PCT/AU98/00783 -44- 2A

K

Upon recognition of this peptide, 3A9 secretes IL-2. IL-2 production was measured in a bio assay which evaluates the proliferation of an IL-2 dependent cell line (CTLL) on the basis of H-thymidine incorporation during DNA synthesis. Presentation of H to 3A9 by B cells was clearly demonstrated by the proliferation of the CTLL and could be specifically blocked with hulgG.

EXAMPLE 7 GENERATION OF THE VARIABLE (V)-KAPPA LIGHT CHAIN ACCORDING TO THE HUMAN LEN PROTEIN SEQUENCE The amino acid sequence of the gene encoding the human myeloma protein LEN was used to generate a variable kappa light chain. This human kappa light chain protein (hereinafter referred to as "kappa") is soluble at relatively high concentrations and has been shown to bind protein L. Kappa was generated from synthetic oligonucleotides by PCR. To facilitate protein purification, a FLAG epitope was added to the N-terminus and a strep-tag to the Cterminus. The nucleotide and amino acid sequence of kappa is shown in SEQ ID NO: and 11, respectively.

EXAMPLE 8 EXPRESSION OF KAPPA IN E.COLI Kappa was cloned into pASK75, allowing inducible expression of kappa into the periplasmic space of E.coli. Expression was induced in logarithmically growing cultures of E.coli strain DH1OB cells with 400ng/ml of anhydro-tetracycline for 4hrs.

EXAMPLE 9 PURIFICATION OF KAPPA PROTEIN FROM THE PERIPLASM OF Cultures were spun down and resuspended in a buffer containing 400mM sucrose on ice.

After 20min cells were pelleted. Kappa was then purified over an anti-FLAG and/or streptavidin column from the periplasmic fraction.

X-

PWO 99/15563 PCT/AU98/00783 EXAMPLE CONFIRMATION OF PROPER FOLDING OF KAPPA AFTER PURIFICATION The proper folding of kappa was demonstrated by its capacity to bind LHL. Kappa was bound to the streptavidin column via its strep-tag. This kappa-loaded column was then shown to bind LHL. The non strep-tag carrying LHL did not bind to the streptavidin column alone.

EXAMPLE 11 GENERATION OF TLHL TLHL was generated from LHL, kappa and synthetic oligonucleotides encoding a linker connecting kappa and LHL by PCR. The linker contained an amino acid sequence corresponding to the tobacco etch virus (TEV) protease recognition/cleavage site. All components were cloned into pASK75 resulting in the following protein sequence: FLAGkappa-linker-TEV-LHL-streptag. Potentially, TLHL could show similar characteristics as CATAB, since one kappa binding site is blocked and two are required for surface immunoglobulin cross-linking. The nucleotide and amino acid sequences of TLHL are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

EXAMPLE 12 EXPRESSION OF TLHL IN TLHL expression was induced in logarithmically growing cultures by addition of 400ng/ml anhydro-tetracycline for >4hrs. TLHL was not secreted into the periplasmic space and caused some cell lysis after induction.

pWO 99/15563 PCT/AU98/00783 -46- EXAMPLE 13 PURIFICATION OF TLHL FROM TOTAL BACTERIAL LYSATE TLHL was purified via its strep-tag over a streptavidin column from total bacterial lysate.

Endotoxin levels were reduced using the washing protocol earlier described.

EXAMPLE 14 CLEAVAGE OF TLHL INTO AND "LHL" WITH TEV TLHL was designed so that the kappa portion of the protein could be cleaved off by the TEV protease. The TEV cleavage would generate two polypeptides, each of 172 amino acids. The identical size of the protein fragments is due to TLHL not being secreted into the periplasmic space of E.coli and, therefore, retaining the ompA signal peptide. Incubation of TLHL with the TEV protease in PBS at room temperature or at 4°C produced therefore, a 19kD band on an SDS-PAGE gel.

EXAMPLE ASSEMBLY OF CATAB-TEV FROM TLHL AND KAPPA BY PCR CATAB-TEV is assembled from TLHL and kappa by PCR. The TLHL and kappa can be linked by different peptides, for example, TNF amino acids 1-31, that are potential target sites for proteolytic antibodies. In this case, the linker includes a recognition sequence for the tobacco etch virus (TEV) protease which allows the generation of LHL from CATAB- TEV in vitro. The nucleotide and corresponding amino acid sequences of CATAB-TEV are shown in SEQ ID NO:3 and SEQ ID NO:4.

EXAMPLE 16 EXPRESSION OF CATAB IN DH10B AND PURIFICATION OVER A STREPTAVIDIN AFFINITY COLUMN VIA STREP-TAG CATAB-TEV is expressed and purified in the same way as TLHL (see above).

WO 99/15563 PCT/AU98/00783 -47- EXAMPLE 17 DEMONSTRATION OF NON-MITOGENIC ACTIVITY OF CATAB-TEV ON B CELLS CATAB-TEV is tested in the already established B cell assays which are used to analyse the mitogenic activity of LHL and LHL.seq.

EXAMPLE 18 REVELATION OF THE MITOGENIC ACTIVITY OF CATAB BY PROTEOLYTIC CLEAVAGE WITH TEV PROTEASE Digestion of CATAB-TEV with the site specific protease from TEV cleaves the covalent bond between LHL and the kappa domains. This cleavage generates the mitogenic compound LHL which is tested in the standardised B cell activation assays.

EXAMPLE 19 USAGE OF CATAB IN SEVERAL MOUSE STRAINS OF THE K-HAPLOTYPE Several mouse strains are immunised by different routes of administration, e.g. intra-splenic, in order to elicit a catalytic antibody response in vivo. The gld and lpr mutant strains are used as they have been shown to have a relatively high incidence of naturally occurring catalytic auto-antibodies, e.g. antibodies with DNAse activity.

EXAMPLE DETECTION OF CATAB SPECIFIC CATALYTIC ANTIBODIES FROM THE SERUM Serum antibodies from immunised mice are purified for example on a LHL affinity column.

Purified antibodies may be incubated with 25 I-labelled CATAB and the proteolytic cleavage is evaluated on PAGE gels. In addition, streptavidin may be used to immobilise CATAB via its C-terminal strep-tag on 96 well ELISA plates. Immobilised CATAB is proteolytically cleaved by incubation with purified catalytic serum antibodies and an N-terminal affinity tag, WO 99/15563 PCT/AU98/00783 -48e.g. flag epitope, is lost. This loss is detected in a sandwich ELISA assay using horse radish peroxidase (HRPO) conjugated antibodies. B cells producing catalytic antibodies can be recovered by standard hybridoma techniques and the catalytic antibodies can be humanised by recombinant DNA technology. For example, "human" antibodies can be derived from humanized mice.

EXAMPLE 21 LHL.seq INDUCED B7-1 EXPRESSION LHL.seq was tested for its ability to activate B cells as compared to stimulation with anti- IgM and anti-kappa. Activation status was measured by the induction of cell surface expression of the activation markers B7-1 and B7-2 and by entry of B cells into cell cycle.

Levels of expression of B7-1 and B7-2 were determined by flow cytometry (FACS) with fluorescence-labelled monoclonal antibodies while entry into cell cycle was monitored by an increase in cell size by Forward Light Scatter (FSC).

The method employed was as follows. Mesenteric lymphnode cells from C3H/HeJ mice were centrifuged in Nycodenz (1.091 g/cm 3 to remove dead cells and red blood cells (rbc).

This was followed by 1 hour adherence on plastic at 37 0 C to remove adherent cells such as macrophages. Lymph node cells were stimulated in triplicate cultures 3x10 5 /well in flat bottom 96-well plates in complete RPMI 10% FCS medium at 37 0 C for 1-3 days.

Upregulation of activation markers on B cells was monitored by gating on B220+Thy 1 cells to identify B cells. Stimulation with LPS (20 pg/ml), polyclonal F(ab) 2 anti-IgM antibodies pg/ml) and anti-kappa antibodies (10 pg/ml) were included as controls. LHL.seq was used at 1 pg/ml. C3H/HeJ mice were used as source of lymphocytes since this particular mouse strain is non-responsive to LPS. The use of this strain in combination with the LPS control effectively precludes the possibility that B cell stimulation induced by LHL.seq were due to LPS (endotoxin) contamination of the bacterially expressed proteins.

FACS analysis showed that this two day stimulation of C3H/HeJ lymph node cells with LPS did not result in B cell activation whereas stimulation with either anti-IgM antibodies, anti- WO 99/15563 PCT/AU98/00783 -49kappa antibodies or LHL.seq did as measured by an increased FSC and upregulation of B7- 2. The characteristic potency of LHL.seq is demonstrated by the strong induction of B7-1 1 expression after incubation. Anti-IgM induces B7-1 on day 2-3 of stimulation.

EXAMPLE 22 LHL.seq INDUCED MHC CLASS II LHL.seq was compared in its potential to ensure proper upregulation of MHC class II on stimulated B cells. Anti-IgM antibodies (20 pg/ml) as well as LHL. seq (1 pg/ml) blocked with hulgG (500 pg/ml) were included as controls. The method used was as described in Example 21.

Upregulation of MHC Class II molecules on B cells is a prerequisite to receive T cell help in vivo.

Overnight stimulation of C3H/HeJ lymph node cells with anti-IgM antibodies as well as LHL.seq did result in increased FSC and upregulation of MHC class II. LHL.seq's activities were completely blocked by addition of 500 pg/ml hulgG to the cultures.

EXAMPLE 23 LHL.seq INDUCED PROLIFERATION IN A DOSE DEPENDENT FASHION Serial dilutions of LHL.seq were used to stimulate B cell proliferation. The experiment demonstrated that LHL.seq's biological properties are similar to conventional B cell mitogens like anti-IgM antibodies. Thus, dose-response curves for stimulation of either mesenteric lymphnode cells from C3H/HeJ and splenocytes from CBA/J were obtained.

WO 99/15563 PCT/AU98/00783 EXAMPLE 24 TLHL INDUCED B CELL ACTIVATION LHL.seq, TLHL and TEV-cleaved TLHL were tested for their ability to activate B cells as measured by the induction of cell surface expression of the activation markers B7-1 (CD86) and B7-2 (CD80) and by entry of B cells into cell cycle. Levels of expression of B7-1 and B7-2 were determined by flow cytometry (FACS) with fluorescence-labelled monoclonal antibodies while proliferation was monitored by an increase in cell size by Forward Light Scatter (FSC) and by 3 H-thymidine-uptake assays.

The method employed as described in Example 21.

Overnight stimulation of C3H/HeJ lymph node cells with LPS did not result in B cell activation whereas stimulation with either anti-IgM antibodies or LHL.seq did as measured by an increased FSC and upregulation of B7-2. The characteristic potency of LHL.seq is demonstrated by the strong induction of B7-1 expression after overnight incubation. Anti- IgM induces B7-1 on day 2-3 of stimulation.

TLHL, however, activated B cells to the same extent as LHL.seq. This was unexpected since it was presumed that blocking one L domain with a covalently linked kappa would prevent crosslinking of immunoglobulin on the B cell surface. Prevention of crosslinking should result in no or significantly lower B cell activation than that achieved with equal amounts of LHL.seq. TEV-cleaved TLHL, which results in omp-kappa (see below) plus the LHL.seq part, gave much lower B cell activation than uncleaved TLHL as indicated by less B7-1 and B7-2 upregulation and lower FSC increase.

Splenocytes fromCBA/J mice were centrifuged in Nycodenz (1.091 g/cm 3 to remove dead cells and rbc. This was followed by 1 hour adherence on plastic at 37 0 C to remove adherent cells. Splenocytes were then stimulated in triplicate cultures at 2x10S/well in flat botforn 96well plates in complete RPMI 10% v/v FCS medium at 37 0 C for 2 days. Cells were pulsed for the last 6 hours with 3 H-thymidine. DNA was then harvested onto glassfibre SWO 99/15563 PCT/AU98/00783 -51 filters and incorporation of 3 H-thymidine was measured in a P-counter.

The results obtained by FACS analysis were confirmed by the proliferation data; TLHL and LHL.seq induced equivalent B cell proliferation while TEV-cleaved TLHL was about less potent.

EXAMPLE TEV-CLEAVED TLHL STIMULATION DATA CONFIRM OMP INDUCED MULTIMERISATION The B cell activation data lead the inventors to the conclusion that both LHL, LHL.seq and TLHL exist in solution as multimeric molecules. While dimeric or oligomeric immunoglobulin-binding molecules such as anti-IgM antibodies induce B cell activation, multimers such as anti-IgD-dextran result in a significantly higher degree of B cell activation. This is also the case with LHL, LHL.seq and TLHL in the above experiments as demonstrated by the extensive upregulation of B7-1 after overnight culture. The multimerisation is facilitated by the ompA signal peptide (omp) It has been published by others that the ompA signal peptide forms multimers in aqueous solution. Evidence for LHL, LHL.seq and TLHL aggregation has also been obtained in HPLC studies.

A new recombinant LHL.seq protein lacking the ompA signal peptide, called LHL-omp, was engineered which also confirms these conclusions (see below).

EXAMPLE 26 TLHL MULTIMERISATION OVERCOMES "KAPPA-BLOCKING" Although one domain should be blocked by kappa in TLHL, the multimerisation mediated by the omp allows several free domains to exist in one multimeric molecule [TLHL],. This will lead to extentive sIg crosslinking and full B cell activation as demonstrated.

WO 99/15563 PCT/AU98/00783 -52- EXAMPLE 27 GENERATION AND ANALYSIS OF LHL-OMP LHL-omp was generated from LHL.seq via PCR with the proofreading polymerase Pfu eliminating the ompA signal sequence.

EXAMPLE 28 AFFINITY COLUMN PURIFICATION OF LHL-OMP Although LHL-omp contains a Strep-tag, it could not be purified via the Streptavidin column using the standard protocol, indicating a lower avidity to the column matrix than that of LHL.seq. This lower avidity confirms the multimerisation of LHL.seq via omp, being the only difference between LHL.seq and LHL-omp. In agreement with this LHL-omp was readily purified over a hulgG affinity column.

EXAMPLE 29 LHL-OMP INDUCED B CELL ACTIVATION The ability of LHL-omp to induce B cell activation was assessed by incubating splenocytes from C3H/HeJ mice for varying periods of time before analysing B7-1 and B7-2 expression levels on B cells as outlined above. The progression of B cells into cell cycle was monitored by FACS and proliferation assays.

Cells were prepared and cultured as described above. LPS (20 pg/ml) and anti-IgM pg/ml) were used as controls.

Stimulation of C3H/HeJ splenocytes with LPS did not result in detectable B cell activation whereas treatment with either anti-IgM antibodies or LHL.seq induced B cell activation during overnight culture; increased FSC and B7-2 upregulation for anti-IgM antibodies and increased FSC and B7-1 and B7-2 expression for LHL.seq. LHL-omp, used at 2 pg/ml, was less potent than LHL.seq in inducing upregulation of B7-1, B7-2 and blasting of B WO 99/15563 PCT/AU98/00783 -53 cells, as indicated by the FSC profile. The unchanged FSC profile indicated that LHL-omp did not induce B cell proliferation. This was confirmed in proliferation assays.

B cells were stimulated simultaneously with LHL-omp and anti-CD40L antibodies (mAb FGK45.5 at a concentration of 0.5 jg/ml). Anti-CD40L antibodies served as a partial substitute for T cell help. The combination of sIg and helper T cell like signaling achieved good levels of B cell activation and proliferation. This could especially be demonstrated when using LHL-omp at a concentration of 125 ng/ml. 125 ng/ml induced no B cell activation on its own, however, when used in combination with the anti-CD40L antibody, which by itself is also of low potency, B7-1, B7-2 and FSC upregulation were achieved.

Suggesting that LHL-omp and anti-CD40L antibodies can act synergistically.

EXAMPLE UTILISING OMP TO DESIGN A NOVEL MULTIMERIC

MITOGEN

Experimental data obtained show that the signal peptide from the outer membrane protein A (ompA) of E. coli induces aggregation of the recombinant proteins LHL.seq and TLHL. The ompA signal peptide (omp) is usually cleaved off once the protein reaches its destination, the bacterial periplasmic space. In the case of LHL, LHL.seq and TLHL, however, the secretion into the periplasm is impaired. All three proteins remain in the cytoplasm and the omp peptide forms their N-terminal part. The N-terminal omp peptide induces multimerisation as demonstrated by the potentiation of their biological activity as compared to the recombinant protein LHL-omp and TEV-cleaved

TLHL.

The observation that omp induces multimerisation allows the design of simpler molecules with the same desired biological function as LHL, TLHL and CATAB. For this purpose we propose the following protein design. Above results demonstrate that the proteins described are not secreted into the periplasmic space. It should therefore be possible to produce proteins that have an omp peptide as their N-terminal part and L or HL as their C-terminal part. As omp allows the formation of multimers, this should result in the formation of [ompL],, hereafter called ompL, or [ompHL], where n is equal or larger than 2.

VWO 99/15563 PCT/AU98/00783 -54- EXAMPLE 31 MULTIMERISATION OF OMPL AND DESIGN OF FV-CATAB Multimerisation of ompL generates a protein complex that should allow crosslinking of surface immunoglobulins in a similar fashion to LHL or LHL.seq. OmpL itself, however, is a relatively simple monomeric protein which needs only a single blocking entity. This blocking domain will be the below described scdsFv resulting the fusion protein ompL-linker-TEVscdsFv (Fv-catAb). The reverse of this configuration, scdsFv-TEV-linker-Lomp (pFvcatAb) will also be generated, as this might allow for periplasmic secretion of pFvcatAb.The latter pFv-catAb requires the functional multimerisation and biological activity of Lomp, a protein with the reverse fusion order of ompL and the omp peptide at its Cterminal. All described recombinant proteins are tested in the experimental systems outlined above.

EXAMPLE 32 REDESIGN OF THE L DOMAIN BLOCKING ENTITY Two potential problems are associated with the use of the LEN kappa light chain as a blocking domain for L. First, proteins (ie. LHL, LHL.seq and TLHL) are not secreted into the periplasmic space during expression in E. coli, which might cause folding problems in the kappa portion. Secondly, there are no direct means of purifying proteins with potentially correctly folded kappas in the described system, as antibodies against kappa would be bound by LHL.seq.

In order to allow for purification of correctly folded growth factor precursors, the blocking entity was therefore redesigned. Kappa will be replaced by a single chain (sc) antibody which is stabilised by an internal disulphide bridge (disulphide bridge stabilised, ds). This scdsFv will be derived from the extensively described plasmacytoma McPc603 [Freund et al. Biochemistry 33: 3296-3303, 1994] with anti-phosphorylcholine specificity. The phosphorylcholine-binding ability will facilitate the purification of correctly folded recombinant proteins via a phosphorylcholine affinity column.

WO 99/15563 PCT/AU98/007 8 3 EXAMPLE 33 POTENTIAL USE OF LHL/CATAB DERIVATIVES IN HUMANS In order to enable production of catalytic antibodies in humans, slight modifications of the constructs need to be performed. The T cell epitope has to be exchanged for an "universal T cell epitope" which will be recognised by T cells in the majority of humans in conjunction with their more diverse MHC class II molecules.

EXAMPLE 34 GENERATION OF LgL The periplasmic secretion of LHL (see PCT/AU97/00194, filed 26 March 1997) fusion proteins like TLHL and others demonstrated that the H in LHL was quantitatively cleaved during transport. This made the purification of full-length products from the periplasmic space or the culture supernatant more difficult. In order to circumvent this proteolytic cleavage, the H-linker was replaced with a Glycine-Serine linker. This linker consists of a quadruple repeat of four glycine followed by one serine, (GGGGS)x4. In addition the proteins were fused to a hexa-his-Tag at their C-terminus to allow their purification over a nickel-chelate-column (Fig.1).

EXAMPLE STRUCTURE, ANALYSIS AND PURIFICATION OF LgL From expression studies with ompL (OHL) the inventors demonstrated that the insertion of the H-linker sequence between ompA and L allowed secretion of L-proteins into the periplasm. In order to direct the expression of LgL into the periplasmic space, the ompA signal sequence as well as the H-linker sequence were therefore added to the N-terminus of the protein. This protein was named OHLgL (Fig.1).

OHLgL was expressed in E.coli strain DH10B by overnight induction with 400 zg/1 anhydrotetracycline in non-buffered TB-media at room temperature. Cells were harvested WO 99/15563 PCT/AU98/00783 -56and incubated in 500mM sucrose, PBS on ice for 30min. Cells were pelleted and LgL was purified from the supernatant containing the periplasmic proteins over a hulgG or a nickel-chelate column. LgL containing fractions (Fig. 2) as analysed on 20% w/v PHAST-gels were concentrated. LgL was further purified via a Superose 12 sizing column in PBS. The HPLC Superosel2 sizing profile was used to determine the concentration of LgL in the final eluate according to the absorbance at 280nm (Fig. LgL containing fractions were again analysed on 20% w/v PHAST-gels and if necessary pooled for B cell activation assays (Fig.4).

EXAMPLE 36 B CELL ACTIVATION POTENTIAL OF LgL LgL was tested for its ability to activate B cells as compared to stimulation with anti-IgM and Lomp. Activation status was measured by the induction of cell surface expression of the activation markers B7-1 and B7-2 and by entry of B cells into cell cycle. Levels of expression of B7-1 and B7-2 were determined by flow cytometry (FACS) with fluorescence-labelled monoclonal antibodies while entry into cell cycle was monitored by an increase in cell size by Forward Light Scatter (FSC).

FACS were performed as follows. Mesenteric lymph node cells from C3H/HeJ mice were centrifuged in Nycodenz (1.091 g/cm 3 to remove dead cells and red blood cells (rbc). This was followed by 1 hour adherence on plastic at 37 "C to remove adherent cells such as macrophages. Lymph node cells were stimulated in triplicate cultures at 3x 10 5 /well in flat bottom 96-well plates in complete RPMI 10% v/v FCS medium at 37 "C overnight.

Upregulation of activation markers on B cells was monitored by gating on B220' Thy- cells to identify B cells. Stimulation with LPS (20/g/ml) and polyclonal F(ab) 2 anti-IgM antibodies (20 /g/ml) were included as controls. LgL was used at 1-10 gg/ml. C3H/HeJ mice were used as source of lymphocytes since this particular mouse strain is non-responsive to LPS. The use of this strain in combination with the LPS control effectively precludes the possibility that B cell stimulation induced by LgL is due to LPS (endotoxin) contamination of the bacterially expressed protein.

WO 99/15563 PCT/AU98/00783 -57- The results of the FACS analysis are as follows. Stimulation of C3H/HeJ lymph node cells with LPS did not result in B cell activation whereas stimulation with either anti-IgM antibodies or LgL did as measured by upregulation of B7-1 and B7-2. The characteristic potency of LgL is demonstrated by the strong induction of B7-1 expression already after overnight stimulation. Anti-IgM induces B7-1 on day 2-3 after stimulation EXAMPLE 37 GENERATION OF ccMTLgL ccMTLgL was generated by cloning the disulphide linked single chain Fv from McPc603 in place of the H sequence in OHLgL. ccM and LgL were separated by a glycine-serine linker and the TEV cleavage signal as used before in TLHL. A FLAG-tag was used between the ompA and ccM for purification purposes. The sequence of the individual protein domains was therefore as follows: O-FLAG-ccMTLgL-6xhis (Fig.6). The nucleotide sequence and corresponding amino acid sequence for ccMTLgL is set forth in SEQ ID NOs: 15 and 16, respectively.

EXAMPLE 38 STRUCTURE, ANALYSIS AND PURIFICATION OF ccMTLgL ccMTLgL was expressed in E.coli strain DH10B by overnight induction with 400/g anhydrotetracycline in non-buffered TB-media at room temperature. Cells were pelleted and ccMTLgL was purified from the concentrated supernatant over the Ca" dependent FLAG Ml affinity column. This FLAG Ml affinity column only purifies correctly processed free FLAG peptide at the N-terminus of a recombinant protein. ccMTLgL containing fractions (Fig.7) as analysed on 20% w/v PHAST-gels were concentrated to 500/1 in 10.000MW cut off spin concentrator. ccMTLgL was further purified via a Superosel2 sizing column in PBS. The HPLC Superosel2 sizing profile was used to determine the concentration of ccMTLgL in the final eluate according to the absorbance at 280nm (Fig.8). ccMTLgL containing fractions were again analysed on 20% w/v PHAST-gels and if necessary pooled for B cell activation assays (Fig.9). The correct formation of the inter-domain disulphide I I WO 99/15563 PCT/AU98/00783 -58bond was shown by running ccMTLgL on 20% w/v PHAST-gel under reducing and non-reducing condition before and after cleavage with TEV (Fig. EXAMPLE 39 TEV CATALYSIS INDUCED B CELL ACTIVATION BY ccMTLgL of ccMTLgL in 140ul of PBS were incubated with 50 Units TEV protease at 4°C overnight. Complete cleavage into ccMT and LgL was verified on a 20% w/v PHAST-gel (Fig. Mesenteric LN cells (prepared as above) were stimulated overnight with controls (anti-IgM, LPS, LOMP, LgL and 2.5U TEV protease alone; all with and without hulgG) as well as ccMTLgL and 10p/g/ml ccMTLgL cleaved with TEV.

Results are shown in Figure 11. ccMTLgL by itself gives no B cell stimulation whereas ccMTLgL cleaved with TEV shows B cell stimulation with upregulation of B7-1.

These results were reproduced three times. The same results were also obtained when TEV protease were added in situ to the o/n B cell cultures (Fig. 12). Demonstrating that the in situ cleavage of ccMTLgL has the desired effect of liberating a B cell mitogen. This mimics the action of a catalytic antibody expressed by a B cell.

EXAMPLE UTILISING OMP TO DESIGN A NOVEL MULTIMERIC MITOGEN ompL (Fig. 13) is secreted into the periplasmic space. The ompA signal peptide is, therefore, processed and cleaved off. ompL can be purified on a hulgG column. ompL fractions from hulgG column are concentrated over a Millipore concentrator and are further purified over a Superose-12 HPLC sizing column. ompL does not multimerise and,therefore, runs as a monomeric protein at approximately WO 99/15563 PCT/AU98/00783 -59- Lomp is the reverse of ompL, carrying a modified ompA signal peptide at the C-terminus of LH. Lomp is expressed intracellularly and purified via hulgG and Superose-12 as described for ompL. ompL multimerises as predicted and elutes from the HPLC column in the void volume at >670kD.

ompL and Lomp were tested for their ability to activate B cells. As measured by the induction of cell surface expression of activation markers and by entry into cell cycle. The method is as described above.

FACS analysis showed that this two day stimulation of lymph node cells with LPS did not result in B cell activation whereas stimulation with either anti-IgM antibodies or Lomp did as measured by an increased FSC and upregulation of B7-2. The characteristic potency of Lomp is demonstrated by the strong induction of B7-1 expression after incubation.

Lomp activity was blocked by the addition of 500 /g/ml soluble hulgG into the culture.

ompL has no activity in FACS or proliferation assays.

EXAMPLE 41 RE-DESIGN OF THE L DOMAIN BLOCKING ENTITY A single chain Fv of McPc603 [scMcPc603] is expressed into the periplasmic space of E.

coli DH10B. scMcPc603 can be purified on a L-column (Fig. 15). scMcPc603 is properly folded because it binds to the L domain. scMcPc603 can be utilised as a blocking entity for L in a catab construct. In one example, Fv-catAb is used (Fig. 14).

t WO 99/15563 PCTIAU98/00783 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

t' WO 99/15563 PCT/AU98/00783 -61- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: (other than US) AMRAD OPERATIONS PTY LTD (US only) KOENTGEN, Frank; SUESS, Gabriele M; TARLINTON, David M; and TREUTLEIN, Herbert R (ii) TITLE OF INVENTION: CATALYTIC ANTIBODIES AND A METHOD OF PRODUCING SAME (iii) NUMBER OF SEQUENCES: 16 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: INTERNATIONAL APPLICATION FILING DATE: 18-SEP-1998 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PO9306 FILING DATE: 19-SEP-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: HUGHES, DR E JOHN L REFERENCE/DOCKET NUMBER: EJH/EK (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 TELEX: AA 31787 WO 99/15563 PCT/AU98/00783 -62- INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 549 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..549 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

ATG

Met 1 AAA AAG ACA Lys Lys Thr ATC GCG ATT GCA GTG GCA CTG GCT GGT Ile Ala Ile Ala Val Ala Leu Ala Gly 10 TTC GCT Phe Ala ACC GTA GCG Thr Val Ala AAA GCG AAC Lys Ala Asn

CAG

Gin GCC GCT CCG AAA Ala Ala Pro Lys

GAT

Asp 25 AAC ACG GAA GAA Asn Thr Glu Glu GTC ACG ATC Val Thr Ile GCA GAA TTC Ala Glu Phe CTG ATC TTT GCA Leu Ile Phe Ala

AAT

Asn 40 GGT AGC ACA CAA Gly Ser Thr Gin

ACT

Thr 144 192 AAA GGT Lys Gly ACC TTC GAA AAA Thr Phe Glu Lys

GCG

Ala 55 ACC TCG GAA GCT Thr Ser Glu Ala

TAT

Tyr GCG TAT GCA GAT Ala Tyr Ala Asp

ACT

Thr TTG AAG AAA GAC Leu Lys Lys Asp

AAT

Asn 70 GGT GAA TAT ACT Gly Glu Tyr Thr

GTA

Val 75 GAT GTT GCA GAT Asp Val Ala Asp

AAA

Lys GGT TAC ACC CTG Gly Tyr Thr Leu

AAC

Asn ATC AAA TTC GCG Ile Lys Phe Ala AAA GAA GCG ACC Lys Glu Ala Thr AAC CGT Asn Arg AAC ACC GAC Asn Thr Asp TGG GGT GGT Trp Gly Gly 115

GGT

Gly 100 TCC ACC GAC TAC Ser Thr Asp Tyr

GGT

Gly 105 ATC TTA CAG ATC Ile Leu Gin Ile AAC TCT CGT Asn Ser Arg 110 GCG AAC CTG Ala Asn Leu CTG ACC CTG AAA Leu Thr Leu Lys

GAA

Glu 120 GAA GTC ACG ATC Glu Val Thr Ile

AAA

Lys 125 ATC TTT Ile Phe 130 GCA AAT GGT AGC Ala Asn Gly Ser

ACA

Thr 135 CAA ACT GCA GAA Gin Thr Ala Glu

TTC

Phe 140 AAA GGT ACC TTC Lys Gly Thr Phe 432

GAA

Glu 145 AAA GCG ACC TCG Lys Ala Thr Ser

GAA

Glu 150 GCT TAT GCG TAT Ala Tyr Ala Tyr

GCA

Ala 155 GAT ACT TTG AAG Asp Thr Leu Lys

AAA

Lys 160 GAC AAT GGT GAA Asp Asn Gly Glu

TAT

Tyr 165 ACT GTA GAT GTT Thr Val Asp Val GAT AAA GGT TAC Asp Lys Gly Tyr ACC CTG Thr Leu 175 AAC ATC AAA Asn Ile Lys

TTC

Phe 180 GCG GGT TA Ala Gly W;O 99/15563 PCT/AU98/00783 -63- INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 182 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 Thr Val Ala Gln Ala Ala Pro Lys Asp Asn Thr Glu Glu Val Thr Ile 25 Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gin Thr Ala Glu Phe 40 Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala Asp 55 Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys 70 75 Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu Ala Thr Asn Arg 90 Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gin Ile Asn Ser Arg 100 105 110 Trp Gly Gly Leu Thr Leu Lys Glu Glu Val Thr Ile Lys Ala Asn Leu 115 120 125 Ile Phe Ala Asn Gly Ser Thr Gin Thr Ala Glu Phe Lys Gly Thr Phe 130 135 140 Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala Asp Thr Leu Lys Lys 145 150 155 160 Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys Gly Tyr Thr Leu 165 170 175 Asn Ile Lys Phe Ala Gly 180 WO 99/15563 PCT/AU98/00783 -64- INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1491 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1491 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

ATG

Met 1 AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly TTC GCT Phe Ala ACC GTA GCG Thr Val Ala ATG ACC CAG Met Thr Gin GCC GAC TAC AAG Ala Asp Tyr Lys GAT GAC GAC AAG Asp Asp Asp Lys GAT ATC GTG Asp Ile Val GAG CGT GCC Glu Arg Ala TCT CCA GAC TCC Ser Pro Asp Ser

CTG

Leu GCT GTG TCT CTG Ala Val Ser Leu ACC ATC Thr Ile AAT TGC AAG TCC Asn Cys Lys Ser CAG AGT GTT TTA Gin Ser Val Leu TAC AGC TCC AAC AGC Tyr Ser Ser Asn Ser GGT CAG CCT CCT AAG Gly Gin Pro Pro Lys

AAG

Lys AAC TAC CTG GCT Asn Tyr Leu Ala

TGG

Trp 70 TAC CAG CAG AAA Tyr Gin Gin Lys

CCA

Pro CTG CTC ATT TAC Leu Leu Ile Tyr GCA TCT ACC CGT Ala Ser Thr Arg

GAA

Glu TCC GGC GTT CCT Ser Gly Val Pro GAC CGT Asp Arg TTC AGT GGT Phe Ser Gly CTC CAG GCT Leu Gin Ala 115

AGC

Ser 100 GGT TCT GGT ACA Gly Ser Gly Thr

GAT

Asp 105 TTC ACT CTC ACC Phe Thr Leu Thr ATC AGC AGC Ile Ser Ser 110 TAT TAC AGT Tyr Tyr Ser GAA GAT GTG GCA Glu Asp Val Ala

GTT

Val 120 TAT TAC TGC CAG Tyr Tyr Cys Gin ACC CCG Thr Pro 130 TAC TCC TTC GGT Tyr Ser Phe Gly

CAG

Gin 135 GGT ACC AAA CTG Gly Thr Lys Leu

GAA

Glu 140 ATC AAA CGC TCC Ile Lys Arg Ser 384 432 480 528

GGT

Gly 145 AGC GGT GGC GGT Ser Gly Gly Gly

GGT

Gly 150 TCT GGT GGT GGT Ser Gly Gly Gly

GGG

Gly 155 AGC TCT GGT GGT Ser Ser Gly Gly

GGC

Gly 160 TCT GGT GGT GGT Ser Gly Gly Gly

AGC

Ser 165 GAA AAC CTG TAC Glu Asn Leu Tyr

TTC

Phe 170 CAG GGT GGT AGC Gin Gly Gly Ser GCC GAA Ala Glu 175 GAA GTC ACG Glu Val Thr AAA GCG AAC CTG Lys Ala Asn Leu

ATC

Ile 185 TTT GCA AAT GGT Phe Ala Asn Gly AGC ACA CAA Ser Thr Gin 190 576 ACT GCA GAA Thr Ala Glu 195 TTC AAA GGT ACC Phe Lys Gly Thr

TTC

Phe 200 GAA AAA GCG ACC TCG GAA GCT TAT Glu Lys Ala Thr Ser Glu Ala Tyr i- ffi4 t- a WO 99/15563 WO 9915563PCT/AU98/00783 65 GCG TAT Ala Tyr 210 GCA GAT ACT TTG Ala Asp Thr Leu

AAG

Lys 215 AAA GAC AAT GGT Lys Asp Asfl Gly

GAA

Glu 220 TAT ACT GTA GAT Tyr Thr Val Asp

GTT

Val1 225 GCA GAT AAA GGT Ala Asp Lys Gly

TAC

Tyr 230 ACC CTG AAC ATC Thr Leu Asn Ile

AAA

Lys 235 TTC GCG GGT AAA Phe Ala Giy Lys

GAA

Glu 240 GCG ACC AAC CGT Ala Thr Asn Arg

AAC

Asn 245 ACC GAC GGT TCC Thr Asp Gly Ser

ACC

Thr 250 GAC TAC GGT ATC Asp Tyr Gly Ile TTA CAG Leu Gln 255 ATC AAC TCT Ile Asn Ser AAA GCG AAC Lys Ala Asn 275

CGT

Arg 260 TGG GGT GGT CTG Trp Gly Gly Leu AGC GCC GAA GAA Ser Ala Glu Glu GTC ACG ATC Val Thr Ile 270 GCA GAA TTC Ala Glu Phe 720 768 816 864 912 960 CTG ATC TTT GCA Leu Ile Phe Ala

AAT

Asn 280 GGT AGC ACA CAA Gly Ser Thr Gin AAA GGT Lys Gly 290 ACC TTC GAA AAA Thr Phe Giu Lys

GCG

Ala 295 ACC TCG GAA GCT Thr Ser Glu Ala

TAT

Tyr 300 GCG TAT GCA GAT Ala Tyr Ala Asp

ACT

Thr 305 TTG AAG AAA GAC Leu Lys Lys Asp

AAT

Asn 310 GGT GAA TAT ACT Gly Glu Tyr Thr

GTA

Vali 315 GAT GTT GCA GAT Asp Val Ala Asp

AAA

Lys 320 GGT TAC ACC CTG Gly Tyr Thr Leu

AAC

Asn 325 ATC AAA TTC GCG Ile Lys Phe Ala

GGT

Gly 330 AAA GAA AGC GGT Lys Giu Ser Giy GGC GGT Gly Gly 335 GGT TCT GGT Gly Ser Gly AGC GAA AAC Ser Giu Asn 355 GGT GGG AGC GGC Gly Gly Ser Gly

GCC

Ala 345 GGT GGT GGC TCT Gly Gly Gly Ser GGT GGT GGT Gly Gly Gly 350 GGC GGT GGT Gly Gly Gly CTG TAC TTC CAG Leu Tyr Phe Gin

GGT

Gly 360 GGT GGC GGT GGC Gly Gly Gly Gly

AGC

Ser 365 GGT GAT Gly Asp 370 ATC GTG ATG ACC Ile Val Met Thr

CAG

Gin 375 TCT CCA GAC TCC Ser Pro Asp Ser

CTG

Leu 380 GCT GTG TCT CTG Ala Val Ser Leu

GGC

Gly 385

AGC

Ser GAG CGT GCC ACC Glu Arg Ala Thr TCC AAC AGC AAG Ser Asn Ser Lys 405

ATC

Ile 390 AAT TGC AAG TCC Asn Cys Lys Ser

AGC

Ser 395 CAG ACT GTT TTA Gin Ser Val Leu

TAC

Tyr 400 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 AAC TAC CTG GCT Asn Tyr Leu Ala

TGG

Trp 410 TAC CAG CAG AAA Tyr Gin Gin Lys CCA GGT Pro Gly 415 CAG CCT CCT Gin Pro Pro GTT CCT GAC Val Pro Asp 435

AAG

Lys 420 CTG CTC ATT TAC Leu Leu Ile Tyr

TCG

Trp 425 GCA TCT ACC CGT Ala Ser Thr Arg GAA TCC GGC Giu Ser Gly 430 TTC ACT CTC Phe Thr Leu CGT TTC ACT GGT Arg Phe Ser Gly

AGC

Ser 440 GGT TCT GGT ACA Gly Ser Gly Thr ACC ATC Thr Ile 450 AGC AGC CTC CAG Ser Ser Leu Gin

GCT

Ala 455 GAA CAT GTG GCA Glu Asp Val Ala

GTT

Val1 460 TAT TAC TGC CAG Tyr Tyr Cys Gin

CAG

Gin 465 TAT TAC ACT ACC Tyr Tyr Ser Thr

CCG

Pro 470 TAC TCC TTC CGT Tyr Ser Phe Gly

CAG

Gin 475 GGT ACC AAA CTG Gly Thr Lys Leu

GAA

G iu 480 WO 99/15563 WO 99/ 5563PCT/AU98/00783 -66- ATC AAA CGC AGC GGT AGC GCT TGG CGT CAC CCG CAG TTC GGT GGT TAA Ile Lys Arg Ser Gly Ser Ala Trp Arg His Pro Gin Phe Gly Gly* 485 490 495

TA

INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 496 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 1488 1491 Met Lys Lys Thr Ala Ile Ala Ile Ala Vai 1 Thr Met Thr Lys Leu Phe Leu Thr Gly 145 Ser Giu Thr Ala Val 225 Val1 Thr Ile Asn Leu Ser Gin Pro 130 Ser Giy Vai Ala Tyr 210 Ala Ala Gin Asn Tyr Ile Gly Ala 115 Tyr Gly Gly Thr Giu 195 Ala Asp Gin Ser Cys Leu Tyr Ser 100 Giu Ser Giy Gly Ile 180 Phe Asp Lys 5 Ala Pro Lys Ala Trp Gly Asp Phe Gly Ser 165 Lys Lys Thr Giy Asp Asp Ser Trp 70 Ala Ser Vai Gly Gly 150 Glu Ala Gly Leu Tyr 230 Tyr Ser Ser 55 Tyr Ser Gly Ala Gin 135 Ser Asn Asn Thr Lys 215 Thr Lys Leu 40 Gin Gin Thr Thr Val 120 Gly Gly Leu Leu Phe 200 Lys Leu Asp 25 Ala Ser Gin Arg Asp 105 Tyr Thr Gly Tyr Ile 185 Giu Asp Asn 10 Asp Val Val Lys Glu 90 Phe Tyr Lys Gly Phe 170 Phe Lys ASfl Ile Ala Asp Ser Leu Pro 75 Ser Thr Cys Leu Gly 155 Gin Ala Ala Gly Lys 235 Leu Asp Leu Tyr Giy Giy Leu Gin Giu 140 Ser Giy Asn Thr Giu 220 Phe Ala Lys Gly Ser Gin Val Thr Gin 125 Ile Ser Gly Gly Ser 205 Tyr Ala Gly Asp Giu Ser Pro Pro Ile 110 Tyr Lys Gly Ser Ser 190 Glu Thr Gly Phe Ala Ile Val Arg Ala Asn Ser Pro Lys Asp Arg Ser Ser Tyr Ser Arg Ser Gly Gly 160 Ala Glu 175 Thr Gin Ala Tyr Val Asp Lys Giu 240 Leu Gin 255 Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile WO 99/15563 PCT/AU98/00783 -67- Ile Asn Ser Lys Lys Thr 305 Gly Gly Ser Gly Gly 385 Ser Gin Val Thr Gin 465 Ile Ala Gly 290 Leu Tyr Ser Glu Asp 370 Glu Ser Pro Pro Ile 450 Tyr Lys Asn 275 Thr Lys Thr Gly Asn 355 Ile Arg Asn Pro Asp 435 Ser Tyr Arg Arg 260 Leu Phe Lys Leu Gly 340 Leu Val Ala Ser Lys 420 Arg Ser Ser Ser Ile Glu Asp Asn 325 Gly Tyr Met Thr Lys 405 Leu Phe Leu Thr Gly 485 Phe Lys Asn 310 Ile Gly Phe Thr Ile 390 Asn Leu Ser Gin Pro 470 Ser Trp Gly Gly Leu Ala Ala 295 Gly Lys Ser Gin Gin 375 Asn Tyr Ile Gly Ala 455 Tyr Ala Asn 280 Thr Glu Phe Gly Gly 360 Ser Cys Leu Tyr Ser 440 Glu Ser Trp Thr 265 Gly Ser Tyr Ala Ala 345 Gly Pro Lys Ala Trp 425 Gly Asp Phe Arg Ser Ser Glu Thr Gly 330 Gly Gly Asp Ser Trp 410 Ala Ser Val Gly His 490 Ala Thr Ala Val 315 Lys Gly Gly Ser Ser 395 Tyr Ser Gly Ala Gin 475 Pro Glu Gin Tyr 300 Asp Glu Gly Gly Leu 380 Gin Gin Thr Thr Val 460 Gly Gin Glu Thr 285 Ala Val Ser Ser Ser 365 Ala Ser Gin Arg Asp 445 Tyr Thr Phe Val 270 Ala Tyr Ala Gly Gly 350 Gly Val Val Lys Glu 430 Phe Tyr Lys Gly Thr Glu Ala Asp Gly 335 Gly Gly Ser Leu Pro 415 Ser Thr Cys Leu Gly 495 Ile Phe Asp Lys 320 Gly Gly Gly Leu Tyr 400 Gly Gly Leu Gin Glu 480 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1032 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1032 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 <ffi r~ WO 99/15563 W099/5563PCT/AU98/00783 68 ACC GTA GCG Thr Val Ala ATG ACC CAG Met Thr Gin GCC GAC TAC A-AG Ala Asp Tyr Lys GAT GAC GAC AAG Asp Asp Asp Lys GAT ATC GTG Asp Ile Val GAG CGT GCC Glu Arg Ala TCT CCA GAC TCC Ser Pro Asp Ser

CTG

Leu 40 GCT GTG TCT CTG Aia Vai Ser Leu

GGC

Gly ACC ATC Thr Ile AAT TGC A-AG TCC Asn Cys Lys Ser

AGC

Ser CAG AGT GTT TTA Gin Ser Val Leu AGC TCC AAC AGC Ser Ser A-sn Ser

A-AG

Lys AAC TAC CTG GCT Asn Tyr Leu Ala

TGG

Trp 70 TAC CAG CAG AAA Tyr Gin Gin Lys

CCA

Pro 75 GGT CAG CCT CCT Gly Gin Pro Pro

A-AG

Lys CTG CTC ATT TAC Leu Leu Ile Tyr

TGG

Trp GCA TCT ACC CGT Ala Ser Thr Arg

GAA

Giu 90 TCC GGC GTT CCT Ser Gly Vai Pro GAC CGT Asp Arg TTC A-GT GGT Phe Ser Giy CTC CAG GCT Leu Gin Ala 115

AGC

Ser 100 GGT TCT GGT ACA Giy Ser Giy Thr TTC ACT CTC ACC Phe Thr Leu Thr ATC AGC AGC Ile Ser Ser TAT TAC AGT Tyr Tyr Ser GAA GAT GTG GCA Giu Asp Vai Ala

GTT

Vali 120 TAT TAC TGC CAG Tyr Tyr Cys Gin

CAG

Gin 125 144 192 240 288 336 384 432 480 528 576 624 672 ACC CCG Thr Pro 130 TAC TCC TTC GGT Tyr Ser Phe Gly

CAG

Gin 135 GGT ACC AAA CTG Gly Thr Lys Leu GAA ATC AA-A CGC Glu Ile Lys Arg 140 AGC TCT GGT GGT Ser Ser Gly Giy

TCC

Ser

GGC

Gly 160

GGT

Gly 145 AGC GGT GGC GGT Ser Gly Gly Giy

GGT

Gly 150 TCT GGT GGT GGT Ser Gly Gly Gly

GGG

Gly 155 TCT GGT GGT GGT Ser Gly Gly Gly

AGC

Ser 165 GAA AAC CTG TAC Giu Asn Leu Tyr

TTC

Phe 170 CAG GGT GGT AGC Gin Gly Gly Ser GCC GAA Ala Giu 175 GAA GTC ACG Giu Val Thr ACT GCA GA-A Thr Ala Giu 195 AAA GCG AAC CTG Lys Ala A-sn Leu

ATC

Ile 185' TTT GCA A-AT GGT Phe Ala Asn Giy AGC A-CA CAA Ser Thr Gin 190 GAA GCT TAT Giu Ala Tyr TTC A-A-A GGT ACC Phe Lys Gly Thr

TTC

Phe 200 GAA AAA GCG ACC Giu Lys Ala Thr

TCG

Ser 205 GCG TAT Ala Tyr 210 GCA GAT ACT TTG Ala Asp Thr Leu

A-AG

Lys 215 AAA GAC AAT GGT Lys Asp Asn Gly

GAA

Glu 220 TAT ACT GTA GAT Tyr Thr Val Asp

GTT

Val1 225

GCG

Ala GCA GAT A-A-A GGT Ala Asp Lys Gly A-CC AA-C CGT AAC Thr Asn Arg Asn 245

TAC

Tyr 230 A-CC CTG A-AC ATC Thr Leu Asn Ile

AA-A

Lys 235 TTC GCG GGT AAA Phe Ala Gly Lys

GAA

G lu 240 A-CC GAC GGT TCC Thr Asp Gly Ser GAC TAC GGT ATC Asp Tyr Gly Ile TTA CAG Leu Gin 255 768 ATC A-AC TCT Ile Asn Ser AAA GCG A-AC Lys Ala A-sn 275

CGT

A-rg 260 TGG GGT GGT CTG Trp Gly Giy Leu

ACC

Thr 265 AGC GCC GAA GAA Ser Ala Giu Glu GTC A-CG ATC Val Thr Ile 270 GCA GA-A TTC Ala Giu Phe 816 CTG A-TC TTT GCA Leu Ile Phe Ala

A-AT

A-sn 280 GGT AGC ACA CAA Gly Ser Thr Gin 864 WO 99/15563 PCT/AU98/00783 -69- AAA GGT ACC TTC GAA AAA GCG ACC TCG GAA GCT TAT GCG TAT GCA GAT Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala Asp 290 295 300 ACT TTG AAG AAA GAC AAT GGT GAA TAT ACT GTA GAT GTT GCA GAT AAA Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys 305 310 315 320 GGT TAC ACC CTG AAC ATC AAA TTC GCG GGT AAA GAA AGC GCT TGG CGT Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu Ser Ala Trp Arg 325 330 335 CAC CCG CAG TTC GGT GGT TAA TA His Pro Gln Phe Gly Gly 340 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 343 amino acids TYPE: amino acid TOPOLOGY: linear 912 960 1008 1032 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Lys Thr Thr Met Thr Lys Leu Phe Leu Thr Gly 145 Ser Glu Thr Val Thr Ile Asn Leu Ser Gln Pro 130 Ser Gly Val Ala Ala Gin Asn Tyr Ile Gly Ala 115 Tyr Gly Gly Thr Glu 195 Gin Ser Cys Leu Tyr Ser 100 Glu Ser Gly Gly Ile 180 Phe Ala Ala Pro Lys Ala Trp Gly Asp Phe Gly Ser 165 Lys Lys Ile Ala Ile Ala Asp Asp Ser Trp 70 Ala Ser Val Gly Gly 150 Glu Ala Gly Tyr Ser Ser 55 Tyr Ser Gly Ala Gin 135 Ser Asn Asn Thr Lys Leu 40 Gin Gin Thr Thr Val 120 Gly Gly Leu Leu Phe 200 Asp 25 Ala Ser Gin Arg Asp 105 Tyr Thr Gly Tyr Ile 185 Glu Val 10 Asp Val Val Lys Glu 90 Phe Tyr Lys Gly Phe 170 Phe Lys Asp Ser Leu Pro 75 Ser Thr Cys Leu Gly 155 Gin Ala Ala Asp Leu Tyr Gly Gly Leu Gin Glu 140 Ser Gly Asn Thr Ala Leu Ala Gly Lys Gly Ser Gin Val Thr Gin 125 Ile Ser Gly Gly Ser 205 Asp Glu Ser Pro Pro Ile 110 Tyr Lys Gly Ser Ser 190 Glu Phe Ala Ile Val Arg Ala Asn Ser Pro Lys Asp Arg Ser Ser Tyr Ser Arg Ser Gly Gly 160 Ala Glu 175 Thr Gin Ala Tyr .ssa. r .d WO 99/15563 PCT/AU98/00783 Ala Tyr Ala Asp Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp 210 215 220 Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu 225 230 235 240 Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gin 245 250 255 Ile Asn Ser Arg Trp Gly Gly Leu Thr Ser Ala Glu Glu Val Thr Ile 260 265 270 Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gin Thr Ala Glu Phe 275 280 285 Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala Asp 290 295 300 Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys 305 310 315 320 Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu Ser Ala Trp Arg 325 330 335 His Pro Gin Phe Gly Gly 340 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 600 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..600 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT 48 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 ACC GTA GCG CAG GCC GAC TAC AAG GAC GAT GAC GAC AAG GGC GCC GAA 96 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Glu 25 GAA GTC ACG ATC AAA GCG AAC CTG ATC TTT GCA AAT GGT AGC ACA CAA 144 Glu Val Thr Ile Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gin 40 ACT GCA GAA TTC AAA GGT ACC TTC GAA AAA GCG ACC TCG GAA GCT TAT 192 Thr Ala Glu Phe Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr 55 GCG TAT GCA GAT ACT TTG AAG AAA GAC AAT GGT GAA TAT ACT GTA GAT 240 Ala Tyr Ala Asp Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp 70 75 WO 99/15563 PCT/AU98/00783 -71- GTT GCA GAT AAA GGT TAC ACC CTG AAC ATC AAA TTC GCG GGT AAA GAA 288 Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu 90 GCG ACC AAC CGT AAC ACC GAC GGT TCC ACC GAC TAC GGT ATC TTA CAG 336 Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gin 100 105 110 ATC AAC TCT CGT TGG GGT GGT CTG ACC AGC GCC GAA GAA GTC ACG ATC 384 Ile Asn Ser Arg Trp Gly Gly Leu Thr Ser Ala Glu Glu Val Thr Ile 115 120 125 AAA GCG AAC CTG ATC TTT GCA AAT GGT AGC ACA CAA ACT GCA GAA TTC 432 Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gin Thr Ala Glu Phe 130 135 140 AAA GGT ACC TTC GAA AAA GCG ACC TCG GAA GCT TAT GCG TAT GCA GAT 480 Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala Asp 145 150 155 160 ACT TTG AAG AAA GAC AAT GGT GAA TAT ACT GTA GAT GTT GCA GAT AAA 528 Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp Val Ala Asp Lys 165 170 175 GGT TAC ACC CTG AAC ATC AAA TTC GCG GGT AAA GAA AGC GCT TGG CGT 576 Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu Ser Ala Trp Arg 180 185 190 CAC CCG CAG TTC GGT GGT TAA TA 600 His Pro Gin Phe Gly Gly 195 200 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 199 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Glu 25 Glu Val Thr Ile Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gin 40 Thr Ala Glu Phe Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr 55 Ala Tyr Ala Asp Thr Leu Lys Lys Asp Asn Gly Glu Tyr Thr Val Asp 70 75 Val Ala Asp Lys Gly Tyr Thr Leu Asn Ile Lys Phe Ala Gly Lys Glu 90 Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gin 100 105 110 Ile Asn Ser Arg Trp Gly Gly Leu Thr Ser Ala Glu Glu Val Thr Ile 115 120 125 WO 99/15563 PCTIAU98/00783 -72- Lys Ala 130 Asn Leu Ile Phe Ala 135 Asn Gly Ser Lys 145 Gly Thr Phe Glu Lys 150 Ala Thr Ser Glu Thr Gin Thr Ala Glu Phe 140 Ala Tyr Ala Tyr Ala Asp 155 160 Val Asp Val Ala Asp Lys 175 Thr Leu Lys Lys Asp 165 Asn Gly Glu Tyr Thr 170 Gly Tyr Thr His Pro Gin 195 Leu 180 Asn Ile Lys Phe Gly Lys Glu Ser Ala Trp Arg 190 Phe Gly Gly INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Asp Tyr Lys Asp Asp Asp Asp Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 471 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..471 (xi) SEQUENCE DESCRIPTION: SEQ ID

ATG

Met 1 AAA AAG ACA Lys Lys Thr

GCT

Ala 5 ATC GCG ATT GCA GTG GCA CTG GCT GGT Ile Ala Ile Ala Val Ala Leu Ala Gly 10 TTC GCT Phe Ala ACC GTA GCG Thr Val Ala ATG ACC CAG Met Thr Gin

CAG

Gin GCC GAC TAC AAG Ala Asp Tyr Lys

GAC

Asp 25 GAT GAC GAC AAG Asp Asp Asp Lys GAT ATC GTG Asp Ile Val GAG CGT GCC Glu Arg Ala TCT CCA GAC TCC Ser Pro Asp Ser GCT GTG TCT CTG Ala Val Ser Leu 144 ACC ATC Thr Ile AAT TGC AAG TCC Asn Cys Lys Ser AGC CAG AGT GTT TTA TAC AGC TCC AAC AGC Ser Gin Ser Val Leu Tyr Ser Ser Asn Ser 55 192 SWO 99/15563 PCT/AU98/00783 -73- AAG AAC TAC CTG GCT TGG TAC CAG CAG AAA CCA GGT CAG CCT CCT AAG 240 Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys 70 75 CTG CTC ATT TAC TGG GCA TCT ACC CGT GAA TCC GGC GTT CCT GAC CGT 288 Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 90 TTC AGT GGT AGC GGT TCT GGT ACA GAT TTC ACT CTC ACC ATC AGC AGC 336 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 100 105 110 CTC CAG GCT GAA GAT GTG GCA GTT TAT TAC TGC CAG CAG TAT TAC AGT 384 Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr Ser 115 120 125 ACC CCG TAC TCC TTC GGT CAG GGT ACC AAA CTG GAA ATC AAA CGC TCC 432 Thr Pro Tyr Ser Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Ser 130 135 140 GGT AGC GCT TGG CGT CAC CCG CAG TTC GGT GGT TAA TA 471 Gly Ser Ala Trp Arg His Pro Gin Phe Gly Gly 145 150 155 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 156 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ile Val 25 Met Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala 40 Thr Ile Asn Cys Lys Ser Ser Gin Ser Val Leu Tyr Ser Ser Asn Ser 55 Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys 70 75 Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 90 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 100 105 110 Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr Ser 115 120 125 Thr Pro Tyr Ser Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Ser 130 135 140 Gly Ser Ala Trp Arg His Pro Gin Phe Gly Gly 145 150 155 SWO 99/15563 PCT/AU98/00783 -74- INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 540 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..540 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

ATG

Met 1 GAC.TAC AAG Asp Tyr Lys

GAC

Asp 5 GAT GAC GAC AAG Asp Asp Asp Lys

GGC

Gly 10 GCC GAA GAA GTC Ala Glu Glu Val ACG ATC Thr Ile AAA GCG AAC Lys Ala Asn AAA GGT ACC Lys Gly Thr

CTG

Leu ATC TTT GCA AAT Ile Phe Ala Asn AGC ACA CAA ACT Ser Thr Gln Thr GCA GAA TTC Ala Glu Phe TAT GCA GAT Tyr Ala Asp TTC GAA AAA GCG Phe Glu Lys Ala

ACC

Thr 40 TCG GAA GCT TAT Ser Glu Ala Tyr

GCG

Ala ACT TTG Thr Leu AAG AAA GAC AAT Lys Lys Asp Asn

GGT

Gly 55 GAA TAT ACT GTA Glu Tyr Thr Val

GAT

Asp GTT GCA GAT AAA Val Ala Asp Lys

GGT

Gly TAC ACC CTG AAC Tyr Thr Leu Asn AAA TTC GCG GGT Lys Phe Ala Gly GAA GCG ACC AAC Glu Ala Thr Asn

CGT

Arg AAC ACC GAC GGT Asn Thr Asp Gly

TCC

Ser ACC GAC TAC GGT Thr Asp Tyr Gly TTA CAG ATC AAC Leu Gin Ile Asn TCT CGT Ser Arg TGG GGT GGT Trp Gly Gly ATC TTT GCA Ile Phe Ala 115

CTG

Leu 100 ACC AGC GCC GAA Thr Ser Ala Glu

GAA

Glu 105 GTC ACG ATC AAA Val Thr Ile Lys GCG AAC CTG Ala Asn Leu 110 GGT ACC TTC Gly Thr Phe AAT GGT AGC ACA Asn Gly Ser Thr

CAA

Gln 120 ACT GCA GAA TTC Thr Ala Glu Phe

AAA

Lys 125 384 GAA AAA Glu Lys 130 GCG ACC TCG GAA Ala Thr Ser Glu

GCT

Ala 135 TAT GCG TAT GCA Tyr Ala Tyr Ala

GAT

Asp 140 ACT TTG AAG AAA Thr Leu Lys Lys

GAC

Asp 145 AAT GGT GAA TAT Asn Gly Glu Tyr

ACT

Thr 150 GTA GAT GTT GCA Val Asp Val Ala AAA GGT TAC ACC Lys Gly Tyr Thr

CTG

Leu 160 480 528 AAC ATC AAA TTC Asn Ile Lys Phe GGT GGT TAA TA Gly Gly 180

GCG

Ala 165 GGT AAA GAA AGC Gly Lys Glu Ser

GCT

Ala 170 TGG CGT CAC CCG Trp Arg His Pro CAG TTC Gln Phe 175 540 WO 99/15563 PCT/AU98/00783 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 179 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Asp Tyr Lys Asp Asp Asp Asp Lys Lys Lys Thr Gly Asn Trp Ile Glu Asp 145 Asn Gly Ala Gly Leu Tyr Thr Gly Phe Lys 130 Asn Ile Gly Asn Thr Lys Thr Asp Gly Ala 115 Ala Gly Lys Leu Phe Lys Leu Gly Leu 100 Asn Thr Glu Phe Ile Glu Asp Asn Ser Thr Gly Ser Tyr Ala 165 Phe Lys Asn Ile 70 Thr Ser Ser Glu Thr 150 Gly Ala Ala Gly 55 Lys Asp Ala Thr Ala 135 Val Lys Asn Thr 40 Glu Phe Tyr Glu Gin 120 Tyr Asp Gly 25 Ser Tyr Ala Gly Glu 105 Thr Ala Val Gly Ser Glu Thr Gly Ile Val Ala Tyr Ala Gin Tyr Asp Glu Gin Ile Phe Asp 140 Lys Ala Glu Glu Val Thr Ile Thr Ala Val Ala Ile Lys Lys 125 Thr Gly Ala Tyr Ala Thr Asn Ala 110 Gly Leu Tyr Phe Asp Lys Arg Arg Leu Phe Lys Leu 160 Glu Ser Ala Trp Arg His Pro 170 Gin Phe 175 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Ala Trp Arg His Pro Gin Phe Gly Gly I WO 99/15563 WO 9915563PCT/AU98/00783 -76- INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1479 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID CAAAAATCTA GATAACGAGG CTGGCTGGTT TCGCTACCGT

GTGAAGCTGG

TGTGCAACTT

GGGAAGAGAC

TACAGTGCAT

TACCTTCAGA

TACTACGGTA

TCAGGTGGTG

GGTTCCGACA

GTCACTATGA

TTGGCCTGGT

ACTAGGGAAT

CTTACCATCA

AGTTATCCGC

GGTGGTTCTG

CTGTACTTCC

ATCTTTGCAA

TCGGAAGCTT

GTTGCAGATA

GGTGGTTCTG,

GAAGTCACGA

AAAGGTACCT

GACAATGGTG

TGGAATCTGG

CTGGGTTCAC

TGGAGTGGAT

CTGTGAAGGG

TGAATGCCCT

GTACCTGGTG

GCGGTGGTAG

TTGTGATGAC

GTTGCAAGTC

ACCAGCAGAA

CTGGGGTCCC

GCAGTGTGCA

TCACGTTCGG

GTGGTGGTGG

AGGGTGGTGG

ATGGTAGCAC

ATGCGTATGC

AAGGTTACAC

GTGGTGGTGG

TCAAAGCGAA

TCGAAAAAGC

AATATACTGT

GCAA.AAA.ATG

AGCGCAGGCC

AGGAGGCTTG

CTTCAGTGAT

TGCTGCAAGT

TCGGTTCATC

GAGAGCTGAG

CTTCGATGTC

CGGTGGCGGT

ACAGTCTCCA

CAGTCAGAGT

ACCAGGGCAG

TGATCGCTTC

GGCTGAAGAC

TGCTGGGACC

GAGCGGCGCC

CGGTGGCAGC

ACAAACTGCA

AGATACTTTG

CCTGAACATC

TTCTGGTGGC

CCTGATCTTT

GACCTCGGAA

AGATGTTGCA

AAAAAGACAG

GACTACAAGG

GTACAGCCTG

TTCTACATGG

AGAAACAAAG

GTCTCCAGAG

GACACAGCCA

TGGGGCGCAG

GGTTCTGGTG

TCCTCCCTGA

CTGTTAAACA

CCTCCTAAAC

ACAGGCAGTG,

CTGGCAGTTT

AAGCTGGAGC

GGTGGTGGCT

GCTGAAGAAG

GAATTCAAAG

AAGAAAGACA

AAATTCGCG

GGTGGTTCTG

GCAAATGGTA

GCTTATGCGT

GATAAAGGTT

CACTAATAA

CTATCGCGAT

ACGATGACGA

GGGGTTCTCT

AGTGGGTCCG

GTAATAAATA

ACACTTCCCA

TTTATTACTG

GGACCACGGT

GTGGTGGTAG

GTGTGTCAGC

GTGGAAATCA

TGTTGATCTG

GATCTGGAAC

ATTACTGTCA

TGAAACGTGC

CTGGTGGTGG

TCACGATCAA

GTACCTTCGA

ATGGTGAATA

GTAAAGAAC

GTGGTGGTGG

GCACACAAAC

ATGCAGATAC

TGCAGTGGCA

CAAGAGCGAG

GAGACTCTCC

CCAGCCTCCA

TACAACAGAA

AAGCATCCTC

TGCAAGAAAT

CACCGTCTC!C

CGGTGGTGGT

AGGAGAGAGA

AAAGAACTTC

CGGGGCATCC

CGATTTCACT

GAATGATCAT

TAGCGGTGGC

TAGCGAAAAC

AGCGAACCTG

AAAAGCGACC

TACTGTAGAT

TAGCGGTGGC

TTCTGCTGAA'

TGCAGAATTC

TTTGAAGAAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1479 ACACCCTGAA CATCAAATTC GCGGGTAAAG AAGCTCATCA CCATCACCAT SUBSTITUTE SHEET (Rule 26) (ROIAU) WO 99/15563 PCT/AU98/00783 -77- INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 482 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 Thr Val Ala Gin Ala Asp Tyr Lys Asp Asp Asp Asp Lys Ser Glu Val 25 Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu 40 Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe Tyr Met 55 Glu Trp Val Arg Gin Pro Pro Gly Lys Arg Leu Glu Trp Ile Ala Ala 70 75 Ser Arg Asn Lys Gly Asn Lys Tyr Thr Thr Glu Tyr Ser Ala Ser Val 90 Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gin Ser Ile Leu Tyr 100 105 110 Leu Gin Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 115 120 125 Ala Arg Asn Tyr Tyr Gly Ser Thr Trp Cys Phe Asp Val Trp Gly Ala 130 135 140 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val 165 170 175 Met Thr Gin Ser Pro Ser Ser Leu Ser Val Ser Ala Gly Glu Arg Val 180 185 190 Thr Met Ser Cys Lys Ser Ser Gin Ser Leu Leu Asn Ser Gly Asn Gin 195 200 205 Lys Asn Phe Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys 210 215 220 Leu Leu Ile Cys Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 225 230 235 240 Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 245 250 255 Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn Asp His Ser 260 265 270 Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala 275 280 285 SUBSTITUTE SHEET (Rule 26) (RO/AU) <--~t2nj~Sa~sn~ WO 99/15563 WO 9915563PCT/AU98/00783 -78- Ser Ser 305 Ser Ser Giu Thr Gly 385 Gly Ala Gly Leu Tyr 465 His Gly 290 Gly Ala Thr Ala Val1 370 Lys Gly Asn Thr Lys 450 Thr His Giy Gly Glu Gin Tyr 355 Asp Giu Gly Leu Phe 435 Lys Gly Gly Glu Thr 340 Ala Val1 Ala Ser Ile 420 Glu Asp Gly Ser Val1 325 Ala Tyr Ala Ser Gly 405 Phe Lys Asn Ser Giu 310 Thr Glu Ala Asp Gly 390 Gly Ala Ala Gly Lys 470 Gly 295 Asn Ile Phe Asp Lys 375 Gly Gly Asn Thr Glu 455 Giy Leu Lys Lys Thr 360 Gly Gly Gly Giy Ser 440 Tyr Gly Tyr Ala Gly 345 Leu Tyr Gly Ser Ser 425 Glu Thr Gly Phe Asn 330 Thr Lys Thr Ser Ala 410 Thr Ala Val1 Ser Gin 315 Leu Phe Lys Leu Gly 395 Giu Gln Tyr Asp Giu 475 Gly 300 Gly Ile Giu Asp Asn 380 Gly Glu Thr Ala Val 460 Ala Gly Phe Lys Asn 365 Ile Gly Val1 Ala Tyr 445 Ala Gly Gly Ala Ala 350 Gly Lys Gly Thr Giu 430 Ala Asp Gly Gly Asn 335 Thr Glu Phe Ser Ile 415 Phe Asp Lys Giy Gly 320 Gly Ser Tyr Ala Gly 400 Lys Lys Thr Gly His 480 Leu Asn Ile Phe Ala Gly Lys Ala His His His SUBSTITUTE SHEET (Rule 26) (ROIAU)

Claims (14)

1. A method of selectively activating B cells expressing catalytic antibodies, said method comprising administering to a subject a growth factor precursor comprising a recombinant polypeptide chain wherein said polypeptide chain comprises at least one B cell surface molecule binding portion having a multimerizing inducing element at its C- terminal end, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable light chain and variable light chain domains in the growth factor precursor, associate together by intra- and/or inter-domain bonding and substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule, said growth factor precursor being administered for a time and under conditions sufficient to induce B cells activation produce catalytic antibodies specific for said antigen.

A method according to Claim 1 wherein the multimerizing inducing element is a signal peptide.

3. A method according to Claim 2 wherein the signal peptide is from ompA.

4. A method according to Claim 1 or 2 or 3 wherein the peptide portion comprising domains from both a variable heavy chain and variable light chain is a single chain Fv molecule.

A method according to Claim 4 wherein the single chain Fv molecule is 4WN. from McPc6O3 as herein defined. n-sa-~ rs~t-gs-n~ V> P:\Opc\Ejh.m ded\91467-98.kolntgmau.am dcd claims.doc-0901/02

6. A growth factor precursor comprising a recombinant polypeptide chain wherein said polypeptide chain comprises at least one B cell surface molecule binding portion having a multimerizing inducing element at its C-terminal end, at least one T cell surface molecule binding portion capable of providing T cell dependent help to a B cell, an antigen cleavable by a catalytic antibody and a peptide portion comprising domains from both a variable heavy chain and a variable light chain of an immunoglobulin and wherein said variable light chain and variable light chain domains in the growth factor precursor, associate together by intra- and/or inter-domain bonding and substantially prevent the at least one B cell surface molecule binding portion from interacting with a B cell surface molecule such that upon cleavage of said antigen by a catalytic antibody, the peptide comprising said variable heavy chain and variable light chain domain permits the at least one B cell surface molecule binding portion to interact with a B cell surface molecule.

7. A growth factor precursor according to Claim 6 wherein the multimerizing inducing element is a signal peptide.

8. A growth factor precursor according to Claim 7 wherein the signal peptide is from ompA.

9. A growth factor precursor according to Claim 6 or 7 or 8 wherein the peptide portion comprising domains from both a variable heavy chain and variable light chain is a single chain Fv molecule.

A growth factor precursor according to Claim 9 wherein the single chain Fv molecule is from McPc603 as herein defined.

11. The use of the products of catalysis of a growth factor precursor as defined in any one of Claims 6 to 10 to induce B cell mitogenesis to generate catalytic antibodies to a specific antigen.

A composition comprising the recombinant growth factor precursor P:\Opcr\EjhbunmdodWI467-998Lko~ggm.z.mdd dIim0doc-9/01/02 -81 according to any one of Claims 6 to 10 and one or more pharmaceutical carries and/or diluents.

13. A composition comprising a catalytic antibody generated to the growth factor precursor according to any one of Claims 6 to

14. A method according to any one of Claims 1 to 5 or a growth factor precursor according to any one of Claims 6 to 10 or the use according to Claim 11 or a composition according to any one of Claims 12 or 13 substantially as hereinbefore defined with reference to the Figures and/or Examples. o o *oo