Title:  Interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders

United States Patent:  6,350,860

Assignee:   Innogenetics N.V. (Ghent, NL)

Filed:  February 14, 2000

PCT NO:  PCT/EP98/05165

102(e) Date:  February 14, 2000

PCT PUB. Date:  February 25, 1999

Foreign Application Priority Data:  Aug 18, 1997[EP] (97870122);  Jun 18, 1998[EP] (98870139)

The present invention concerns molecules which bind and neutralize the cytokine interferon-gamma. More specifically, the present invention relates to sheep-derived antibodies and engineered antibody constructs, such as humanized single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies which can be used to treat diseases wherein interferon-gamma activity is pathogenic. Examples of such diseases are: septic shock, cachexia, multiple sclerosis and psoriasis.

SUMMARY OF THE INVENTION

It is clear from the prior art as cited above that problems such as suboptimal stability, affinity, clearance rate, specificity, efficacy, and an unwanted carrier effect and HAMA response hamper the successful usage of several therapeutics which, potentially, could neutralize the activity of IFN.gamma.. Also suggested solutions to overcome some of these problems did not result in the development of effective products. Thus, unpredictable and unknown factors still appear to determine the success of these biologicals. Despite these unknown factors, the present inventors have been able to design and develop useful constructs which effectively neutralize IFN.gamma.-activity. Indeed, the constructs have all a surprisingly high affinity for IFN.gamma., they do not provoke a HAMA or related response, and they do not result in a carrier effect. In addition, some of the constructs pass the blood brain barrier, whereas others have a very good clearance rate. Therefore, the present invention aims at providing a molecule which binds and neutralizes interferon-gamma and which is chosen from the group consisting of:

a scFv comprising the humanized variable domain of the monoclonal antibody D9D10

a chimeric antibody comprising the humanized variable domain of the monoclonal antibody D9D10

a diabody comprising the humanized variable domain of the monoclonal antibody D9D10

a multivalent antibody

a ruminant antibody.

The present invention further aims at providing a multivalent antibody chosen from the group consisting of triabodies, tetravalent antibodies, peptabodies and hexabodies.

The present invention also aims at providing a triabody, tetravalent antibody, peptabody and hexabody which comprise 3, 4, 5 and 6 variable domains, respectively, of different anti-interferon-gamma antibodies.

The present invention further aims at providing a triabody as described above which comprises 3 identical variable domains of an anti-interferon-gamma antibody. A preferred variable domain used in the latter constructs is derived from the mouse anti-interferon-gamma antibody D9D10 which is described by Sandvig et al. (1987) and Froyen et al. (1993) or from the sheep anti-interferon-gamma antibody described in the present application. Therefore, the present invention aims at providing a triabody as described above which comprises 3 identical D9D10 scFv's, 3 identical humanized D9D10 scFv's, 3 identical sheep-derived anti-interferon-gamma scFv's or 3 identical humanized sheep-derived anti-interferon-gamma scFv's.

The present invention further aims at providing a tetravalent antibody (called MoTAb I) as described above which comprises 4 identical domains of an anti-interferon-gamma antibody. More specifically, the present invention aims at providing a tetravalent antibody as described above which comprises either 4 identical D9D10 scFv's or 4 identical sheep-derived anti-interferon-gamma scFv's in the format of a homodimer of 2 identical molecules, each containing 2 D9D10 scFv's or 2 humanized D9D10 scFv's or 2 sheep-derived anti-interferon-gamma scFv's or 2 humanized sheep-derived anti-interferon-gamma scFv's, and a dimerization domain, or, a full-size humanized D9D10 antibody or sheep-derived anti-interferon-gamma antibody to which 2 humanized D9D10 scFv's or 2 humanized sheep-derived anti-interferon-gamma scFv's, respectively, are attached at the carboxyterminus (called MoTAb II).

The present invention further aims at providing a peptabody and hexabody as described above which comprise 5 and 6 identical variable domains of an anti-interferon-gamma antibody, respectively. A preferred variable domain used in the latter constructs is derived from the mouse anti-interferon-gamma antibody D9D10 which is described above or from the sheep anti-interferon-gamma antibody described in the present application. Therefore, the present invention aims at providing a peptabody and hexabody as described above which comprises 5 or 6 identical D9D10 scFv's, 5 or 6 identical humanized D9D10 scFv's, 5 or 6 identical sheep-derived anti-interferon-gamma scFv's, or, 5 or 6 identical humanized sheep-derived anti-interferon-gamma scFv's, respectively.

The present invention further aims at providing a molecule as described above, wherein said ruminant antibody is a sheep antibody.

The present invention also aims at providing a molecule as described above, wherein said sheep antibody is a monoclonal antibody. Furthermore, the present invention aims at providing a humanized antibody, a single-chain fragment or any other fragment which is derived from said monoclonal antibody and which has largely retained the specificity of said monoclonal antibody.

Moreover, the present invention aims at providing methods for producing the above-described molecules.

The present invention further aims at providing a pharmaceutical composition comprising a molecule as described above, or a mixture of said molecules, in a pharmaceutically acceptable excipient.

The present invention also aims at providing a molecule or a composition as described above for use as a medicament.

Furthermore, the present invention aims at providing a molecule or a composition as described above for preventing or treating septic shock, cachexia, immune diseases such as multiple sclerosis and Crohn's disease and skin disorders such as bullous, inflammatory and neoplastic dermatosis.

Finally, the present invention aims at providing a molecule as described above for determining interferon gamma levels in a sample.

Claim 1 of 14 Claims

What is claimed is:

1. An isolated molecule which binds and neutralizes interferon-gamma selected from the group consisting of:

a scFv comprising a humanized variable domain, wherein said variable domain comprises amino acids 1-117 and 133-239 of SEQ ID) NO: 85;

a chimeric antibody comprising:

a) a humanized heavy chain variable domain, said heavy chain variable domain having an amino acid sequence as shown in positions 1-117 of SEQ ID NO: 85, and

b) the humanized light chain variable domain, said light chain variable domain having an amino acid sequence as shown in positions 133-239 of SEQ ID NO: 85;

a diabody comprising:

a) a humanized heavy chain variable domain, said heavy chain variable domain having an amino acid sequence as shown in positions 1-117 of SEQ ID NO: 85, and

b) a humanized light chain variable domain, said light chain variable domain having an amino acid sequence as shown in positions 133-239 of SEQ ID NO: 85; and,

a multivalent antibody, wherein said multivalent antibody is selected from the group consisting of a triabody, a tetravalent antibody, a peptabody, and a hexabody, and wherein said multivalent antibody comprises:

a) a humanized heavy chain variable domain, said variable domain comprising amino acids 1-117 of SEQ ID NO: 85; and

b) a humanized light chain variable domain, said variable domain comprising amino acids 133-239 of SEQ ID NO: 85.

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