National Institutes of Health, a Component of the US Dept. of Health & Human Services

Robert Kreitman

Ira H Pastan

Richard Beers

Satoshi Nagata

Masanori Onda

Giuliana Salvatore

David J FitzGerald

Byungkook Lee

Elizabeth Mansfield

George Vasmatzis

Yoshito Numata

James Vincent

David Fitzgerald

Yasuo Tsutsumi

Kenneth Santora

Abhishek Sinha

Methods and compositions relating to anti-CD22 antibodies with high binding affinity, and immunoconjugates comprising the anti-CD22 antibody linked to a therapeutic agent such as aexotoxin or a detectable label. The invention provides diagnostic methods, and means to inhibit the growth of malignant B cells.

Recombinant antibodies and immunoconjugates targeted to CD-22 bearing cells and tumors

The invention provides mutatedexotoxins (PE) that have reduced immunogenicity compared to PEs containing the native sequence. The PEs of the invention have one or more individual mutations of positions of the native sequence of PE that reduce antibody binding to one or more PE epitopes. Nucleic acids encoding the mutated PEs, chimeric molecules comprising them, compositions comprising the chimeric molecules and methods of using them, are also provided.

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Recombinant immunotoxins are fusion proteins composed of the Fv domains of antibodies fused to bacterial or plant toxins. RFB4 (Fv)-PE38 is an immunotoxin that targets CD22 expressed on B cells and B cell malignancies. The present invention provides antibodies and antibody fragments that have improved ability to bind the CD22 antigen of B cells and B cell malignancies compared to RFB4. Immunotoxins made with the antibodies and antibody fragments of the invention have improved cytotoxicity to CD22-expressing cancer cells. Compositions that incorporate these antibodies into chimeric immunotoxin molecules that can be used in medicaments and methods for inhibiting the growth and proliferation of leukemia and lymphoma cells.

Mutated anti-CD22 antibodies with increased affinity to CD22-expressing leukemia cells

Mutated Anti-CD22 antibodies with increased affinity to CD22-expressing leukemia cells

Methods and compositions relating to recombinant anti-CD22 antibodies with high binding affinity, and immunoconjugates comprising the anti-CD22 antibody linked to a therapeutic agent such as aexotoxin or a detectable label. The invention provides diagnostic methods, and means to inhibit the growth of malignant B cells.

The invention provides recombinant immunotoxins that have been modified from a parental immunotoxin to lower liver toxicity. The immunotoxins are created by specifically mutating charged residues in the framework regions of the heavy chain, the light chain, or both, of the antibody portion or antigen-binding fragment thereof of the parental immunotoxin to reduce the pI of the antibody or fragment. In preferred forms, the antibody portion of the parental is an anti-Tac, anti-mesothelin, or anti-LewisY antigen antibody or antigen-binding fragment, and in particularly preferred forms the antibody portion is an M16 dsFv, a St6 dsFv or a Mt9 dsFv, or a sequence that has at least 90% sequence identity to one of these molecules but retain the particular mutations that lower pI without affecting antibody activity. The invention further provides nucleic acids encoding the recombinant immunotoxins of the invention, expression cassettes comprising the nucleic acids, and host cells comprising the expression cassettes. The invention also provides a method for killing a cell comprising an antigen on the surface of the cell, the method comprising contacting the cell with a recombinant immunotoxin of the invention that has an antibody or antigen-binding fragment thereof that binds specifically to the antigen on the surface of the cell, and uses of immunotoxins of the invention for the manufacture of medicaments.

Reduction of the nonspecific animal toxicity of immunotoxins by mutating the framework regions of the Fv to lower the isoelectric point

CD30 is a receptor expressed on cells of Hodgkin's disease and certain leukemias. The extracellular portion of CD30 is cleaved, releasing a form known as sCD30. The invention relates in part to the discovery that a residual, extracellular 'stalk' of CD30 remains after cleavage of sCD30. The stalk provides an advantageous and previously unrecognized target for immunotoxins. The invention provides antibodies that bind to the CD30 stalk or to epitopes destroyed upon the cleavage of CD30 which results in the stalk. The invention further provides new anti-CD30 antibodies that form effective immunotoxins and are particularly suitable for making disulfide stabilized Fv ('dsFv')-immunoconjugates. The dsFv immunoconjugates can be used as reagents to label CD30-expressing cancer cells or to inhibit the growth of CD30-expressing cancer cells. Moreover, the invention provides anti-CD30 antibodies that activate complement-dependent cytotoxicity.

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Potomac, MD, United States of America

Robert Kreitman