The United States of America, As Represented by the Secretary, Department of Health and Human Services

The United States of America As Represented by the Department of Health

The United States of America As Represented by the Secretary of

Genentech, Inc.

Cetus Corporation

Protein Design Labs, Inc.

David J FitzGerald

Ira H Pastan

Vijay K Chaudhary

Cary L Queen

Thomas Alexander Waldmann

Robert Kreitman

Yoram Reiter

Elizabeth Mansfield

Bernard Moss

David B Ring

Randall J Mrsny

Tamio Mizukami

Walter J Laird

Mark C Willingham

Arthur E Frankel

Edward A Berger

Michael J Iadarola

Sankar L Adhya

Thomas R Fuerst

Marian L McKee

Michael J Bjorn

Ann L Daugherty

John Weldon

Jeffrey L Windelhake

Masato Ogata

Janendra Batra

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 mutated, cytotoxic forms ofexotoxin A (PE) comprising a furin cleavage sequence conjugated or fused directly to residues 395-613 of PE or variants of that sequence. These minimal forms of PE are smaller than previous cytotoxic forms of PE, reduce non-specific toxicity, and reduce immunogenicity due to domain II or domain Ib of PE. The invention further provides nucleic acids encoding said PEs, chimeric molecules containing them, and methods of use thereof.

Deletions in domain II of pseudomonas exotoxin a that remove immunogenic epitopes

This invention providesexotoxin A-like chimeric immunogens that include a non-native epitope in the Ib domain ofexotoxin. Methods of eliciting an immune response using these immunogens also are provided.

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This invention provides bioactive conjugates. The bioactive conjugates include: (1) a cell recognition moiety that binds to &#x3b1;macroglobulin receptor &#x3b1;2-MR and (2) a bioactive moiety which: (a) has a biological activity, (b) does not function solely as an immunogen to invoke an immune response and (c) does not have ADP ribosylating activity. The bioactive conjugates of this invention are useful in methods of transporting the bioactive moiety across a polar epithelial membrane. Thus, this invention provides methods for parenteral administration of proteins without injection.

Delivery of proteins across polar epithelial cell layers

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 chimeric proteins comprising a non-toxicexotoxin A sequence and a Type IV pilin loop sequence, wherein the Type IV loop sequence is inserted within the non-toxicexotoxin A. The invention also provides polynucleotides encoding the chimeric proteins, and compositions comprising the polynucleotides or the chimeric proteins. The invention also provides methods for using the chimeric proteins, polynucleotides and compositions of the invention.

The invention pertains to the discovery of novel disulfide linked cell toxins which can ablate NK-1 receptor expressing cells. These toxins are used as pharmaceutical compositions for the ablation of NK1 receptor expressing cells and comprise a substance P (SP)-exotoxin disulfide linked conjugate. The invention also includes methods of making and using these toxins and pharmaceutical compositions.

Disulfide conjugated cell toxins and methods of making and using them

This invention provides protease-activatable Pseudomonas exotoxin A-like (&ldquo;PE-like&rdquo;) proproteins. The proproteins comprise (1) a cell recognition domain of between 10 and 1500 amino acids that binds to a cell surface receptor; (2) a modified PE translocation domain comprising an amino acid sequence sufficiently homologous to domain II of PE to effect translocation to a cell cytosol upon proteolytic cleavage, wherein the translocation domain comprises a cysteine-cysteine loop that comprises a protease activatable sequence cleavable by a protease and wherein the cysteine-cysteine loop is substantially un-activatable by furin; (3) optionally, a PE Ib-like domain comprising an amino acid sequence up to 1500 amino acids; (4) a cytotoxicity domain comprising an amino acid sequence substantially homologous to domain III of PE, the cytotoxicity domain having ADP-ribosylating activity; and (5) an endoplasmic reticulum (&ldquo;ER&rdquo;) retention sequence. The invention also provides methods of using these proproteins for killing target cells.

Protease-activatable pseudomonas exotoxin A-like proproteins

This invention provides protease-activatable Pseudomonas exotoxin A-like (&ldquo;PE-like&rdquo;) proproteins. The proproteins comprise (1) a cell recognition domain of between 10 and 1500 amino acids that binds to a cell surface receptor; (2) a modified PE translocation domain comprising an amino acid sequence sufficiently homologous to domain II of PE to effect translocation to a cell cytosol upon proteolytic cleavage, wherein the translocation domain comprises a cysteine-cysteine loop that comprises a protease activatable sequence cleavable by a protease and wherein the cysteine-cysteine loop is substantially un-activatable by furin; (3) optionally, a PE Ib-like domain comprising an amino acid sequence up to 1500 amino acids; (4) a cytotoxicity domain comprising an amino acid sequence substantially homologous to domain m of PE, the cytotoxicity domain having ADP-ribosylating activity; and (5) an endoplasmic reticulum (&ldquo;ER&rdquo;) retention sequence. The invention also provides methods of using these proproteins for killing target cells.

Polynucleotides encoding protease-activatable pseudomonas exotoxin a-like proproteins

Single chain immunotoxins directed at the human transferrin receptor using PCR based methods are described. Anti-TFR(Fv)-PE40 contains DNA for the antigen binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor fused to the DNA for a 40,000 molecular weight fragment of Pseudomonas exotoxin (PE40). In another fusion protein, DT388-anti-TFR(Fv), DNA for the antigen binding portions of the anti-TFR antibody has been fused to the DNA encoding a truncated form of Diphtheria toxin. In anti-TFR(Fv)-PE40, the single chain antibody precedes the toxin whereas in DT388-anti-TFR(Fv) the single chain antibody is at the carboxyl end of toxin. The proteins encoded by these gene fusions were expressed in E.coli and purified by conventional chromatographic techniques to near homogeneity.

Recombinant immunotoxin composed of a single chain antibody reacting

The present invention describes constructs encoding recombinant scFv-toxin fusion proteins which selectively kill cells bearing appropriate antigens or receptors.

Constructs encoding recombinant antibody-toxin fusion proteins

Improved Pseudomonas exotoxins of low animal toxicity and high cytocidal activity are described. Substitution of positively charged amino acid residues with an amino acid residue without a positive charge provides markedly changed exotoxins. Conjugation of the new exotoxins with suitable targeting agents provides cytocidal specificity for killing desired cellular entities.

Psuedomonas exotoxins of low animal toxicity and high cytocidal activity

A target-specific, cytotoxic, recombinant Pseudomonas exotoxin is described. Such toxins are made by inserting specific recognition molecules at specific cloning sites in at least domain III near the carboxyl terminus of the PE molecule. Various modifications of the carboxyl terminus of the PE molecule to increase cytotoxicity are set forth. Multifunctional, recombinant, cytotoxic fusion proteins containing at least two different recognition molecules are provided for killing cells expressing receptors to which the recognition molecules bind with specificity. Methods for producing novel recombinant PE molecules with specific properties are described.

Target-specific, cytotoxic, recombinant pseudomonas exotoxin

The present invention describes recombinant antibody toxin fusion proteins which selectively kill cells bearing appropriate antigens or receptors.

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Rockville, MD, United States of America

David J FitzGerald