Mutated anti-cd22 antibodies and immunoconjugates
Inventors
Pastan, Ira H. • Ho, Mitchell • Bang, Sookhee
Assignees
US Department of Health and Human Services
Publication Number
US-7982011-B2
Publication Date
2011-07-19
Expiration Date
2024-11-24
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Abstract
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 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 such cancers. Additionally, the invention provides a method of increasing the cytotoxicity of forms of Pseudomonas exotoxin A (“PE”) with the mutation of a single amino acid, as well as compositions of such mutated PEs, nucleic acids encoding them, and methods for using the mutated PEs.
Core Innovation
The invention provides antibodies and antibody fragments with an improved ability to bind the CD22 antigen compared to the previously known antibody RFB4. These improved antibodies are incorporated into recombinant immunotoxins, which are chimeric fusion proteins comprising antibody variable domains fused to bacterial or plant toxins, specifically Pseudomonas exotoxin A (PE) derivatives. The immunotoxins made with these antibodies possess enhanced cytotoxicity against CD22-expressing cancer cells.
The background outlines the problem of hematological malignancies, such as non-Hodgkin's lymphoma and leukemia, which have high morbidity and mortality rates and often do not respond well to conventional therapies. Immunotoxins targeting CD22, such as BL22 (RFB4 dsFv fused to PE38), have demonstrated clinical efficacy but have limitations in binding affinity and cytotoxic potency, especially in cancers with low CD22 expression like chronic lymphocytic leukemia (CLL). Improving binding affinity and toxin cytotoxicity is complicated due to multifaceted antibody-antigen interactions and intracellular routing requirements for toxin activity.
The present invention addresses these challenges by providing antibodies with mutations in complementarity determining regions (CDRs) that increase binding affinity for CD22 beyond that of both RFB4 and an improved antibody called HA22. In addition, the invention introduces a single amino acid mutation at position 490 in PE toxins, replacing arginine with an aliphatic amino acid such as alanine, which increases the cytotoxic potency of the toxin moiety. This mutation can be applied to various PE derivatives to enhance their effectiveness, improving therapeutic outcomes in immunotoxin therapies.
Claims Coverage
The patent claims cover multiple inventive features mainly focused on anti-CD22 antibodies with specific variable region mutations, chimeric molecules comprising these antibodies fused or conjugated to therapeutic or detectable moieties, compositions, methods of using these molecules, and mutated forms of Pseudomonas exotoxin A with increased cytotoxicity.
Anti-CD22 antibody with defined complementarity determining region sequences
An anti-CD22 antibody having a variable light (VL) chain and variable heavy (VH) chain, each comprising three complementarity determining regions (CDRs), wherein VL CDR1 sequence at positions 4-5 is selected from HG, GR, RG, and AR; VL CDR2 and CDR3, VH CDR1, CDR2 and CDR3 have specified sequences (SEQ ID NOs:7,11,12,13,14,16 respectively) with variability at VH CDR3 positions 8-10 selected from THW, YNW, TTW, and STY.
Chimeric molecule comprising anti-CD22 antibody and therapeutic or detectable moiety
A chimeric molecule comprising the above-defined anti-CD22 antibody conjugated or fused to a therapeutic moiety or detectable label, maintaining the same specific CDR sequences and sequence variations as described for the antibody.
Use of mutated Pseudomonas exotoxin A with substitution at position 490
Pseudomonas exotoxin A (PE) or cytotoxic fragments thereof having an aliphatic amino acid substitution (glycine, alanine, valine, leucine, or isoleucine) in place of arginine at the position corresponding to 490 of SEQ ID NO:24, which results in increased cytotoxicity.
Methods of inhibiting growth of CD22+ cancer cells
Methods that involve contacting CD22 positive cancer cells with chimeric molecules comprising the above-defined anti-CD22 antibodies and therapeutic moieties, resulting in inhibition of cell growth.
Methods of detecting CD22+ cancer cells
Methods of detecting CD22-expressing cancer cells in biological samples by contacting the sample with the defined anti-CD22 antibodies conjugated or fused to detectable labels and detecting the presence of bound antibody.
Kits for detection and treatment
Kits comprising containers and anti-CD22 antibodies as defined herein, conjugated or fused to detectable labels or therapeutic moieties for detection or treatment of CD22-expressing cancer cells.
The claims comprehensively cover novel anti-CD22 antibodies with specific CDR mutations conferring increased affinity, their incorporation into chimeric immunoconjugates with therapeutic or detectable moieties, mutated Pseudomonas exotoxin A derivatives with enhanced cytotoxicity, and methods and kits for cancer therapy and diagnosis employing these molecules.
Stated Advantages
Antibodies with improved affinity for CD22 compared to parental antibodies RFB4 and HA22.
Immunotoxins made with these improved antibodies exhibit increased cytotoxicity against CD22-expressing cancer cells.
Single amino acid mutation in Pseudomonas exotoxin A (position 490) doubles the cytotoxic activity of the toxin moiety in immunotoxins.
Increased potency allows for reduced immunotoxin doses in therapy, potentially lowering side effects and achieving better therapeutic outcomes.
Immunotoxins exhibit specificity to CD22-positive cells, with reduced non-specific toxicity to CD22-negative cells such as endothelial cells.
Documented Applications
Use of improved anti-CD22 antibody immunotoxins for detection and targeted killing of CD22-expressing B-cell malignancies, including hairy cell leukemia and chronic lymphocytic leukemia.
Use of mutated Pseudomonas exotoxin A immunotoxins to enhance cytotoxic activity against CD22-positive cancer cells and mesothelin-expressing epithelial cancers such as pancreatic and ovarian cancers.
Diagnostic applications such as in vitro assays and in vivo detection of CD22-positive cells in biological samples using antibodies conjugated to detectable labels.
Purging of CD22-expressing malignant cells from blood or cell cultures using immunotoxins for patients prior to reinsertion.
Therapeutic applications including administration of immunotoxins to inhibit growth and induce remission in hematological malignancies expressing CD22.
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