Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells

a technology of humanized antibodies and immunoconjugates, which is applied in the field of immunoconjugates for diagnostic and therapeutic use in cancer, can solve the problems of increased radiolabel concentration to increase the dosage to the tumor, and limited clinical use of mll2, just as with most other promising murine antibodies, and achieves the effect of lowering the hama reaction

Inactive Publication Date: 2005-05-19
IMMUNOMEDICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017] In another aspect of the invention, there is provided a hLL2 mAb related to mLL2 mAb, in which the complementarity-determining regions (CDRs) of the light and heavy chains of the mLL2 mAb are joined to the framework (FR) sequence of human VK and VH regions, respectively, and subsequently to the human kappa and IgG1 constant region domains, respectively. This humanized antibody retains the B-lymphoma and leukemia cell targeting and internalizaiton characteristics of the parental mLL2 mAb, and can exhibit a lowered HAMA reaction.

Problems solved by technology

However, the toxic side effects associated with chemotherapy and the toxicity to the hematopoietic system from local, as well as whole body, radiotherapy, limits the use of these therapeutic methods.
Increasing the concentration of the radiolabel to increase the dosage to the tumor is counterproductive generally as this also increases exposure of healthy tissue to radioactivity.
The clinical use of mLL2, just as with most other promising murine antibodies, has been limited by the development in humans of a HAMA response.
This can limit the diagnostic and therapeutic usefulness of such antibody conjugates, not only because of the potential anaphylactic problem, but also as a major portion of the circulating conjugate may be complexed to and sequestered by the circulating anti-mouse antibodies.
Such HAMA responses in general pose a potential obstacle to realizing the full diagnostic and therapeutic potential of the mLL2 antibody.

Method used

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  • Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
  • Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
  • Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

Choice of Human Frameworks and Sequence Design for the Humanization of LL2 Monoclonal Antibody

[0068] By comparing the murine variable (V) region framework (FR) sequences of LL2 to that of human antibodies in the Kabat data base (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, U.S. Government Printing Office, Washington, D.C.), which is incorporated by reference, the human REI (FIG. 1A, Sequence ID No. 1) and EU (FIG. 1B, Sequence ID No. 2) sequences were found to exhibit the highest degree of sequence homology to the FRs of VK and VH domains of LL2, respectively. Therefore, the REI and EU FRs were selected as the human frameworks onto which the CDRs for LL2 VK and VH were grafted, respectively. The FR4 sequence of NEWM, however, rather than that of EU, was used to replace the EU FR4 sequence for the humanization of LL2 heavy chain. Based on the results of computer modeling studies (FIGS. 2A and 2B), murine FR res...

example 2

PCR Cloning and Sequence Elucidation for LL2 Heavy and Light Chain Variable Regions

[0071] The variable regions for both heavy (VH) and light (VK) chains of mLL2 (IgG2a) were obtained by PCR cloning using DNA primers as described in general above and in greater detail in Example 3, below. As PCR is prone to mutation, the variable region sequence of multiple individual clones for either the heavy or light chains was determined for six clones and confirmed to be identical prior to use for the construction of the chimeric antibody.

[0072] The PCR products for VK were subcloned into a pBR327-based staging vector, VKpBR, which contained an Ig promoter, a signal peptide sequence and convenient restriction sites to facilitate in-frame ligation of the VK PCR products (FIG. 3A). The PCR products for VH were subcloned into a similar pBluescript-based staging vector, VHpBS (FIG. 3B).

[0073] As noted above, at least six individual clones containing the respective PCR products were sequenced acc...

example 3

PCR / Gene Synthesis of the Humanized V Genes

[0075] The designed sequence for the hLL2 VH domain, the construction of the hLL2 VH domain by long oligonucleotides and PCR, and the staging vector VHpBS containing the hLL2 VH domain are summarized in the sketch shown in FIG. 6.

[0076] For the construction of the hLL2 VH domain, oligo A (149-mer) and oligo B(140-mer) were synthesized on an automated CYCLONE PLUS™ DNA synthesizer (Milligen Bioresearch).

[0077] Oligo A (Sequence ID No. 7 below) represents the minus strand of the hLL2 VH domain complementary to nt 24 to 172.

Sequence ID No. 75′-TAT AAT CAT TCC TAG GAT TAA TGT ATC CAA TCC ATTCCA GAC CCT GTC CAG GTG CCT GCC TGA CCC AGT GCAGCC AGT AGC TAG TAA AGG TGT AGC CAG AAG CCT TGCAGG AGA CCT TCA CTG ATG ACC CAG GTT TCT TGA CTTCAG CC-3′

[0078] Oligo B (Sequence ID No. 8 below) represents the minus strand of the hLL2 VH domain complementary to nt 180 to 320.

Sequence ID No. 85′-CCC CAG TAG AAC GTA ATA TCC CTT GCA CAA AAA TAAAAT GCC GTG TC...

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Abstract

A chimeric LL2 monoclonal antibody is described in which the complementarity determining regions (CDRs) of the light and heavy chains of the murine LL2 anti-B-lymphoma, anti-leukemia cell monoclona lantibody has been recombinantly joined to the human kappa and IgG1 constant region domains, respectively, which retains the immunospecificity and B-cell lymphoma and leukemia cell internalization capacity of the parental murine LL2 monoclonal antibody, and which has the potential of exhibiting reduced human anti-mouse antibody production activity. A humanized LL2 monoclonal antibody is described in which the CDRs of the light and heavy chains have been recombinantly joined to a framework sequence of human light and heavy chains variable regions, respectively, and subsequently linked to human kappa and IgG1 constant region domains, respectively, which retains the immunospecificity and B-lymphoma and leukemia cell internalization capacities of the parental murine and chimeric LL2 monoclonal antibodies, and which has the potential for exhibiting reduced human anti-mouse antibody production activity. Vectors for producing recombinant chimeric and humanized chimeric monoclonal antibodies are provided. Isolated DNAs encoding the amino acid sequences of the LL2 variable light and heavy chain and CDR framework regions are described. Conjugates of chimeric and humanized chimeric LL2 antibodies with cytotoxic agents or labels find use in therapy and diagnosis of B-cell lymphomas and leukemias.

Description

[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0002] This application is a continuation of U.S. Ser. No. 09 / 741,843 filed on Dec. 22, 2000, which is a continuation of U.S. Ser. No. 09 / 127,902 filed on Aug. 3, 1998, now U.S. Pat. No. 6,187,287, which is a continuation of U.S. Ser. No. 08 / 690,102 filed on Jul. 31, 1996, now U.S. Pat. No. 5,789,554, which is a continuation of U.S. Ser. No. 08 / 289,576 filed on Aug. 12, 1994, now abandoned. This application claims only subject matter disclosed in the parent applications and therefore presents no new matter.BACKGROUND OF THE INVENTION [0003] The invention relates generally to immunoconjugates for diagnostic and therapeutic uses in cancer. In particular, the invention relates to recombinantly produced chimeric and humanized monoclonal antibodies directed against B-cell lymphoma and leukemia cells, which antibodies can be covalently conjugated to a diagnostic or therapeutic reagent without loss of antibody binding and internalizatio...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K39/00C12N15/09A61K39/395A61K47/48A61K49/00A61P35/02C07K16/30C12P21/02C12P21/08C12R1/91
CPCA61K38/00A61K47/48569A61K47/4863A61K47/48669C07K2319/00C07K16/3061C07K2317/24C07K2317/41C07K2317/77A61K2039/505A61K47/6851A61K47/6867A61K47/6877A61P35/02
Inventor LEUNG, SHUI-ONHANSEN, HANS
Owner IMMUNOMEDICS INC
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