Patent Application
20160032007
This invention is related to the area of tumor therapy. In particular, it relates to antibody constructs for tumor therapy.
One of the biggest challenges for immunotoxin-based cancer therapy is the limitation of tumor-targeting and internalizing human monoclonal antibodies (mAbs) or antibody fragments, which are capable of delivering recombinant toxins to the cytoplasm of cancer cells effectively and specifically. There is a continuing need in the art to develop such antibodies.
According to one embodiment of the invention a human scFv is provided that binds to an CSPG4 extracellular domain. The extracellular domain is selected from the group consisting of: amino acids 30-640; amino acids 641-1233; amino acids 1234-1586; and amino acids 1587-2222.
These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with
The inventors have developed human scFvs that specifically bind to extracellular domains of CSPG4 and may be internalized by cells. Internalization permits the use of the scFvs as delivery devices to tumors that express CSPG4 and as delivery devices for a payload to the interior of the targeted cells. Thus toxins and other agents that have intracellular targets can be delivered to the cytoplasm.
The human scFvs may bind to any CSPG4 extracellular domain, including amino acids 30-640; amino acids 641-1233; amino acids 1234-1586; and amino acids 1587-2222. Although not required, we utilized antibodies from non-immunized host. Immunized hosts can also be used as a starting material. Moreover, once isolated and identified, scFvs may be subjected to an affinity improvement process. We used a random mutagenesis DNA library that was expressed on yeast cell surfaces. It was initially panned against H350 melanoma cell line expressing CSPG4 on its surface. Later increasingly stringent flow cytometry sorting was used. However, any scheme can be used which selects for variants with increased affinity. Affinity may be improved by a fold of at least 2, 5, or 10.
The initial scFvs that were isolated are shown in SEQ ID NO: 1-9. The affinity matured scFvs are shown in SEQ ID NO: 10-23. These can be further modified for ease of production or increased affinity or stability, for example, without departing from the spirit of the invention.
The antibody fragments are useful for delivering agents to any cancer cells which express CSPG4 on their surfaces. These include melanoma, triple-negative breast cancer, glioblastoma, mesothelioma, osteosarcoma, clear cell renal carcinoma, head and neck squamous cell carcinoma, and sarcoma.
Any toxin or toxic agent that may kill a cell can be attached, whether post-translationally or translationally as a fusion with the scFv molecules. Suitable toxins and toxic agents include but are not limited to Diphtheria toxin, Pseudomonas aeruginosa exotoxin A shigella toxin, derivatives of these toxins, camptothecin, paclitaxel, and Vinca alkaloids,
The antibody fragments of the invention may also be useful for delivering detectable agents to tumor cells. This may facilitate monitoring of therapy or disease progression. It may facilitate early diagnosis. Detectable agents include radiolabeled molecules, fluorescent molecules, dye molecules, and the like.
The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
To conquer the hurdle of tumor-targeting and internalizing antibodies which are capable of delivering recombinant toxins to the cytoplasm of cancer cells effectively and specifically, we established a platform using phage display and yeast display techniques to develop human single chain variable antibody fragments (scFvs) against tumor antigens. First, different domains of the extracellular region of Chondroitin Sulfate Proteoglycan 4 (CSPG4) were displayed on the yeast surface and verified by CSPG4 mouse mAbs 9.2.27 and Mel-14. Then we constructed a human non-immunized scFv phage library, and used this library to select scFvs reactive against the different CSPG4 domains displayed on yeast surface. After multiple rounds of selection, several phage scFvs reactive against different domains of CSPG4 were chosen and analyzed by fluorescence-activated cell sorting (FACS). Finally, phage scFvs showed specific binding to melanoma cancer cell lines H350 and Malme3M.
Note: Y: yeast display; R: round; scFv, single chain variable fragments
We sequenced the phage genomes which displayed useful scFv antibody fragments that we developed using this platform. The sequences are shown in
A random mutagenesis DNA library was generated based on the parental clone D2A-1H10 scFv using error-prone PCR (
PCR generated mutant scFv DNA library was cloned into pYD1 yeast display vector and transformed into EBY100 yeast strain for screening of clones with high affinity to the D2A domain of CSPG4. The first 3 rounds of screening were performed by panning the yeast library against H350 melanoma cell line expressing CSPG4 on its surface (
After 6 rounds of screening, 30 D2A-1H10 mutant scFv yeast clones were randomly picked up for DNA sequencing. Sequencing identified 14 unique D2A-1H10 mutant yeast clones with DNA sequence differences in the frame work and complementarity determining regions (
The top seven D2A-1H10 mutant scFv yeast clones demonstrating highest binding to the D2A domain of CSPG4 by FACS were selected for further characterization. Affinity (KD) of the purified D2A-1H10 mutant scFvs were determined by flow cytometry and by BIAcore (
This invention was made with government support under CA11898-42 awarded by the National Cancer Institute. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61985126 | Apr 2014 | US |
