Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-05-25
2003-11-11
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S325000, C435S366000, C530S387100
Reexamination Certificate
active
06645727
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel antibody compositions, and processes and kits for preparing cell preparations enriched for mesenchymal progenitors.
BACKGROUND OF THE INVENTION
The bone marrow microenvironment regulates both positively and negatively, the proliferation, maturation and differentiation of hematopoietic stem cells (1). The microenvironment is made up of “stromal cells” which are believed to be derived from a pluripotent cell in the marrow. The stromal cell precursor, detected as a colony forming unit fibroblast (CFU-F), is distinct from other hematopoietic stem cells (2). In addition to supporting hematopoietic cells, stromal cells can differentiate into bone, cartilage, muscle and tendon (3, 4) and for these reasons the term mesenchymal stem cell (MSC) has been adopted. Although the CFU-F assay has been used in vitro to correlate with mesenchymal cell potential (5), the relationship between the cells capable of producing CFU-F and MSC has not yet been fully established. The CFU-F assay has been used to evaluate the bone marrow microenvironment in normal donors (6), in diseased states (7, 8) and following chemotherapy (9). There is much clinical interest in these cells and the following clinical applications are being evaluated:
expansion and reinfusion of MSC into patients in an attempt to reconstitute the microenvironment and provide optimal conditions to support hematopoiesis (10)
gene transfer into MSC (11)
repair of mesenchymal tissues (12, 13)
U.S. Pat. No. 5,643,736 describes markers on mesenchymal cells and a positive selection (method for isolating the cells). Negative selection of mesenchymal progenitors has used anti-CD45 alone or in combination with anti-glycophorin A (Verfaillie et al. 1998). However, the inventors have found that Immunoadsorption with anti-CD45 depletes mesenchymal precursors present in bone marrow as assayed in the CFU-F (colony forming unit fibroblast) functional assay.
In view of the foregoing, there is a need in the art to develop novel methods to prepare cell preparations enriched for mesenchymal progenitors.
SUMMARY OF THE INVENTION
The inventors have developed antibody compositions for use in preparing cell preparations highly enriched for mesenchymal progenitor cells in a negative selection protocol. The antibodies in the antibody composition are specific for selected markers associated with cells that are other than mesenchymal progenitor cells thereby allowing them to be removed from the cell preparation. In particular, the present inventors have found that an antibody composition containing antibodies specific for the antigens (a) CD2 and/or CD3 and/or CD5 and/or both CD4 and CD8; (b) CD66b and/or CD16 and/or CD11b and/or CD15; and (c) CD19 and/or CD20 and/or CD21 and/or CD22 and/or CD24 and/or Ig gives a cell preparation highly enriched for mesenchymal progenitor cells. This antibody composition may be generally referred to herein as the mesenchymal progenitor enrichment composition or cocktail. Optionally, the mesenchymal progenitor enrichment composition additionally includes antibodies to CD14, CD33, CD34, CD38, CD56, IgE or glycophorin A.
In a preferred embodiment, the mesenchymal progenitor enrichment composition comprises antibodies specific for the antigens (a)CD2 and/or CD3 and/or CD5 and/or both CD4 and CD8; (b) CD66b and/or CD16 and/or CD11b and/or CD15; (c) CD19 and/or CD20 and/or CD21 and/or CD22 and/or CD24 and/or Ig; (d) CD14; and (e) CD33 and/or CD38.
The enrichment and recovery of mesenchymal progenitor cells using the antibody compositions of the invention in a negative selection technique has many advantages over conventional positive selection techniques. Highly enriched cell preparations can be obtained using a single step. The cells obtained using the antibody composition of the invention are not labeled or coated with antibodies or modified making them highly suitable for many uses.
The present invention also relates to a negative selection process for enriching and recovering mesenchymal progenitor cells in a sample comprising: (1) reacting the sample with an antibody composition containing antibodies capable of binding to the antigens (a) CD2 and/or CD3 and/or CD5 and/or both CD4 and CD8; (b) CD66b and/or CD16 and/or CD11b and/or CD15; and (c) CD19 and/or CD20 and/or CD21 and/or CD22 and/or CD24 and/or Ig under conditions permitting the formation of conjugates between the antibodies and cells in the sample having the antigens (a) CD2 and/or CD3 and/or CD5 and/or both CD4 and CD8; (b) CD66b and/or CD16 and/or CD11b and/or CD15; and (c) CD19 and/or CD20 and/or CD21 and/or CD22 and/or CD24 and/or Ig on their surfaces; (2) removing the conjugates; and (3) recovering a cell preparation which is enriched in mesenchymal progenitor cells.
In a specific embodiment, the mesenchymal progenitor enrichment composition may be used in an immunorosetting method wherein the cells to be depleted are rosetted with red blood cells or erythrocytes. In such an embodiment, each of the antibodies in the mesenchymal progenitor enrichment composition is linked to an antibody that binds to erythrocytes.
Accordingly, the present invention provides a negative selection immunorosetting method for enriching and recovering mesenchymal progenitor cells in a sample containing the mesenchymal progenitor cells, erythrocytes and undesired cells comprising: (1) contacting the sample with an antibody composition comprising (i) antibodies capable of binding to the antigens (a) CD2 and/or CD3 and/or CD5 and/or both CD4 and CD8; (b) CD66b and/or CD16 and/or CD11b and/or CD15; and (c) CD19 and/or CD20 and/or CD21 and/or CD22 and/or CD24 and/or Ig linked to (ii) at least one antibody that binds to the erythrocytes, under conditions to allow immunorosettes of the undesired cells and the erythrocytes to form; and (2) separating the immunorosettes from the remainder of the sample to obtain a sample enriched in mesenchymal progenitor cells.
The present invention also relates to a kit useful in preparing a cell preparation enriched in mesenchymal progenitor cells comprising antibodies specific for the antigens (a) CD2 and/or CD3 and/or CD5 and/or both CD4 and CD8; (b) CD66b and/or CD16 and/or CD11b and/or CD15; and (c) CD19 and/or CD20 and/or CD21 and/or CD22 and/or CD24 and/or Ig, and instructions for preparing a cell preparation enriched in mesenchymal progenitor cells.
The invention further relates to cell preparations obtained in accordance with the processes of the invention. The invention still further contemplates a method of using the antibody compositions of the invention in negative selection methods to recover a cell preparation which is enriched in mesenchymal progenitor cells.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.
REFERENCES:
patent: 5486359 (1996-01-01), Caplan et al.
patent: 5643736 (1997-07-01), Bruder et al.
patent: 5733542 (1998-03-01), Haynesworth et al.
Allay, J.A. et al.,Human Gene Therapy. 8:1417-1427 (1997).
Bruder, S.P. et al.,J. Cell. Biochem.. 64:278-294 (1997).
Majumdar, M.K. et al.,J. Cell. Physiol. 176:57-66 (1998).
Pittenger, M.F., et al.Science. 284:143-147 (1999).
Reyes, M. et al.Blood92:725a (1998).
Clarke Emer
Thomas Terry
Belyavskyi M.
Bereskin & Parr
Chan Christina
Gravelle Micheline
StemCell Technologies Inc.
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