Status – approved; marketed

Organizations involved:

Miltenyi Biotec GmbH – World mark.

Baxter Hyland Immuno – Manuf.; R&D; Tech.; Patent dispute

Nexell Therapeutics, Inc. – R&D; Tech.; Patent dispute; Former

CellPro, Inc. – R&D; Tech.; Former

Becton Dickinson & Co – Patent dispute

Johns Hopkins Univ. – R&D; Tech.; Patent dispute

VimRx Phar-maceuticals Inc. – Former

Description: Isolex 300 systems are automated devices using a highly specific CD34 stem cell murine monoclonal antibody (MY-10; MY10; 9C5 Mab; Isolex antibody) and ovine (sheep) anti-mouse antibody-coated paramagnetic beads to target, capture, and isolate hematopoietic CD34+ stem cells from peripheral blood, with these cells later returned to the patient. The system includes use of the MY-10 murine monoclonal antibody, cultured in vivo using the mouse ascites method, having high affinity for the CD34 antigen on the surface of human stem cells; and sheep (ovine) immune globulin containing high titers of anti-mouse MY-10 antibodies (obtained from murine MY-10 monoclonal antibody vaccinated sheep) covalently bound to microscopic paramagnetic polystyrene beads or microspheres.

These devices are used as part of autologous peripheral blood stem cell (PBSC) transplantation and related autologous bone marrow transplantation (BMT) procedures. After processing (CD34+ cell capture/isolation), the concentrated CD34 stem cells are used for autologous trans-plantation (administration back to their donor), usually after high dose cancer chemotherapy and/or radiation treatment which kills hematopoietic (blood cell-forming) stem cells in the bone marrow (i.e., the device is used to extract and later replenish stem cells killed by cancer chemotherapy and/or radiation therapy). The device achieves a high yield and purity of extracted CDC34+ stem cells, with the selected cell surfaces virtually free of antibodies and paramagnetic beads; and also reduces the number of circulating tumor cells returned to the patient. Positive selection of normal CD34+ marrow stem cells uses the MY-10 monoclonal antibody (Isolex antibody) with specificity for an epitope of the CD34 glycoprotein antigen on the surface of human lymphohematopoietic progenitor cells (CD34+ stem cells). Cells expressing the CD34 antigen include essentially all unipotent and multipotent human hematopoietic colony-forming cells, with return of these cells facilitating recovery of the bone marrow and associated generation/renewal of hemopoietic and immune cells (that would otherwise be compromised by cancer chemotherapy/radiotherapy).

Peripheral blood cells, including stem cells, are collected from the patient by apheresis prior to chemotherapy and/or radiation treatments (i.e., the patient’s blood is filtered through an apheresis device, which removes cellular components and returns fluid portion, plasma, to the patient). In the Isolex system, the stored cells are mixed with the MY-10 monoclonal antibody which binds the CD34-positive cells. Following washing to remove unbound monoclonal antibody (MAb), the stem cell-MY-10 Mab complex-rich cell suspension is mixed with Dynabead M-450 Sheep Anti-Mouse IgG, a 10 mL suspension of 4 x 108 (400 million) paramagnetic beads/microspheres coated with covalently-bound anti-mouse MY-10 sheep (ovine) immune globulin (fractionated from plasma from sheep vaccinated with the MY-10 murine antibody, with purification including immune affinity chromatography). The CD34+ cell–MY-10 Mab complexes bind to the anti-mouse MY-10 antibody-bound paramagnetic spheres, and the resulting the CD34+ cell/MY-10 Mab/anti-mouse IgG-paramagnetic bead (bead-target cell rosette) complexes are concentrated using a magnetic field. Following washing to remove unbound non-target cells, the desired CD34+ stem cells are cleaved from the MY-10 Mab bead complex by mixing with a 8-amino acid releaser peptide (an octapeptide; PR34+ Stem Cell Releasing Agent; synthetic acetyl-Gln-Gln-Gly-Trp-Pro-Lys-Asp-OH), originally identified through high-throughput screening of a combinatorial phage display library of 1010 (10 billion) peptides. A prior version of Isolex used a proteolytic enzyme, chymopapain, to release bead-bound cells. The CD34+ cells are released from the antibodies/beads, which are retained by a magnetic field. The CD34+ cells are washed to remove residual mouse and sheep antibodies and collected. The resulting concentrated CD34+ stem cells are viable (living), intact, and free of both residual antibodies and magnetic particles. The isolated CD34+ stem cells are stored frozen. Several days after the patient completes chemotherapy and/or radiation treatments, the CD34+ stem cells are defrosted and rein-fused (transplanted) back into the patient. During the next few weeks, the stem cells migrate back to bone marrow and renew patient’s production of the blood’s cellular components, including lymphocytes, red blood cells, and platelets.

The Isolex 300i system is fully automated, integrating all steps in the stem cell selection process into one continuous process taking less than three hours. The device is relatively small (32 inches high, 16 inches depth, 24.5 inched width), easily fitting on a desk top, and weighs 71 pounds. The device and associated software are currently at Version 2.5. The Isolex 300i system is fully closed, with a spinning membrane for automated cell washing, and sterile filtered buffer and reagent pathways. Peptide release and cell washing remove residual antibody reagents below detectable levels. Using of Isolex, over 99.9% of CD34+ T-lymphocytes are typically removed for concentration. A yield of CD34+ stem cells over 60% is obtained, with purity over 90%. There is no evidence of sensitization (HAMA; human anti-mouse antibodies) in patients against the murine monoclonal antibody used.

Isolex Stem Cell Reagent Kits sold for use with the Isolex 300i device include sufficient reagents for processing of up to 8 x 1010 (80 billion) stem cells. The kits include a vial of Isolex anti-CD34 Monoclonal Antibody (MY-10) in 2.5 mL sterile buffered saline.; one vial of Dynabead M-450 Sheep Anti-Mouse IgG, a 10 mL nonpyrogenic suspension of ovine IgG against mouse MY-10 protein bound to 4 x 108 (400 million) magnetic beads/spheres in phosphate buffered saline with 0.1% Albumin (Human) as stabilizer; and one 20 mL vial of PR34+ Stem Cell Releasing Agent in phosphate buffered saline. The Dynabead paramagnetic microspheres are obtained from Dynal (Oslo, Norway).

The basic technological problem (advance) with Isolex involves how to release positively selected cells from the affinity matrix once they have been isolated from the non--selected cells. This was initially resolved using the enzyme chymo-papain (see the Patent section for further information). An synthetic octapeptide is now used as the release agent.

Nomenclature: CD34+ Stem Cell Selector [BIO]; Isolex [TR]; Isolex 300 & 300i Magnetic Cell Selection System [FDA]; Stem Cell Selection System [FDA for CEPRATE SC]; CEPRATE SC [TR formerly used]

Companies.: CEPRATE SC (later become Isolex) was originally developed by CellPro, Inc. It is currently manufactured by Baxter Healthcare Corp. and marketed worldwide by Miltenyi Biotec GmbH.

CellPro lost a patent dispute in 1998 with Baxter Health-care Corp. See the Tech. transfer section for further information. The court ruled that CellPro’s CEPRATE SC device infringed CD34 antibody patents, e.g., U.S. 5,081,030, assigned to Johns Hopkins University and exclusively licensed by the university to Baxter Healthcare and Becton Dickinson Co. Cell-Pro subsequently sold all of its assets, including CEPRATE, to Nexell Therapeutics, Inc. (Wilmington, DE), a joint venture of VimRx Phar-maceuticals Inc. (Wilmington, DE) and Baxter Healthcare Corp., and filed for bankruptcy (Chapter 11). CellPro was forced to do this after spending about $175 million over six years, primarily for development of CEPRATE. Baxter Healthcare then contributed the patent properties it had licensed from Johns Hopkins University to the Nexell joint venture. Thus, Baxter won a patent dispute against the original device developer, CellPro, and formed a joint venture, Nexell, with another company, VimRx, for development of its successor product (Isolex). CEPRATE SC devices remained on the market, with worldwide marketing rights (to devices in stock) granted by Nexell to Baxter (until the similar Isolex system from Nexell was approved).

Nexell Therapeutics assumed full control of the Isolex system in July 1999 though its acquisition of the Immunotherapy Division of Baxter Healthcare, and Baxter continued to exclusively market the product. This agreement was terminated in Nov. 1999, with Nexell assuming worldwide marketing, and Baxter sublicensing to Nexell its rights related to CD34 monoclonal antibody. Baxter continued to manufacture the components and systems, provided servicing/repair support, and received royalties on Nexell’s sales of Isolex.

In Aug. 2001, Nexell sold/returned all worldwide marketing and distribution rights and responsibilities to Baxter for about $4.3 million plus unspecified royalties on sales. In late 2002, Nexell Therapeutics closed shop and liquidated its business, with Baxter Health-care receiving its remaining assets. Baxter assumed exclusive manufacturing and worldwide marketing of Isolex products.

In Sept. 2004, Baxter transferred to Miltenyi Biotec GmbH exclusive rights for five years to distribute, market and sell Baxter’s cellular therapy products worldwide, including Isolex, with the exception of China, Japan, Korea and Taiwan. Baxter continues to manufacture Isolex.

FDA class: Medical Device PMA

Approvals: Date = 19961206, first approval for CEPRATE SC; PMA BP940001; Indication = for the processing of autologous bone marrow to obtain a CD34+ cell enriched population which is intended for hematopoietic support after myelo-ablative chemotherapy

Date = 19980730; PMA supplement for CEPRATE SC; Indication = for the selection of peripheral blood progenitor cells and tumor purging

Date = 19990702; first approval of semi-automated Isolex 300 & and fully-automated 300i Magnetic Cell Selection Systems, PMA numbers BP970001 & BP970001/01; Indication = for processing autologous peripheral blood progenitor cell (PBPC) products to obtain a CD34+ cell enriched population intended for hematopoietic reconstitution after myeloablative therapy in patients with CD34-negative tumors

Date = 20000515; PMA supplement; approval of “next generation” version 2.5 upgrade for the current Isolex 300i System (from version 1.12), involving new instrument software and modifications to the hardware and disposable set (launched in July 2000)

Indications: [full text of the "INDICATIONS AND USAGE” section of product insert/labeling]:

The lsolex 300 and lsolex 300i Magnetic Cell Selection Systems are indicated for processing autologous peripheral blood progenitor cell (PBPC) products to obtain a CD34+ cell enriched population intended for hematopoietic reconstitution after myeloablative therapy in patients with CD34-negative tumors. lsolex processing reduces the number of non-CD34+(non-target) cells, including tumor cells, in the autograft compared with unselected PBPC. Clinical studies have not determined whether use of the Isolex 300 or 300i systems will alter progression-free or overall survival.

It is recommended that sufficient peripheral blood be collected to provide at least 2 x 106 CD34+ cells per kilogram of patient body weight after CD34+ cell selection. Infusion of fewer cells has been associated with delayed time to platelet engraftment. (See WARNINGS).

Tech. transfer: The product insert cites U.S. patent numbers 4,714,680; 4,965,204; 5,035,994; 5,081,030; 5,130,144; 5,443,451; 5,460,493; and 5,536,475.

See the Companies section for background about CEPRATE, CellPro, and a dispute and licensing concerning CD34 monoclonal antibody patents assigned to Johns Hopkins University. [Johns Hopkins University has also licensed CD34 antibody technology for other stem cell-related uses to SyStemix Inc., now part of Novartis Pharma AG, and to Applied Immune Systems Inc., now part of Sanofi Aventis S.A.].

U.S. patent 5,130,144 (also 4,965,204), “Human stem cells and monoclonal antibodies,” by C.I. Civen, July 14, 1992, assigned to Johns Hopkins University, covers antibodies specific for MY-10 (MY10), a stage-specific antigen that is detectable on normal, human marrow or blood colony-forming cells and immature lymphoid precursor cells, but not on normal, mature human lymphoid and myeloid cells. The antigen is not lineage dependent, appearing on a spectrum of lympho-hematopoietic progenitor cells. A mouse hybridoma producing MY-10 monoclonal antibody was deposited with the American Type Culture Collection (ATCC) as ATCC Accession No. HB-8483.

U.S. patent 5,081,030, “Release of cells from affinity matrices,” by C.I. Civin, January 14, 1992, assigned to Johns Hopkins University, describes aspects of CEPRATE SC and the Isolex systems. This patent describes positive stem cell selection using MY-10 CD34 monoclonal antibodies with immunomagnetic microspheres to isolate CD34-positive marrow cells. Chymopapain enzyme is used to release stem cells from magnetic microspheres coated with MY-10 MAB (specific for CD34). The isolated CD34+ cells are particularly useful for bone marrow transplantation. The exemplary claim (#1) states, “A method for releasing animal cells bound to antibodies specific for the MY10 epitope which comprises treating the antibody bound animal cells with chymopapain and separating viable cells from said antibodies.”

U.S. 6,017,719 and 5,968,753, “Positive and Positive/Negative Cell Selection Mediated by Peptide Release,” assigned to Nexell Therapeutics cover the cell releasing octapeptide that releases the CD34+ cells.

U.S. 5,443,451, “Peristaltic pumping assembly,” and U.S. 5,460,493, “Organizer frame for holding an array of flexible tubing in alignment with one or more peristaltic pump rotors,” are assigned to Baxter International Inc.

Medical: The Isolex system allows autologous rather than allogeneic peripheral blood stem cell (PBSC) transplant grafts and similar bone marrow transplantation or BMT, both often associated with graft vs. host disease (GVHD; rejection) despite immune suppressant prophylaxis. GVHD is apparently mediated by T lymphocytes in the grafted cell population. The risk of GVHD has limited allogeneic PBSC and BMT to use only in highly fatal diseases, and even then, only for patients with HLA-matched donors, usually siblings. Autologous PBSC and BMT, using the patient’s own stem cells, can avoid most of the problems associated with allogeneic transplants. In autologous PBSC and BMT, however, it is necessary to reintroduce only desirable cell populations free of diseased cell populations (e.g., occult tumor cells) to avoid re-introduction of the disease. The most desirable cell suspension for PBSC is one that is substantially free of cell:receptor complexes.

Market: Average Wholesale Price (AWP) is not available (product not in 2005 or 2004 Red Book).

Nexell reported year 2000 revenues of $18.2 million, of which about three-quarters ($13.7 million) came from sales of Isolex and its disposables and reagents. This represented the first full year of sales to end-users by Nexell, rather than to Baxter (which accounted for sales through 1999). Over 90% of sales were in the U.S. and European Union. Recent sales have not been reported by Baxter.