WO1994008621A1 - Immunological purging of tumor cells from bone marrow using microspheres and monoclonal antibodies - Google Patents
Immunological purging of tumor cells from bone marrow using microspheres and monoclonal antibodies Download PDFInfo
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- WO1994008621A1 WO1994008621A1 PCT/US1993/009891 US9309891W WO9408621A1 WO 1994008621 A1 WO1994008621 A1 WO 1994008621A1 US 9309891 W US9309891 W US 9309891W WO 9408621 A1 WO9408621 A1 WO 9408621A1
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- monoclonal antibody
- tumor cells
- cells
- microspheres
- marrow
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3061—Blood cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- TECHNICAL FIELD This invention relates to the immunologic purging of tumor cells from bone marrow using a unique combination of monoclonal antibodies and microspheres, and to a method of treating persons having B cell lymphoma by the autologous treatment and transplantation of bone marrow which has been purged of tumor cells by use of said unique combination.
- telomeres a technique of particular utility in assaying for occult cells in bone marrow.
- PCR polymerase chain reaction
- Several publications [18-28] have shown that PCR amplification can be used to detect the inter- chromosomal translocation involving the bcl-2 proto- oncogene on chromosome-18 and the immunoglobulin heavy chain locus on chromosome-14. The translocation occurs in approximately 85% of patients with follicular NHL and
- determining whether marrow is PCR positive or PCR negative allows a more accurate assess ⁇ ment of the efficacy of the materials used to purge bone marrow. This accuracy, in turn, permits a more accurate assessment of a patient's disease-free survival prospects.
- MmAb monoclonal antibodies
- the CmAb data included herein indicates that there is a highly significant correlation between the ability to purge bone marrow to PCR negativity for the bcl-2 translocation and disease-free survival of a patient after ABMT. However, only about 50% of marrow samples have been found purgable to PCR negativity using CmAb. Since the data presented herein indicates that the use of MmAb significantly increases the percentage of samples which can be purged to PCR negativity (to 90-100%), it is believed that the use of MmAb may significantly improve the disease-free survival statistics.
- the invention discloses a unique method for immunologically purging tumor cells from the bone marrow of a patient having B cell lymphoma for the purpose of therapeutic autologous bone marrow transplantation.
- the method comprises the steps of:
- the method entails the use of antibodies which conjugate to the tumor cells and microspheres, preferably magnetic microspheres of size in the range about 0.3 to about 5.0 microns, coated with goat anti-mouse immunoglobulin(s) (Ig) when then conjugate to the antibodies.
- Ig goat anti-mouse immunoglobulin(s)
- Goat anti-rabbit and rabbit anti-mouse Igs may also be used according to the invention.
- Goat anti- mouse Ig is preferred.
- the microspheres, with antibodies and tumor cells conjugated thereto, were then separated from the marrow sample.
- This unique method of using a plurality of monoclonal antibodies and microspheres has been found to remove tumor cells from bone marrow to PCR negativity without the use of complement. Furthermore, whereas the use of a plurality of selective monoclonal antibodies and complement has been found to purge about 50% of tumor containing bone marrow samples to PCR negativity, 90-100% of tumor containing samples have been purged to PCR negativity using a plurality of monoclonal antibodies and microspheres.
- the monoclonal antibodies may be bound to the micro- spheres, or to microspheres conjugated or coated with immunoglobulins or other substances, before contact with tumor cell containing bone marrow.
- the antibodies are to be conjugated to the microspheres before contact with tumor cell containing samples, it may be necessary to insert a bridging group of about 1-20 atoms long between the surface of the microsphere and the antibody. This in turn may require washing the microspheres after a purging cycle and combining the washing with a bulk sample in order to minimize the loss of non-tumor cells.
- Fig.l is a graph showing the efficacy of immunologic purging of Raji cells with various complement and/or antibodies as assessed by clonogenic cell growth;
- Figs. 2A and 2B is the Southern Blot analyses of bcl-2 translocation sequences amplified by polymerase chain reaction (PCR) before and after treatment according to the invention
- Fig.3 is a graph of the actuarial probability of disease free survival after ABMT in 114 persons with B- cell NHL;
- Figs. 4A-4C is a graph of the actuarial probability of disease free survival after ABMT subclassified by disease status at ABMT;
- Figs. 5A-5D is graph of the actuarial probability of disease-free survival after ABMT subclassified by bone marrow involvement at ABMT;
- Fig. 6 is the Southern Blot analyses of bone marrow (A) and multiple peripheral blood samples (B-G) taken from two patients for bcl-2 translocation sequences as amplified by polymerase chain reaction.
- Figs. 7A-7C illustrate detection by Southern Blot analysis of the bcl-2 translocation sequences amplified by polymerase chain reaction.
- Fig. 8 illustrates that PCR analysis is capable of detecting one lymphoma cells on 10 normal mononuclear cells following MmAb-4 treatment.
- clonogenic tumor cells might be re-infused with autologous bone marrow and contribute to patient relapse.
- This invention enables effective immunologic purging using a unique combination of microspheres and selective monoclonal antibodies. This combination is capable of
- the bone marrow was PCR negative in 57 of 114 patients who had a PCR amplifiable breakpoint involving the bcl-2 translocation at the time of bone marrow harvest.
- Protocol eligible minimal disease criteria included (1) whether there was complete remission (CR) or partial remission (PR) of tumor masses two centimeters or less and (2) whether there was bone marrow infiltration of 5% or less by tumor cells in the initial protocol, but less than 20% of the intertra- becular space in subsequent protocols. All cases were documented by immunotyping to express Bl (CD20).
- Bone marrow was re-infused following immunologic purging using complement and a tumor removing sufficiency of a plurality of anti-B cell and anti-CALLA (anti-Common Acute Lymphoblastic Leukemia Antigen) monoclonal antibodies.
- the presence of a sufficiency of monoclonal antibodies is determined by pretreatment tests which determine the approximate amount of tumor cells present in the bone marrow. Rabbit complement was preferred. PCR negativity was typically achieved after three treatment or tumor cell purging cycles. Consequently, in all cases, purged bone marrow was transplanted only after three marrow treatment or purging cycles. Vials of marrow from all patients were cryopreserved before and after purging.
- Bone marrow was harvested from the iliac crest under general anesthesia and collected in RPMI media (Whitakker, Piscataway, NJ) containing preservative free heparin. The harvested bone marrow was concentrated to yield a buffy coat and washed on a Cobe 2991 cell washer. The mononuclear cell fraction was isolated by centrifu- gation over Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and the cells resuspended at 2 x 10 cells/ml in RPMI 1640 containing 0.5% fetal bovine serum (FBS, Hycone Lab, Logan UT).
- FBS Hycone Lab, Logan UT
- the cells were incubated with a tumor removing sufficiency, preferably a saturating concentra ⁇ tion, of the monoclonal antibodies for a time in the range of 15 minutes to about 1 hour, preferably for about 15 minutes, at 4°C.
- a saturating concentration for each antibody is determined from the number of tumor cells present and the antigen density on the cells.
- Rabbit complement (3-4 week old rabbit serum, Pel Freeze Inc., Brown Deer, WI) was added at predetermined dilutions for each lot and incubated with target cells for 30 minutes at 37°C in the presence of deoxyribonuclease (Sigma, St. Louis, MO) at 2.5 mg/ml to prevent cell clumping.
- the amount of complement added is dependent on the volume of the sample is in the range 1/1 to 1/10 v/v.
- the cells were pelleted by centrifugation and the procedure repeated twice for a total of three treatments in vitro with the monoclonal antibodies and rabbit complement.
- the cells were washed three times, resuspended in autologous serum and 10% dimethylsulfoxide (Sigma, St. Louis, MO) and cryopreserved according to the method of Takovarian et al., N. Engl. J. Med, 316: 1499-1505 (1987).
- lymphoma cell lines Raji and DHL-6 were used in evaluating the invention.
- Raji is a human Burkitt's lymphoma B cell line expressing CD20, CD10 and B5 anti ⁇ gens.
- Raji cells were grown in RPMI 1640 medium contain ⁇ ing 10% heat inactivated FBS, 2% 1-glutamine, 1% sodium pyruvate and 1% penicillin and streptomycin.
- DHL-6 is a human B cell line containing a bcl-2 translocation and was received from Dr. A. Epstein (Univ. So. Calif., Los Angeles, CA).
- the monoclonal antibodies used according to the invention are an anti-CD (anti-Bl) monoclonal antibody, a monoclonal antibody specific for activated B cells and B cell lymphoma (anti-B5), an anti-CDlO (anti-J5) mono ⁇ clonal antibody and an anti-CD19 (anti-B4) monoclonal antibody.
- the antibodies used in accordance with the invention must induce lysis of tumor cells in the bone marrow in the presence of complement.
- the antibodies used herein all induce lysis in the presence of rabbit complement.
- Anti-Bl is an IgG2a murine monoclonal antibody specific for a 35,000 dalton cell surface glycoprotein present on normal and malignant cells.
- the Bl antigen i found on all B cells isolated from peripheral blood, lymph node, spleen, tonsil and bone marrow. This antige is also found on 50% of CALLA positive acute lymphoblas- tic leukemias (ALL); but not normal T cells, monocytes, granulocytes or tumors of these lineages.
- ALL CALLA positive acute lymphoblas- tic leukemias
- monocytes monocytes
- granulocytes or tumors of these lineages The Bl antige is an integral component of the B cell membrane and is also found on some null cell lymphomas and non-T cell
- the anti-Bl monoclonal antibody was prepared by P Stashenko et al., "Characterization of a human B lympho ⁇ cyte-specific antigen", J. Immunol., 125: 1678 et seq. (1980) and L. M. Nadler et al., "A unique cell surface antigen identifying lymphoid malignancies of B cell origin", J. Clin. Invest., 67: 134 et seq. (1981). Any monoclonal antibody which reacts with the Bl antigen in the same manner as anti-Bl may be used in practicing the invention. Complement mediated lysis is dependent upon the complement mediated cytolytic activity of the mono ⁇ clonal antibody.
- Anti-B5 is a murine IgM monoclonal antibody described in U.S. Patent No. 4,692,405.
- the B5 antigen has a molecular weight of 75,000 daltons and is expresse on a small number of activated B cells in unstimulated lymph node, spleen and tonsil. It is not expressed on resting B cells isolated from peripheral blood, lymph node, spleen or tonsil. Resting spleenic B cells activated in vitro with protein A, anti-Ig, Epstein-Barr virus or pokeweed mitogens demonstrate the appearance of B5 antigen.
- B5 is found on a wide variety of B cell neoplasms and the majority of B-Cll, Burkett's lymphomas nodular poorly differentiated lymphocytic lymphomas, diffuse poorly differentiated lymphocytic lymphomas, diffuse large cell lymphomas, hairy cell leukemias, and diffuse well differentiated lymphocytic lymphomas. B5 is not expressed on non-T cell Alls, T cell or myeloid leukemic cells, normal T cells, monocytes, granulocytes, red blood cells or platelets. It has, however, been noted on some non-hemopoietic malignancies, especially small cell lung carcinomas. Any monoclonal antibody which reacts with the B5 antigen in the same manner as anti-B5 may be used in practicing the invention.
- Anti-J5 is an IgG2a murine monoclonal antibody directed against the human Common Acute Lymphoblastic Leukemia Antigen (CALLA) which has a molecular weight of about 100,000 daltons.
- CALLA Common Acute Lymphoblastic Leukemia Antigen
- the antigen is found on tumor cells from 80% of patients with non-T cell ALLs and 40- 50% of patients with chronic myelocytic leukemia in blast crisis. This antigen is present on a small number of cells in normal bone marrow and in fetal liver.
- the J5 antigen is also found on the tumor cells from some B cell and T cell lymphomas, including poorly differentiated nodular lymphocytic lymphoma, Burkett's lymphoma and T cell lymphoblastic lymphoma, as well as normal renal tubular and glomerular epithelial and breast myoepihtelial cells.
- the anti-J5 monoclonal antibody was derived from the hybridization of mouse NS/l-AG cells with spleen cells from BALB/cJ mice immunized with tumor cells from a patient with CALLA positive non-T cell ALL by J.
- Anti-B4 (anti-CD19) is a murine IgG monoclonal
- the antibody recognizes the B4 antigen which is present on all B cells isolated from lymphoid organs and on approximately 5% of normal adult bone marrow cells. In addition, the B4 antigen is expressed on greater than 95% of non-T cell acute lympho- blastic leukemias, on 90% of B cell lymphomas and B cell chronic lymphocytic leukemias.
- anti-Bl, anti-B5, anti-B4 and anti-J5 monoclonal antibodies are commercially available from Coulter Immunology Division, Coulter Corporation, Hialeah, Florida.
- Bone marrow was obtained from healthy volunteer donors and anticoagluted with preservative free heparin.
- the mononuclear cell fraction was isolated by Ficoll- Hypaque density gradient centrifugation and cells irradiated to 40 Gy at 11.1 Gy/min ( Cs, Gamma cell, Atomic Energy of Canada, Ottawa, Canada) before they were mixed with lymphoma cells in purging experiments.
- the lymphoma cell line Raji was added to irradiated normal marrow mononuclear cells in a 1:20 ratio and suspended in media at 2 x 10 cells/ml.
- the cell suspensions were treated with the monoclonal antibodies and complement combination for a total of three treatments using the same protocol used for the marrow harvest samples.
- the cells were then washed three times and plated in a limiting dilution assay [33] . Each sample was serially
- DNA was heated to 96°C for 10 minutes to destroy proteinase K activity before amplification. Nested oligonucletide amplification was performed at both the
- PCR amplification was performed for 25 cycles in a Perkin Elmer Cetus thermal cycler (Cetus, Emeryville, CA) in 50 ml of buffer containing 50 mM KC1, lOmM Tris-Cl, 2.25 mM MgCl , 0.01% gelatin, 1.5 mg of DNA, 20nM of oligonucleotide primers, 200 mM each of dATP, dCTP, dGTP and dTTP, 1.5 U Taq polymerase (Cetus, Emeryville, CA).
- the initial amplification was performed using oligonucleotides 5'CAGCCTTAAACATTGATGG(Seq 1) for the
- a 5 ml aliquot of the amplified mixture was made to 50 ml and re-amplified for 30 cycles using oligonucleotides internal to the original primers,
- DNA was blotted onto Zeta-probe blotting mem ⁇ branes (Bio Rad, Richmond, CA) and bcl-2 specific DNA detected by hybridization overnight with P-labelled oligonucleotide probes, 5'CCCTCCTGCCCTCCTTCCG3' (Seq 7) for the MBR, and 5'GGACCTTCCTTGGTGTGTTG3' (Seq 5) for the
- Oligonucleotides were radiolabelled with ( P)ATP using T4 polycucleotide kinase (New England Biolabs, Beverly, MA) according to the manufacturer's instructions.
- Control tests were performed with each amplification using a weak positive control consisting of DNA from a
- the ability to purge tumor cells from normal bone marrow by using monoclonal antibodies was demonstrated with an in vitro model.
- the hybridoma cells line Raji was added to normal donor bone marrow mononuclear cells in a 1:20 ratio.
- This suspension was then treated with the anti-B cell monoclonal antibodies anti-Bl and anti- B5, with anti-J5, either singly or in combination, in the presence of complement.
- the cell suspension was treated a total of three times in order to deplete Raji clonogenic cells.
- Fig. 1 indicates that treatment of the Raji containing suspension with complement alone or with the combination anti-(B5+Bl+J5) without complement did not significantly reduce the fraction of clonogenic cells in the suspension sample.
- antibody and component lysis was capable of inducing approximately three logs (10 ) of cell kill.
- the combinations anti-Bl + anti-J5 + complement and anti-Bl + anti-B5 + anti-J5 + complement both reduced the fraction of clonogenic cells to about 10 .
- the latter combination is preferred.
- samples from 10 patients whose harvested marrow contains cells with translocation of the bcl-2 oncogene were analyzed to determine whether these cells could be depleted to PCR negativity by immunogenic purging.
- Each of these bone marrows was treated with a cocktail comprising anti-Bl plus anti-B5 plus anti-J5 plus complement.
- Samples were analyzed for the bcl-2 translocation by PCR before and after each of the three rounds of monoclonal antibodies plus complement treatment.
- Fig. 2a The results obtained with three representative patients from this group are shown in Fig. 2a. Patients 1 and 2 reached PCR negativity following the third round of treatment while patient 3 remained PCR positive for the bcl-2 translocation.
- Negrin et al. Blood, 77:654-660 (1991) [43] have noted that immunologic purging of lymphoma cell line could result in PCR negativity, although these authors also noted that there was a difference among lymphoma cell lines with regard to susceptibility to purging.
- results described herein were obtained from analysis of the treatment of patients who had a docu ⁇ mented PCR amplifiable breakpoint for the bcl-2 trans ⁇ location and for whom both pre- and post-lysis samples were available for analysis.
- the bcl-2 translocation was identified by PCR in the diagnostic tissue of 125 patients and the requisite samples were available for 114 of these cases.
- pre- and post-lysis samples were also available from 47 patients who had B-cell NHL with no PCR amplifiable translocation involving the bcl translocation. These 47 samples were used as controls.
- DNA was extracted from the pre- and post-lysis samples and PCR was performed at the MBR and mcr of bcl-2 to assess whether residual cells with the bcl-2 trans ⁇ location were present at harvest and following compl- ment/antibodies purging.
- PCR was performed at the MBR and mcr of bcl-2 to assess whether residual cells with the bcl-2 trans ⁇ location were present at harvest and following compl- ment/antibodies purging.
- Each of the pre- and post-lysis samples was analyzed three times and was blinded with regard to clinical outcome or to whether a bcl-trans ⁇ location had previously been identified for that patient.
- PCR detected bone marrow infiltration in the pre-lysis marrow in all of the 114 cases for whom a bcl-2 translocation was detected in the diagnostic tissue.
- Immunologic purging resulted in the loss of detectable PCR product in the post-lysis samples of 57 of the 114 patients involved herein.
- PCR of the bcl-2 translocation was positive after purging.
- the results from nine represen ⁇ tative pre- and post-lysis samples is shown in Fig. 2b.
- Lanes 1-4 represent cases where lymphoma cells with the bcl-2 translocation could not be detected by PCR in the post-lysis sample.
- Lanes 5-9 are representative of patients who remained PCR positive after lysis. Although equal amounts of DNA were added to each PCR reaction, the ability to purge to PCR negativity did not appear to correlate with the intensity of the PCR signal in the pre-lysis sample.
- Fig. 3 indicates that DFS after ABMT correlates strongly with whether the patient purged PCR negative or positive after lysis (p,0.00001).
- DFS disease free survival
- Eleven patients had overt histologic bone marrow involvement (10-20% of the intertrabecular space) at the time of bon marrow harvest. Only two of these patients became PCR negative after purging.
- PC negativity is shown to correlate with increased DFS. I is unclear as to why the bone marrow of all patients cannot be purged to PCR negativity. It is within the scope of the claimed invention that other anti-Bl and anti-J5 monoclonal antibodies, and other monoclonal antibodies specific to activated B cells and B cell lymphomas will increase the percentage of patients whos bone marrow may be purged of tumor cells.
- peripheral blood cells with the bcl-2 translocation after the ABMT procedure has been carried out was found to correlate with the ability to purge the bone marrow of tumor cells.
- analyses were carried out t determine whether there were detectable lymphoma cells i the peripheral blood of patients at the time of ABMT. Twenty-five patients were involved in the study. Blood samples were also tested six months after ABMT. Multipl blood samples were obtained from each patient and analyzed by PCR. The results from the analysis of bone marrow and multiple blood samples from two representati patients, patients 1 and 8 from Fig. 2b, are shown in Fig. 6.
- the tumor cell containing marrow was harvested from five non-Hodgkin's lymphoma patients. Aliquots from all patients were fir treated with complement and the monoclonal antibodies anti-Bl, -B5 and -J5. After three rounds of treatment described above, aliquots from two of the five patients reached PCR negativity. Additional aliquots were then treated in the same manner, but with the anti-B4 mono ⁇ clonal antibody added to the combination. The same results, two of five aliquots reaching PCR negativity, were obtained. The aliquots reaching PCR negativity we from the same source in both tests.
- Example 2 Further Comparison of Complement Induced Lysis And Magnetic Sphere Depletion Of Tumor Cells From The Bone Marrow Samples Of Lymphoma Patients. Combinations of three (anti-Bl, -B5 -and -J5) and four (anti-Bl, -B4, -B5 and -J5) monoclonal antibodies were prepared. Harvested bone marrow from lymphoma patients was obtained and divided. One portion of the marrow was purged of tumor cells using complement and the three monoclonal antibody combination as previously described. The mononuclear cell fraction from the remainder of the harvested marrow was isolated by Ficoll- Hypaque density centrifugation and divided into four fractions. The four fractions were treated separately.
- Two fractions of the centrifuged marrow were incubated at about 4°C for about 30 minutes, with a saturating concen- tration of three monoclonal antibodies and two were incu ⁇ bated with four antibodies.
- the antibody incubation time may be in the range of about 15 minutes to about 1 hour.
- a three antibody and a four antibody incubate were depleted of tumor cells by complement lysis using a sufficient concentration of rabbit complement from the serum of 3-4 week old rabbits (Pel-freeze, Brown Deer, WI).
- the cells were incubated with complement at about 37°C for a time in the range of about 15 minutes to about 1 hour, preferably about 30 minutes.
- the cells were washed in media containing deoxyribonuclease (Sigma, St. Louis, MO) at a final concentration of 2.5 mg/ml. This prevented cell clumping and ensured that DNA from the lysed cells was not co- purified during DNA extractions.
- the cells were pelleted by centrifugation and the procedures were twice repeated for a total of three treatment cycles. Before and after each of the three treatment cycles, an aliquot of cells was collected from each fraction and genomic DNA was isolated by cell lysis with non-ionic detergents and proteinase K (Sigma, St. Louis, MO). Nested oligonucleotide amplification was performed at the MBR or mcl region of the bcl-2/Ig
- the magnetic microspheres used according to the invention may be any magnetic microspheres such as magnetic polystyrene latex spheres, magnetic dextran or gelatin microspheres and similar microspheres.
- the term microspheres or immunologically accept ⁇ able substrate includes magnetic or non-magnetic parti- cles of suitable size and of any shape; for example, round, cubic, rectangular, or other shapes.
- the magnetic particles may be enclosed by a coating material such as polystyrene, or gelatin or the particles may be embedded in the coating.
- the size of the magnetic microspheres used in the invention may range from about 0.3 microns to about 5 microns.
- Non-magnetic equivalents to the mag ⁇ netic species described herein may also be used according to the invention. Appropriate separation techniques are selected when such non-magnetic particles are used.
- the monoclonal antibodies used in the invention may be attached to the microspheres by techniques known in the art before the microspheres are mixed with the bone marrow samples.
- the monoclonal antibodies may be attached to immunoglobulin coated microspheres prior to mixing the microspheres with the tumor cell containing marrow.
- Another example is the use of a carboxylate-diamine couple as a bridge between the antibody and the micro- sphere. If the microsphere has pendent groups such as an amine, a carboxy group or a thiol group, the antibody may be attachable to the microsphere without further functionalization of either species.
- Non-porous magnetic microspheres (sometimes called beads) of generally monodispersed or uniform size in the range of about 0.3 to about 5.0 microns are conditioned for binding of a combination of monoclonal antibodies thereon by pre-coating the microspheres with rabbit or goat anti-mouse immunoglobulin (GAM or RAM) as follows. First, 250 mg of beads are dispersed in 3 ml of distilled water and sonicated for 2-3 minutes. The mixture of beads in water is then cooled for several hours at 4°C. The beads are then magnetically separated and the water discarded.
- GAM goat anti-mouse immunoglobulin
- the beads are resuspended in 5mg of RAM or GAM and diluted with 500 microliters of phosphate- buffered saline (PBS) . This mixture is incubated at room temperature and is mixed for 4-5 hours. Thereafter the beads are washed six times with 4 ml portions of PBS containing 1% bovine serum albumin (BSA). The beads are then resuspended in 4 ml of PBS-1% BSA and mixed.
- PBS phosphate- buffered saline
- a quantity of 5x10 of the resuspended beads is pipetted into a siliconized test tube.
- the beads are magnetically separated from the liquid which is dis- carded.
- the beads are then resuspended in PBS and the combination of three or four selective monoclonal anti ⁇ bodies as described above is added to the suspension such that the concentration of antibody combination totals approximately 0.5 mg/ml of solution.
- the solution containing beads and antibodies is then incubated at a temperature in the range of 10-30°C, for about one hour.
- the beads are then washed a plurality of times with PBS- 1% BSA and resuspended in the same.
- Bone marrow containing tumor cells are harvested, concentrated, washed, fractionated and suspended in RPMI medium as described above for complement mediated lysis.
- the antibody containing magnetic beads prepared according to Example 3 are then added to the marrow suspension and the resulting mixture is incubated for a time in the range of about 10 minutes to about 1 hour, preferably for about 15 minutes, at a temperature of about 4°C.
- the magnetic spheres are then removed, and the cells are washed and pelleted. The procedure is twice repeated for a total of three treatments.
- the bone marrow cells are then washed and tested for tumor cells by PCR.
- Microsphere use produces a significant improvement of unexpected magnitude in tumor cell deple ⁇ tion.
- MmAb-3 or MmAb-4 mono- clonal antibodies
- all PCR detectable tumor cells were purged from aliquots of twenty-five (25) different tumor cells containing bone marrow samples.
- CmAb-3 monoclonal antibodies
- CmAb-4 monoclonal antibodies
- the addition of a fourth monoclonal antibody followed by complement mediated lysis (CmAb-4) purged the marrow of an additional five (5) patients, bringing the total to 16 out of 25.
- MmAb also was found to be specific because there was no loss of committed mye- loid progenitor cells.
- the microsphere results suggests that the use of microspheres will be superior to comple ⁇ ment mediated lysis, and the lack of non-specific toxicity to myeloid progenitor cells predicts that microsphere use will not impair engraftment.
- the follow ⁇ ing describes further testing using microspheres and monoclonal antibodies to remove tumor cells from bone marrow samples. Materials and Methods.
- Bone marrow was obtained from twenty-five patients with B cell NHL after Human Protection Committee validation and informed consent. Immunotyping documented that all lymphomas expressed CD20. All patients had achieved a protocol eligible minimal disease level following induction or salvage chemotherapy at the time of bone marrow harvest. Minimal disease criteria includes either a complete remission (CR) or a partial remission (PR) to tumor masses of 2 cm or less and marrow infiltration of less than 20% of the intertrabecular space. Bone marrow aspirates and biopsies were obtained one month before marrow harvest to assess lymphomatous infiltration and to confirm that the bcl-2 translocation could be detected by PCR amplification. All samples contained a PCR detectable bcl-2 translocation.
- Patient characteristics are summarized as follows. Twenty patients had follicular small cleaved cells, one patient had follicular mixed small and large cells and four patients had diffuse small cell histology. Nine patients achieved CR following induction or salvage chemotherapy and the remaining patients achieved a protocol eligible PR as defined herein. Fifteen patients had a prior history of morphologic bone marrow infiltra- tion ranging from local infiltrates to 90% of the inter ⁇ trabecular space. Eleven patients had morphological evidence of bone marrow infiltration at the time of bone marrow harvest. All twenty-five patients had residual detectable lymphoma cells in their harvested bone marrow when it was assessed by PCR. Twentyteen patients had a a translocation involving the major breakpoint region (MBR) of bcl-2 and six patients had a translocation at the minor cluster region (mcr).
- MLR major breakpoint region
- mcr minor cluster region
- microspheres or beads as used herein is descriptive of a particle of 0.1 to 5 microns in its major dimension and is inclusive of spherical, cubic, and rectangular particles as well as particles having other shapes. Preferred particles are about 0.3 to 5.0 microns.
- the particles may be made of a polymeric mater- ial such as polystyrene, polyacrylate, polymethacrylate, polyester, a styrene-divinylbenzene copolymer. poly- phenylene oxide and other polymers, copolymers or substances such as gelatin, dextran or an aminodextran, or may be made of a substance which is coated with these polymers, copolymers or substances.
- the microspheres may be magnetic or non-magnetic. Magnetic microspheres are preferred for their ease of separation. Magnetic microspheres may be polymeric particles which have a magnetic material embedded in a polymer matrix or they may comprise a magnetic nucleus or core having a polymeric coating. An examples of gelatin coated particles may be found in U.S. Patent No. 5,062,991.
- the magnetic beads used to treat the twenty-five patients' bone marrow were purchased from Advanced
- the anti-B cells monoclonal antibodies have been previously described herein.
- the three antibody combin- ation (m Ab-3) was a mixture of murine anti-Bl, anti-B5 and anti-J5 monoclonal antibodies.
- the four antibody combination (mAb-4) was formed by adding an anti-B4 mono ⁇ clonal antibody to the three antibody combination. Excess saturating concentrations of the monoclonal anti- bodies were used.
- the anti-T cell monoclonal antibodies used as controls were specific to CD2, CD3, CD4 and CD28, and were obtained from Dr. C. Morimoto, Dana-Farber Cancer Institute, Boston, Massachusetts. Other anti-T cell monoclonal antibodies having the indicated CD specificities may be used in their place.
- the anti-T cell monoclonal antibodies were isotype matched to the anti-B cell monoclonal antibodies. Excess saturating concentrations of the antibodies were used.
- the estimated bead-to- tumor cell ratio was in the range of about 250-500.
- the samples were incubated with the beads for 30 minutes at 4°C. After incubation, the magnetic beads were collected for 15 minutes using a magnetic particle concentrator (Dynal, Great Neck, New York) or other means of magnetic separation. A depleted bone marrow cell sample was transferred to a clean vessel and any residual magnetic beads contained therein were collected during a second 15 minute magnetic separation.
- the treated bone marrow cells were pelleted by centrifugation, washed three times and the tumor cell depletion or purging repeated for a total of three cycles.
- a sample of cells was collected and genomic DNA was isolated by cell lysis with non-ionic detergents followed by treatment with proteinase K (Sigma, St. Louis, Missouri).
- PCR amplification of any tumor cells present in a sample was conducted as described elsewhere herein. In the present analysis, 50 L of samples were used and the cell line RL was used in place of DHL-6.
- the cell line RL was a gift from Dr. W. Urba, National Cancer
- RL has a PCR amplifiable translocation at the major breakpoint.
- Other cell lines which have a similar translocation may be used in place of RL. No cell line was available with a translocation at the minor break ⁇ point region.
- PCR amplification was also performed using oligonucleotides for the human B cell activation antigen B7 in order to confirm that extracted DNA could be amplified by PCR in all samples.
- Bone marrow samples from eight of the twenty-five patients were assayed for hematopoietic progenitor cell growth both before purging and after the third purging cycle using the CmAb-4 and MmAB-4 procedures. The samples were blinded from the person who was conducting the colony assays. Granulocyte-macrophage colony-forming units (CFU ) were assayed by a standard procedure using
- GM were harvested on days 7 and 14, and 0.3% agar overlayers were dried onto glass slides and stained with Gill's hematoxylin (Sigma, St. Louis, Missouri). CFU were
- bone marrow having PCR detectable tumor cells was obtained from twenty-five patients and was treated using the MmAB procedure to determine if an improvement could made in the number of samples which could be depleted below the level of PCR tumor cell detection as compared to the CmAB procedure.
- the results of the PCR analysis of samples from two representative patients are shown in Figs. 7A-7B.
- patient 6 had residual PCR detectable tumor cells following three cycles of CmAb-3 treatment.
- all PCR detectable tumor cells were removed after three rounds of CmAb-4 treatment. Depletion using MmAB-3 and MmAb-4 resulted in the removal of all PCR detectable tumor cells after two rounds of treatment.
- Fig. 7C shows the results obtained by PCR amplification using oligonucleotides for the B7 gene of this patient. The results confirm that PCR amplifiable DNA was extracted from each of the samples.
- patient 7 had no PCR detectable lymphoma cells following the third round of treatment using CmAB-3 or CmAb-4.
- analogous MaAb treatment was used, no PCR detectable tumor cells were found after the second treatment cycle.
- the purging of bone marrow samples using microspheres appears to be specific and does not result in the loss of antibody-free cells through conjugation such cells to the microspheres. In repetitive experi- ments, when bone marrow samples were treated through three cycles using anti-T cell monoclonal antibodies and
- PCR analysis is capable of detecting one lymphoma cell in
- Table 3 gives the results of the PCR amplification at the major and minor breakpoint regions of the bcl-2 translocation after each of the three CmAb-3, CmAb-4, MmAb-3 and MmAb-4 treatment cycles for the bone marrow samples from all twenty-five patients. After the third cycle of CmAb-3 treatment, only 11 out of 25 samples
- CmAb-4 treatment of marrow samples increased the number samples having no PCR detectable tumor cells after three cycles to 16 out of 25 (64%).
- the five additional patients whose samples could be purged of residual lymph- oma cells by four, but not three, monoclonal antibodies are indicated by an asterisk after the patient number.
- H owever regardless of whether three or four monoclonal antibodies were use, three CmAb treatment cycles were required before any of the samples were purged of PCR detectable tumor cells.
- p 0.256, Fisher exact test.
- results shown herein indicate that tumor cell depletion using a plurality of monoclonal antibodies and microspheres is significantly more efficient than the procedure using a plurality of monoclonal antibodies and complement mediated lysis.
- results also indicate that three rounds of MmAb depletion is required, that re- infusion of the MmAb depleted marrow may produce superior results compared to CmAb depleted marrow and that the lack of non-specific toxicity to myeloid progenitor cells in an indication that microsphere use will not impair engraftment.
- the failure of CmAb purging to remove all tumor cells can be explained and attributed to three possible mechanisms.
- the clonogenic tumor cells might not express the surface antigens expressed by a majority of tumor cells.
- modulation of one or more of the surface antigens f ol l owing monoclonal antibody attachment to its ligand might limit complement mediated lysis.
- a subgroup of patients may have lymphomas which are intrinsically more resistant to complement mediated lysis. Since, when the same number of monoclonal anti- bodies was used in both procedures, the microsphere procedure was capable of depleting residual lymphoma cells in all cases whereas complement mediated lysis procedure failed to completely remove tumor cells, the third mechanism is believed the most likely.
- the approach of the claimed invention is that if the relapse rate significantly decreases when tumor-free marrow is re-infused, this is a strong indi ⁇ cator that marrow derived cells contribute to relapse.
- the long-term CmAb results shown in Fig. 3 indicates that disease-free survival after ABMT correlates strongly with whether the patient"s marrow purged PCR negative or positive. In the group that purged negative, only 4 out of 57 patients relapsed after as long as 8 years.
- Magnetic microspheres are prepared according to U.S. Patent No. 5,062,991 and monoclonal antibodies are conjugated there to using bridging groups 1-20 atoms long. Bone marrow containing tumor cells are harvested, concentrated, washed, fractionated and suspended in RPMI medium as described above for complement mediated lysis. The antibody containing magnetic beads are then added to the marrow suspension and the resulting mixture is incubated for an experimentally determined time, possibly 10 minutes to about 2 hours, at a temperature of about 4°C. The magnetic spheres are then removed, and the cells are washed and pelleted. The procedure is twice repeated for a total of three treatments. The bone marrow cells are then washed and tested for tumor cells by PCR. Alternatively, the magnetic microspheres prepared according to Patent No. 5,062,991 may be coated with GAM and used as described in Example 2.
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JP6510304A JPH08502489A (en) | 1992-10-19 | 1993-10-18 | Immunological purging of tumor cells from bone marrow using microspheres and monoclonal antibodies |
AU53616/94A AU681605B2 (en) | 1992-10-19 | 1993-10-18 | Immunological purging of tumor cells from bone marrow using microspheres and monoclonal antibodies |
EP93923903A EP0671950A4 (en) | 1992-10-19 | 1993-10-18 | Immunological purging of tumor cells from bone marrow using microspheres and monoclonal antibodies. |
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US20100124741A1 (en) * | 2008-11-18 | 2010-05-20 | Quest Disgnostics Investments Incorporated | METHODS FOR DETECTING IgH/BCL-1 CHROMOSOMAL TRANSLOCATION |
US20200399593A1 (en) * | 2018-02-14 | 2020-12-24 | Universidad Del Pais Vasco/Euskal Herriko Unibertsitatea | Method for obtaining a spermatozoid cell population with improved fitness |
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US4692405A (en) * | 1985-03-05 | 1987-09-08 | Dana-Farber Cancer Institute, Inc. | Monoclonal antibodies to antigen on activated human B-cells and assay therefor, protein antigenic determinant therefor and method of making same |
WO1990010692A1 (en) * | 1989-03-15 | 1990-09-20 | University Of Florida | Monoclonal antibody for use in detection and treatment of childhood leukemia |
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US20100124741A1 (en) * | 2008-11-18 | 2010-05-20 | Quest Disgnostics Investments Incorporated | METHODS FOR DETECTING IgH/BCL-1 CHROMOSOMAL TRANSLOCATION |
US20200399593A1 (en) * | 2018-02-14 | 2020-12-24 | Universidad Del Pais Vasco/Euskal Herriko Unibertsitatea | Method for obtaining a spermatozoid cell population with improved fitness |
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CA2147010A1 (en) | 1994-04-28 |
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