Transplantation Human Hematopoietic Progenitor ...

Evidence for Absence of Toxicity of T101 Immunotoxin on Human Hematopoietic Progenitor Cells Prior to Bone Marrow Transplantation L. Douay, N. C. Gorin, M. Lopez, et al. Cancer Res 1985;45:438-441. Published online January 1, 1985.

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[CANCER RESEARCH

45, 438-441, January 1985]

Evidence for Absence of Toxicity of T101 Immunotoxin on Human Hematopoietic Progenitor Cells Prior to Bone Marrow Transplantation1 L. Douay,2 N. C. GorÃ¬n,M. Lopez, P. Casellas, M. C. Liance, F. K. Jansen, G. A. Voisin, C. Baillou, J. P. Laporte, A. Najman, and G. Duhamel Laboratoire de DiffÃ©renciation Cellulaire, FacultÃ©de MÃ©decineSaint-Antoine [L. D., N. C. G., C. B., J. P. L, A. N., G. D,], and UnitÃ©de Cryobiologie, Centre National de Transfusion Sanguine [N. C. G., M. L] and UnitÃ©ÃŒNSERM U 23 [G. A. V.], HÃ´pital Saint-Antoine, Paris, France, and Centre de Recherches Clin-Midy-Sanofi, Rue du Professeur Joseph Blayac, 34082 Montpellier Cedex, France [P. C., M. C. L, F. K. J.]

ABSTRACT

the T101 monoclonal antibody, in view of its possible application to the In vitro treatment of bone marrow in patients with T-cell cancers prior to autologous bone marrow transplantation.

T101 -ricin A-chain immunotoxin is a hybrid molecule made up of the T101 monoclonal antibody bound to the A-chain of ricin. It specifically destroys cells expressing the cell surface T65 antigen. We have designed a preclinical study to evaluate its possible use for the in vitro treatment of T-cell hematological

MATERIALS AND METHODS Immunotoxin T101. T101-IAT (Centre de Recherches Clin-Midy-San ofi, Montpellier, France) is an hybrid molecule made up of the A-chain

cancers prior to autologous bone marrow transplantation. The data presented here show that conditions previously defined to produce high tumor cell killing, /.e., a 20-hr incubation at 37Â°in the presence of T101-ricin A-chain immunotoxin up to 10~7 M in

subunit of ricin linked to the T101 monoclonal antibody described by Royston. T101-IAT contains a mean of 2.5 A-chains per antibody mole cule (4). T101, an lgG2a globulin, reacts with the T65 antigen present on the surface of all normal T-cells, most thymocytes, T-cells of acute lymphocytic leukemias, and leukemic T-cell lines (22) and also on B-cells

a 10 rnw ammonium chloride solution, do not affect the In vitro proliferative capacity of human hematopoietic stem cells studied by means of semisolid medium cultures (granulocyte-macrophage progenitors, burst-forming units-erythrocyte) and contin uous liquid cultures (pre-granulocyte-macrophage progenitors). Therefore, autologous bone marrow transplantation with T101ricin A-chain immunotoxin-treated graft should be feasible.

of chronic lymphocytic or prolymphocytic leukemias (23). The T101 binds the T65 differentiation antigen which allows the A-chain of ricin to cross the membrane. Then the A-chain penetrates the cytoplasm, inactivates ribosomes, and blocks protein synthesis irreversibly, inducing cell death (16). Patient Population and Bone Marrow Collection. Bone marrow samples were harvested by aspiration from iliac spines in patients either with T-cell cancers in complete remission or with solid tumors without

INTRODUCTION

bone marrow involvement at the time of collection in the surgery room for autologous bone marrow transplantation. Bone Marrow Treatment with T101-IAT. Volumes of 50 or 100 ml of

Monoclonal antibodies have been used recently in an attempt to purge leukemic bone marrows, collected in remission, of their few residual tumor cells, (21, 27). Indeed, many of the obstacles limiting their use in vivo, such as the presence of circulating antigens, antigenic modulation, reactivity with normal cells, or immune response to murine antibody, may be circumvented through the carefully controlled manipulation of the reagents in vitro. However, these techniques require important bone marrow processing and the use of heterologous complement, the non specific toxicity of which may result in the loss of normal hema topoietic stem cells. Monoclonal antibodies may be now used as carriers of various cytotoxic agents. Specific procedures to conjugate to antibodies chemotherapeutic agents (1,10), lipid vesicles loaded with either drugs or toxins (17), or plant toxins (28) such as ricin A-chain (2,

freshly aspirated marrow were used for the following in vitro studies. The total bone marrow (i.e., without any cell separation) was incubated in a 150-ml bag (GAMBRO DF 1000, Heschingen, West Germany) at 37Â°in a 10 mw ammonium chloride solution with increasing dosages of T101-IAT,

in molarity, with

temperature inside the freezing chamber was sharply diminished. A particular attention was devoted to the third slope in order to keep a cooling rate slower than -5Â°/min after the transition phase (7,13). The marrow was then stored in the gas phase of liquid nitrogen at -196Â°. After thawing in a waterbath at 37Â°,specimens were drawn for in vitro

' This work was carried out with the help of Grant 6113 from the ARC (Villejuif, France) and INSERM Grant 129022. 2 To whom requests for reprints should be addressed, at Laboratoire d'HÃ©ma

assays of the cryopreserved stem cells. Culture of CFUc. CFUc assay was performed in semisolid agar by a modification of the technique of Pike and Robinson (19). The basic medium was McCoy's Medium 5A without serum (Gibco/Biocult) supple

tologie, CHU Saint-Antoine, 27, rue de Chaligny, 75012 Paris, France. 3 The abbreviations used are: T101-IAT, T101-ricin A-chain immunotoxin; FCS, fetal calf serum; BFUc, granulocyte-macrophage progenitors; BFUe, burst-forming unit-erythroblast; CSA, colony-stimulating activity. Received July 18, 1984; accepted October 3, 1984.

RESEARCH

as final A-chain concentration

Glasgow, Scotland), 20% dimethyl sulfoxide (Assistance Publique, Paris, France), and 10% matched human serum. The final concentrations were 10% for dimethyl sulfoxide and 5% for serum. This mixture was then frozen according to previously described techniques (6). Bone marrow was frozen in the vapor phase of liquid nitrogen in a Nicool biological freezing system (Air Liquide, Grenoble, France). The freezing rate was -1Â°/min until the release of the heat of fusion at which time the

11,15,16, 20, 25) are presently available. We report here the results of a preclinical study designed to evaluate the possible toxicity on human hematopoietic stem cells of T101-IAT,3 consisting of the A-chain subunit of ricin linked to

CANCER

expressed

gentle continuous shaking. Marrow samples were drawn at various intervals (1 to 20 hr) for hematopoietic stem cell assay. Bone Marrow Freezing Technique. After the incubation period, bone marrow was mixed with an equal volume of freezing medium consisting of TC 199 with glutamine and salts of Earie's medium (Gibco/Biocult,

mented with 30% decomplemented

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FCS (Gibco/Biocult).

CSA was sup-

T101-RICIN

IMMUNOTOXIN

AND HUMAN PROGENITOR

CELLS

plied by 10% human placental conditioned medium (kindly provided by Dr. R. Berthier, CEN, Grenoble, France), produced according to the method of Schlunk and Schleyer (24). The same batches of PCS and human placental conditioned medium were used for all of the studies. The mononuclear bone marrow cells were isolated over a Ficoll-sodium metrizoate gradient (d = 1.077; Lymphoprep; Nyegaard, Oslo, Norway). They were cultured in 35- x 10-mm Petri dishes (Coming), using 2 x 105

Table 1 Influence of experimentalconditions on bone marrow CFUcand BFUeproliferation Total bone marrows were incubated at 37Â°with agitation in the presenceor not

bone marrow cells/ml of medium containing equal volumes of 0.6% agar and McCoy's Medium 5A 2x (Gibco/Biocult) to achieve a final serum

Â±13Â°ND" Â±23 64Â±3 67 Â±14 ND 10 1 90 Â±24 93 Â±16 ND 10 ND 2 ND 89 Â±16 115Â±16 ND 10 3 66 Â±19 65 Â±6 96 Â±20 113 Â±23 420Recovery 0 104 Â±24 111 Â±31 69 Â±20 58 Â±28 10 106 Â±34 161 Â±58 52 Â±32 28 Â±10 0 10CFUc100 100 Â±22BFUe100 106 Â±53Recovery 33 Â±9BFUe73 32 Â±16 * Expressed by comparison with the number of progenitors detected per ml of

Of 10IT1MNH.CI.

after incubation* Incubatkx (hr)0

concentration of 15%. Three Petri dishes were plated for each assay. The cultures were incubated for 10 days at 37Â°in a humidified 5% C02 atmosphere. The colonies were examined on the Petri dishes by means of an inverted microscope, and counts were made of colonies containing more than 50 cells. The results were expressed in terms of the number of colonies per ml of marrow. Culture of BFUe. BFUe assay was performed in methylcellulose by a modification of the technique of Iscover and Sieber (14). Mononuclear cells (2 x 105) isolated as above were cultured in triplicate in 0.9%

(%)NH,CI

after cryopreservation1' (%)CFUc73

(rriM)0

marrow at time 0 without NK>CI. 6 Expressed by comparison with the number of progenitors detected after incubation. '" Mean Â±S.D. of 3 to 9 separate experiments. " ND, not done.

methylcellulose in a medium (Gibco/Biocult) containing 20% PCS, 1% bovine serum albumin (Sigma), 20% NCTC (Eurobio, Paris, France) and 1 unit pork erythropoietin (CNTS, Paris, France). Cultures were incubated for 14 days at 37Â°in a humidified 5% CO2 atmosphere. Colonies were

Table 2 Influence of T101-IATtreatment on bone marrow CFUcand BFUeproliferation Total bone marrows were incubated at 37Â°with agitation for 4 or 20 hr in the presenceof 10 mw NH4CIwith increasingdoses of T101-IAT.

examined on the Petri dishes through an inverted microscope, and counts were made of colonies containing more than 40 cells. The erythroid nature of the cells in colonies was confirmed by examination of picked colonies stained with May-Grunwald-Giemsa. The results were ex

(%)CFUc96 treatment8

pressed in ternis of the number of colonies per ml of marrow. Continuous Liquid Cultures. Long-term liquid cultures were estab

after

after treatment and cryopreservation" (%)CFUc70

10mu + Incubation(hr)420T101-IATdose(u) NH,CI10-"IO"io-710-Â«10-'10-Â«io-710-Â«Recovery 4c93+

lished using a modification (8) of the technique of Dexter ef a/. (5). Bone marrow buffy coat cells (108) from normal donors were suspended in 1

889+ Â±1191 Â±1087 Â±1887 Â±688 Â±14113Â±1497 Â±1788

ml of a medium (Gibco/Biocult) supplemented with 20% FCS. Five ml of this suspension were put into Falcon 25-sq cm tissue culture flasks, and the suspension was incubated at 37Â°in a 5% CO2 atmosphere. Cultures

Â±2585 Â±1587 Â±1860 Â±446

3ND^72 + Â±1429 1629 Â±

Â±4186 Â±2028 1434 Â± Â±2193 Â±4577 Â±1537 Â±14NDND Â±1417Â±3BFUe33 Â±1857 Â±2231 Â±16BFUe93Â±7Recovery

were maintained by weekly demipopulation with addition of fresh me dium. Under these conditions, an uniform adherent layer spread within 3 to 4 weeks. Then, this primary culture was depopulated of all suspension cells and irradiated at 30 Gy. A new allogenic inoculum of bone marrow cells was added onto the adherent layer. Cultures were maintained by replacing all the medium weekly, without demidepopulation. At the same time, suspension cells were assayed for CFUc as described above.

8 Expressed by comparison with the number of progenitors detected at time 0 with untreated marrow. "Expressed by comparison with the number of progenitors detected after treatment. c Mean Â±S.D. of 3 to 9 separate experiments. " ND, not done.

RESULTS

ing freezing and thawing of incubated marrow to the recovery of cryopreserved nonincubated marrow. CFUc recovery of marrow incubated for 20 hr with 10 mw NH4CI dropped to 33 Â±9%. BFUe were somewhat more sensitive with a recovery of 32 Â± 16%. Marrows were then incubated in those same conditions with increasing doses of T101-IAT, ranging from 10~9 to 10~6 M.

The sensitivity of normal progenitors was evaluated by their ability to proliferate. Results were expressed in terms of the percentage of stem cells recovered per ml of manipulated mar row, by comparison with nonmanipulated marrow. The results shown represent the mean Â±S.D. of 3 to 9 determinations for Results are shown on Table 2. No effect of T101-IAT on progen each point. Sequential studies with incubation of the marrow for periods up to 20 hr were done for each individual patient. In itor cells proliferation was observed after a 4-hr incubation up to 10~6 M. After a 20-hr incubation, toxicity was seen at the 10~6 M these experiments, the mean number of colonies detected in level. BFUe progenitors were more sensitive to T101-IAT than controls were, respectively, for CFUc and BFUe: 124 Â±38 and 149 Â±52/2 x 105 mononuclear cells or 1420 Â±340 and 1603 Â± was CFUc. After cryopreservation, CFUc and BFUe recovery 633/ml bone marrow. These values represent what is usually again was reduced but in a way similar to that observed previ expected for normal marrow in our experience. ously for marrows either nontreated or NH4CI incubated. The ability of T101-IAT treated marrow to maintain myeloConditions previously shown to produce high tumor cell killing were tested (5). Table 1 shows the recovery of CFUc and BFUe poiesis in continuous marrow culture was tested. Frozen bags of marrows incubated with 10~8 M T101-IAT for 20 hr at 37Â°in after the incubation of the marrow at 37Â°for a period up to 20 hr in the presence of 10 HIM NH4CI. As indicated, NH4CI does not alter the viability of CFUc or BFUe. Moreover, a slight increase in CFUc and BFUe is observed. To test the influence of these experimental conditions further on the sensitivity of progenitor cells to cryopreservation, we then compared the recovery follow CANCER

RESEARCH

a 10 PPM NH4CI solution were maintained over the primary irradiated allogenic adherent layer. No impairment in CFUc gen eration (Table 3) was seen in the T101-IAT-treated group. More over, a small but significant improvement in the extent of CFUc production was evident at Weeks 1, 2, and 3, as compared to VOL. 45 JANUARY

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Table 3 Continuousliquid culture results Frozen bags of bone marrows treated with 10"* M T101-IAT in the presence of 10 mM NH4CIfor 20 hr at 37Â°with agitation were maintained over a primary irradiated allogenicadherent layer (see "Materials and Methods"). CFUcgeneration is compared to nontreated frozen marrow. CFUc/ml culture Wk inculture0

frozen marrows938 Â±691a

653 Â±611 705 Â±593 105 Â±92 27 Â±10 5T101-IAT-treated 1 Â±1Nontreated " Mean Â±S.D. of 3 separate experiments. 1 234

Â±935 325 + 109 491 Â±382 48 Â±13 36 Â±7 1.5 Â±1.5

of CFUc which hence are maintained longer in culture. Subse quently, T-cells regenerate from pluripotential stem cells, as shown from pluripotential stem cells in semisolid medium culture analysis (4), which explains at Week 4 the secondary drop in CFUc. Therefore, these experimental conditions for T101-IAT allow the in vitro treatment of bone marrow potentially contaminated with residual leukemic cells prior to autologous bone marrow transplantation. A trial using T101-IAT in vitro has been initiated in 4 patients with T-cell cancers.4 All patients have engrafted

the control marrows. However, there was no difference in the duration of the culture. CFUc were maintained up to 5 weeks, and nucleated cells were maintained up to 7 weeks. DISCUSSION This study was designed to evaluate the potential toxicity of T101-IAT on human hemopoietic stem cells under conditions previously defined to produce high tumor cell killing. The efficacy of T101-IAT has been measured previously by inhibition of protein synthesis through the uptake of [14C]leucine into CEM

within normal limits, in spite of sensitization of progenitors to cryoinjury, which may be related to the incubation procedure itself and not to the action of T101-IAT. The use of T101 immunotoxin A-chain may also be extended to the setting of allogenic bone marrow transplantation, through the elimination of T-cells for prevention of graft-versi/s-host disease (12). Monoclonal antibodies as carriers for toxins are a promising tool for a selective removal of leukemic cells which may signifi cantly increase the clinical effectiveness of antibodies in vitro, without any nonspecific toxicity.

cells (human T-leukemic cell line expressing T65) (3) and by the evaluation of the surviving cells determined by cloning assay in agar (4). The optimal conditions for a reliable and highly efficient elimination of leukemic cells from autologous graft were estab lished as follows (3, 4). A 99.90% elimination of leukemic cells among a 10-fold excess of bone marrow cells was obtained after a 4-hr incubation at 37Â°in a 10 mM NH4CI solution with doses of 10~8 M ITT101. However, after a 20-hr incubation, elimination rose to 99.99%. We therefore selected this duration for cleansing the marrow. NH4CI (10 mM) has been shown to have a highly potentiating effect. It acts on the immunotoxin intemalization process by increasing intralysosomal pH which blocks the lysosomal pathway of protein degradation (3). Therefore, the acti vation can be attributed to an inhibition of protein degradation. Unfractionated bone marrows were used rather than mono-

ACKNOWLEDGMENTS We wish to thank M. C. Leguen and D. Bardinet for their expert technical assistance and M. Veillard for typing the manuscript.

REFERENCES

nuclear cell suspension for practical purposes, mainly to prevent a loss of stem cells which might result from additional steps such as spinnings and washings and because it has been shown that erythrocytes and granulocytes do not bind T101-IAT nonspecif-

1. Amon, R., Hurwitz, E., and Sela, M. Antibodies as carriers for oncostatic materials. Recent Results Cancer Res., 75: 236,1980. 2. Blythman, H. E., Casellas, P., Gros, O., Gros, P., Jansen, F. K., Paolucci, F., Pau, B., and Vidal, H. Immunotoxins:hybrid moleculesof monoclonalantibod ies and a toxin subunit specifically kill tumor cells. Nature (Lond.), 290: USHO, 1981. 3. Casellas, P., Bourrie, B., Gros P., and Jansen, F. K. Kinetics of cytotoxicity induced by immunotoxins: enhancement by lysosomotropic amines and carboxyfc ionophore. J. Biol. Chem., 259: 9359-9365,1984. 4. Casellas, P., Canat, X., Fauser, A. A., Gros, 0., Poncelet, P., and Jansen, F. K. Optimal eliminationof leukemicT cells from human bone marrow with T101 ricin A chain immunotoxin. Blood, in press, 1984. 5. Dexter, R. M., Allen, T. D., and Lajtha, L. G. Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell. Physiol. 97: 335-344, 1977. 6. Douay, L., Gorin, N. C., David, R., Stachowiak, J., Salmon, C., Najman, A., and Duhamel, G. Study of granulocyte macrophage progenitor (CFUc) pres ervation after slow freezing of bone marrow in the gas phaseof liquid nitrogen.

ically (3). Our results have shown that the incubation period at 37Â°up to 20 hr in the presence of 10 mM NH4CI does not affect the in vitro proliferation of committed progenitor cells. Moreover, the slight decrease of CFUc and BFUe within 1 hr, immediately followed by a secondary increase, may be attributed to a trig gering into DMA synthesis. T101-IAT itself has no significant toxicity up to 10~7 M. These results are consistent with those of Teatle and Royston (26), demonstrating the absence of T65 antigen on normal CFUc and BFUe. They also confirm the results of Casellas ef a/. (4) showing the lack of nonspecific toxicity of T101-IAT on the Daudi cell line (T65 negative) up to 10~7 M. The

4N. C. Gorin, L. Douay, J. P. Laporte, R. Zittoun, B. Rio, J. Jansen, P. Casellas, P. Poncelet, M. C. IJance, G. A. Voisin, M. Lopez, C. Salmon, G. Le Blanc, J. Deloux, R. David, J. Stachowiak, A. Najman, and G. Duhamel. Autologous bone marrow transplantation with marrow decontaminated by immunotoxin T101 in the treatment of leukaemia and lymphoma. First clinical observations, submitted for publication.

slight sensitivity of BFUe may be related to the absence of Tcells, the presence of which could be required for their in vitro proliferation in semisolid media culture (18). RESEARCH

Since committed stem cells are an indirect reflection of the self-renewing capacity of the totipotent stem cells, we have studied the behavior of T101-IAT-treated and frozen marrows in continuous liquid cultures. Results demonstrate the lack of tox icity on pre-CFUc progenitors of T101-IAT at doses used in clinics (10~8).4 Indeed, a greater number of CFUc colonies was detected at Weeks 1, 2, and 3 in cultures of treated marrows as compared to controls. It has been demonstrated (9) that deple tion of some T-lymphocyte subsets may significantly alter the balance between differentiation and proliferation of CFUc possi bly because of a modification in CSA level. Therefore, one may postulate that initial destruction of relatively mature T-cells by T101-IAT, leading to CSA level depression, slows differentiation

frozen marrows975

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Exp. Hematol., 70: 360-366,1982. 7. Douay, L, Gorin, N. C., and Duhamel, G. Rote critique de la vitesse de refroidissement au-delÃ de la libÃ©rationde la chaleur de fusion, pour la conservation des cellules souches hÃ©matopoÃ©tiques. C. R. Acad. Sci. Paris, 294 (III): 103-106,1982. 8. Douay, L., Gorin, N. C., Najman, A., Leguen, M. C., and Duhamel,G. Contin uous human bone marrow culture. Sensitivity to chemotherapeuticagents of probable pluripotential stem cells. Application to autologous bone marrow transplantation. Exp. Hematol., 11 (Suppl. 14): 7,1983. 9. Francis, G. E., Guimaraes,J. E. T., Granger, S., Bemey, J. J., Tidman, I. V., Wing, M. A., Janossy, G., and Hoffbrand, A. V. Distinct T lymphocyte subjects affect granulo-monocyticdifferentiation and proliferation.Stem Cells, 792:7687. 10. Chose, T., and Nairn, A. H. Antibody-linked cytotoxic agents in the treatment of cancer: current status and future prospects. J. Nati. Cancer Inst.. 67: 658, 1978. 11. Gilliland,D. G., Steptewski, Z., Collier, R. J., Mitchell, K. F., Chang, T. H., and Koprowski, H. Antibody-directedcytotoxic agents: use of monoclonalantibody to direct the toxin of toxin A chains to colorectral carcinoma cells. Proc. Nati. Acad. Sci. USA, 77: 4539-4543,1980. 12. Gilmore, M. J., Prentice, H. G., Price-Jones,E., Blacklock, H. A., Tidman, N., Schey, S., Goldstein, G., Janossy, G., and Hoffbrand, A. V. Allogenic bone marrow transplantation: the monitoring of granulocyte-macrophagecolonies following the collection of bone marrow mononuclear cells and after the subsequent in vitro cytolysis of OKT4 positive lymphocytes. Br. J. Haematol., 55: 587-593, 1983. 13. Gorin, N. C., Douay, L., David, R., Stachowiak, J., Parlier, Y., Oppenheimer, M., Najman, A., and Duhamel, G. Delayed kinetics of recovery of haematopoiesis following autologous bone marrow transplantation. The rote of exces sively rapid marrow freezing rates after the release of fusion heat. Eur. J. Cancer Clin. Oncol., 79: 485-491,1983. 14. Iscove, N. N., and Sieber, F. Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture. Exp. Hematol., 3: 3243, 1975. 15. Jansen, F. K., Blythman, H. E., Carriere, D., Casellas, P., Diaz, J., Gros, P., Hennequin, J. R., Paolucci, F., Pau, B., Poncetet, P., Richer, G., Salhi, S. L., Vidal, H., and Voisin, G. A. High specific cytoxicity of antibody-toxin hybrid molecules (immunotoxins)for target cells. Immunol. Lett., 2:97-102,1980. 16. Jansen, F. K., Blythman, H. E., Carriere, D., Casellas, P., Gros, O., Gros, P.,

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RESEARCH

17. 18. 19. 20.

CELLS

Laurent, J. C., Paolucci, F.. Pau, B., Poncetet, P., Richer, G., Vidal, H., and Voisin, G. Immunotoxins: hybrid molecules combining high specificity and potent cytotoxicity. Immunol. Rev., 62: 185-216,1982. Leserman, L. D., Barbet, J., Kourilsky, F., and Weinstein, J. N. Targeting to cells of fluorescent liposomes covalentlycoupled with monoclonalantibody or protein A. Nature (Lond.),288: 602, 1980. Nathan, D. G., Chess, L., Hillman, D. G., Clarke, B., Breard, J., Merzlter, E., and Housman, D. E. Human erythroid burst forming unit: T cell requirement for proliferationin vitro. J. Exp. Med., 747: 324-338,1978. Pike, B. L., and Robinson, W. A. Human bone marrow colony growth in agar gel. J. Cell. Physiol., 76: 77-84,1970. Raso, V., and Griffin, T. Specific cytotoxicity of a human immunogtobulindirected Fab'-ricin A chain conjugate. J. Immunol., 725: 2610-2616,1980.

21. Ritz, J., Sallan,S. E., Bast, R. C., Upton, J. M., Nathan, D. G, and Schlossman, S. E. Autologous bone marrow transplantationin CALLA positive ALL following in vitro treatment with J5 monoclonal antibodies and complement. Blood, 58 (SupplV175a, 1981. 22. Royston, I., Majda, J. A., Barid, S. M., Meserve, B. L., and Griffiths, J. C. Monoclonalantibody specificfor humanT lymphocytes:identificationof normal and malignantT cells. Blood, 54 (Suppl. 1)- 106a, 1979. 23. Royston, I., Majda, J. A., Baird, S. M., Meserve, B. L., and Griffiths, J. C. HumanT cell antigens defined by monoclonalantibodies: the T 65,000 antigen of T cells (T65) is also found on chronic lymphocytic leukemia cells bearing surface immunoglobulin.J. Immunol., 725: 725-731, 1980. 24. Schlunk, T., and Schteyer, M. The influence of culture conditions on the production of colony-stimulatingactivity by human placenta. Exp. Hematol.,fl: 179-184,1980. 25. Seon,B. K. Specifickillingof humanT leukemiccells by immunotoxinsprepared with ricin A chain and monoclonalanti T cell leukemiaantibodies.Cancer Res., 44: 259-264,1984. 26. Taette, R., and Royston, I. Human T cell antigens defined by monoclonal antibodies. Absence of T 65 on committed myeloid and erythroid progenitors. Blood, 56: 943-946, 1980. 27. Wells, J. R., Billing R., Herzog, P., Feig, S. A., Gale R. P., Terasaki, P. I. and Cline, M. J. Autotransplantation after in vitro immunotherapyof lymphoblastic leukemia. Exp. Hematol., 7 (Suppl.).-164-169,1979. 28. Wiels, J., Junquas, S., Dujardin, P., Le Pecq, J-B., and Tursz, T. Properties of immunotoxinsagainsta glycolipidantigenassociated with Burkitt's lymphoma. Cancer Res., 44: 129-133, 1984.

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