From www.bloodjournal.org by guest on December 22, 2014. For personal use only. Normal Progenitors Antibody Human Pluripotential Do Not Express BA-2: Implications By Analysis of poietic surface of cell tion in the sia. The has cells and tion with mine is if the antigen T HE that certain notypes features importance or antibodies monoclonal antigens animal From and have Departments also raised and Supported VA. in part Institutes ofHealth. CA28526 from supported structures. efficacy by (enter, the Veterans and k8вЂ™ in par! the March Presented in Federationfor the at Clinical American Texas, December Clinical Research Address reprint Oncology versity 18, 1982; part of the Research, Society 7, of NIH. the /981, Department Kentucky MEdical conditioning of Center, of Ill., 0006/4971/82/6006O0l & Stratton. Inc. 2$0l.00/0 antibodies previously dalton for for recogantibody treatment most autologous for immunotherapy reported.вЂ™3 This cell surface 70% of non-T, (ALL) and of useful in ex bone marrow of human antibody cell line antibody polypeptide non-B a small binds (p24) acute BA-2 by has been to a 24,000- approximately in lymphocytic population neo- leukemias of normal bone marrow lymphoid progenitors. Both in order to better define the expression of the p24 antigen during normal lymphohematopoietie differentiation and because possible antibody requires to relevant normal marrow hematopoietic precurthe interactions of monoclonal (such as normal we have explored MATERIALS Marrow Award J.J. was in San abstract American marrow of these I 2 normal into 9, 1981. Antonio. form in C. Ash, Medicine, M.D., Room Rose Street, normal for bone consent the was 1640 containing over Ficoll-Hypaque mm METHODS volunteer ranged 3-5-mI syringes. 10% calf marrow Interface mononuclear g. were and cells age drawn in RPMI FCS), I .077), in RPMI-I0% donors The informed three-fold (RPMI-lO% (density and resuspended After aspirates diluted gradients normal subjects. I 8 to 45. were serum and as control from bone Samples fetal donors served subjects at 400 3 times, paid transplantation obtained, heparinized 25-30 AND Samples Hematologically layered centrifuged were collected, FCS. Hemaiology/ MS 681, Lexington, Uni- Monoc/onal BA-2 by Grune agent perhaps plasia.вЂ™#{176}вЂ™2 The production of monoclonal immunization with a pre-B-ALL Grants Netherlands November Meetings, 800 monoclonal transplantation. washed 40536. /982 vivo specific express are of 12, 1982. published to Robert and leukemia. this not that antibody BA-2 with committed granulocyte-macrophage and erythroid progenitors as well as the human pluripotential hemopoietic progenitors, the CFU- 1981. requests Division, Chicago, and 29:76/A, July Meetings Hematology that do cells that in National Investigator of the Fund, accepted as a potent ALL for show and and the ofNew Midwest progenitors suggest lymphocytic results tissues sors), boundation. Submitted These Minn. Institute Wi/heirnina and BA-2 cytotoxicity lines. on hemato- contrast. of School Administration Cancer Queen Pediatrics, AM24O27from is recipient National normal In therapeutic use of any monoclonal prior demonstration of nontoxicity of use ofMinnesota Minneapolis. (вЂ�A21737. T. W.L. the possibility Physiology, University Medical CA25097, the of Medicine, Medicine/Pathology. CA23021. with has underthe develop- antibodies directed at neoplasia-associated in the treatment of several malignancies the Medicine correlate surface of the cell represented by BA-2 marrow with method. hematopoietic sites of these cellular pheof T and B may of these cell demonstrations studies5 Laboratory defined arrestвЂќ reactivity either BFU-E. of neoplastic of some neoplasms34 of understanding mental expression Moreover, recent 1310 BA-2 no D. Zanjani complement-mediated human serve Esmail by potent antigenic may deter- antibody structures differentiationвЂ™2 the clinical scored the :) to showed ALL-derived nized (CFLJ-GEMM) monoelonal antigenic utilized (CFLJ-GM. immunologically reflect вЂњmaturation may lymphocyte Ky. RBC by the pluripotential of we and progenitors normal line cells has facilitated the study of gene expression in the processes and consideration of potential cliniof such antibodies in the classification of hematologic neoplasia. The recogni- differentiation and selected concerning cal application and treatment Cancer neopla- lymphoid questions tion exhibited ox progenitors surface applica- monoclonal LeBien, BA-2 clinical cell W. CFU-E). rosette-separa- to Tucker poietic study and recognized human PRODUCTION and normal this coupled hematopoietic against of (p24) on hemato- a pre-B-ALL cytotoxicity represented committed In Kersey, biologic of with indirectly H. of hematologic previously. antibody John to basic characterization complement-dependent Committed Hematopoietic the p24 Antigen Detected by Monoclonal for Immunotherapy of Lymphocytic Leukemia of both to potential immunization reported Jansen, determinants treatment and by Jan relevance and production BA-2 been has differentiation diagnosis antibody C. Ash, antigenic progenitor study Robert and tion of BALB/c ascitic Antibodies is an antibody fluid female was collected, of the IgG3 mice with allowed Blood, subclass produced the NALM-6-Ml to clot, and stored by immunizacell line.вЂ™3 at BA-2 - 70#{176}C, and Vol. 60. No. 6 (December), 1982 From www.bloodjournal.org by guest on December 22, 2014. For personal use only. ABSENCE used in this 0.45-jz lots OF ANTIGEN study used study was approximately structures,вЂ™7 and used immunofluorescence with the sheep J, A. Hansen Wash.) respectively. Monoclonal human lineage,20 B-cell receptor,вЂ™8 and BA-I, monoclonal to platelet-megakaryocyte-specific ter a gift from Dr. Cytotoxicity Bone FCS marrow were for mm antibody, agitation, obtained from screened in this at Pelfreez at (the and cytotoxicity. a one-step 37#{176}C)was cytotoxicity mm. was was pre- with 9.6, human used with a and hemo- cells, in antibody, reduced marrow at with start 5% CO, protein A published.23 One part Fine Chemicals, in 0.9% in 0.9% NaCI NaCI and 10% FCS thereafter x 106/ml for 30 mm, then washed twice in cold FCS. The added in equal oRBC:marrow volumes cell at I 50 g for 10 mm, The was pellet of rosetting Hypaque gradient face pellet to approximately centrifugation RBCs lysed from and the cell fractions pellet clonal Desired with (SPA- taken to to Ficoll- g, 15#{176}C).Intercontaminating Tris-buffered and appropriately cell 0.83% diluted. cytotoxicity or control ascitic binding populations or fluid before rosetting was at the red large consist an cells identifiable can derived) distinct cells. cell be seen. can The be with a morphological within individual by Wright-Giemsa histochemical also colonies types of and вЂњmixedвЂќ in which (CFU-GM of specific 15. Hemoglo- color, derived) and of by direct staining stains.вЂ™4 The 6 mega- content of these mixed colonies analysis a functionally вЂњmixedвЂќ only areas Culture glycoprotein they appeared to be discrete (lIb-Illa) of were scored as single colonies in CFU-E system were was cultured aliquots colonies complex Colonies of plates. clot samples 0.1-mI with specific Assayfor plasma has also been confirmed by monoclonal antibody Tab, which lineage.2224 when nonconfluent In some (indicated used at 2-6 containing scored 1.5 as x I0 IU previously cells/mI Ep/mI, in quadrupliand benzidine- on day 7. according al.27 mixed of monoand after conditioned experiments. with target BFU-E of 1.5 staining. 0.2 ml medium, mixed, 5% CO, experiments in Results), to a microwell Briefly, were Assay preliminary separately flat-bottomed were scored was used to assess marrow on day as morphologically megakaryocyte-platelet positive DulbeccoвЂ™s added enumerated derived) small selective dishes condi- of phytohemag- Ep was (BFU-E been demonstrated use of other Microculture for Analyses t-dependent monoclonal interface 400 collected, elements presence have culture (media modified by their of nonhemoglobinized and by Fauser x 10вЂ™ mononu- of I I U erythropoie- magnification colonies time and presence and colonies (CFU-GEMM at this recognizes cate as at 37#{176}C in an atmosphere recognizable erythroid arrangement Marrow used at centrifuged subjected mm, then temperature. aliquot remainder then to 10 x a fluorescence- of0.25-2 in the of nonhemoglobinized The 30 sites assay described incubated humidity, elements the the aliquots in 35-mm 5% PHA-LCM at 40-200x вЂњpureвЂќ karyocyte at 4#{176}C was mixed, at room an (25-30 were at suspension 100:1), washed immunofluorescence antibodies complemen and fractions Immunofluorescence cell for 20 mm the I- cells of BA-2 suspension resuspended, and in RPM and resuspended marrow incubated cell NH4CI, Indirect the gently media was ox RBCs a I :50 dilution were immunofluorescence 0.5 mixture mononuclear SPA-oRBC and cells, The and resuspended Marrow with ratio then counting and of I 09/mI. at of 2.5 x 10 4M CrCI3 ox RBCs. 3 times incubated in RPMI-l0% N.J.) the Staph-protein-A-coated were 106/ml 10 parts methods of Staphylococcal Piscataway, washed washed to a concentration a solution with packed I hr at 30#{176}C, and (SPA-oRBCs) 20-24 was mixed 1 part of and BA- and on in the presence leukocytes easily colonies were of binding BFU-E, Concentrations cultured blood in situ periphery mixed cytotoxicity method of obtaining BA-2-depleted cell populations was adopted from (Pharmacia mg/mI incubated of complement-dependent were Plates heterogeneity results studies, an alternate 2-enriched marrow previously cells and high with for with epi-illumination. antibodies intensity of the methylcellulose colonies, those All saturation CFU-GEMM, Granulocyte-macrophage Separation per slide. Ploem or F(abвЂ™)2 (FACS). was used. of culture. possible, was continued with to achieve Va.) and examination 30% fetal calf serum, IscoveвЂ™s and 5 x l05M 2-mercaptoethanol; recognized to confirm equipped immunofluorescence by human glutinin), medium, when incubation tin (Ep) in quadruplicate I-mI containing 0.9% methylcellulose, cellular for 60 mm minimally clear binized indicated incubated MessnerвЂ™4.вЂќ and, mounting, counted Assayfor and flatter In order were cell sorter observation to maximize as (i.e., and lots W6/32, against incubation was this procedure. Rosette by activated second was (Springville, Pa.) by washing, sufficient at 4#{176}C. After FITC-GAMG Laboratories microscope 200 cells tioned used cytotoxicity experiments, lysis of rabbit from BA-2, of with continu- Arks.) toxicity cell volumes Complement specific simultaneously specific the incubation preliminary added utilized: addition (including two-step some and complement 60 nonspecific this In by (Rogers, antibodies in RPMI-lO% equal for maximum minimal x l06/ml and incubation, 37#{176}Cfor laboratory progenitors; fluorescent Culture CFU-GM lat- also used. 4#{176}C with followed Biologicals of monoclonal Results, IIb-IIIa2вЂ™22 The 30 mm (FITC-GAMG). Meloy used. for fluorescein-isothiocyanate-conju- (Cochranville, were determined incubated with Ig from concentrations N.J.), and stained at 4#{176}C, followed At least Drs. with cells of which binds Tab, were at 20-24 30 ( I 0% final concentration) gentle poietic reactive Laboratories Zeiss 106/ml) anti-mouse Cappel which from goat conjugates reacting gifts were either mm (Raritan, antibody San Antonio) mononuclearcells diluted OKT-3) OKT-3,вЂ™9 x cells A modificationвЂ™6 incubated complement panel 9.6, 10-25 obtained or for Assays appropriately ous N.Y.). were glycoprotein R. McEver, Westbury, G. Goldstein antibody and and these C (through experiments (at gated and Lab antibodies cells washing, Monoclonal Sera the monoclonal a fluid HLA-A,B, from pl9; through ascitic mg/ml. Corporation, structure (Seattle, 1-5 in cytotoxicity erythrocyte antigenic passage of BA-2 recognizing Scientific included the T-cell after purchased as controls 1311 CELLS concentration antibody was Chemical antibodies recognizes IgG an IgG2a STEM dilutions The in this Accurate Other HUMAN filter. W6/32, framework ON at appropriate Nalgene antibody p24 and and high Ep/mI modification26 cells (at 0.2 ml FCS, 6 replicates microtiter after I 0 days and CFU-E IU of evaluating CFU-GM of the 5 x ml assayed 0.2 pipetted of Iscove ml et leukocyte after into the wells of a Onard, Calif.) Colonies at 37#{176}C in an atmosphere of In the W6/32 were also cultured and method lOS/mI), and 0.4 ml of 2% methylcellulose of 0.1 plate (Falcon, of incubation humidity. the separation steps alone were cultured rosetting in this system appropriate experiment, in the presence fixation and From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 1312 ASH Table 1 . Reactivity With of Monoclonal Normal Marrow Antibody Mononuclear U, U, 1:1000 > 3.5 1:200 a) 4.5 Combined 10 -2 10 -3 10 Antibody -4 10 -5 10 of complement cells marrow mononuclear immunofluorescence at least I : I 0,000 marrow mononuclear Approximately mononuclear immunofluorescence marrow samples. mononuclear Considerations data calculated presented cultures using in this at individual the StudentвЂ™s t paper represent data points. the mean p Values of that incubations test. conditions results of cytotoxicity leukemic cell lines with effective BA-2 and with experiments demonstrating complement, under target cell lines, 2. Effect of Monoclon cells, BA-2 Addutuvest Media + CвЂ™(1:5)t Media + CвЂ™(l:lO) Media + CвЂ™(l:lO)t Media + CвЂ™(l:lO) + Media + CвЂ™(l:lO) + W6/32(1:5000) Media 4 W6/32(1:50)(noCвЂ™) Media + CвЂ™(l:lO) -+ Media + CвЂ™(l:lO) + Media + CвЂ™(l:lO) * on Nor mal Marrow cells + Media + CвЂ™(l:lO) CвЂ™(l:lO) complement 2.7 34.5 1.5 38.3 В± 0 during brightly The on the of either number Hematopoietic 10вЂ™ CFU-E 2.1 1.5 187 В± 7 33 В± 1.5 3 165 В± 15 29 В± 4 В± 4 178 В± 12 31 В± 3.5 0 0 0 1.5 В± 2.3 3.5 35 В± 2.1 0 В± 2.1 172 В± 22 11 31 В± 5 21 33.5 В± 3.1 В± 1.7 39 В± 4 169 В± 41 В± 4.5 177 В± BA-2(1:100) 13.7 В± 1.8 33 В± 2.9 185 В± 17 37 159В±23 2.3 38 В± 1.8 172 В± В± 2.5 36.5 В± 5 179 В± 1 2.3 1.5 В± В± В± 2.9 37.5В±3.1 1 36 В± 60-mm controls. clonigenic В± В± 15.1В±3 compared В± 14.3 adult no pluripoten- CFU-GM 12.5 a normal effective cells, Progenitors BA-2(1:50) with immuno- Cells BA-2(1:25) as described incubation. by of BA-2 В± experiment of of BA-2 conditions 13.1 incubation. to dilutions number under <0.5 <0.3 14 by indirect cyto- cytolysis 0 0 W6/32(1:500) 11.9 cytotoxicity present 2 В± BA-2(1:800) Data of a representative tTwo-step 37 В± 12.1 + BA-2(1:400) + and median 27) small round enumerated BFU-E 13.5 12.7 Media+CвЂ™(1:1O)+BA-2(1:200) Media antibody colony formation by BA-2 and complement, to both media and complement-incubated The efficacy of this system for abrogating BA-2 W6/32(1:50)t (age 18-45, cells were and stained optimal effect CFU-GEMM Mediaalone + with (Trypan-blue tial (CFU-GEMM derived) or committed hematopoietic precursors was observed (Table 2). Table 2 shows detailed data or a representative experiment, demonstrating the absence of significant inhibition of presumably al Antibody for adult BA-V of ascites. Colonies! Incubation incubation incubation. viable = and complement significant dependent in part on the density of the p24 antigen on the respective cell lines. The binding of monoclonal antibody BA-2 to normal Table of total 4%-5% (4.5 В± 0.8, n 9) of cells were BA-2 positive by The for accomplishing for cytolysis of BA-2вЂ™ cells from I), are highly effective in of ALL cells. The efficiency of normal marrow (Table accomplishing cytolysis cytolysis varied between samples. fluorescence showed good agreement with that determined by specific cell lysis using complement-dependent cytotoxicity (Table I). When marrow mononuclear cells were incubated were RESULTS Figure 1 shows with ALL-derived 60-mm marrow cytotoxicity cells was evaluated and complement-dependent Statistical (-s-). culture after 15 В± Concentration toxicity. marrow All 9 adult one-step enumeration by remaining Fig. 1 . Cytotoxicity of BA-2 for selected ALL-derived cell lines. The symbols designate the percent cell lysis calculated from the number of viable (Trypan-blue excluding) cells remaining following a one-step incubation with complement and antibody (in the increasing dilutions indicated). Cell lines tested included NALM-6-Ml (-#{149}--). HPB-Null (- x -). reh NALM-6-B (-A-). NALM-1 (-0-). Combined data of three experiments. quadruplicate with control, lysis determined cell excluding) complement. -6 5 data of determinations percent Specific 5В±6 1.3 В± 13 В± 8В±6 5В±1.7 1:25 tAs 6 4В±0.5 1:50 0 Percent ( + ) CвЂ™-Cytotoxicityt 0.7 В± BA-2 Cells Percent ( + ) Immunofluorescence -J ET AL. 34В±2.5 15 33.7 В± 3 8 31.3 В± 1.9 donor. in Materials and Methods. Dilutions listed refer to final concentrations of antibody and/or From www.bloodjournal.org by guest on December 22, 2014. For personal use only. ABSENCE OF ANTIGEN viability (and/or p24 ON HUMAN accomplishing poietic progenitors identifies antigens demonstrated by STEM 1313 CELLS cytolysis) Table of hemato- in the presence of an antibody that expressed on their cell surfaces is the nearly total absence of colony murine not by the reduction ofviable antigens after incubation IgG class shown). monoclonals, In 5 separate OKT-3 and 9.6 (mean percentage GEMM, BFU-E, ony formation, ment-incubated I :20-I inhibition of CFU-E, and CFU-GM determined with respect control, was zero at BA-2 : I 000). This contrasted with CFU-GEMM, 98% for BFU-E, 99% to obtain BA-2-depleted cell populations; these subjected to hematopoietic the results experiment Table with 4. Effect of W6/32 Rosette 2 <1 В± 7 <3 CFU-GM 18 В± 4 CFU-E 74В±6 population cells, celIs Percent rosetting Percent of total recovered so that Colonies/ 1 ment for BA-V immunofluorescence inhi- the and progenitor pellet. The pellet cells constituted only cell fractions cells of the enrich- was, however, demonstrated by analysis showing random distri- ofother T (OKT-3, 9.6) and B(BA-I) antigencells between interface and pellet compared to bution bearing significant (p 0.001 < ) concentration of BA-V cells in the pellet. Moreover, the appearance of small numbers of hematopoietic progenitor cells in the pellet BA-2 rosette of progenitors cell fractions following similar to the numbers sham following absence rosetting of antibody. which four all The with was pellet SPA-oRBC results progenitors separation in the in the of 5 experiments in (CFU-GEMM, BFU-E, 3 shows CFU-E, rosette SPA-oRBC CFU-GM) were assayed after BA-2 rosetting were confirmed by 6 additional experiments the microwell in which CFU-GM alone were cultured assay; all experiments demonstrated Separation and bulk of hematopoietic 2-depleted interface An additional on Normal Marrow progenitors cell fractions. experiment Hemat opoietic to be in the was performed, in the BA- using Progenitors Interface Pellet >90 >90 >90 25 45 15 separation 39 59 1BA-2 17 cells BFU-E 13.4 CFU-GM 20.3 CFU-E Experiment BA-V lW6/32 rosette of assayable 20.5% В± 4.6% of BA-2-enriched pelleted in 5 similar experiments. The specificity cells after the bulk contained a number of cells nonspecifiin the rosetting and/or sedimentation procedure, 1W6/32 1BA2 cells experiment. BA-V. to the BA-2-enriched cell population cally trapped CFU- (W6/32 1BA-2 by immunofluorescence was 4.5% contained compared Unseparated Percent representative marrow <3 12В±6 (data separation. The interface cell population was highly depleted of BA-V cells compared to the prerosetting marrow, with <0.5% residual BA-2 cells detected in a 200-cell count of interface cells postdepletion. As shown in Table 4, the BA-2-depleted interface cell Table Cells В± 8 SPA-ox RBC rosetting. and BA-2-enriched populations were then cell culture. of a representative 21.5 1 25 tPrerosetting 97% for CFU-GM derived colony formation at W6/32 dilution of I :500). In order to further confirm the apparent absence of binding of BA-2 to hematopoietic progenitors, rosette separation with BA-2 indirectly bound to ox RBCs was utilized marrow Pellet <0.5 BFU-E W6/32 and of 99% for for CFU/E, on Normal Progenitors CFU-GEMM derived colto compledilutions of the consistent bition of colony formation observed with complement (mean percentage inhibition Separation Colonies/ formed, the number of hematopoietic colonies of each class remaining after BA-2 and complement incubation was unaffected over a wide concentration of antibody Rosette Hematopoietic %BA-2 per- similarly of BA-2 Interface T cells bearing with two other experiments Effect Marrow formation by marrow after incubation with the antiHLA antibody W6/32 and complement; this antibody-complement treatment served as a positive control in each experiment with BA-2. In companion experiments, the specificity of cytoloysis was further demonstrated the respective 3. 17.9 cultured in microassay. CFU-GEMM numbers В± В± В± 5.3 4.5 4.3 are too low for detection 1W6/32 BA-2 1W6/32 1BA-2 fW6/32 1BA-2 in this system. 6.9 В± 1.1 8.6 В± 3.5 19.8 В± 4.0 1.3 В± 4.0 16.1 В± 3.5 16.7 В± 3.0 33.8 В± 5.0 4.0 В± 60.0 В± 10.6 20.3 74.8 В± 14.0 3.1 В± В± 1.0 6.3 1.0 the From www.bloodjournal.org by guest on December 22, 2014. For personal use only. ASH 1314 ET AL. microculture assay, as a positive control for the rosetting experiments. Because BA-V lymphoid cells might theoretically be inherently more rosettable than hematopoietic progenitor cells, we performed W6/32 roset- age in pediatric marrows2t), it does not appear to bind antigenic sites present on either the human pluripotential hemopoietic progenitor (the CFU-GEMM) or committed hemopoietic progenitors (BFU-E, CFU-E, ting to determine whether hematopoietic progenitors could indeed by positively selected by antibodydirected rosetting with an antibody that recognized and cells known antigens to be expressed on such CFU-GM). constitute very Because small hematopoietic progenitor minority populations within marrow and are not morphologically distinguishable from other small lymphoid cells, indirect methods such progenitor cells (Table 4). The overall efficiency of rosetting was less with W6/32 than with BA-2 (i.e., while >90% of marrow mononuclear cells were W6/3V by immuno- as those utilized here must be applied to this analysis. The use of antibody and complement-dependent cytotoxicity has been widely applied to similar studies,34 fluorescence, and our data to hemopoietic However, between rosetting only some 45% of these those progenitors pellet and interface in numbers roughly tion of cells that with the results rosetted. This obtained cells rosetted). assayed were distributed fractions after W6/32 equivalent to the propor- with rosetting, BA-2 showed no evidence of toxicity progenitors in conditions suitable for cytotoxicity contrast against the such cells. in which complement was in marked BA-2 effecting with with HLA-A,B,C There are an antibody (W6/32) locus presumably present recognized disadvantages cytotoxicity,34 however, including on of the the- essentially all BA-V cells are removed from the interface population, while the large majority of progenitors remain in the BA-2 interface cell population. oretical These results demonstrate that hematopoietic progenitors can be positively selected by rosetting with an antibody (such as W6/32) against antigens that they express, and strengthen our conclusion that hematopoietic progenitors do not express the p24 antigen. density. Moreover, failure of cytotoxicity may reflect inefficient complement fixation rather than absence of surface antigens. Therefore, we took pains to explore BA-2 binding by another method independent of complement-cytotoxicity. The experiments with BA-2 concern with monoclonal antibody BA-2 have demonstrated that this antibody binds a cell surface antigen (p24) expressed at an early stage of normal lymphoid than 50% of TdT cell development; indeed greater normal marrow cells expressed the p24 antigen.вЂ™3вЂ™28 The demonstration that BA-2 binds strongly to most non-T, non-B ALL and most preB-ALL suggests that BA-2 primarily defines a human lymphoid progenitor cells rosetting therefore cell/leukemia associated anti- The antigen p24, identified by BA-2, chemically communis, ity columns change after cm suggest chains are labeling with a lipophilic nitrene gests that, like the p24 is a nonintegral common ALL cell surface early of direct have system fore ship B-cell stages, shares differentiation. the entire a common During early lymphohematopoietic cellular origin,30вЂ™3вЂ™ it is of biologic importance to define of such early lymphoid developmental and there- early hemopoietic differentiation events. The availability of in vitro clonal assays for committed hemopoietic progenitors26вЂ™32вЂ™33 and the recent development of a system for identification of a human pluripotential hemopoietic progenitor6 now facilitate studies of this kind. Our binds cells adult studies demonstrate to a small within marrow normal population marrow mononuclear clearly that, while of lymphoid (approximately cells and a greater BA-2 percent- not of support our antigenic progenitors. sites reagent which is immuno- or 32P sug- antigen protein.35 been monoclonal this structure. carried (cALLA), Although out, other antibodies36вЂ™37 most Published data on cell binding specificities and molecular weight determinations of the antigens immunoprecipitated by these antibodies338 are similar to those observed with BA21328 (T. W. LeBien et al., unpublished data). The published report of nonreactivity of monoclonal DuALL-l with CFU-GM37 is also in agreement with our results with BA-2 in this study. It should also be noted that, while BA-2/p24 expression can conveniently be thought of as primarily defining cell/lymphoid leukemia cell progenitor 4%-5% comparisons recently described likely also recognize the relationmarkers to anti- in some detail. Its failure to bind to Ricinus Lens culinaris, and concanavalin-A affinand the absence of any molecular weight treatment with glycosidases or tunicamythat N-asparagine linked oligosaccharide not present on the antigen.35 Its lack of these leukemic cells presumably displayindicative of вЂњarrested developmentвЂќ at stages have interactions surface antigen the molecular structure has now been studied gen,вЂ™3вЂ™24 with ing structures developmental may further conclusion that BA-2 does not recognize present on normal marrow hemopoietic DISCUSSION studies progenitor gens inaccessible to antibody-complement or be resistant to lysis because of low SPA-oRBC Previous that expressed human variably platelets, on some neuroblastoma, a primitive phenotype, activated and lymphoid p24 is lymphocytes, some epithelial- From www.bloodjournal.org by guest on December 22, 2014. For personal use only. ABSENCE OF ANTIGEN derived neoplasmsвЂ™3 data). The demonstration expression to reports ON HUMAN (T. with in this antibodies, 1315 et al., unpublished study of absence differentiation The Whether the antibody is bation or coupled to a ricin,9вЂ™43 its potential for BA-V neoplasia seems of p24 and/or pattern from that have which CELLS progenitors is in contrast other antigenic structures lymphoid neoplasia.31вЂ™392 differs significantly (Ia) STEM W. LeBien on hematopoietic concerning certain associated phoid BA-2 p24 attractive as an agent for ex vivo marrow in autologous bone marrow transplantation. lym- of reactivity of of anti-H LA-DR been reported to react The cells GM.42 markers was may tion/neoplasias topoietic tivity found These examples associated with with tion on early determinant biologic selec- within by p24/ the implications cells of these that lymphocytic leukemia. The very appropriate complement cytotoxic nontoxic events BA-2 BA-2 might be in autolo- mixed colonies it seems after BA- unlikely are that affected by of BA-2 positive cells. The question of p24 is expressed at the level of the committed progenitor using recently human megakaryocyte taken cell (CFU-Mk) for hematopoietic antibody treatment. be important monoclonal of developed to examine techniques well after studies, for assay of have been under- progenitors,22 this might reconstitution Additional question. ACKNOWLEDGMENT can be potently We would at concentrations that are hematopoietic progenitors. applica- treatment treatment, cell lines and normal that, in the presence of an source, to leukemic cells to normal marrow results marrow cells clinical in megakaryocytopoiesis megakaryocyte is not found different nonlymphoid BA-2. as for CFU-GEMM-derived removal whether on normal hematopoietic progenitors suggests that this monoclonal antibody may be clinically useful as a selective immunotherapeutic agent for treatment of experiments with ALL-derived marrow clearly demonstrate instance, 2 and complement developmental expression of that BA-2 recognizes an anti- on neoplastic monoclonals for early to the basic on some in considering expression of p24 on human platelets and determination of the point in megakaryocyte-platelet differentiation at which p24 expression occurs. Inasmuch as we have observed usual numbers of megakaryocytes hema- demonstrated of p24 conditioning gous marrow transplant for neuroblastoma as well as for ALL. Also of potential clinical importance is the CFU- to the relative progenitors studies in defining p24, the demonstration genie be expressed in contrast for lymphoid with of such useful, suggest that developmental both B- and T-cell differentia- also progenitors, BA-2. In addition to be reactive expression may also be important CFU-GM,39 BFU-E,вЂ™#{176}and CFU-GEMM.4вЂ™ A recently described monoclonal antibody (R FB-1) produced against a T-lymphoblastic leukemia and reactive with T-lineage used with complement incutoxic effector molecule like selective immunotherapy of manifest, and is especially excellent like to thank technical preparation Maria assistance, McGinnis and and Bernetta Daniel R. Boue Kambeitz for help for with of the manuscript. REFERENCES I Kersey . J, Kung JH, mia D: Academic, origins 516:193, 1978. 3. Sallan Coral F, 4, Kersey Ritz JH, with Science TW, Miller Conference. and the Biophys R, OвЂ™Brien surface Acta C, Hitchcock antigens: lymphoblastic Hurwitz DR. Prognostic leukemia. 72:746, Blood 207:68, DM, Science 8. Scheinberg apy 42:44, 1982 9. Krolick kaemia gous bone ME, Gajl-Peczals- Leikin S: marrow of phenotypes and 12. B-cell prognoses. Med Nowinski antibodies RC: against Mouse a thymus leukemia differentiation 1980 land Maloney hybridoma 58:78, Z, Herlyn carcinoma MF, Koprowski in nude mice antibody. Cancer Res 40:717, 1980 7. Young WW Jr. Hakamori S: Therapy H: Inhibiby monoclonal lymphoma cell targeting model ES: and therCancer killing Implications Nature McKillop system. Selective conjugates: antibody 295:604, J, Levy in a patient SF: of leukemia Miller, RA, 306:517, with Res of leu- for autolo1982 R: In vivo T-cell effects leukemia. Maloney with Utilization and lymphoma. DG, Warnke monoclonal of monoclonal Blood R, Levy anti-idiotype antibodies 59:1 , 1982 R: Treatment antibody. of N EngI Kersey JH, LeBien M: p24: progenitor antibody. bone TW, Med Messner marrow, Abramson A human cell J Exp 14. Fauser AA, CS, Newman leukemia-associated surface structure 153:726, identified with mono- 1981 HA: Granuloerythropoietic peripheral R, Sutherand lympho- blood, and cord colonies blood. AA, Messner HA: Identification in Blood 1978 I 5. Fauser J 1982 R, Greaves 52:1243, of mouse of low antigenic 1981 lymphoma human Vitetta DG, J, Scholossman hemopoietic clonal JW, transplantation. RA, I I . Ritz Childhood in a mouse by antibody-toxin Miller of murine Blood Uhr Selection 1981 M: Leukemic antibody KA, cells to glycolipid: 21 1:487, DA, Strand by monoclonal 13. MR. Steplewski of colorectal PF, 1979 Tam monoclonal R, Nesbit Coccia Heterogeneity ID, antigen. Herlyn Cell acute LeBien D, Path of growth Leuke- antibodies in vivo. in the treatment therapy 6. J, Gelber SF: childhood 5, Bernstein tion Marker Biochim with monoclonal variants Cell W (ed): of gene expression leukaemias. J, Pesando leukemia-lymphoma: J Clin in Knapp G: Patterns human Schlossman in Hammond Am A, 10. SE, implications 55:395, 1980 ka, of 5, Bleyer p 453 MF, Janossy cellular P. Siegel of the Leukemia 1981, M, Jansen leukemia/lymphoma: heterogeneity, Proceedings 2. Greaves K, Nesbitt H, Coccia lymphoblastic phenotypic Markers, GajI-Peczalsha G, Sather Acute define London, S, TW, P, Goldstein Hammond markers LeBien of megakaryocytes, From www.bloodjournal.org by guest on December 22, 2014. For personal use only. ASH 1316 macrophages, and containing 16. Ash RC, hemopoietic 17. eosinophils granulocytes Detrick stem Parham antibody RA, cells Zanjani CJ, associated Studies Bodmer studies 58:309, Use ofa of HLA-A, with 30. J the E rosette J, Brown T-lymphocyte receptor. M, J Exp Siadak surface Med A, pro- Kung clonal PC, Goldstein antibodies Science Immunol CS, McEver of Growth JH, T cell surface LeBien cells TW: of human NL, platelet A monoclonal J membrane PW: Isolation glycoprotein hybridoma and deficient antibody. in McEver of pure RP, and McGinnis mixed human by immunofluorescent 23. Gronowicz l):lI7A, Lindquist D, Zanjani megakaryocyte labeling with colonies monoclonal A, Melchers Ig of a given type F: A plaque assay for all or class. Eur J Immunol 6:588, 24. Ash RC, pluripotential themia vera: 69:1112, 25. Detrick Direct Zanjani ED: progenitors evidence of stem In vitro studies (CFU-GEM M) cell involvement. of human in polycy- J Clin Invest Zanjani ED, colony formation dence on erythropoietin. Jansen J, effective stimulate granulocyte formation by Effect JD, J Clin in NN, normal Hoffman R, Wasserman by polychthemia Veenhof globulin 27. Iscove, Lutton the vera Invest 59:841, WFJ, Goselink treatment of precursor Senn and cells. Exp leukemic human media from Ery- Depen- mitted 29. Hematol does 9: 1028, McCulloch bone EA: marrow human leukocytes. FJ, LeBien TW: not 1981 Kersey JH, progenitor Bollum cells in human bone JH, Nakagawa 5, Mirato Immunol 64:192, Fay JW, Jones NH, Metzgar and MJ, Blood to Megzger RS: antigen common HE, RJ: (sub- C, HLA-A, K, Minowada monoclonal 155:949, 1982 by and a monoclonal leukemia APC, human of probable Delia D, Meyers bone PA, marrow pluripotential Edwards cells. DA, specific Ferrone for immature Youle across 289:68, 1981 stem MJ: from Neville DM, lethalGVHD anti-Thy-l.2 coupled Expression of hemato- hemopoietic Kersey histocompatibility by pretreatment to the toxin M: glycophorin SM, Janossy G: A human 1981 major Greaves multipotential I 27:2269, RJ, and 5, Cline on human J Immunol PAW, HLA-ABC, Nature CL, B antigens antibody ofmice BF: granulocyte1981 lymphoblastic Sheridan HLA-DR, Fevre transplantation Protection RS, Haynes Characterization Continuous differentiation. Vallera EL, glycoprotein 1981 defined acute Broxmeyer surface JH, and T lineage marrow Reinherz Res (in press) J, Sieff erythroid et 1980 of cell Fitchen JH, Biochim dalton monoclonal with Kersey JM, cells for publication) TW, Cell Pesando poietic progenitor cells. Blood 59: 188, 1982 42. Bodger MP, Francis GE, Delia D, Granger in Kersey BA-2. of a 26,000 characterization Robinson la-like TW, of leukemia-associated Colony Ontogenic marrow. T cells. Winchester 55:682, 1974 characteristic 305: 17, 1981 antibody LM, Borowitz Ia antigen of the chroma- 83:399, antigenic LeBien monoclonal a 24,000-dalton Leuk colony Analyses 1982 J: Expression MAS, N, Blood 43. CJ, of lymphoid LeBien Moore 41. Erythroid Physiol J Med 1976 Erythropoietic 1974 and affinity characterization by the elicited cells. during Anti-thymocyte anemia N, of Expression 37:1, 1971 Brashem Jones 40. and normal characterization of human bone marrow forming progenitor cells. Blood 58: 1047, Jacobsen 1977 aplastic JE, LR: marrow: HM: JS, Till of conditioned bone JL: of marrow: The DR. Nadler MN, distribution cells. N EngI T, human MJ: genetic Strominger 73:1288, KH: human Cline Ritz on activated cultures: Sci USA A. J Cell KA, 701:318, SF, 39. three surface by gel filtration Sutherland Schlossman (cALL) 1982 throid 26. RA, hemopoietic and antibod- 1970 RE, the F, Winterhalter cells. Acta 38. on Acad of mouse RA, p24 defined Hercend for 21:281, Humphreys Natl stem Biophys (Du-All-l) I 976 Proc Foon antigen Phenotypic macrophage antibody L, molecules M R: Biochemical 37. Levine 1981 E, Coutinho secreting M, JA, Newman 36. J Clin Chess monoclonal 1982 Chimerism Biol on agarose-concanavalin Fitchen 35. Dcv Sieber of hematopoietic anti- JG: mice. for erythropoietin tography lineage. Res 6:299, AD, Curtis JE, McCulloch EA: of human marrow. Blood 44:659, NN, requirement Mono- antigens. B lymphocyte Majerus a monoclonal B1ood58(Suppl Tab. SF: 1980 RC, identified studies cells. in cultures Greaves using Ash human Baenziger the lnvest66:131l, Schlossman 1981 thrombasthenia 22. Kersey with RP, quantitation 28. human Iscove 34. reactive 126:83, 21. distinctive EL, 1979 Abramson (BA-I) G, Reinberz defining 206:347, 20. body culture. Ia-like cell lines with Leuk Gilman SF, of 32. Tepperman colonies in cultures 33. 153:207, TG, Schlossman Distribution leukemic P. Hasen of a human BA-3. in tetraparental 31. monoclonal B, C antigens. lymphoma and Wegmann formation 19. cells BA-2, systems 1981 I981 ED: of human ies BA-I, of pluripotential Blood WF: J: Analysis marrow 1979 1979 18. Kamoun M, Martin Nowinski R: Identification tein ED: bone 53:1023, in vitro. in structural 123:342, of human Blood (CFU-GEMM) P, Barnstable (W6/32) Immunol in colonies and erythroblasts. ET AL. ricin. JH: barriers cells Bone V. of donor cells J Exp Med From www.bloodjournal.org by guest on December 22, 2014. For personal use only. 1982 60: 1310-1316 Normal human pluripotential and committed hematopoietic progenitors do not express the p24 antigen detected by monoclonal antibody BA-2: implications for immunotherapy of lymphocytic leukemia RC Ash, J Jansen, JH Kersey, TW LeBien and ED Zanjani Updated information and services can be found at: http://www.bloodjournal.org/content/60/6/1310.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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