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DNA-TR-90-66

Radiation-Induced Hemopoietic and Immune Dysfunction

Rainer Storb, M.D.Friedrich Schuening, M.D.Fred Hutchinson Cancer Research Center1124 Columbia StreetSeattle, WA 98104

June 1991

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Radiation-Induced Hemopoietic and Immune Dysfunction C - DNA 001-86-C-0 '92PE - 62715SH

6. AUTHOR(S) PR - RMTA - RJ

Rainer Storb, M.D. and Friedrich Schuening, M.D. WU - DHO11990

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Fred Hutchinson Cancer Research Center1124 Columbia StreetSeattle, WA 98104

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13. ABSTRACT (Maximum 200 words)

'"his work was aimed at investigating radiation-induced hemopoietic and immunedysfunction in the dog model. One project was to produce monoclonal antibo-dies directed against canine hemopoietic precursor cells and select for plu-ripotent stem cells. Antibodies obtained reacted with different myeloid anderythroid precursor cells; unfortunately, none of the antibodies were specif-ic for pluripotent hemopoietic stem cells. Positive selections for thesecells were performed using magnetic beads and an antibody against class IT-

antigen. Transplantation of class II positive marrow cells into otherwise le-thally irradiated dogs led to sustained recovery in only 1 of 5 dogs.

A second project established and investigated long-term marrow cultures inthe dog. Culture conditions were studied and optimized, and marrow cells weretransplanted into otherwisz lethally irradiated dogs to investigate stem cellsurvival in long-term cultures. Engraftment was observed only with short-termmarronw cultur,:!.

14. SUBJECT TERMS 1I' NU&CACQ " nim'AtFq

Pluripotent Stein Cell TBI 60

Hemopoietic Growth Factors Monoclonal Antibody 16. PRICE CODE

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296-102

PREFACE

Dogs are provided by the Center's canine breeding program and bybreeding facilities licensed by the U.S. Department of Agriculturewho have contracted to provide pedigreed dogs for research purposes.Dogs are housed in individual cages or runs according to the spacerecommendations of the "Guide for the Care and Use of LaboratoryAnimals." The Center's facility is accredited by the AmericanAssociation for Accreditation of Laboratory Animal Care and is aU.S.D.A. registered research facility (#91-25). Animal facilities arecleaned by trained personnel on a daily basis. Animals are fed oncedaily and have continuous access to fresh, uncontaminated drinkingwater. All investigators are physicians or veterinarians and theyare assisted by a chief animal technician who has been working inthe canine research program for 25 years and has extensiveexperience in animal care and husbandry. All experimental dogs areseen twice daily by one of these individuals. Separate clinicalcharts are maintained on each dog. All animals that dieunexpectedly or that are humanely euthanized undergo careful autopsywith subsequent review of histological specimens by trainedpathologists. Carcasses are incinerated. Experimental protocolscovering all the procedures performed are'discussed at regular staffmeetings and, after being approved by the principal investigator andsenior staff members, are submitted to the Institutional Animal Careand Use Committee for approval. All research activities andprocedures described above are conducted according to the principlesenunciated in the "Guide for the Care and Use of Laboratory Animals"prepared by the Institute of Laboratory Animal Resources, NationalResearch Council.

I.,.

iii

TABLE OF CONTENTS

Section Page

Preface ....... .... ...................... ii

Conv2rsion Table ...... .................. iv

List of Illustrations ..... ................ vii

List of Tables ....... ................... ix

1 Monoclonal Antibodies Directed AgainstCanine Hemopoietic Precursor Cells . ......... 1

1.1 Monoclonal antibody 16-151-S3, recognizingmature myeloid and erythroid caninehematopoietic cells as well as CFU-GM but notpluripotent stem cells .... .............. 1

1.2 Monoclonal antibodies recognizing mature andimmature myeloid canine hemopoietic cells butnot mature canine lymphocytes ..... ......... 2

1.3 Radioimmune precipitation of DM5 antigens . . . 3

2 Positive Selection for Canine Marrow Stem CellsUsing Separation with Magnetic Beads .. ... ........ 5

3 Long-term Culture of Canine Bone Marrow Cells . . . . 3

3.1 Comparison of long-temn marrow culturesestablished with identical culture conditions 8

3.2 Comparison of different culture temperatures 8

3.3 Comparison of different culture media and sera 10

3.4 Comparison of different amounts of hydro-cortison ....... ................... 12

3.5 Comparison of different amounts of marrow cellsused for recharging long-term marrow cultures 12

3.6 Comparison of recharging long-term marrowcultures with autologous or allogeneic DLA-nonidentical marrow cells ... ........... 12

3.7 Ultrastructural analysis of canine long-termmarrow cultures ..... ................ 16

3.8 Autologous transplants of marrow cells culturedin canine long-term marrow cultures ...... 16

v

LIST OF ILLUSTRATIONS

Figure Page

1 Cumulative weekly CFU-GM in the nonadherent cellfraction of canine long-term marrow culturesrecharged with autologous or allogeneic DLA-nonidentical marrow cells after 1, 2, or 3 weeks 15

2 Effect of recombinant human IL-3 on CFU-GMproduction in long-term marrow cultures ....... .. 20

3 Effect of recombinant human G-CSF on CFU-GM productionin long-term marrow cultures ... ............ 21

4 Effect of recombinant human GM-CSF on CFU-GMproduction in long-term marrow cultures ....... .. 22

5 Effect of recombinant canine G-CSF on CFU-GMproduction in long-term marrow cultures ....... .. 23

6 Stimulation of canine CFU-GM colony formation byrecombinant human IL-3 ..... ............... 27

i Effect of continuous IV infusion of recombinant humanIL-3 on peripheral blood neutrophil counts of-fivenormal dogs ........ ..................... 28

8 Effect of continuous IV infusion of recombinant humanIL-3 on the platelets of five normal dogs ...... . 29

9 Effect of recombinant human GM-CSF on canine CFU-GMcolony formation ...... .................. 32

10 Effect of continuous IV infusion of recombinant humanGM-CSF on peripheral blood of four normal dogs . . . 34

11 Effect of intermittent SQ injections of recombinanthuman GM-CSF on peripheral blood counts of two normaldogs ......... ........................ 36

12 Effect of twice-daily SQ injections of recombinanthuman G-CSF on peripheral blood counts of two normaldogs ......... ........................ 38

13 Peripheral blood neutrophil changes in dogs after400 cGy of TBI ....... ................... 39

14 Peripheral blood neutrophil changes in dogs after400 cGy TBI and recombinant human G-CSF from day 0to day 20 ........ ..................... 42

vii

LIST OF ThfLES

Table Page

1 Comparison of canine long-term marrow culturesestablished at identical culture conditions .....

2 Comparison of two different culture temperatures incanine long-term marrow culture ...... ........... 10

3 Comparison of different culture media in caninelong-term marrow culture .... .............. 11

4 Comparison of different amounts of hydrocortisone incanine long-term marrow culture .. ........... . 13

5 Comparison of different amounts of marrow cells usedfor recharging canine long-term marrow cultures . . . 16

6 Stimulation of canine CFU-GM colony formation byrhIL-3 ......... ....................... 26

7 Bone marrow hisotology before and after two weeks ofcontinuous intravenous infusion of recombinant humanIL-3 .......... ........................ 30

a Stimulation of canine CFU-GM colony formation byrhGM-CSF . . . . . . . . . . . . . . . . . . . . . . 33

9 Survival of dogs after 4 Gy of TBI without or withrhG-CSF treatment starting either immediately orseven days after TBI ..... ................ 40

10 Recovery of peripheral blood counts after TBI andtransplantation of marrow from DLA-identical littermatewithout and with subsequent rhG-CSF .......... . 45

11 Mortality, GVHD, lethal GVHD and graft failure afterTBI and transplantation of marrow from DLA-identicallittermate without and with subsequent rhG-CSF . . . 47

ix

SECTION 1

MONOCLONAL ANTIBODIES DIRECTED AGAINST CANINEHFMOPOIETTC PRECURSOR CELLS

1.1 MONOCLONAL ANTIBODY 16-151-S3, RECOGNIZING MATURE MYELOID ANDERYTHROID CANINE HEMATOPOIETIC CELLS AS WELL AS CFU-G I BUT NOTPLURIPOTENT STEM CELLS.

Generation of monoclonal antibody 16-151-S3 followed theprocedure initially developed by Koehler and Milstein (Nature 1975,256: 495) and modified by Galfre and Milstein (Meth for Enzymol1981, 73:1). Marrow cells were aspirated trom both humeri of theirradiated dogs six days after TBI (total dose 9.2 Gy, dose rate0.07 Gy per minute) without subsequent marrow transplant. Formarrow aspiration, dogs were anesthetized by intravenous injectionof sodium pentobarbital at 24 mg/kg. The aspiration sites on bothhumeri of the dogs were shaved and aseptically cleansed with apovidone-iodine scrub and an alcohol rinse. Marrow cells obtainedsix days, after TBI were used as antigen for the immunization ofmice. At this time after TBI, most hemopoietic cells are destroyedexcept for a small number of radiation-resistant cells, some ofwhich are thought to play a role in marrow graft rejection.Originally, we planned to use this antigen for fusion in order toobtain monoclonal antibodies against cells mediating graftrejection. One of the clones derived from this fusion, 16-151-S3,produced monoclonal antibodies which reacted strongly (98%) withgranulocytes as shown by immuiofluorescence analysis with the cellsorter FACS-440. Monoclonal antibodies of clcne 16-151-S3 alsobound strongly to marrow cells (54%) and monocytes (59%) but not tolymph node lymphocytes (0%). Determination of antibody class byimmunodiffusion (Ouchterlony) technique showed this antibody to beIgG2,. To further determine which type of marrow cells monocionalantibody 16-151-S3 was reacting with, we sorted marrow cells intomonoclonal antibody positive and neqative cells using the FACS-440cell sorter. Cytospins of the positively sorted marrow cells showed87% neutrophils, 7% myelocytes, 2% promyelocytes, 1% nyeloblasts, 1%pronormoblasts, 2% normoblasts, and no lymphocytes, whereascytospins of the antibody-negative marrow cells contained 18%lymphocytes, 4% normoblasts, and 78% erythrocytes. When positivelyand negatively sorted marrow cells were cultured in CFU-GM assay,only monoclonal antibody positive marrow cells showed growth ofCFU-GM colonies.

We then investigated whether monoclunal antibody 16-151-S3 reactswith pluripotent hematopcietic stem cells. This was done bytreating marrow cells in vitro with monoclonal antibody and rabbitcomplement before infusion into lethally irradiated dogs (9.2 Gytotal dose, dose rate 0.07 Gy per minute). U studied whetherin vitro treatment interfered with the ability of autob gous marrowto protect dogs against radiation-indaced marrow apldsia. Two dogs

fractions using the FACS-440. Unsorted, monoclonal antibody-positive and monoclonal antibody-negative marrow cells were thenassayed for CFU-GM colony growth. DM5-positive marrow cells did notproduce CFU-GM colonies, whereas DM6 and DM10-positive marrow cellsproduced CFU-GM colonies. DM7- and DM8-positive marrow cells aswell did not produce any CFU-GM colonies.

We then investigated whether one of th-e antibodies, DM5,recognizes canine pluripotent stem cells. This was done by treatingmarrow cells in vitro with DM5 antibody and rabbit complement beforeinfusion into lethally irradiated (920 cGy) marrow donors. Two dogswere entered into this experiment and received 6 and 8 x 107 marrowcells, respectively, treated with DM5 and complement. In bothexperiments no DM5-positive marrow cells could be detected by FACSanalysis after treatment with antibody and complement. Recovery ofwhite blood cells an1 platelet counts was comparable to the recoveryin control dogs re iving untreated autologous marrow transplants.We concluded from these experiments that DM5 seems not to react withcanine pluripotent stem cells. As DM5 binds to 33% of marrow cells,but not to pluripotent stem cells, it may be useful to pre-enrichfor stem cells by treating marrow with antibody a:.1 complement.

1.3 RADIOIMMUNE PRECIPITATION OF DM5 ANTIGENS.

After SDS-PAGE under nonreducing conditions, three bands wereseen in the DM5 immunoprecipitate of marrow cells with apparentmolecular weights of 19, 21 and 23 kD. Under reducing conditions,an additional small molecular weight band was observed that migratedbeyond the 14.3 kD molecular weight. Occasionally a faint band of16-17 kD was seen under reducing conditions. To address thepossibility that the small molecilar weight component represented adisulfide-) inked polypeptide which associated, in a nonspecificmanner, witi gp 1 9 ,27 ,2 3 Dm at the time of cell lysis, cells werelabeled and incubated for 10 minutes in 10 mM Iodacetamide (Sigma)in PBS prior to detergent solubilization. Results identical tothose obtained under reducing conditions were obtained when thisalkylating aaent was used to prevent artifactual disulfideassociations that can occur during cell solubilization. When DM5RIPs were analyzed by 2-dimensional PAGE under reducing conditions,gp 19,21,23"5 all migrated at a pI of 5.9, and a small molecularweight fragment had a pI of 6.2. This observation suggests that thethree larger components remained associated during detergentsolubilization of marrow cells and during the first isolectricfocusing dimension of 2D-PAGE as well, but were separated only bySDS denaturation. Alternatively, the three freely solublecomponents may possess a high degree of structural homology, butdiffer in the relative contribution to apparent molecular mass ofuncharged substituents, such as neutral oligosaccharides. Todetermine if the three molecules (19, 21, and 23 kD) wereglycosylation variants of each other, N-Glycanase digestion wasperformed on immunoaffinity isolated molecules. N-Glycanasecompletely removes N-linked oligosaccharides, including high mannoseclains from glycoproteins. After overnight digestion, all threespecies resolved to a single 15 kD band This result suggests that

3

SECTION 2

POSITIVE SELECTION FOR CANINE MARROW STEM CELLSUSING SEPARATION WITH MAGNETIC BEADS

As described previously (Blood 69:165-172, 1987), we have shown thatcanine pluripotent hemopoietic stem cells express a class II antigenas recognized by the antibody 7.2. We have demonstrated that7.2-positive marrow cells, separated with FACS, can rescue otherwiselethally irradiated marrow donors. As the positive sort of a marrowtransplant with FACS is very time-consuming and expensive, we wereinterested to find out whether we can use the magnetic beadtechnique for positively sorting marrow transplants. We firstestablished in vitro the positive separation method using magneticbeads coated with sheep antimouse antibody (Dyna beads M-450) and aneodymium-iron-boron magnet (Magnet Development Ltd, Swindon, Wales,England). We used the anti-class II antibody 7.2 for positive bonemarrow separation, which we have shown to be expressed on caninepluripotent hemopoietic stem cells. All separation steps were doneon ice or at 40C. Bone marrow cells were first separated on aFicoll-Hypaque gradient, washed, and counted. They were thenincubated with the anti-class II antibody 7.2 for 20 minutes, washedtwice with RPMI containing 10 percent fetal calf serum. To test howmany cells were viable and how many viable cells were rosette-forming, i.e., binding three or more beads per cell, approximately25,000 to 50,000 cells were pipetted into a well of a round-bottommicroplate. Excess number of beads (10-20 times the number ofcells) were added. The cell bead mixture was spun down in a coldcentrifuge for one minute at 1200 rpm, and afterwards resuspended byadding one or two droplets of ethidium bromide acridine orangesolution to the cell bead mixture. The suspension was then placedin a Burker hemocytometer and counted in a fluorescence microscopeto determine the relative number of rosette-forming cells.Rosette-forming cells were defined as cells which have bound threeor more beads. Live cells stained green and dead cells stained red.Approximately 1000 cells were counted to obtain a fairly accuraterelative number of rosette-forming cells. After the relative numberof rosette-forming cells had been established, the amount of beadsneeded was calculated according to the formula: number of totalmononuclear cells x percent of rosette-forming cells equals thenumber of target cells. For example, if a ratio of beads to targetcells of 3:1 was wanted, beads at the amount of 3 times the numberof target cells needed to be added to the marrow cells, which wereset up at a concentration of 40,000,000 - 60,000,000 per ml. Afterhaving added the beads to the cell suspension, the bead/cell mixturewas centrifuged for one minute at 1200 rpm. After centrifugation,the volume was increased by adding 3-5 cc of RPMI containing 10%fetal calf serum and DNAse. The cell suspension was carefully mixedand the tube containing the cell/bead mixture was then laid sidewayson the magnet. After two to three minutes, the tube and the magnetwere turned around so that the magnet was now on top of the tube.The unattached supernatant containing the negative population wasaspirated carefully. The remaining positive cell population wasthen resuspended gently and the number of cells binding three ormore beads counted. This separation step with the magnet was

5

The numbers of 7.2-positive nucleated marrow cells infused into thethree dogs after TBI were between 0.03 and 0.06 x 108 mononuclearcells/kg body weight. These numbers were 5-10 times less than theone given to the dog who showed sustained and complete hemopoieticrecovery. The low cell number transplanted may explain why we didnot see any signs of hemopoietic recovery in these last three dogs.The low number of cells transplanted was due to the low amount ofcells obtained at the time of marrow aspiration and to the lownumber of cells recovered after cell separation with the magneticbeads. It therefore seems necessary to further improve the numberof cells originally aspirated, as well as the number of cellsrecovered after cell separation with the magnetic beads.

7

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Figure 1. Cumulative weekly CFU-GM in the nonadherent cell fraction ofcanine long-term marrow cultures recharged with autologous orallogeneic DLA-nonidentical marrow cells after 1, 2, or 3 weeks.Cultures were started by incubating 6 x 10' MNC from the samemarrow aspiration in 75 cm 2 canted-neck flasks at 2 x 106MNC/ml in RPMI-1640 medium supplemented with 20%prescreened heat-inactivated horse serum. Flasks were eithernot recharged (*) or boosted after 1, 2, or 3 weeks with 2 x 10'MNC derived either from the same donor as used for establishingthe stromal cell layer (0, 1-week boost; A, 2-week boost; and 0,3-week boost) or from a DLA-nonidentical unrelated donor(6, 1-week boost; A, 2-week boost; and U, 3-week boost). Atweekly intervals nonadherent cells were plated in triplicate inCFU-GM assay. Each point represents the cumulative number ofweekly CFU-GM.

need to be autologous compared to the cells of the adherent layer orwhether they can be allogeneic DLA-nonidentical. Cultures werestarted by incubating 6 x 107 MNC from the same marrow aspiration inculture flasks. Flasks were either not recharged or boosted after1, 2, or 3 weeks with 2 x 10

7 MNC derived either from the same donorused for establishing the stromal cell layer or from a DLA-nonidentical unrelated donor. Results are shown in Figure 1. Whencultures were recharged one week after establishing the stromallayer, the flask recharged with autologous marrow showed a highersum of weekly CFU-GM counts in the nonadherent cell fractioncompared to the flask boosted with DLA-nonidentical marrow (at week20, the week before the first flask became contaminated, 796,337

15

long-term marrow culture seem to lose their ability to protect dogsagainst irradiation induced marrow aplasia. This finding was incontrast to our previous finding that we could grow CFU-GM coloniesfrom long-term cultures for up to 31 weeks. Most probablypluripotent hemopoietic stem cells seem to lose their ability forself-renewal rather quickly under the present long-term cultureconditions by developing into committed precursor cells. On theother side, these precursor cells, though committed, were able toproduce CFU-GM for tup to eight months.

To further clarify the contrasting results of our in vitro and invivo studies, we then addressed the question whether failure ofengraftment of autologous marrow cells cultured for more than sixdays might be due to the lack of accessory cells which may notsurvive in long-term marrow cultures. For instance, it has beendescribed that lymphocytes are rapidly lost in long-term marrowcultures (Mauch et al., Blood 63:112, 1984). We therefore usedautologous lymph node lymphocytes as possible source of accessorycells. Marrow cells were cultured in long-term culture for eightdays, harvested and infused into the lethally irradiated marrowdonor together with autologous lymph node lymphocytes. Tiue numberof cultured mononuclear marrow cells infused was 17 x 106 per kg bodyweight and the number of lymph node lymphocytes was 6 x 106 per kgbody weight. The dog never showed any signs of engraftment and diedon day 13 after transplantation due to sepsis. A second dogreceived as well marrow cells cultured for eight days in long-termmarrow culture, but in this experiment stem cell-free peripheralblood lymphocytes were used as possible source of accessory cellsassuming that this would be more physiological. To obtain peripheralblood lymphocytes depleted of hemopoietic stem cells, we firstseparated blood cells by density gradient centrifugation overFicoll-Hypaque. Interface cells were then treated with hemolyticbuffer (0.155 M per 1 of ammonium chloride) and separated by adiscontinuous albumin density gradient as described by Dicke(Transplantation 6:562, 1968). The peripheral blood leukocytes fromfraction five of albumin density gradient are depleted of hemo-poietic stem cells insofar as they failed to achieve hemopoieticreconstitution after lethally irradiation (Schuening et al., Blood69:165, 1987). Marrow cells were cultured in long-term cultures foreight days, harvested, and then infused into the lethally irradiatedmarrow donor together with autologous peripheral blood lymphocytesfrom fraction five of the albumin density gradient. The number ofcultured mononuclear marrow cells infused was 76 x 106 per kg bodyweight and the number of PBL from fraction five was 9 x 106 per kgbody weight. The marrow cell number transplanted always results insustained engraftment when using fresh autologous marrow cells.However, similar to the previous experiment where we gave autologouslymph node cells in addition to marrow cells cultured for eightdays, this dog never showed any signs of engraftment as well anddied on day 12 due to marrow aplasia. This result confirmed ourprevious observation and showed that failure of engraftment usingmarrow cells cultured for more than six days in long-term marrowculture is probably due to insufficient numbers of pluripotenthemopoietic stem cells rather than to lack of accessory cells.

18

In summary, using the current culture conditions, we obtainedsustained engraftment of marrow cells cultured in long-term marrowculture in one of one dog receiving marrow cells cultured for fourdays and in three of five dogs transplanted with autologous marrowwhich had been cultured for six days in long-term marrow culture.These results are inferior to those obtained in mouse and man. Inmice, sustained marrow constitution has been seen after transplanta-tion of marrow cultured in long-term marrow culture for three weeks(Transplantation 35:624, 1983). Patients receiving marrow which hasbeen kept in long-term culture for ten days have shown successfulengraftment as well (Lancet 1:294, 1986). Our current results indogs need to be improved. We therefore started to investigatewhether the addition of hemopoietic growth factors to long-termmarrow cultures improve the transplantation results.

3.9 EFFECT OF RECOMBINANT HEMOPOIETIC GROWTH FACTORS ON CANINELONG-TERM MARROW CULTURES.

The effect of recombinant hemopoietic growth factors on caninelong-term marrow cultures was tested using the following experi-mental setting. Identical 75 cm2 tissue cultures flasks wereinoculated with 60 x 106 bone marrow mononuclear cells in 30cc mediaand boosted with 60 x 106 marrow buffy coat mononuclear cells oneweek later. Fifty percent of the culture media was replaced weeklyand growth factors of interest were also added at weekly intervals.A CFU-GM assay was performed weekly on nonadherent cells. Inrepeated long-term cultures with marrow obtained from differentnormal beagles, we observed a stimulatory effect with recombinanthuman IL-3 on CFU-GM production in a dose-dependent fashion withoutevidence of stem cell exhaustion over an 8-week interval (Figure 2).This is consistent with the hypothesis that IL-3 affects a multi-potential hemopoietic precursor cell capable of at least limitedself-renewal.

The effect of recombinant human G-CSF has been variable. At 10-'gram per ml, we have inconsistently observed a stimulatory effect onCFU-GM production. However, at doses of 10.8 and 106 gram per ml, asignificant negative effect on CFU-GM production was clearly evident(Figure 3). This suggests that G-CSF at high doses causesdifferentiation of committed progenitor cells to beyond the CFU-GMstage. At low doses, some effect on early progenitors towardself-renewal, or limited differentiation is probable.

The effect of recombinant human GM-CSF has paralleled that observedwith recombinant human G-CSF. At 1010 gram per ml, markedstimulation was seen in CFU-GM production (Figure 4). However,doses of 104 and 106 gram per ml induced marked inhibition of colonyproduction. Results obtained with recombinant canine G-CSF weresimilar to the results obtained with recombinant human G-CSF. Wefound an approximately 60% cumulative increase in CFU-GM productionover 8 weeks in flasks recharged weekly with recombinant canineG-CSF at doses of 1012 and 10' g/ml compared to controls (Figure 5).However, there was a markedly reduced cumulative CFU-GM productionin the flasks receiving the higher doses of recombinant canine G-CSF

19

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21

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Figure 5. Cumulative weekly CFU-GM in the nonadherent cell fraction of caninelong-term marrow cultures recharged with autologous marrow cellsafter 1 week and fed weekly with recombinant canine G-CSF at10 .6 g/ml (6-& ), 10-8g/ml (v-v), 10-'Og/ml n_).Control was fed withmedium only (0-0), and 10 2g/ml (0-0).

23

SECTION 4

STIMULATION OF CANINE HEMATOPOIESIS BY RECOMBINANT HUMAN IL-3

4.1 STIMULATION OF CANINE CFU-GM COLONY FORMATION BY RECOMBINANTHUMAN IL-3.

Recombinant human IL-3 (rhIL-3) was added to CFU-GM cultures atdifferent concentrations. Culture plates without any addition ofgrowth factor served as negative controls and plates with 10%postendotoxin dog serum as positive controls. In five separateexperiments, an up to 29-fold stimulation of canine CFU-GM colonyformation by rhIL-3 was observed compared to the negative controls(Figure 6). In a separate experiment, rhIL-3 was either incubatedfor 30 minutes with anti-rhIL-3 antiserum (diluted 1:200) or heat-inactivated at 100 0C for 30 minutes before adding the growth factorto the culture plates. The unmanipulated rhIL-3 stimulated CFU-GMcolony formation up to seven-fold compared to the negative controls.rhIL-3 treated with either anti-rhIL-3 antiserum or heat-inactivation did not show any stimulatory activity compared to thecolony growth of the negative control (Table 6). Anti-rhIL-3antiserum by itself, did not have any effect on CFU-GM colonygrowth. These results are consistent with the notion that rhIL-3specifically interacts with canine CFU-GM.

4.2 STIMULATION OF HEMATOPOIESIS IN DOGS BY CONTINUOUS INTRAVENOUSINFUSION OF RhIL-3.

Five dogs were treated with 30, 60 (two dogs) 120 and 240 4grhIL-3 per kg/day given as continuous intravenous infusion for 14days. rhIL-3 was well tolerated and no systemic toxicity wasobserved over the dose range studied. No stimulation ofhematopoiesis was seen at the dose of 30 Mg/kg per day (Figure 7).At 60, 120, and 240 gg/kg per day, neutrophil counts reached levelsby day 14 which were two times higher than the counts of two controldogs receiving either 60 gg heat-inactivated rhIL-3 per kg/day for14 days (range indicated by two horizontal lines) or 10 Mg humanserum albumin per kg/day. After discontinuation of rhIL-3,neutrophil counts remained increased for 10 to 14 days beforereturning to the counts of the control dogs. Peripheral monocyteswere increased up to three-fold. No significant changes wereobserved in eosinophil, lymphocyte, reticulocyte or hematocritlevels. The effect of rhIL-3 on platelets is shown in Figure 8. At30 and 60 gg rhIL-3 per kg/day, the platelet counts did not changecompared to the control dog receiving 60 gg heat-inactivated rhIL-3per kg/day (range indicated by two horizontal lines). The dogreceiving 120 Mg/kg per day showed a slight increase of plateletsabove the upper limit of the control seven days after thediscontinuation of rhIL-3. At a dose of 240 Mg rhIL-3 per kg/day,platelet counts were increased two-fold above the control five daysafter stopping the rhIL-3.

Marrow biopsies were taken before and after 14 days of rhIL-3infusion and slides were evaluated without knowledge of treatment

25

225

200

175 4

Z i5o

c)125 4

.00

_ 75-4

2510

L. 0 0 0 0 0 0U, 0 0 0 0 0 Wi(n, 0 0 0 0 U10 0 0 0 CL

C 0 0 W

Figure 6. Effect of recombinant human IL-3 (rhIL-E) on canine CFU-GM colorwformation. 3 x 10' mononuclear marrow cells (MNC) depleted ofmonocytes and T-lymphocytes have been cultured per plate for 10

days in agar medium without and with addition of rhlL-3 atdecreasing concentrations. Cultures were plated in triplicate. Datarepresent mean value ± standard deviation of five separateexperiments.

27

PLAT COUNTS IL-3 * 30 /kg/d y

.6 q/ g/doy

900000- -- - A 6 0q/.g/day

o 1 2 0pq/kcg/day800000 240og/kg/doy

700000'I

600000'E

400000 /j ooooo ,._.

1 00000"

0 7 14 21 28

Day

F'gure 3. Effect of continuous (14-day) intravenous infusion of rhlL-3 given at 30,60, 190 or 240 pg/kgidav on the peripheral blood platelets )f fivenormal dogs. The two horizontal lines indicate the range ot peripteralblood counts of a control dog given 60 pg heat-inactivated (!00 "C 30'rhlL-3 per kg/day for 14 days.

29

SECTION 5

STIMULATION OF CANINE HEMATOPOIECIS BY RECOMBINANTGRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR

5.1 STIMULATION OF CANINE CFU-GM COLONY FORMATION BY RECOMBINANTHUMAN GM-CSF.

Recombinant human GM-CSF (rhGM-CSF) was added to the CFU-GMcultures at different concentrations. Culture plates without anyaddition of growth factor served as negative control and plates with10% postendotoxin dog serum as positive control. In two separateexperiments, an up to tenfold stimulation of canine CFU-GM colonyformation by rhGM-CSF was observed compared to the negative control(Figure 9). In a separate experiment, rhGM-CSF was either incubatedfor 30 minutes with anti-rhGM-CSF antiserum (diluted 1:100) orheat-inactivated at 100 0C for 30 minutes before adding the growthfactor to the culture plates. The unmanipulated rhGM-CSF stimulatedCFU-GM colony formation up to fourfold compared to the negativecontrol. rhGM-CSF treated with either anti-rhGM-CSF antiserum orheat-inactivation did not show any stimulatory activity above thecolony growth in the negative control (Table 8). Anti-rhGM-CSFantiserum by itself did not have any effect on CFU-GM colony growth.The results are consistent with the notion that rhGM-CSFspecifically interacts with canine CFU-GM.

5.2 STIMULATION OF HEMATOPOIESIS IN DOGS BY CONTINUOUS IV INFUSIONOF RhGM-CSF.

Three dogs were treated with 10, 30, or 90 Ag rhGM-CSF perkg/day by continuous IV infusion for 14 days. RhGM-CSF weretolerated and no systemic toxicity was observed over the dose rangestudied. Dogs showed increased neutrophil counts within 24 hours ofstarting the infusion (Figure 10a). By day 14, neutrophil countsreached levels that were three to six times higher than thepreinfusion counts and those of two control dogs receiving eithercontinous IV infusion of 10 Ag human serum albumin per kg/day for 14days (range indicated by two horizontal lines) or 30 Mg heat-inactivated rhGM-CSF per kg/day. The marrows in rhGM-CS'-treateddogs on day 14 were hypercellular with myeloid hyperplasia andleft-shifted granulocytopoiesis. After discontinuation of rhGM-CSF,the neutrophil counts returned to control levels within 3 to 7 days.Whereas neutrophil counts increased at all three dosages of rhGM-CSFto about the same extent, monocyte and lymphocyte counts weremarkedly increased (eight and two- to fourfold, respectively) onlyat the two higher rhGM-CSF dosages (Figures 10b and c). Eosinophiland reticulocyte counts and hematocrits during rhGM-CSF infusionwere similar to the preinfusion levels and the levels in the controldogs receiving human serum albumin or heat-inactivated rhGM-'SF.

The three dogs receiving active rhGM-CSF showed declines in plateletcounts with nadirs ranging from 5,000 to 15,000/mm3 (Figure 10d).After discontinuing the infusion of rhGM-CSF, platelet counts

31

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+1 +14-

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-4a-

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increased rapidly above preinfusion levels. By comparison, theplatelet counts of a saline control remained virtually unchanged,whereas the counts of a human serum albumin control decreased after10 days of infusion to about 100,000/mm3. The control dog receivingheat-inactivated rhGM-CSF also had a decrease of platelets to about100,000/mm3, but this decrease occurred 4 days after rhGM-CSFinfusion. Bone marrow histology on day 14 of rhGM-CSF infusionshowed normal number and morphology of megakaryocytes. Preliminaryresults of platelet survival studies using Cr-labeled autologousplatelets suggest that platelet survival is decreased to 50% ofnormal in the first week after starting rhGM-CSF infusion comparedto normal survival times in dogs given saline or human serumalbumin.

5.3 STIMULATION OF HEMATOPOIESIS IN DOGS BY INTERMITTENTSUBCUTANEOUS INJECTION OF RhGM-CSF.

One dog was treated with 30 Ag rhGM-CSF per kg/day and two dogsreceived 180 g rhGM-CSF per kg/day by intermittent subcutaneousinjections three times a day for 14 days. The subcutaneousadministration of rhGM-CSF was well-tolerated and no systemictoxicity was observed over the dose range studied. rhGM-CSF at thedose of 30 Mg/kg per day SQ increased the neutrophil counts twofoldcompared to the preinfusion counts and those of a control dogreceiving 10 gg human serum albumin per kg/day by continuous IVinfusion for 14 days (range indicated by two horizontal lines)(Figure Ila). Monocyte and lymphocyte counts were not increased(Figure llb and c). A SQ dose of 180 Mg rhGM-CSF per kg/dayincreased the number of neutrophils fivefold above control (Figurella), and monocyte and lymphocyte counts were increased fivefold andfourfold, respectively (Figure llb and c). Compared to resultsobtained by continous IV infusion of 30 Mg rhGM-CSF per kg/day, theintermittent SQ injection of the same amount of rhGM-CSF seemed tobe less effective in stimulating peripheral blood counts (Figurella-c). Platelet counts decrease to only about 100,000/mm 3 when30 Mg/kg per day of rhGM-CSF was given SQ (Figure l1d). With a doseof 180 Mg/kg per day, platelets declined to 10,000/mm 3 similar towhat was seen with continuous IV infusion of 30 Mg/kg per day.

35

SECTION 6

STIMULATION OF CANINE HEMATOPOIESIS BY RECOMBINANT HUMANGRANULOCYTE COLONY-STIMULATING FACTOR

6.1 EFFECTS OF RECOMBINANT HUMAN G-CSF IN NORMAL DOGS.

Two dogs were treated with either 10 or 100 Ag recombinanthuman G-CSF (rhG-CSF)/kg per day subcutaneously twice a day for 14days. RhG-CSF was well-tolerated and no systemic toxicity wasobserved. Both dogs showed increased peripheral blood neutrophilcounts within 24 hours of starting the injections of the growthfactor (Figure 12a). By day 14, neutrophils reached levels thatwere eight to ten times higher than the preinfusion counts and thoseof two control dogs receiving either 60 Mg heat-inactivated rhIL-3per kg/day for 14 days (range indicated by two horizontal lines) or10 Mg human serum albumin per kg/day. After discontinuation ofrhG-CSF the neutrophil counts returned to control levels withinthree days. Peripheral blood monocytes were increased four- tosixfold (Figure 12b). Lymphocyte counts remained unchanged at 10 4grhG-CSF per kg/day and increased threefold at 100 gg rhG-CSF perkg/day (Figure 12c). No significant changes were observed inplatelet counts (Figure 12d), eosinophil, reticulocyte or hematocritlevels. Marrow biopsies were taken before and after 14 days ofrhG-CSF injections, and slides were evaluated without knowledge oftreatment modality. Compared to the normocellular marrow histologybefore rhG-CSF treatment, the marrows of the rhG-CSF-treated dogswere hypercellular with myeloid hyperplasia and left-shiftedgranulopoiesis.

6.2 EFFECTS OF RhG-CSF IN DOGS AFTER LETHAL TBI.

In studies funded by a separate grant, we have investigated thesurvival of dogs given TBI, no marrow infusion and optimalsupportive care. We found that recovery of dogs is uneventful after200 cGy of TBI administered at a rate of 7 or 10 cGy per minute.Approximately 75% of dogs die of marrow aplasia and peripheralpancytopenia after 300 cGy of TBI. All dogs died following 400 cGyor higher doses of TBI. All dogs survived when given autologousmarrow transplantation following TBI. We also found that dogs given450 cGy TBI and DLA-identical marrow transplants showed transientallogeneic engraftment followed by rejection of the graft. One ofthese dogs was killed with IV sodium pentobarbital for purposes ofobtaining tissue samples, one died with intermittent infection, andthree survived with autologous marrow recovery as documented byblood genetic markers. We saw a similar phenomenon even after600 cGy TBI. While 4 out of 7 dogs receiving this dose of TBI andDLA-identical marrow transplants showed sustained engraftment, oneof the three dogs rejecting the marrow graft survived withautologous recovery. These findings show that endogenoushematologic recovery is possible even after an otherwise lethal doseof TBI of up to 600 cGy if dogs are given support through atransient DLA-identical marrow graft.

37

1 00000

E

0000

100-

10

0 7 14 21 2

Day

e-eCe506 f, it C518 C--.520 .--- C 53 0 -*-C581

Figure 1 3. Peripheral blood neutrophil changes in dogs after 400 c~y of TBI(day 0).

39

Five other dogs were irradiated with 400 cGy TBI at 10 cGy perminute as before and, in addition, were treated with 100 Ag rhG-CSFper kg/day subcutaneously twice a day for 21 days, starting withintwo hours after TBI. Four dogs showed complete and sustainedhematopoietic recovery and are now surviving 120 to 180 days afterTBI with normal periphonra] blood couints (Fiqure 14). One dog, C726,died on day 19 after TBI because of accute bacterial pneumoniasecondary to marrow aplasia. On the day of his death, peripheralblood neutrophil count was zero and marrow histology at autopsyshowed 5% of normal marrow cellularity with few myeloid precursorcells but no erythroid or megakaryocytic precursor cells (Table 9).

A third group of five dogs was irradiated with 400 cGy TBI at 10 cGyper minute as before, and then treated with 100 Ag rhG-CSF perkg/day subcutaneously twice a day from day 7 until day 20 after TBIor until death. All five dogs died between days 17 and 20 after TBIwith infections secondary to marrow aplasia (Figure 15). In threedogs, marrow histology at autopsy showed no signs of hematopoiesis,and in two dogs 5% of normal marrow cellularity was seen containingmyeloid precursor cells but no erythroid or megakaryocyticprecursors (Table 9).

This study demonstrated that treatment with rhG-CSF allows sustainedendogenous hematopoietic recovery in dogs after otherwise lethal TBiand also indicated that the time interval between TBI and start ofrhG-CSF treatment is important.

After these encouraging results, a group of 10 dogs was irradiatedwith 500 cGy TBI at 10 cGy per minute and then treated with either100 Mg rhG-CSF per kg/day or 10 gg recombinant canine G-CSF(rcG-CSF) per kg/day subcutaneously twice a day for 21 days,starting within 2 hours after TBI. The dose of 10 gg rcG-CSF perkg/day was found to be equivalent biologically to the dose of 100 AgrhG-CSF per kg/day when given to normal dogs. Three dogs showedcomplete and sustained hematopoietic recovery and have beensurviving for more than 90 days after TBI with normal peripheralblood counts (Figure 16).

6.3 EFFECT OF RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATINGFACTOR ON HEMATOPOIETIC RECOVERY AFTER DLA-IDENTICAL LITTERMATEMARROW TRANSPLANTS IN DOGS.

Marrow transplant candidates either have no marrow function tobegin with, as in aplastic anemia, or their marrow function isirradicated by the chemoradiotherapy aimed at destroying theunderlying hematologic malignancy. Despite prompt infusion ofHLA-identical marrow after completion of the conditioning regimen,patients remain pancytopenic for several weeks before the graftachieves full function. Typically, 15 to 44 (median 24) days willgo by before granulocyte counts reach 1,000/mm 3 during which timepatients are at risk for bacterial or fungal infections with anoverall incidence of 20% to 25%, and a case mortality rate of 25% to50%. Shortening the period of neutropenia after marrow grafting islikely to result in a reduction of the risk of infections. A

41

100000 C-CSF-------

10000

EE

S 1000C,

0~100

z10

0 5 10 15 20 25

DayI~ ~~ 7-- 783 75 C781 ---- C788 C863

Figure 15. Peripheral blood neutrophil changes in dogs after 400 cGy of TBI(day 0) and twice-daily SQ injections of 100/pg rhG-CSF/kg/day fromday 7 after TBI until death. Shaded area indicates the range of theperipheral blood neutrophils of the control dogs not receiving rhG-CSFafter TBI.

43

preclinical study in nonhuman primates and two clinical studiesusing rhG-CSF after autologous marrow transplantation have shownsignificant acceleration of granulocyte recovery compared to thecontrols. The usefulness of rhG-CSF after allogeneic human marrowgrafts has so far been investigated in only one non-randomizedstudy, at least in part because of concerns of side effects such asan increased risk of graft-versus-host disease or late graftfailure. We therefore studied the effect of rhG-CSF administeredafter transplantation of marrow from DLA-identical littermates inthe dog model.

Table 10. Recovery of peripheral blood counts after TBI andtransplantation of marrow from DLA-identical littermatewithout and with subsequent rhG-CSF.

Number of Days to AchieveSpecified Levels

PeripheralBlood rh 25% of 50% of 75% of

Variable Count G-CSF Dogs Dogs Dogs p-value'

Neutrophils 500/mm3 8 10 15 .08+ 6 7 7

1000/mm3 - 11 14 16 .03+ 7 8 8

Monocytes 50/mm3 15 28 35 .0008+ 9 13 17

100/mm3 - 29 49 105 .002+ 13 17 22

Lymphocytes 250/mm3 - 10 15 22 .03+ 8 9 13

500/mm' 21 31 37 .01+ 10 15 27

Platelets 20,000/mm3 13 20 37 .68+ 22 26 31

Logrank test.

Table 10 and Figure 17 summarize the results on hemopoieticrecovery. Neutrophil counts in the rhG-CSF treated dogs reached500/mm3 at a median of seven days and 1000/mm3 at 8 days aftertransplantation. This was significantly faster than neutrophilsrecovery in the controls who reached values of 500 and 1000/mm3 at

45

medians of 10 and 14 days, respectively (log rank test: p<0.08 and<0.03, respectively). The median time to reach 50 and 100monocytes/mm3 in G-CSF-treated dogs was 13 and 17 davs compared to 28and 49 days in the control group (log rank test: p<0.0008 and<0.002, respectively). Lymphocyte counts reached 250 and 500/mm3 byday 9 and 15 in rhG-CSF-treated dogs compared to 15 and 31 days inthe control dogs (log iank test: p<0.03 and <0.01, respectively).Platelet recovery to 20,000/mM3 was not significantly differentbetween the two groups (log rank test: p<0.68) with the median timesbeing 20 days for the control and 26 days for the experimentalgroup.

Table 11 summarizes the results on mortality, GVHD and graftfailure. Three of ten G-CSF-treated dogs died before day 100compared to five of fourteen control dogs (log rank test: p=0.80).Causes of death in the experimental group were GVHD (two dogs) andgraft failure (one dog) and in the control group pneumonia (twodogs), graft failure (two dogs) and thrombocytopenic hemorrhage (onedog). Four of nine rhG-CSF-treated dogs with sustained engraftmentdeveloped persistent GVHD as documented by typical clinical symptoms(skin rash, diarrhea, jaundice) and histological findings at autopsycompared to one of twelve control dogs, but this difference was notstatistically significant (two-tailed Fisher's exact test: p=0.12).Two of the four G-CSF-treated dogs with GVHD died because of thiscomplication compared to none of the control dogs (two-tailedFisher's exact test: p=0.17). One of ten rhG-CSF-treated dogsshowed graft failure compared to two of fourteen control dogs(two-tailed Fisher's exact test: p=l.00). No unusual toxicitieswere seen in dogs receiving rhG-CSF.

Table 11. Mortality, GVHD, lethal GVHD and graft failure after TBIand transplantation of marrow from DLA-identicallittermate without and with subsequent rhG-CSF.

Dogs

Variable Control rhG-CSF Treated p-valueDeath before 100Daysall caues 5/14 (36%) 3/10 (30%) .80,days (all causes)

GVHD2 1/12 (8%) 4/9 (44%) .12'

Lethal GVHD 0/12 (0%) 2/9 (22%) .17'

Graft Failure 2/14 (14%) 1/10 (10%) 1.003

Logrank test.

2 The dogs which died of graft failure were not included in the

comparison of incidence of GVHD and lethal GVHD.3 2-tailed Fisher's exact test.

47

DISTRIBUTION LIST

DNA-TR-90-66

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