Method for improving autologous transplantation

There is disclosed a method for autologous hematopoietic cell transplantation of patients receiving cytoreductive therapy, comprising: (1) obtaining hematopoietic progenitor cells from bone marrow or peripheral blood from a patient prior to cytoreductive therapy; (2) expanding the hematopoietic progenitor cells ex vivo with an ex vivo growth factor selected from the group consisting of interleukin-3 (IL-3), steel factor (SF), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 (IL-1), GM-CSF/IL-3 fusion proteins and combinations thereof, to provide a cellular preparation comprising an expanded population of progenitor cells; and (3) administering the cellular preparation to the patient concurrently with or follwoing cytoreductive therapy. The inventive method optionally comprising a preliminary treatment with a recruitment growth factor to recruit hematopoietic progenitor cells into peripheral blood and a subsequent treatment with an engraftment growth factor to facilitate engraftment and proliferation of hematopoietic progenitor cells administered in the cellular preparation. The invention further provides a hematopoietic progenitor cell expansion media composition comprising cell media, an ex vivo growth factor, and autologous serum.

TECHNICAL FIELD OF THE INVENTION 
The present invention relates generally to methods for autologous 
hematopoietic cell transplantation in patients undergoing cytoreductive 
therapies, and particularly to methods in which bone marrow or peripheral 
blood progenitor cells are removed from a patient prior to 
myelosuppressive cytoreductive therapy, expanded in ex vivo culture in the 
presence of a growth factor, and then readministered to the patient 
concurrent with or following cytoreductive therapy to counteract the 
myelosuppressive effects of such therapy. The present invention further 
relates to a culture media comprising one or a plurality of growth factors 
for expanding progenitor cells in ex vivo culture. 
BACKGROUND OF THE INVENTION 
Cancers are generally treated with various forms of cytoreductive 
therapies. Cytoreductive therapies involve administration of ionizing 
radiation or chemical toxins which are cytotoxic for rapidly dividing 
cells. Side effects of such therapy can be attributed to cytotoxic effects 
upon normal cells and can usually limit the use of cytoreductive 
therapies. A frequent side effect is myelosuppression, or damage to bone 
marrow cells which gives rise to white and red blood cells and platelets. 
As a result of myelosuppression, patients develop cytopenia which are 
blood cell deficits. As a result of cytopenias, patients are exposed to 
increased risk of infection and bleeding disorders. 
Cytopenia is a major factor contributing to morbidity, mortality, and 
under-dosing in cancer treatment. Many clinical investigators have 
manipulated cytoreductive therapy dosing regimens and schedules to 
increase dosing for cancer therapy, while limiting damage to bone marrow. 
One approach involves bone marrow transplantations in which bone marrow 
hematopoietic progenitor cells are removed before a cytoreductive therapy 
and then reinfused following therapy to rescue bone marrow from toxicity 
resulting from the cytoreductive therapy. Progenitor cells may implant in 
bone marrow and differentiate into mature blood cells to supplement 
reduced population of mature blood cells. 
High-dose chemotherapy is therapeutically beneficial because it can produce 
an increased frequency of objective response in patients with metastatic 
cancers, particularly breast cancer, when compared to standard dose 
therapy. This can result in extended disease-free remission for some even 
poor-prognosis patients. Nevertheless, high-dose chemotherapy is toxic and 
many resulting clinical complications are related to infections, bleeding 
disorders and other effects associated with prolonged periods of 
myelosuppression. 
Currently, a human recombinant granulocyte macrophage-colony stimulating 
factor (GM-CSF) analog protein (sargramostim) is available in the U.S. for 
accelerating hematopoietic recovery following bone marrow transplantation. 
Sargramostim treatment has resulted in a reduction of many complications 
associated with bone marrow transplantation. 
The existence of both marrow borne and circulating hematopoietic stem cells 
has been demonstrated using a variety of experimental studies and cell 
culture techniques. Two colony stimulating factors, GM-CSF and granulocyte 
colony stimulating factor (G-CSF), have been shown to increase the 
frequency of circulating hematopoietic progenitor or stem cells. Several 
studies (Gianni et al., Lancet 334:589 (1989); Siena et al., Blood 74:1905 
(1989); and Molineux et al., Blood 76:2153 (1990)) describe in vivo 
administration of GM-CSF to increase the transplantation potential and 
frequency of primitive progenitor cells in a population of peripheral 
blood cells obtained from patients with tumors. These procedures represent 
attempts to rescue chemotherapy-induced suppresion of bone marrow by 
administering GM-CSF in vivo to recruit bone marrow progenitor cells into 
peripheral blood and then later administering harvested hematopoietic 
progenitor cells to patients. 
More specifically, Gianni et al. describe a clinical study in which 
patients received high dose cyclophosphamide (7 g/m.sup.2) and were 
transplanted with autologous peripheral blood progenitor cells and 
autologous bone marrow cells. Patients who were treated with GM-CSF as a 
progenitor cell recruitment agent, prior to harvesting peripheral blood 
progenitor cells, recovered more quickly from cytopenia than patients 
whose peripheral blood progenitor cells were not recruited by GM-CSF. Thus 
GM-CSF administration increased the number of peripheral blood progenitor 
cells. This protocol resulted in more rapid hematopoietic recovery in 
tested patients than in control patients who received chemotherapy without 
autologous bone marrow transplantation but with peripheral blood 
progenitor cell support. 
Cancer patients treated with high dose chemotherapy and autologous bone 
marrow transplantation who received subsequent GM-CSF treatment have shown 
faster myeloid recovery than similarly treated historical controls (Brandt 
et al., N. Engl. J. Med. 318:869 (1988) and Nemunatis et al., Blood 72:834 
(1988)). Studies have shown that the time to achieve a minimum granulocyte 
count of 0.5.times.10.sup.9 /l after cytoreductive therapy was shorter in 
patients receiving GM-CSF. Granulocyte count increases were most 
pronounced during GM-CSF infusion. After discontinuation of GM-CSF, 
leukocyte counts in treated patients fell to control levels (Brandt et 
al., supra). 
GM-CSF is also useful for autologous bone marrow transplantation following 
cytoreductive therapy. Socinski et al., Lancet 331:194 (1988) reported 
that GM-CSF administration after cytotoxic chemotherapy expands a 
circulating pool of hematopoietic progenitor cells by approximately 
60-fold. Others have reported that human mononuclear cells circulating in 
the circulating blood, particularly during recovery from 
chemotherapy-induced myelosuppression, have been used to successfully 
reconstitute hematopoiesis after fully myeloablative (complete bone marrow 
toxicity) treatments (See, e.g., Bell et al., Hematol, Oncol.5:45 (1987)). 
Mason et al., Proc. Amer. Assoc. Cancer Res. 32:193 (1991), reported that 
in vitro interleukin-3 (IL-3) alone or in combination with interleukin-6 
(IL-6) increased the number of colony forming progenitors from human blood 
progenitor cells two fold in vitro. Mason et al. also reported that GM-CSF 
did not expand the colony forming progenitor population in vitro. 
Accordingly, autologous hematopoietic cell transplantation has proven to 
be a valuable technique to speed recovery from cytoreductive therapies. 
Improvements in autologous hematopoietic cell transplantation can further 
speed recovery from cytoreductive therapies and even allow the use of 
higher and more effective doses in cytoreductive therapies. This invention 
provides an improvement in autologous hematopoietic cell transplantation. 
SUMMARY OF THE INVENTION 
The invention is a method for conducting autologous progenitor cell 
transplantation, comprising: (1) obtaining hematopoietic progenitor cells 
from a patient prior to cytoreductive therapy; (2) expanding the 
hematopoietic progenitor cells ex vivo with an ex vivo growth factor 
selected from the group consisting of granulocyte macrophage-colony 
stimulating factor (GM-CSF), interleukin-3 (IL-3), steel factor (SF), 
GM-CSF/IL-3 fusion proteins, and combinations thereof, to provide a 
cellular preparation comprising increased numbers of hematopoietic 
progenitor cells; and (3) administering the cellular preparation to the 
patient in conjunction with or following cytoreductive therapy. 
Interleukin-1 (IL-1.alpha. or IL-1.beta.) can also be used as an ex vivo 
growth factor when used together with at least one other growth factor. 
Progenitor cells may be obtained from peripheral blood harvest or bone 
marrow explants. 
The inventive method optionally comprises a preliminary in vivo procedure 
comprising administering a recruitment growth factor to the patient to 
recruit hematopoietic progenitor cells into peripheral blood prior to 
their harvest, wherein the recruitment growth factor is selected from the 
group consisting of GM-CSF, SF, G-CSF, IL-3, GM-CSF/IL-3 fusion proteins, 
and combinations thereof. 
The inventive method optionally comprises a subsequent in vivo procedure 
comprising administering an engraftment growth factor to the patient 
following autologous transplantation of the cellular preparation to 
facilitate engraftment and augment proliferation of engrafted 
hematopoietic progenitor cells from the cellular preparation. The 
engraftment growth factor is selected from the group consisting of GM-CSF, 
IL-3, SF, GM-CSF/IL-3 fusion proteins and combinations thereof. 
The present invention further includes a progenitor cell expansion media 
comprising cell growth media, autologous serum, and a growth factor 
selected from the group consisting of SF, IL-1, IL-3, GM-CSF, GM-CSF/IL-3 
fusion proteins, and combinations thereof with the proviso that IL-1 must 
be used in combination with at least one other growth factor.

DETAILED DESCRIPTION OF THE INVENTION 
Growth factors can be used in vivo to induce hematopoietic progenitor cells 
in bone marrow to proliferate and to mobilize such hematopoietic 
progenitor cells into peripheral blood. Hematopoietic progenitor cells 
harvested from peripheral blood can be used for hematopoietic rescue 
therapy of patients treated with cytoreductive agents. The present 
invention involves ex vivo treatment of hematopoietic progenitor cells 
from peripheral blood or bone marrow with growth factors to increase their 
numbers prior to infusion or transplantation. In addition, growth factors 
can be used to facilitate engraftment and proliferation of transplanted 
hematopoietic progenitor cells following transplantation. Hematopoietic 
reconstitution of a patient undergoing cytoreductive therapy can reduce 
the incidence of infection and bleeding complications of patients treated 
with high doses of cytoreductive therapies, such as myelosuppressive 
cancer chemotherapeutic agents or high doses of radiotherapy. 
The present invention involves ex vivo and/or in vivo administration of 
growth factors in connection with autologous bone marrow or peripheral 
blood progenitor cell transplantation. The ex vivo growth factor, 
recruitment growth factor and engraftment growth factor are selected from 
the group consisting of GM-CSF, IL-3, SF, IL-1, GM-CSF/IL-3 fusion 
proteins, and combinations thereof. Of the foregoing, a combination of SF 
and a GM-CSF/IL-3 fusion protein is preferred for the ex vivo growth 
factor. Moreover, IL-1 (comprising IL-1.alpha. or IL-1.beta.) should be 
administered in combination with at least one other growth factor and 
preferably as an ex vivo growth factor. 
Granulocyte macrophage-colony stimulating factor (GM-CSF) is commercially 
and clinically available as an analog polypeptide (Leu.sup.23) under the 
trademark LEUKINE.RTM.. The generic name for recombinant human Leu.sup.23 
GM-CSF analog protein expressed in yeast is Sargramostim. Cloning and 
expression of native sequence human GM-CSF was described in Cantrell et 
al., Proc. Natl. Acad. Sci. U.S.A 82:6250 (1985). 
Interleukin-3 (IL-3) occurs in two major allelic variations. The 
predominant human allele encodes an IL-3 molecule having a proline residue 
at position 8 of the mature polypeptide which is described in WO88/04691 
published on Jun. 30, 1988, and in U.S. patent application Ser. No. 
07/004,466, filed on Jan. 20, 1987, the disclosure of which is 
incorporated by reference herein. The other IL-3 allele has a serine 
residue at position 8 of the mature polypeptide. 
Steel factor (SF) has also been called mast cell growth factor (MGF), stem 
cell factor (SCF), and Kit Ligand (KL). All of the names for this factor 
refer to the ligand for the c kit proto-oncogene. SF has been described in 
a series of seven papers in the Oct. 5, 1990 issue of Cell (Williams et 
al., Cell 63:167, 1990; Copeland et al., Cell 63:175, 1990; Flanagan and 
Leder, Cell 63:185, 1990; Zsebo et al., Cell 63:195, 1990; Martin et al., 
Cell 63:203, 1990; Zsebo et al., Cell 63:213, 1990; Huang et al., Cell 
63:225, 1990; and Anderson et al., Cell 63:235, 1990). Expression of 
various recombinant forms of SF has been described in U.S. patent 
application Ser. No. 07/586,073, filed Sep. 21, 1990 and U.S. patent 
application Ser. No. 07/713,715 filed Jun. 12, 1991, the disclosures of 
which are incorporated by reference herein. SF has been found to stimulate 
proliferation and recruitment of early myeloid and lymphoid lineage 
progenitor cells and possibly even the most primitive hematopoietic stem 
cells. 
Interleukin-1 has been found to exist in two forms, IL-1.alpha. and 
IL-1.beta. (March et al., Nature 315:641, 1985). Both IL-1.alpha. and 
IL-1.beta. bind to IL-1 receptors (Type I and Type II) to transduce 
signal. IL-1.alpha. is active in both precursor and mature forms, whereas 
IL-1.beta. is only active in mature form but not in a precursor form 
(March et al., supra). IL-1 also include active fragments and analogs with 
altered amino acid sequences and derivatives, such as fusion proteins 
having an IL-1 component and IL-1 biological activity (Mosley et al., 
Proc. Natl. Acad. Sci., USA 84:4572, 1987). 
Fusion protein comprising GM-CSF and IL-3 components are described in U.S. 
patent application Ser. No. 07/567,983 filed Oct. 14, 1990, the disclosure 
of which is incorporated by reference herein. A particular GM-CSF/IL-3 
fusion protein (PIXY321) has been found to interact with GM-CSF receptors 
and/or IL-3 receptors (Curtis et al., Proc. Natl. Acad. Sci. USA 88:5809, 
1991). PIXY321 is a GM-CSF/IL-3 fusion protein having Leu.sup.23 
Asp.sup.27 Glu.sup.34 hGM-CSF/Gly.sub.4 Ser Gly.sub.5 Ser/Pro.sup.8 
Asp.sup.15 Asp.sup.70 hIL-3. Thus, this protein comprises a 
triply-substituted GM-CSF domain fused to a doubly-substituted Pro.sup.8 
IL-3 domain via a linker or spacer domain comprising glycine and serine 
residues. Preferably, hematopoietic progenitor cells are expanded ex vivo 
using an effective amount of a growth factor comprising a combination of 
SF and a GM-CSF/IL-3 fusion protein (such as PIXY321). 
The growth factors used in the methods of the present invention are 
polypeptides. If recruitment or engraftment growth factors are employed, 
normal routes of in vivo polypeptide administration are preferred, 
including subcutaneous, intravenous (iv), intraperitoneal (ip), 
intramuscular (im), and intralymphatic (il). Most preferably in vivo 
administration of a growth factor is subcutaneous. 
Ex vivo use of a growth factor is by direct addition to cultures of 
hematopoietic progenitor cells from peripheral blood or bone marrow in 
physiological buffer or culture medium. Preferred progenitor cell 
expansion medium is, for example, minimal essential medium supplemented 
with autologous serum and antibiotics. Progenitor cell expansion media, 
according to the present invention, comprises one or a plurality of ex 
vivo growth factors in culture medium, such as minimal essential medium 
supplemented with autologous serum and possibly antibiotics. Other culture 
media include, for example, Hanks, McCoys, RPMI 1640 minimal essential 
media (MEM) and others, and include from 1% to 20% autologous serum and 
possibly antibiotics. 
Preferred in vivo dosages of recruitment or engraftment growth factors are 
from about 10 .mu.g/kg/day to about 800 .mu.g/kg/day for SF; from about 1 
.mu.g/kg/day to about 100 .mu.g/kg/day for GM-CSF and for IL-3; and from 
about 1 .mu.g/kg/day to about 100 .mu.g/kg/day for GM-CSF/IL-3 fusion 
proteins. Preferred ex vivo growth factor concentrations in progenitor 
cell expansion media are from about 1 ng/ml to about 10 .mu.g/ml for SF, 
and from about 10 ng/ml to about 200 .mu.g/ml for GM-CSF, IL-3, IL-1 and 
GM-CSF/IL-3 fusion proteins. 
Progenitor cells may be obtained from human mononuclear cells obtained from 
bone marrow and peripheral blood. Progenitor cells may be separated from 
peripheral blood, for example, by density gradient centrifugation such as 
a Ficoll Hypaque.RTM. system. Another means for separating hematopoietic 
progenitor cells obtained from bone marrow or peripheral blood involves 
separating with antibodies that recognize a stage-specific antigen on 
immature human hematopoietic progenitor cells. One example of an antibody 
recognition method for separating human hematopoietic progenitor cells is 
described in Civin, U.S. Pat. No. 5,035,994 the disclosure of which is 
incorporated by reference herein. 
Once hematopoietic progenitor cells are obtained by a particular separation 
technique, they may be stored in cryogenic conditions or expanded ex vivo 
according to the present invention. Stored cells may later be rapidly 
thawed and expanded ex vivo according to the present invention. 
Hematopoietic progenitor cells treated ex vivo with growth factors are 
readministered to patients by autologous transplantation. Cells are 
cultured ex vivo in the presence of a growth factor for at least one day 
and no more than two weeks. Cells can be stored and retain viability 
either prior to expansion with growth factor or after expansion with 
growth factor. Cell storage is preferably under cryogenic conditions such 
as liquid nitrogen. Cultured cells are washed before being administered to 
the patient. Expanded cells are administered following completion of 
cytoreductive therapy or up to 72 hours after completion of cytoreductive 
therapy. Cell administration usually is by infusion over 2 to 5 days. 
Preferably, from about 10.sup.7 to about 10.sup.9 expanded mononuclear 
cells/kg (approximately 10.sup.5 expanded progenitor cells/kg) are 
administered to the patient for an autologous transplantation. 
Preferably, the hematopoietic progenitor cells are cultured ex vivo for 
approximately one week. Preferably, the growth factor is a combination of 
SF and a GM-CSF/IL-3 fusion protein. Most preferably, the GM-CSF/IL-3 
fusion protein is PIXY321. Preferably, the recruitment growth factor and 
the engraftment growth factor is a combination of SF and a GM-CSF/IL-3 
fusion protein. 
In a preferred embodiment, the method of the present invention comprises in 
vivo treatment of the patient prior to cytoreductive therapy (after 
recovery from any previous cytoreductive therapy) with a recruitment 
growth factor, ex vivo expansion of hematopoietic progenitor cells 
(obtained from the patient's peripheral blood and/or bone marrow) with a 
combination of a GM-CSF/IL-3 fusion protein and SF, and in vivo treatment 
of the patient with an engraftment growth factor after administration of 
the cellular preparation (ex vivo treated hematopoietic progenitor cells). 
In vivo administration of a recruitment growth factor helps to stimulate 
proliferation of more primitive hematopoietic progenitor cells in bone 
marrow and to recruit hematopoietic progenitor cells into peripheral 
blood. Peripheral blood progenitor cells and/or bone marrow cells are 
expanded and later used for hematopoietic rescue following cytoreductive 
therapy. The patient may be treated with an engraftment growth factor 
beginning one to three days following the last administration of the 
cellular preparation. The engraftment growth factor facilitates 
engraftment and proliferation of hematopoietic progenitor cells. 
Hematopoietic rescue helps reduce patient morbidity associated with 
myelosuppressive or myeloablative cytoreductive therapy protocols as 
manifest in decreased infections, susceptibility to infections and 
bleeding disorders. 
Hematopoietic progenitor cells obtained from human peripheral blood from 
normal volunteers were expanded ex vivo by culturing in progenitor cell 
expansion media comprising selected single growth factors or combinations 
of growth factors. Culturing hematopoietic progenitor cells ex vivo in the 
presence of selected growth factors resulted in expansion of myeloid 
lineage and erythroid lineages progenitor cells as determined by colony 
assays for myeloid (GM-CSF) and erythroid (BFU-e) components. Example 1 
herein presents data from a series of experiments utilizing hematopoietic 
progenitor cells cultured in different progenitor expansion media. Data 
presented in Example 1 demonstrate increases in expansion indeces of 
myeloid and erythroid components. Progenitor cell expansion media 
comprising PIXY321, IL-3 or SF growth factors was able to expand myeloid 
and erythroid progenitor cell populations to a greater extent than media 
without added growth factor. Media comprising a combination of SF and 
PIXY321 consistently produced myeloid and erythroid progenitor cell 
expansion greater than media coprising SF alone, PIXY321 alone, 
IL-1.alpha. alone, G-CSF alone, IL-3 alone or even the combination of SF 
and IL-3 or G-CSF and SF. Another particularly effective growth factor 
combination was IL-1.alpha. and PIXY321 for both myeloid and erythroid 
prenitor cell expansion however, this combination did not maintain high 
numbers of myeloid progenitor cells. Accordingly, the claimed method for 
expanding hematopoietic progenitor cells in vitro with an ex vivo growth 
factor or combination of growth factors can improve expansion of at least 
myeloid and erythroid lineage populations of hematopoietic progenitor 
cells obtained from human peripheral blood. Moreover, the growth factor 
combination of PIXY321 and SF was particularly effective. 
The present invention comprises ex vivo treatment of hematopoietic 
progenitor cells from peripheral blood or bone marrow with a growth 
factor. The growth factor is selected from the group consisting of GM-CSF, 
IL-3, SF, IL-1, GM-CSF/IL-3 fusion proteins and combinations thereof. Ex 
vivo progenitor cell expansion in media containing a growth factor is 
capable of expanding the number of hematopoietic progenitor cells 
originally harvested from peripheral blood or bone marrow and improves the 
ability of the expanded population of progenitor cells to engraft and 
proliferate in bone marrow and other hematopoietic tissue when later 
administered in an autologous transplantation. 
This ability to significantly expand a population of hematopoietic 
progenitor cells for autologous transplantation provides an improved 
autologous hematopoietic cell transplantation technique to allow for high 
doses or more intensive cytoreduction therapies. Ex vivo treatment with 
the growth factor improves hematopoietic rescue of the patient following 
myeloablative or myelosuppressive cytoreductive therapy regimens. 
Treatment of hematopoietic progenitor cells obtained from peripheral blood 
or bone marrow further allows for higher dosing of cytoreductive 
therapeutic agents while reducing the risk of infection and bleeding 
disorders to the patient. 
Therefore, the method of hematopoietic rescue by autologous 
transplantation, according to the present invention, helps to reduce 
morbidity (infection and bleeding disorders) and myelotoxicity (bone 
marrow toxicity) associated with higher doses of cytoreductive therapy and 
constitutes an improvement over current autologous hematopoietic cell 
transplantation techniques. The following examples illustrate in vitro 
data of hematopoietic progenitor cell expansion and various clinical 
therapeutic procedures for hematopoietic rescue and autologous 
hematopoietic cell transplantation. 
EXAMPLE 1 
This example illustrates a comparison of expansion ratios for human 
hematopoietic progenitor cells expanded ex vivo with progenitor expansion 
media comprising different growth factors or growth factor combinations or 
media without added growth factor in a series of experiments. The human 
hematopoietic progenitor cells were obtained from peripheral blood of 
normal volunteers. 
Human peripheral blood was obtained from normal, healthy, volunteers via 
veinipuncture and collected in a heparinized tube. Progenitor cells were 
obtained from peripheral blood by density gradient centrifugation on 
Histopaque.RTM. (Sigma, St. Louis) and a mononuclear layer of cells were 
obtained. The mononuclear cells, containing a population of human 
hematopoietic progenitor cells were washed twice in phosphate buffered 
saline (PBS) and viable cells counted by trypan blue due exclusion. 
Ex vivo cultures were made from approximately 10.sup.7 viable cells in 10 
ml of Super McCoys medium supplemented with 20% fetal bovine serum. It 
should be noted that fetal bovine serum was substituted for autologous 
serum in this experiment because the cells expanded in this experiment 
would not be readministered to their original donors. Cells were cultured 
and expanded in petri dishes incubated at 37.degree. C. in an atmosphere 
of 7% CO.sub.2, 8% O.sub.2, 85% air. Culture media were replaced on day 4 
with new growth factor(s). 
Growth factors were added to media at concentrations according to Table 1: 
TABLE 1 
______________________________________ 
Growth Factor Concentration 
______________________________________ 
PIXY321 100 ng/ml 
SF 1 .mu.g/ml 
IL-3 100 ng/ml 
IL-1.alpha. 100 ng/ml 
G-CSF 100 ng/ml 
______________________________________ 
Progenitor cells in culture tend to be nonadherent. For each colony assay, 
50% of nonadherent cells in each culture were obtained. Cells were 
separated from media by centrifugation, washed twice and viable cells 
counted by trypan blue due exclusion. 
Two colony assays were performed. A GM-CFU assay (Lu et al., Exp. Hematol. 
13:989, 1985) measured a myeloid component of the progenitor cell 
population and BFU-e assay (Lu et al., supra) measured an erythroid 
component. Viable cells were plated in a methyl cellulose cloning media 
(Terry Fox Labs, Vancouver, B.C.) in the presence of PIXY321 (GM-CFU) or 
PIXY321 plus erythropoietin (BFU-e). The number of myeloid or erythroid 
colonies were counted and this number was divided by the number of cells 
plated into each well to determine a colony-forming capacity (CFC) 
incidence. CFC incidence was multiplied by total cell number to determine 
CFC number per culture. Each CFC number was compared to a day 0 CFC number 
to determine an expansion ratio for each progenitor expansion media 
tested. 
Myeloid and erythroid component cell expansion was determined after 4 and 8 
days of incubation. An expansion number of 1 means that there was no 
expansion of colony number, whereas an expansion number of 2 means that 
the number of colonies doubled from the day 0 number. 
A first experiment compared myeloid and erythroid expansion of 
hematopoietic progenitor cells cultured and expanded hematopoietic 
progenitor cell expansion media comprising McCoys media plus 20% FCS and 
supplemented with PIXY321, SF or a combination of PIXY321 and SF. 
TABLE 2 
______________________________________ 
Day 4 Day 8 
Growth Factor 
GM-CFU BFU-e GM-CFU BFU-e 
______________________________________ 
Media only 0.58 0.74 0.63 0.89 
PIXY321 1.41 2.19 4.29 13.63 
SF 1.59 0.76 2.49 4.89 
PIXY321 + SF 
3.77 8.33 12.68 22.65 
______________________________________ 
Data from the first experiment show that both PIXY321 and SF improved 
myeloid and erythroid progenitor cell expansion. The combination of 
PIXY321 and SF showed a more-than-additive expansion of myeloid erythroid 
cells. 
A second experiment compared myeloid and erythroid progenitor cell 
expansion when hematopoietic progenitor cells were cultured in the 
presence of PIXY321, SF and IL-1.alpha.. In this experiment, the 
concentration of mononuclear cells added to media was decreased from 
10.sup.6 cells/ml in the first experiment to 4.times.10.sup.5 cells/ml in 
this experiment. 
TABLE 3 
______________________________________ 
Day 4 Day 8 
Growth Factor GM-CFU BFU-e GM-CFU 
______________________________________ 
Media 0.875 0.32 3.50 
IL-1.alpha. 0.74 0.21 1.80 
SF 1.375 0.42 4.26 
PIXY321 2.70 1.90 19.01 
IL-1.alpha. + SF 
1.50 0.80 2.11 
IL-1.alpha. + PIXY321 
8.17 3.12 5.84 
SF + PIXY321 7.31 4.43 32.64 
SF + PIXY321 + IL-1.alpha. 
5.40 4.95 5.25 
______________________________________ 
The data from the second experiment show expansion of myeloid and erythroid 
lineage progenitor cells when expanded with PIXY321 alone as a growth 
factor but not with either SF alone or IL-1.alpha. alone as growth 
factors. Growth factor combinations IL-1.alpha.+PIXY321, SF+PIXY321, and 
SF+PIXY321+IL-1.alpha. provided striking expansions in both myeloid and 
erythroid lineage progenitor cell numbers on day 4, however the day 8 
results showed improvement only for SF+PIXY321 and for PIXY321 alone. 
A third experiment compared myeloid progenitor cell expansion on days 4 and 
8 for cultures containing growth factors PIXY321, SF, IL-3 and 
combinations. 
TABLE 4 
______________________________________ 
Day 4 Day 8 
Growth Factor GM-CFU GM-CFU 
______________________________________ 
Media 0.50 0.74 
PIXY321 2.18 4.83 
SF 1.25 1.08 
IL-3 1.99 3.60 
PIXY321 + SF 4.01 7.05 
IL-3 + SF 2.65 4.22 
______________________________________ 
These data show that a growth factor combination of PIXY321 and SF provide 
greater myeloid progenitor cell expansion than a growth factor combination 
of IL-3 and SF. 
A fourth experiment compared myeloid progenitor cell expansion on days 4 
and 8 for cultures containing growth factors PIXY321, SF, G-CSF and 
combinations. The concentration of mononuclear cells added to media was 
10.sup.6 cells/ml. 
TABLE 5 
______________________________________ 
Day 4 Day 8 
Growth Factor GM-CFU GM-CFU 
______________________________________ 
Media 0.76 0.80 
PIXY321 2.69 3.94 
SF 1.84 0.76 
G-CSF 0.96 1.42 
PIXY321 + SF NA 3.56 
PIXY321 + G-CSF 5.02 12.50 
SF + G-CSF 3.36 1.79 
PIXY321 + SF + G-CSF 
6.34 3.57 
______________________________________ 
NA indicates that there were problems with the assay. 
These data show that combinations with G-CSF were not as effective as the 
combination of PIXY321 and SF. 
A summary of the four experiments confirms the utility of addition of a 
growth factor or a combination of growth factors to media to increase 
expansion of progenitor cells cultured ex vivo. 
EXAMPLE 2 
This example illustrates a clinical protocol providing myeloablative 
chemotherapy and peripheral blood progenitor cell expansion according to 
the present invention. The clinical protocol is designed to evaluate the 
effectiveness of hematopoietic reconstitution of bone marrow with 
peripheral blood derived (and GM-CSF recruited) hematopoietic progenitor 
cells that are expanded ex vivo with with a growth factor combination of 
SF and PIXY321. 
After full recovery from any previous cycle of chemotherapy (leukocyte 
count of greater than 3000/mm.sup.3 and platelet count of greater than 
100,000/mm.sup.3) GM-CSF (Sargramostim) bid is administered sc, at a dose 
of 5 mcg/kg/day. On days 6, 8 and 9 of GM-CSF recruitment growth factor, 
peripheral blood is collected. Leukophoresis is performed using a 9 liter, 
3 hour treatment with collection from set to obtain a preferential 
mononuclear cell collection from peripheral blood to form the population 
of hematopoietic progenitor cells. Cell counts of greater than 
2.times.10.sup.8 /kg are normally obtained from each peripheral blood 
collection. Autologous serum is also obtained from each patient to use in 
a progenitor cell expansion media. 
Hematopoietic progenitor cells obtained from peripheral blood and subject 
to leukophoresis are assayed for colony forming activity (CFU-GM, 
CFU-GEMM, BFU-e and Blast Cell) and phenotyped for CD34, CD33 and CD7. Ex 
vivo cultures are started with cell concentration of about 
5.times.10.sup.5 mononuclear cells/ml in McCoys medium supplemented with 
10% autologous serum, 100 ng/ml SF and 100 ng/ml PIXY321. Cells are 
cultured for 12 days with media changes on days 4 and 8. Cells are 
evaluated thrice weekly for cell counts, differential, progenitor cell 
capability and phenotype. After 12 days of expansion, nonadherent cells 
are harvested, washed to remove growth factor and cryopreserved in 
autologous serum in liquid nitrogen at a concentration range of 10.sup.5 
to 2.times.10.sup.7 cells/ml. 
Each patient undergoes a high dose, myeloablative chemotherapy protocol 
comprising cyclophosphamide (1875 mg/m.sup.2) infused over 1 hour on each 
of three successive days, cis-diaminodichloroplatinum (55 mg/m.sup.2 /day) 
infused continuously over three days (total dose 165 mg/m.sup.2 over 72 
hours) and BCNU on the last day of cytoreductive therapy at 600 mg/m.sup.2 
infused at a rate of 5 mg/m.sup.2 /min. 
Two days after the last day of high dose myeloablative chemotherapy, each 
patient is administered his or her expanded progenitor cells. The cells 
are thawed rapidly and infused iv at a rate of 10 cc/min of 10.sup.6 
cells/ml. The cells are administered in equal aliquots on each of three 
successive days. 
Beginning about three hours after the last cell infusion, GM-CSF is 
administered (iv or sc) at a dose of 12 mcg/kg/day for a total of 7 days. 
GM/CSF is further administered for another 14 days at a dose of 6 
mcg/kg/day (iv or sc). 
This procedure combines the antitumor benefits of myeloablative high dose 
chemotherapy with the ability of the present inventive method to expand 
hematopoietic progenitor to improve bone marrow, hematopoietic and 
immunologic rescue of patients. 
EXAMPLE 3 
This example illustrates a patient treatment schedule with autologous 
transplantation for hematopoietic rescue of myelotoxicity when 
myelotoxicity is caused by treatment with a group of cytoreductive cancer 
chemotherapeutic agents causing myeloreduction but not myeloablative 
results. Patients with solid tumors, such as small cell lung carcinoma 
(SCLC), colon carcinoma, or melanoma are pre-treated for one to seven days 
with SF at a dose of 100-4000 .mu.g/m.sup.2 /day, and PIXY321 at a dose of 
5-250 .mu.g/m.sup.2 /day. After pre-treatment, peripheral blood is 
collected and hematopoietic progenitor cells are isolated by a 
leukophoresis technique, such as one described in Kessinger et al., Blood 
74:1260, 1989. 
The progenitor cells are cultured and expanded for 7-14 days in McCoys 
medium, 10% autologous serum, SF at 1 .mu.m/ml and PIXY321 at 100 ng/ml. 
Media is changed every 4 days. During culturing of the peripheral 
blood-derived progenitor cells, patients undergo cytoreductive therapy at 
doses up to 50% higher than could otherwise be tolerated in the absence of 
hematopoietic rescue. 
After completion of cell expansion, cultured progenitor cells are washed to 
remove culture medium and growth factors and suspended in buffered saline. 
Supplemented with autologous serum, expanded progenitor cells are 
reinfused to the same patient (autologous transplantation) to effect a 
hematopoietic rescue from high dose cytoreductive chemotherapy. 
EXAMPLE 4 
This example illustrates a patient treatment schedule with autologous 
transplantation for hematopoietic rescue of myelotoxicity caused by 
cytoreductive treatment. Patients are pre-treated for 1-7 days with GM-CSF 
at a dose of 50-3000 .mu.g/m.sup.2 /day. After pretreatment, both 
peripheral blood and bone marrow are collected and hematopoietic 
progenitor cells are isolated. The isolated progenitor cells are cultured 
with SF and PIXY-321 as described in Example 3. Patients under 
cytoreductive therapy and the cultured hematopoietic progenitor cells are 
readministered in an autologous transplantation. 
EXAMPLE 5 
This example illustrates another patient treatment schedule with autologous 
transplantation for hematopoietic rescue of myelotoxicity caused by 
cytoreductive cancer therapy agents. The patient treatment schedule 
follows the schedule of Example 3, except bone marrow is removed instead 
of peripheral blood progenitor cells. 
EXAMPLE 6 
This example illustrates another patient treatment schedule with autologous 
transplantation for hematopoietic rescue of myelotoxicity caused by 
cytoreductive cancer therapy agents. The patient treatment schedule 
follows the schedule of Example 4 except only bone marrow is removed 
instead of both bone marrow and peripheral blood progenitor cells. 
EXAMPLE 7 
This example illustrates another patient treatment schedule with autologous 
transplantation for hematopoietic rescue of myelotoxicity caused by 
cytoreductive cancer therapy agents. The patient treatment schedule 
follows the schedule of Example 3, except both bone marrow and peripheral 
blood progenitor cells are removed, instead of only peripheral blood 
progenitor cells. 
EXAMPLE 8 
This example illustrates a patient treatment schedule with autologous 
transplantation of ex vivo stimulated hematopoietic progenitor cells 
followed by subsequent in vivo administration of an engraftment growth 
factor to improve implantation and proliferation of hematopoietic 
progenitor cells. Patients with solid tumors are pretreated for seven days 
with GM-CSF (10 .mu.g/kg/day) or PIXY321 (10 .mu.g/kg/day). Peripheral 
blood (two pints) or bone marrow is removed. Hematopoietic progenitor 
cells are separated by density gradient centrifugation using standard 
techniques. Separated mononuclear cells are cultured in minimum essential 
media supplemented with 10% autologous serum, pyruvate, 
penicillin-streptomycin-glutamine and growth factor (100 ng/ml SF and 100 
ng/ml PIXY-321) to form a culture of expanded hematopoietic progenitor 
cells. The cells are cultured in a humidified atmosphere (5% CO.sub.2) at 
a density between 0.1.times.10.sup.5 and 5.times.10.sup.5 cells/ml. The 
cultures are fed periodically, as needed, with medium and growth factor. 
The cultures are maintained for one to two weeks. 
The cells in culture are harvested, washed, and resuspended in a 
physiologic buffer supplemented with autologous serum. Approximately 
10.sup.7 to 10.sup.9 cells/kg are readministered to the patient one to 
three days following cytoreductive therapy. 
The patients are further treated in vivo with an engraftment growth factor 
(50 .mu.g/kg/day SF and 25 .mu.g/kg/day PIXY321) for 7 to 21 days 
beginning two days following readministration of expanded progenitor cells 
(cellular preparation).