WO1999030723A1 - Use of human umbilical cord blood for adoptive therapy - Google Patents

Abstract

Human Umbilical Cord Blood (HUCB) cells have shown promising advantages over bone marrow (BM) cells for transplantation. This study indicates that in addition to their use for transplatation (i.e. engraftment), another potential clinical use for HUCB cells is as an adjuvant therapy to enhance both endogenous hematopoietic reconstitution and immunocompetence of the host.

Description

USE OF HUMAN UMBILICAL CORD BLOOD FOR ADOPTIVE THERAPY

DOMESTIC PRIORITY CLAIM

The priority is claimed of U.S. Provisional Application No. 60/067,459 filed on December 4, 1997, which is hereby incorporated by reference herein in its entirety.

GOVERNMENTAL SUPPORT

The present invention relates to the restoration of the hematopoietic and immune systems of a host, and more particularly to the practice of a method for achieving these objectives that does not involve engraftment of stimulatory agents or cells.

BACKGROUND OF THE INVENTION

The relatively slow progress in the area of gene therapy for life-threatening diseases such as leukemia and other genetic disorders makes bone marrow (BM) transplantation the treatment of choice. Unfortunately, BM transplantation is plagued with major clinical complications, especially graft-verses-host disease (GVHD). Measures to prevent GVHD in BM transplant patients have been attempted by depletion of particular BM cell populations (e.g. T cells), and also by transplantation of only BM stem cells. There are, however, disadvantages with these strategies, such as the requirement for considerably large amounts of donor BM cells, graft failure and reocurrence of malignancy [1,2]. The clinical complications that are associated with BM transplantation imply a requirement for alternative strategies. Human Umbilical Cord Blood (HUCB) is a rich source of hematopoietic stem cells and, compared to BM, it has more repopulation capabilities [3,4]. In fact, HUCB cells are now being widely used for transplantation in a variety of diseases, including patients undergoing therapy for hematologic disorders [5-7]. Successful engraftment of HLA mismatched. HUCB cells has been reported, accompanied by little or no symptoms attributable to GVHD [8]. Perhaps, this lack of a requirement for an exact donor-recipient HLA match may be partly responsible for the successful use of HUCB in BM transplantation [9]. This may be partly explained by the immunologic immaturity and reduced functional properties of T-cells and other immune cells present in HUCB [10-14]. Another major advantage for use of HUCB cells is their low incidence of infection by cytomegalovirus and Epstein-Barr virus, bodi of which are associated with severe complications in BM transplantation [15, 16]. However, despite the advantages of HUCB over BM cells for transplantation, there is some evidence which indicates that HUCB cells engraft slower than BM

cells [iη.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method is disclosed for promoting the growth of the hematopoietic cells of a host comprising administering to the host an effective amount of human umbilical cord blood cells, active fragments thereof, mimics thereof and agonists thereof. The method of the invention contemplates the circumstance where said host has previously undergone therapeutic treatment that has caused myelosuppression. so that the method comprises a method for restoring the host hematopoietic system.

In a particular embodiment of the invention, the human umbilical cord blood cells, active fragments thereof, mimics thereof and agonists thereof, may be administered to the host by injection. One of the advantages of the invention is that this route of administration does not result in adverse sequelae such as the development of graft versus host disease. Rather the human umbilical cord blood cells promote the stimulation of the host progenitor cells to reconstitute their numbers.

A further aspect of the invention comprises the administration of human umbilical cord blood cells as a form of adjuvant therapy to restore the immune system of a host that may have been suppressed as by exposure to chemotherapy e.g. incident to cancer treatment. Again, the adverse results of cell transplant therapy may be avoided while achieving the salutary results of restoration of the normal immune response of the host.

A still further aspect of the invention contemplates a course of therapy wherein said human umbilical cord blood cells, active fragments thereof, mimics thereof and agonists thereof are co-cultured ex vivo with a quantity of the hematopoietic cells of said host, and the hematopoietic cells so treated are thereafter reintroduced into the host. For example bone marrow cells of the host may be withdrawn from the host and co-cultured with HUCB cells and then re-introduced to the host. Even this therapy would avoid the introduction of cells from a donor and the possibilities for rejection or other complication that attend such transplantation.

Accordingly, the present invention extends to the use of human umbilical cord blood cells as a part of an adjuvant therapy given with HLA-matched bone marrow for patients who require such transplantation. A further application would be to administer the human umbilical cord blood cells as an adjuvant therapy to patients who receive high dose chemotherapy or radiotherapy and either autologous or allogeneic bone marrow cells.

A yet further aspect of the invention would be to apply the administration of human umbilical cord blood cells to treat patients who suffer from hematopoietic and/or immunologic deficiencies resulting from genetic abnormalities, as well as those conditions that may arise from disease-related or trauma-related deficiencies, such as, by way of non-limiting example, the following: trauma - to stimulate endogenous hematopoiesis; radiation injury' - e.g. nuclear accidents; infectious diseases which cause myelosuppression and/or immunosuppression; innate (genetic) or acquired immunodeficiency diseases; innate or acquired (drug-induced) anemias; induction of tolerance for solid organ transplants; and non-malignant diseases associated with aging, e.g. osteoporosis.

Accordingly, it is a principal object of the invention to provide a method for treating deficiencies in hematopoietic and immune condition that is efficient and efficacious.

It is a further object of the invention to provide a method as aforesaid that reduces the likelihood of host rejection.

Other objects and advantages will become apparent from a review of the ensuing description which proceeds with reference to the following illustrative drawings. DESCRIPTION OF THE DRAWINGS

Figure 1. WBC counts in peripheral blood and CFU-GM in the BM and spleen of HUCB- injected mice. NK cell-depleted SJL/J mice were lethally irradiated and ώen injected with either HUCB cells or vehicle. At various times, either peripheral blood WBC counts were determined (A) or mice were sacrificed and die number of CFU-GM in the BM and spleen was determined in clonogenic assays (B) as described in Materials and Methods. The number of CFU-GM colonies in die BM and spleen of normal, age-matched mice was 68 ±4 and 30 ±6 respectively; n=9, ±SE.

Figure 2. Erythropoietic indices in peripheral blood of HUCB-injected mice. NK cell-depleted SJL/J mice were lethally irradiated and then injected with either HUCB cells or vehicle. At various times, a sample of peripheral blood was obtained for determination of RBC counts, and Hgb and Hct levels.

Figure 3. CFU-GM and WBC in peripheral blood in HUCB-injected mice that received 8.0 Gy irradiation. NK cell-depleted SJL/J mice were irradiated with 8.0 Gy and then injected with either HUCB cells or vehicle. At various time periods mice were either A) bled for WBC counts or, B) sacrificed for CFU-GM determination in the spleen and BM.

Figure 4. Allogeneic responses by lymphoid cells from HUCB-injected lethally irradiated SJL/J mice. NK-depleted SJL/J mice (H-2^S) were lethally irradiated and dien injected with either HUCB mononuclear cells or syngeneic BM. At day 17 after injection, lymph node cells from these mice were stimulated with 7-irradiated allogeneic spleen cells obtained from either Balb/c (H-2 ) or Balb.B (H-2¹⁵) mice. The responses of lymph node cells obtained from age-matched, unirradiated control mice are shown for comparison. Cell proliferation, shown on the y-axis as cpm xlO³,- was based on the amount of pHQTdR incorporated during die last 16-18 h of a 96 h incubation. Details of the procedure are described in the Materials and Methods.

Figure 5. Proliferative responses to T- and B- cell mitogens by splenocytes from HUCB- injected mice mat were give a lower dose radiation. NK cell-depleted SJL/J mice were irradiated with 8.0 G and ti en injected with either HUCB cells or vehicle. At 3 and 9 wks after injection, splenocytes were cultured with either Con A or LPS for 72 h. During the final 16-18 h, cell proliferation was determined by pHjTdR incorporation. The Δ cpm are represented on die y- axis. Details of the procedure are described in the Materials and Methods.

Figure 6. Proliferative responses to Mouse Mammary Tumor Virus superantigen (MMTV) by lymphoid cells from HUCB-injected SJL/J mice that were given a lower dose radiation. Lymph node cells were obtained from mice that were irradiated widi 8.0 Gy and then injected with either HUCB cells or vehicle. Cells were cultured with vSAg-expressing 7-irradiated RCS tumor cells for 96 h. During the final 16-18 h, cell proliferation was determined by pH]TdR incorporation. The Δ cpm axe represented on the y-axis. Details of the procedure are described in the Materials and Methods.

Figure 7. Effect of HUCB cells on 5-FU treated BM mononuclear ceils in LTC-IC assays. Human BM stromal cells were irradiated (150 Gy) one day prior to co-culture with 5-FU treated BM mononuclear cells with or without 7-irradiated (100 Gy) HUCB mononuclear cells (10, 8, 5, 2, 1/well). At various time periods, cells from each well were tryps in ed and analyzed for CFU-GM in shoπ term clonogenic assays. The change (Δ) in CFU-GM is represented at each time poin_t as the mean (±SD) of four different experiments. In each of the four experiments, cultures were performed in duplicate. At each time period, die number of colonies obtained for cultures at each of the 5 cell densities was totalled and die numbers of CFU-GM/26 5FU- cells are represented on the y-axis. CFU-GM were not detected in cultures containing only 7- irradia_ted HUCB mononuclear cells. Details of the procedure are described in die Materials and

Methods.

DETAILED DESCRIPTION OF THE INVENTION

A compelling problem that continues to frustrate clinicians is the untoward side effects that accompany conventional forms of treatment which result in myelosuppression in their patients. Since many therapeutic agents target cells of the hematopoietic system, efforts to identify ways to restore die hematopoietic cells which are damaged in padents mat receive these treatments are of paramount importance. We are exploring the potential use of Human Umbilical Cord Blood (HUCB) cells for adoptive ±erapy in such patients. There is increasing clinical use of HUCB cells to treat patients suffering from many diseases for which restoration of the hematopoietic system is required. In most hospitals, HUCB is still considered a medical waste product and is routinely discarded. However, the use of HUCB cells has numerous medical, practical and economic advantages over bone marrow for these purposes.

In most of the instances where HUCB cells have been transplanted to patients, great care is usually taken to select a donor with a close histocompatibility match to the recipient to ensure engraftment of the transplanted cells. However, our preliminary studies, using an experimental mouse model, suggest mat in addition to engraftment, transplanted HUCB cells may also facilitate endogenous hematopoiesis by the recipient's own progenitor cells. In this model, mice are rescued from the lethal effects of high-dose irradiation by injection of HUCB. Within several weeks after injection of HUCB cells, these mice are not only reconstituted hematopoietically, but they are fully immunocompetent. Without such therapy, irradiated mice succumb due to hematopoietic or immunologic failure. Since there is little evidence for the presence of either human cells or even human DNA in these mice, long-term survival of these animals is not due to engraftment of HUCB cells. Rather, injection of HUCB cells into these lethally irradiated mice leads to stimulation of me recipients own surviving progenitor cells to begin the process of hematopoietic reconstitution. We have also established an in vitro model (modified LTC-IC assay) using human 5-Fluorouracil (5-FU) treated bone marrow cells co-cultured with irradiated HUCB and, despite allogeneic differences, HUCB cells are still able to promote accelerated colony formation (CFU-GM) from the 5-FU treated marrow progenitor cells. Although others are using HUCB in lieu of BM with the hope of engraftment, this new mechanism to stimulate endogenous hematopoiesis has, heretofore, not been described.

Thus, our preliminary results suggest the exciting possibility that even despite histocompatibility mismatches, HUCB cells (or their products) can be used as an adjuvant therapy in padents to help replenish the hematopoietic and immunologic progenitor cells that are damaged as a result of conventional forms of treatment, such as radiation or chemotherapy. We are now beginning to define the mechanisms by which HUCB cells mediate their hematopoiesis-enhancing effects, and we believe the results of our investigation will help to establish a new form of adjuvant therapy that is beneficial to many patients in need of hematopoietic/immunologic reconstitution due to various disease-related or treatment-induced deficiencies. We previously reported d at HUCB cells can increase die survival of lethally irradiated SJL/J mice compared to non-injected mice [18, 19]. Long-term engraftment of the HUCB cells did not appear to be responsible for survival, suggesting that other mechanisms were operative. We therefore investigated possible mechanisms by which HUCB cells might enhance endogenous hematopoietic reconstitution. In the present study, we took advantage of die fact diat HUCB cells do not permanendy engraft in the SJL/J mice, and used tiiis model to determine odier functions for HUCB cells. We specifically examined whether HUCB cells can enhance endogenous hematopoietic reconstitution by residual, radioresistant host BM cells in irradiated SJL/J mice. We have also determined whedier HUCB cells can function as an immune adjuvant. This was addressed by studying the responses of lymphoid cells obtained from HUCB-injected mice to: 1) T-cell and B-cell polyclonal activators, 2) alloantigens and, 3) a syngeneic B-cell lymphoma that stimulates through a mouse mammary rumor viral-encoded superantigen, Mtv-29 (vSAg) [20]. A clinical relevance for the results obtained in the mouse model has also been addressed in this study using a stem ceil assay.

Materials and Methods

Cytokines and antibodies

Recombinant murine granulocyte-macrophage colony stimulating factor (rMuGM-CSF) was kindly provided by die Immunology Department of Genetics Institute (Cambridge, MA). Murine monoclonal fluorescein isothiocyanate (FΙTC)-conjugated anti-human CD34 (IgGl), murine monoclonal phycoerythrin (PE)-conjugated anti-human CD38 (IgGl) and, PE- and FITC- conjugated isotype controls were purchased from Caitag Laboratories (Burlingame, CA). Anti- CD45, CD3 and CD10 were conjugated to FITC and anti-CD19, CD 14 and CD56 were conjugated to PE. All were obtained from Becton Dickinson Immuncytometry Systems (San Jose, CA).

Human Umbilical Cord Blood

Approximately 15-50 ml of HUCB was collected into citrate phosphate dextrose (Sigma, St Louis, MO). Deliveries were routine, and subjects had no underlying disease or infection. The collection and use of HUCB for. this study was reviewed and approved by die Institutional Review Board of UMDNJ-New Jersey Medical School, Newark, NJ. Mononuclear cells were separated by Ficoll Hypaque (Sigma) density gradient centrifugation within 24 h of collection.

Adoptive Transfer

Female SJL/J mice, 6-8 wks, were obtained from me Jackson Laboratories (Bar Harbor, ME) and housed in d e AAALAC-accredited Research Animal Facility at UMDNJ-New Jersey Medical School, Newark, NJ. Mice were depleted of natural killer (NK) cells by retro-orbital injection (i.v.) of 100 μl rabbit anti-asialo GM1 (Wako Pure Chemicals, Osaka, Japan). Two weeks beyond tiiis injection, NK cells remain undetectable, based on a cytotoxicity assay that utilizes splenic effector cells and die NK-susceptible target cells, YAC-1 [18,21].

Twenty four hours later, mice were irradiated either lethally (9.5 Gy) or subletiially (8.0 Gy) by a cesium source (Mark 1 model 68-A-3 gamma irradiator, J.L. Shepherd, San Fernando, CA). After 1-2 h,. mice were injected i.v. witii 10⁷ HUCB mononuclear cells resuspended in PBS. Control mice were injected with comparable volume of PBS (vehicle control). Mice were then boused in a laminar flow environment in sterile cages with sterile bedding, food and water. At various-times diereafter, mice were analyzed for routine peripheral blood indices, lymphocyte functional assays, and granulocyte-macrophage colony-forming units (CFU-GM) in BM and spleen.

Immunofluorescence assays

The phenotypic profde for the expression of CD34, CD38, CD45, CD3, CD19, CD14, CD 10 and CD56 in HUCB mononuclear cells was determined in random samples. Cells were labeled for 30 mins at 4°C with specific fluorescein (FITC)- or phycoerythrin (PE)-conjugated antibodies. After labeling, cells were washed to remove unbound antibodies, fixed by resuspending in 1 % paraformaldehyde, and ti en analyzed by FACScan.

Clonogenic assays

Single cell suspensions from mice were prepared from either the femurs or spleens and then used in clonogenic assays for CFU-GM as described [22]. Briefly, cells were resuspended in culture medium and tiien plated in duplicate in methylcellulose at 10 /plate in a total volume of 1 ml. Due to the low numbers of cells recovered in non-injected, ledially irradiated mice, for these cultures, the total numbers of recovered cells from two femurs (up to 3x10 ) were plated in a single dish. Cultures were supplemented with 4 U of rMuGM-CSF. Colonies >20 cells were enumerated at day 10 of culture. Lymphocyte Responses

A single cell suspension of responder cells (R) was prepared from the lymph nodes or spleen of die HUCB-injected or non-injected SJL/J mice. Responder cells were cultured witii stimulators (S) obtained from either syngeneic 7-irradiated (7.5 Gy) B-lymphoma cells expressing vSAg (R/S =4), 7-irradiated (2.5 Gy) allogeneic (H2^b) spleen cells (R/S = 1) or the following mitogens: concanavalin A (Con A) at 1 μg/ ϊ; lipopolysaccharide (LPS) at 5 g/ml. Botii mitogens were purchased from Sigma. Cultures stimulated widi lymphoma cells were incubated for a total of 96 h and diose stimulated with eitiier allogeneic cells or mitogens were incubated for a total of 72 h. Cell proliferation was based on die incorporation of 1 μCi tritiated diymidine (specific activity 1.9 Ci mM; A ersham Life Inc., Arlington Height, IL). ³H-TdR incorporation was determined by harvesting cells onto glass fiber filters with an automated harvester. For each experiment, stimulation was performed in triplicate. The background cpm (responder cells alone or responder + stimulator cells alone) was subtracted from the cpm of stimulated cultures (A cpm).

BM stroma

BM aspirate was obtained from die posterior iliac crest of normal healthy volunteers. Samples were immediately placed into Iscove's medium (Life Technologies, Grand Island, NY) containing 50 U/ml preservative-free heparin. Informed consent was obtained from each donor according to d e guidelines of the Institutional Review Board of UMDNJ-New Jersey Medical School, Newark, NJ.

BM aspirate cells (4xl0⁶) were cultured in 12-well plates (Corning Costar, Cambridge, MA) in.a total volume of 2 ml medium which consisted alpha minimal essential media (α-MEM) (Life Technologies) containing 12.5 % FCS (Hyclone Laboratories, Logan, UT), 12.5 % horse serum (Hyclone Laboratories), 10^"7M hydrocoπisone (Sigma), 10^"*M 2-ME (Sigma) and 1.6 mM glutamine (Cellgro, Mediatech) (stromal medium). Cultures were incubated for 3 days at 33°C after which die mononuclear cells (BMNC) were separated from die non-adherent population by Fi oll-Hypaque density gradient centrifugation. BMNC were replated into culture flasks, which were reincubated with weekly 50% change of medium until confluency occurred.

Long-term culture-initiating cell assay (LTC-IC)

Confluent BM stroma prepared in 12-well plates were irradiated widi 150 Gy that was delivered by a cesium source (Mark 1 Model 68-A-3). After 24 h, non-adherent cells were replaced with fresh media containing quiescent BM mononuclear cells (1-10/well). Parallel cultures consisted of wells with 10⁵ 7-irradiated (100 Gy) HUCB cells. The radiation dose was established in LTC-IC assays with HUCB cells that were subjected to various dose of radiation (30-150 Gy). HUCB cells diat were given less dian 100 Gy proliferated in culture. During the culture period, 50% stromal medium was replaced weekly. At various time periods, cells from each well were trypsi ized and cultured in duplicate in short term BM cultures.

Quiescent BM mononuclear cells were prepared by incubating cells with 5-Fluorouracil (5-FU) (Hoffman La Roche Inc. , Nudey, NJ). This drug preferentially kill cells in cycling phase, while the quiescent population remains viable [23]. Cells (10⁷) were resuspended in 5 ml α-MEM containing 20% FCS and 200 μg/ml 5-FU for 7-10 days. The cycling states of die cells were determined by pulsing 10⁵ cells with 1 μC pHjTdR (35 Ci/mM, ICN Biomedicals Inc. , Irvine, CA) for 24 h. fHJTdR incorporation was determined as described for lymphocyte responses. By day 7, die dpm plateau at 215 ±24.

Results

We have previously shown [18,24] diat a majority of lethally irradiated, HUCB-injected mice survived for a significant period beyond die time when non-HUCB-injected mice succumbed. Indeed, 40% of HUCB-injected mice survived until 180 days, compared to 0% survival in irradiated non-HUCB-injected mice. Since evidence of permanent engraftment of HUCB cells was not obtained in tiiese mice, it appeared d at endogenous hematopoietic repopulation was responsible for the long-term survival. We therefore addressed die mechanism by. which such endogenous reconstitution might occur in HUCB-injected mice.

Phenotypic and Localization properties of injected HUCB cells

The phenotypic profde within die samples of HUCB mononuclear cells used in die study were determined in seven randomly selected samples. We measured die distribution of progenitor/stem cells (CD34), more matured progenitors (CD34/CD38), T-ceil (CD3), B-cell (CD 19), Thyl (CD45) and NK cells (CD56). The phenotypic distribution witiiin the mononuclear fractions used in our studies (Table 1) was consistent widi published reports [25].

NK cells can affect hematopoietic activity in cord blood cells [26]. However, in this study, we did not attempt to engraft HUCB cells in the mice. Therefore, d e significance of the 4 % NK cells witiiin die HUCB cells was not a concern for this particular model. Furthermore, the human NK cells would be irrelevant to the long-term endogenous hematopoietic reconstitution in the mice, since the repopulating cells are not of human origin [18,24]. However, we previously observed that recipient murine NK cells can affect endogenous hematopoietic reconstitution in HUCB-injected mice [24]. Therefore, recipient mice were depleted of NK cells prior to transfer of HUCB cells.

We next determined d e initial anatomic localization of die HUCB cells following i.v. injection. ^J1Cr-labeled HUCB cells were injected into mice that had received 9.5 Gy and on days 1 and 2, groups of animals were sacrificed, and die radioactivity in various tissues was determined as a percentage of the injected cpm (Table 2). Control irradiated mice received syngeneic ^5lCr-labeled BM cells. Following i.v. injection of HUCB cells, the highest percentage of injected cpm (50 %) was found in the liver on day 1, and tiiis percentage was essentially unchanged on day 2. When syngeneic BM cells were injected, high counts (15 %) were also found in die liver, but by day 2, tiiis dropped to about 9% of injected cpm. Differences between HUCB-injected and syngeneic BM-injected recipients were also noted on day 1 for spleen (2.5% vs 7.2%), BM (0.14% vs 0.5 %), and lung (0.07% vs 0.7%). However, by day 2, the differences between these groups in these organs decreased.

Effects on hematopoietic activity by HUCB in lethalty-irradiated mice

Due to the radioresistance of NK cells, and tiieir influence on hematopoiesis [27,28], mice were injected with an anti-NK antibody 24 h prior to administration of letiial radiation (9.5 Gy). This was followed by injection wid eitiier HUCB mononuclear cells or vehicle. At various time periods up to 3 weeks, peripheral blood indices were determined at selected interval.;. In botii HUCB-injected and vehicle-injected mice, tiiere was a precipitous drop in the WBC count to a nadir on day 10 (Figure 1A). After day 10, however, HUCB-injected mice showed accelerated return of WBC in comparison to non-injected mice. Indeed, by days 15-18, die peripheral blood WBC counts were 2200/μl in HUCB-injected mice, compared to only 500/μl in non-injected mice (Figure 1A). The WBC counts in normal age-matched controls were 8.8±0.6/_/ιl (n=26, ±SE). In contrast (Figure 2), erydiropoietic related peripheral blood cell parameters exhibited little, if any decreases at day 5 in comparison to die levels observed in age- matched controls (n=26, ±SE; Hct: 32± 1 %; Hgb: 11.6±0.4g; RBC: 7±0.2xlO^s/_/χl). There was a slight decrease in these parameters in botii groups of mice on day 10. Beyond day 10, however, whereas die red cells indices continued to fall in die non-HUCB-injected mice, these values increased to normal levels in mice that received HUCB cells.

We next studied myelopoiesis in the spleen and BM from botii groups of mice (HUCB- injected and non-injected) using clonogenic cultures that contained 10⁵ cells/ml. Based on die localization patterns, we cultured cells diat were obtained from the liver, spleen, and BM. In BM and spleen, tiiere was an increase in CFU-GM at day 10 d at continued dirough days 15-18 (Figure IB). By this time, the numbers of CFU/10⁵ cells (60 and 40 in BM and spleen, respectively), were approaching the levels measured in tissues from normal age-matched control SJL/J mice (68±4 for BM and 30±6 for spleen, ±SE; n=9). At none of diese time intervals, however, was CFU-GM detected in cells taken from the livers of diese mice. CFU-GM were also undetectable at diese times in BM or spleens of vehicle-injected mice, despite die plating of up to 3x10 cells per culture.

We ruled out die possibUity that die injected HUCB cells might contribute to the number of CFU-GM measured in the clonogenic assays. HUCB cells were used for CFU-GM assays in which rMuGM-CSF was added. No colony growti occurred when HUCB cells were included with rMuGM-CSF, but they responded well to rhuGM-CSF in these assays (data not shown).

Therefore, it is unlikely diat die injected HUCB are a direct source of any of the CFU-GM assayed wid die cells obtained from e mice in these experiments. Overall, these results indicate that HUCB cells are clearly involved in die process of hematopoietic recovery observed in the 7-irradiated mice, and appear to enhance the ability of surviving murine stem cells to begin endogenous repopulation.

Hemazopoietic activity in lower dose ^-irradiated mice

We next determined if die period in which HUCB potentiates hematopoietic recovery could be shortened in mice mat received a lower dose of radiation. Our rationale here was that at a lower dose, die surviving murine hematopoietic cells would include totipotent and multipotent cells compared to only totipotent cells remaining in letiialiy irradiated mice. Thus, following HUCB cell injection, die recovery period should be shorter. A secondary reason for using a lower dose was to be able to analyze the non-injected controls for a longer period of time, since at 9.5 Gy, practically all of the vehicle- injected mice succumbed between 2 and 3 weeks after irradiation. For diese experiments, NK-depleted mice were irradiated widi 8.0 Gy and tiien injected i.v. widi 10⁷ HUCB cells. At various time periods, WBC levels and CFU-GM in BM and spleen were determined. Although WBC levels and CFU-GM in HUCB-injected mice were greater than in vehicle- injected animals, die differences were not statistically significant (p >0.5) (Figures 3 and 3B). These results indicate diat with a lower dose of radiation, the level of endogenous reconstitution determined by the levels of progenitors and differentiated cells are comparable for mice that were injected widi eitiier HUCB cells or vehicle.

Recovery of lymphocyte function in lethally irradiated, HUCB-injected mice

Prompted by die accelerated recovery of WBC and CFU-GM seen in lethally irradiated mice injected widi HUCB cells, we also determined die recovery of lymphocyte function as measured by e ability of spleen cells to mount in vitro proliferative responses to murine alloantigens.

Three weeks after lethal irradiation and injection with eitiier syngeneic BM or HUCB cells, SJL (H-2¹) splenic responder cells were stimulated in mixed lymphocyte reaction (MLR) widi irradiated Balb.B (H-2^b) or Balb/c (H-2^*1) stimulator cells. As shown in Figure 4, spleen cells from HUCB-injected mice proliferated in response to alloantigens to a similar degree as spleen cells taken from syngeneic BM-injected mice. None of die mice that received 9.5 Gy irradiation alone survived for 3 weeks in this series of experiments. These results demonstrate that injection of HUCB cells into lethally irradiated mice also influences lymphopoiesis, causing a recovery of mature, alloantigen-responsive lymphocytes within a time period similar to that observed for erythroid and myeloid compartments (Figures 1A, IB and 2).

Recovery of lymphocyte function in mice that were given a lower dose radiation

In mice irradiated with 8.0 Gy, since no significant differences were observed in the myelopoietic compartment between the HUCB-injected mice irraαiated with die lower dose and die non-injected controls (Figures 3 A and 3B), we determined if HUCB cells can influence trer immunocompetence levels. The data presented in Figure 5 indicate diat at 3 weeks after administration of 8.0 Gy, responses to die polyclonal lymphocyte activators Con A (T-cell) and LPS (B-cell) are more prominent in irradiated mice that received HUCB cells dian in mice that were irradiated, but not injected widi HUCB cells. At 9 weeks after irradiation, T-cell responses to Con A were comparable in botii groups of mice, but B-cell responses to LPS in the HUCB- injected mice were closer to die response of age-matched unirradiated controls than, _mj_ce that only received irradiation (Figure 5).

In Figure 6, the proliferative response of cells from these same mice to a syngeneic B- cell Iymphoma was measured. Tsiagbe, et al [29] have shown that the response of SJL lymphoid cells to diese Iymphoma cells is stimulated by die expression of a mammary tumor provirus (Mtv)-encoded superantigen (vSAg) on the tumor cells (Mtv-29). Furthermore, the Mtv-29 vSAg stimulates T-helper (TH) cells that use a specific β-chain (VB16) in their T-cell receptor [30]. The data in Figure 6 show that the presence of tumor-responsive VB16+ TH cells is comparably low in bodi groups of mice at 3 weeks after radiation. At 9 weeks, however, the VB16+ TH cell response to syngeneic Iymphoma cells is significantly reconstituted only in the irradiated mice that received HUCB cells, although the response was not the same as that of age- matched normal control mice.

Overall, these results suggest that the ability to mount polyclonal T- and B- cell responses is reconstituted earlier in 8.0 Gy irradiated mice that receive HUCB cells. This is in keeping with the results seen at tiiis dose with the recovery of cells of other hematopoietic lineage (i.e. erythroid and myeloid). However, die results using more specific stimuli, such as the ability to mount a clonally restricted TH cell response to Mtv-vSAg, suggest that the return of selected, antigen-specific lymphocyte subsets proceeds more rapidly in irradiated mice diat also receive HUCB cells. To what degree tiiis is true for other clonal populations of antigen-specific lymphocytes remains to be fully determined.

Effects of irradiated HUCB cells in LTC-IC cultures

Although die rescue of lethally irradiated mice by injection of HUCB cells demonstrated the biological relevance of the model, we could not exclude d e possibility diat some of the observed hematopoietic-inducing results were due to a "xenogeneic" effect. Therefore, we next determined if die ability of HUCB cells to stimulate hematopoiesis in the xenogeneic murine model could also be demonstrated with allogeneic human hematopoietic stem cells, using the LTC-IC assay.

Modified LTC-IC assays were performed with quiescent human BM cells in the presence or absence of 7-HUCB mononuclear cells. Beginning on day 10, cells from each well were trypsinized and the number of CFU-GM was determined in short term clonogenic assays. As shown in Figure 7, the presence of 7-HUCB cells considerably shortened die period by which die quiescent (5-FU treated) human stem cells proliferated. CFU-GM were detected by day 15 in 7-HUCB-containing cultures and maximal CFU-GM were observed at day 30. In contrast, in control cultures without HUCB, CFU-GM were not detected until day 40. Furthermore, it took twice as long (60 days) for d e control cultures to reach maximal levels of colony formation compared to die time for HUCB-containing cultures. No CFU-GM were observed in parallel culmres with 7-HUCB cells alone. These results show that HUCB cells can potentiate human hematopoiesis by a mechanism that does not require their own proliferation, and suggest die potential clinical benefits of using HUCB cells therapeutically. Discussion

HUCB cell ώerapy rescues mice from irradiation deadi [18]. It appears that most of die injected HUCB cells initially localize to the liver (Table 2), although the significance for this localization has yet to be determined. The data indicate diat HUCB cells mediate die recovery of die endogenous hematopoietic and immunologic systems in NK-depleted, Iedially irradiated SJL/J mice. Therefore, this mouse model is a potentially useful experimental system to study a heretofore unrecognized property of HUCB cells in clinical application. Although the HUCB cells injected into lethally irradiated SJL/J mice may initially survive and provide transient protection- from acute radiation damage, long-term engraftment of HUCB cells in diese mice is unlikely [18, 19,24]. Nonetheless, ou results using the SJL/J model show d at HUCB cells can provide significant benefits for enhanced hematopoietic reconstitution by endogenous stem ceils in lethally irradiated mice (Figures 1A, IB, 3A and 3B). However, tiiere was not a significant effect on hematopoiesis following injection of HUCB cells into mice that received a lower dose (8.0 Gy) of irradiation. There are two possible explanations for these results. First, there may be survival of a sufficient number of stem/progenitor cells in these mice to initiate endogenous hematopoiesis without a need for exogenous stimulation. Secondly, although not tested for, ic is possible diat HUCB-injected mice can mount an immune response which destroys die HUCB ceils before tiiey can fully perform their hematopoiesis-enhancing function. Moreover, these two mechanisms are not mutually exclusive.

Our results also show that HUCB cells exhibit an adjuvant-like activity for reconstitution of selected immune responses (Figures 4, 5, and 6). Especially significant is the enhancement of antigen-specific responses by mice that received HUCB cells (Figure 6). These latter results are especially important since, in addition to d e advantages of HUCB cells over BM cells for transplantation, ou results suggest additional clinical benefits of using HUCB cells. The adjuvant-like functions of HUCB cells suggest that they can potentially be used in situations where immune stimulation may be necessary, such as patients wi cancer or infectious disease. Furthermore, with regard to application in humans, the immunologic adjuvant property of HUCB gives diese cells a dual role, since they can simultaneously engraft and diminish die immunosuppression that can lead to secondary opportunistic infections.

Cell surface markers on HUCB cells may partially explain the combined hematopoietic and immune adjuvant effects observed in tiiis study. Compared to BM, MHC Class II molecules are more densely expressed on HUCB stem cells [31]. Recent studies indicate that MHC Class II is involved in autologous hematopoietic reconstitution in subleuially irradiated dogs [32]. This suggests that part of the hematopoietic effects observed by HUCB cells could be attributed to die high expression of MHC Class II molecules on their stem cells. In addition, CD 10 expression in HUCB cells might also be important, since this cell surface marker has an endogenous endopeptidase activity that can utilize as its substrate, several peptides diat are relevant to hematopoiesis [33-35].

In vitro, irradiated (7-) HUCB cells enhance the proliferation of human stem cells (Figure 7). Despite their inability to proliferate, this effect could be mediated by die release of early acting cytokines by the 7-HUCB cells. However, Santois, et al [36] have shown d at HUCB cells do not exhibit a dramatic-difference in tiieir ability to produce relevant hematopoietic cytokines when compared to peripheral blood mononuclear cells. Therefore, it is more likely that the 7- HUCB cells stimulate the BM stroma to produce cytokines which in turn are capable of upregulating stem cell activiry. The mechanisms by which tiiis stem cell activation occurs is a current focus of our ongoing investigation, since the interaction between HUCB and stromal cells appears important to botii die immune adjuvant, as well as the hematopoietic enhancing effects observed in HUCB-injected mice.

In addition to inducing endogenous stem cell proliferation (Figure 7), injection of HUCB ce'ls also leads to reconstitution of differentiated hematopoietic cells as judged by the reappearance of WBC in the peripheral blood of lethally irradiated mice (Figure 1A). This suggests that the presence of HUCB cells not only influences the induction of stem cell proliferation, but also their differentiation. In fact, tiiis is supported by the results of the LTC-IC cultures, where we observed botii an accelerated appearance and increased quantity of CFU-GM (Figure 7) in cultures containing 7-HUCB cells. The reason for the decrease at later time periods in the number of CFU-GM in cultures with 7-HUCB is not readily apparent, but could be due to cell deatii. Trypan blue dye exclusion indicated good viability of cells in these cultures, however, a time-dependent induction of apoptosis cannot be excluded at this time. This area of investigation is currently being addressed, and will provide further insight into the mechanisms of HUCB-mediated endogenous, hematopoietic reconstitution.

The results of this study can be significantly useful for application to the current problems associated with die continued use of allogeneic BM transplantation for treatment of many hematopoietic disorders [37]. Complications associated with GVHD continue to be a medical challenge. Although depletion of T-cells from the donor BM has reduce die incidence of GVHD, loss of this cell population can have negative effects on engraftment, such as an increased rate of reoccurrence of disease [38,39]. This study suggests diat HUCB cells could very well be replacing the engra tment benefits of having T-cells present, but without dieir deleterious effects of potentiating GVHD. Associated with a lower risk of developing GVHD appears to be a less stringent need for exact donor-recipient HLA matching when HUCB is used for transplantation [40], benefits that have been attributed to die immaturity of HUCB cells [40].

Our results suggest yet another unrecognized benefit of using HUCB cells in lieu of BM for such patients, namely, the ability of HUCB cells to stimulate endogenous hematopoietic repopulation. Because the HUCB cells do not show long-term engraftment in the mouse model, we were able to focus our analysis on this function of HUCB, exclusively. However, in situations- here HUCB transplants have been performed in human patients, engraftment of the transplanted cells would actually mask the hematopoiesis-enhancing function of the transplanted HUCB cells. Indeed, since care is usually taken to match the donor-recipient for HLA loci, the hematopoiesis-enhancing function of HUCB would be difficult to measure, and would go largely unnoticed.

In summary, the data presented in tiiis study indicate diat HUCB cells facilitate the ability of radioresistant endogenous stem cells to reconstitute the hematopoietic and immunologic systems of lethally irradiated SJL/J mice. Although the mechanisms by which this occurs are yet to be fully determined, the data suggest that HUCB can be used in novel treatment regimens to stimulate endogenous repopulation in patients who currendy require BM transplantation. Furthermore, these results suggest that HUCB may also be of potential therapeutic value for immune stimulation. If, as our data suggest, these properties of HUCB cells also occur following transplantation in humans, our observations would be highly significant in lieu of the shortage of human donors, and the widespread controversy regarding xenotransplantation. Table 1. Phenotypic distribution in cord blood mononuclear cells.

HUCB mononuclear cells were labeled with either FITC- or PE- conjugated monoclonal antibodies. The percentages of labeled cells were determined by FACScan.

Table 2. Distribution of ^slCr-Iabeled HUCB or BM cells in irradiated SJL/J mice.

Post % Injected/Organ (cpm)

Injection Spleen Liver BM Lung Blood

HUCB ceUs

24 h 2.5±0.9 50.0±12.3 0.14±0.04 0.07±0.02 l.O± l.S

(n=5)

48 h 2.1±0.9 48.0±10.0 0.13±0.05 0.06±0.03 0.07±0.07 (n=6)

BM cells

24 h 7.2±2.7 15±1.6 0.5±0.2 0.7±0.3 0.6±0.2 (n=3)

48 h 4.1±0.6 9.3±1.5 0.2±0.03 0.3±0.1 0.8±0.1

(n=5)

Mice were lethally irradiated 1 day prior to intravenous injection with ⁵¹Cr-Iabeled HUCB cells (1.5xl0⁷ cells = 2.6x10* cpm) or ^Cr-labeled BM cells (10⁷ cells = 0.5xl0⁶ cpm). Labeling was performed by incubating cells (10⁷) with 200 μCi ⁿCr in 1 ml volume for 90 min. At 24 and 48 h post-injection, animals were sacrificed and the radioactivity present in various tissues determined. The following is a list of documents related to the above disclosure and particularlv to the experimental procedures and discussions. The documents should be considered as incorporated by reference in their entirety.

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This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A method for promoting the growth of the hematopoietic cells of a host comprising administering to said host an effective amount of human umbilical cord blood cells, active fragments thereof, mimics thereof and agonists thereof.

2. The method of Claim 1 wherein said host has previously undergone therapeutic treatment that has caused the myelosuppression of said host, and said method comprises a method for restoring the host hematopoietic system.

3. The method of Claim 1 wherein said human umbilical cord blood cells, active fragments thereof, mimics thereof and agonists thereof, are administered by injection into said host.

4. The method of Claim 1 wherein said human umbilical cord blood cells, active fragments thereof, mimics thereof and agonists thereof are co-cultured ex vivo with a quantity of the hematopoietic cells of said host, and said hematopoietic cells so treated are thereafter reintroduced into said host.

5. A method for restoring the immune response of host, comprising the method of Claim 1.