WO1991018615A1 - Human peripheral blood cells in an immunocompromised host - Google Patents

Abstract

Human peripheral blood cell circulation is achieved in xenogeneic immunocompromised host with introduction of human fetal bone marrow into the host after irradiation of the host to substantially deplete the host of endogenous hematopoietic cells. The host remain viable for extended periods of time while demonstrating substantial levels of human blood cells in the peripheral blood.

Description

HUMAN PERIPHERAL BLOOD CELLS

IN AN IMMUNOCOMPROMISED HOST

INTRODUCTION Technical Field The field of this invention is the production of human hematopoietic cells.

Background

The blood cell system comprising the diverse cells emanating from a single stem cell is essential to the survival and well being of mammals. The cells serve to carry oxygen, cleanse the body of debris, inhibit the proliferation of pathogens, monitor for neoplastic cells, and provide a wide variety of factors essential for their own and the growth of other cells. Recently, chimeric mice were developed, where human fetal tissue was introduced into CB-17 scid/scid mice to provide the chimeric SCID-hu mouse. The mice were shown to maintain for extended periods of time, human fetal lymph node and thymus and provide for peripheral blood cells, when supplied with a source of hematopoietic stem cells from fetal liver. While in some instances, the maintenance of human peripheral blood cells could be extended for long periods of time, in all instances the human cells were only a very small proportion of the total number of peripheral blood cells, frequently substantially fewer than one percent. Furthermore, the circulating cells in these animals were shown to be T Cells.

In many instances, it would be desirable to have a relatively high proportion of the peripheral blood cells as human cells' and desirably have a significant proportion of the total number of hematopoietic cells being human cells. Further, it would be desirable to have circulatory myelomonocytic cells and red blood cells, besides lymphoid cells.

For many applications, it would be of substantial advantage to have a high absolute number of human cells. These include the opportunity for immunization to obtain a strong immune response from the human cells, studies of various diseases and their effect on the various hematopoietic cells and studies of drugs against various diseases and their effect on the hematopoietic cells. There is, therefore, substantial interest in providing for ways in which a non-human mammal may be provided with human blood cells for testing, research, and as a source of blood cells of the various lineages.

Relevant Literature EPA 0 322 240 describes the introduction of human fetal tissue in a CB-17 scid/scid mouse. See references cited therein.

SUMMARY OF THE INVENTION Xenogeneic bone marrow is introduced into an endogenous bone marrow depleted genetically immunocompromised mammalian host, where the bone marrow may be present as bone tissue or dispersed bone marrow, particularly from a fetal source. The fetal bone tissue may be conveniently introduced into the peritoneal cavity or the bone marrow may be injected into the long bone of the immunocompromised host.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Non-human mammals, particularly small mammals under 25kg, are provided having human peripheral blood cells present as at least two percent (2%) of the total peripheral blood cells, preferably at least five percent (5%) of the total peripheral blood cells and more preferably at least about fifteen percent (15%) . The enhanced level of human cells may be achieved by depletion of endogenous bone marrow, particularly stem cells, conveniently by irradiating the host with at least a sublethal dosage of radiation, generally being at least about 200 rad and varying with the size and nature of the host. The blood cells are then reconstituted with human bone marrow, conveniently as human bone or bone marrow cell suspensions, particularly from a fetal source. The human bone marrow source may be introduced before and after ablation to enhance the survival rate of the host upon ablation, particularly by irradiation.

Various immunocompromised hosts may be employed, where the host being immunocompromised is as a result of a natural mutation, breeding, transformation of embryonic cells to provide a transgenic mouse, or the like. Of particular interest are hosts which lack a functional lymphoid lineage. The lack of the immune system may be as a result of a lack of a particular organ, a genetic defect, such as an incompetent reco binase or regulatory gene regulating the expression of the recombinase, transport of slg and T cell receptors to the surface, non-functional major histocompatibility antigens, or the like. Of particular interest are CB-17 scid/scid mice and mice derived therefrom which may have further enhanced immunodeficiency, e.g. lack of natural killer (NK) cells.

The host is irradiated or treated in any convenient manner to substantially ablate the endogenous bone marrow. Conveniently, X-irradiation may be employed where the level of radiation will be selected to provide at least substantially complete ablation of the hematopoietic cell system, while maintaining a reasonable, preferably a high, proportion of viable hosts. With each type of host, the particular level of irradiation may be selected by screening for the level of remaining viable hematopoietic cells for the efficiency of the ablation. Other techniques for bone marrow ablation which may be employed by themselves or in combination include cytotoxic drugs, immunotoxins, antibodies to lymphokines and growth factors, antibodies to natural killer cells, etc. The selection of cytotoxic drugs would be based on their ability to clear quickly from the lymphatic and blood circulatory systems, so as to rapidly be reduced to a non-cytotoxic level, prior to or shortly after the administration of the human bone marrow.

The human bone marrow will normally be fetal bone marrow and may be employed as bone marrow slices, fragments, chunks, or the like, coming from various parts of the fetus, such as the femur, tibia, humerus or rib. The longest dimensions of human bone will generally be in the size range of about 8mm to 2cm, where the bone may be fragmented along the long axis, usually in not more than about four parts. Usually, the total volume of fetal bone which is introduced into the host will vary with the size of the host. For mice, the volume will generally be in the range of about 40mm³ to 1,200mm³. Alternatively, human fetal bone marrow cell suspensions may be injected into long bones, such as the femur or tibia. Generally the volume of the bone marrow suspension will be in the range of about 10ml to 50ml for a mouse.

The age of the host will generally be at least neonatal and usually not more than about six months, more usually not more than three months. The particular age is not critical to this invention, but younger animals are more convenient to handle and are more economical.

The site of introduction of the fetal bone portions may be interperitoneal, subcutaneous, mammary fat pad, or other convenient site.

After introduction of the bone marrow, the host is grown in accordance with conventional ways, feeding the host as appropriate, either with or without the presence of antibiotics in the feed. Conveniently, about 0.2% to 1.2% of sulfamethoxazole/trimethoprim (Septra) may be provided in the feed. As appropriate, additional introductions of the human fetal bone marrow may be made, usually not more than about two additional explants, generally at four to twenty week intervals.

The host may receive a variety of growth factors and cytokines, either native to the host or human. Interleukins, colony stimulating factors, hormones, or the like may be added, such as IL-1, IL-3, IL-6, and G-CSF. Generally, the amount of the additive will vary, depending upon the particular additive, the host, and the like. For example, in the case of IL-3 0.5 - 5 μg/day may be administered. In the subject host, it is found that the bone tissue shows variable cellularity from 10 to 80%. In addition, the human cells comprise a significant proportion of the blood cells in circulation in the peripheral blood. The cells may include members of the lymphoid, myelomonocytic and erythroid lineages. The cells may be detected by employing a wide variety of antibodies which are commercially available for the detection of human markers on blood cells, such as CD-3, -A , -8, -10, -13, -15, -19, -20, -33, -41, -45, -59, HLA, or the like. Conveniently, a blood sample may be taken and assayed employing a fluorescence activated cell sorter. Alternatively, the cells may be lysed and a Western blot employed. Other techniques which may find use for the quantitative determination of a particular cell type include the polymerase chain reaction, gel electrophoresis, HPLC, or the like. In addition to the bone marrow, other human organs may also be present, particularly thy us, lymph node, skin, liver, pancreas, tonsil, appendix, epithelium, kidney, etc. One or more mesenteric or peripheral lymph nodes may be introduced. The various organs may be introduced conveniently in the kidney capsule, mammary fat pad, particularly the fourth mammary fat pad for a mouse, the popliteal fossa, or other convenient site where the organ may be vascularized, lymphatic vessels connected and maintained by the host for a reasonable period of time, usually at least four weeks, preferably at least six weeks. Thus, the non-human mammal may provide for a significant portion of the human hematopoietic system with the continued regeneration of stem cells and generation of more than fifty percent (50%) of the different types of committed or mature cells. In this manner cells of the myelomonocytic lineage, such as macrophages, mast cells, neutrophils (PMNs) , eosinophils, basophils, monocytes, megakaryocytes, etc. or the lymphoid lineage, B lymphocytes, T lymphocytes, NK cells, ADCC cells, LAK cells, TIL cells, etc. may be propagated, studied or used for various purposes. The subject host may find a wide variety of uses by virtue of the significant presence of circulating human blood cells in the non-human host. The host may be employed for the production of human monoclonal antibodies or immortalized T lymphocytes. The host may be immunized in accordance with conventional ways with an appropriate immunogen, which may be injected intravascularly at an appropriate site. Of particular interest is injection at a site which is drained by a human lymph node introduced into the host. The immunized host may receive one or more booster shots, followed by removal of a lymphoid organ, e.g. lymph node, followed by immortalization, conveniently by fusion with an appropriate yeloid cell or transfection with Epstein-Barr virus. Alternatively, the cells may be cloned by any convenient means, at limiting dilution to provide for individual clones, and the supernatants of the individual clones may be screened for the binding affinity of the antibodies present in the host.

Alternatively, the host may be used for studying T and B lymphocyte interactions, in determining the manner of stimulation of T and B lymphocytes and the mechanism for the immune reaction. Individual lymphocytes may be cloned to evaluate their role in disease protection against disease, identify slg or T cell receptor variable regions, isolate the DNA encoding the variable regions, or the like. Various immunogens may be screened for their abilities as vaccines in producing antibodies which may be effective in neutralizing pathogens and the cells or DNA provide a source of the variable regions associated with the response.

Pathogens of interest are those particularly associated with humans, such as HIV, HTLV-I, and - II, human papilloma virus, cytomegalovirus, Epstein- Barr virus, hepatitis B virus, non-A, non-B hepatitis, chlamydia, malaria (Falciparum) etc. Also, the subject system may be used in the study of cancer in attempting to develop cytotoxic cells specific for a particular cancer type.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL Bone pieces were transplanted into either untreated mice (6-10 weeks old) or mice (6-10 weeks old) pre-treated with radiation from a cesium 137 source. The mice are treated with either whole body irradiation or irradiation of the long bones. Mice treated with whole body irradiation receive 200 to 400 rads on a single dose. Alternatively, the mice are treated with 600 rads after shielding of the thorax and abdomen with a lead shield. The mice are anesthetized with Nembutol prior to shielded irradiation. The mice were CB-17.5 scid/scid mice. In addition, some of the mice were treated with exogenous human IL-3 in two lμg doses per day. The fetal bone pieces are prepared from fetuses of 18-22 week gestation by first removing all of the soft tissues and cartilagenous portions from the bone. The bones are then cross sectioned at 2 - 5 mm intervals. The mice are prepared by injection ip of Nembutol. Once the mice are fully anesthetized, a 1 cm incision is made in the skin as well as the underlying peritoneum. The bone fragments are then placed into the peritoneal cavity randomly. Approximately 25mm to 1.5cm of a femur, tibia or humerus is implanted into each mouse. The mice which are irradiated are irradiated within 24 hours of the subsequent implantation of the fetal bone.

After two to six weeks, the percentage of human cells in the peripheral blood is determined by employing monoclonal antibodies specific for human hematopoietic cell markers. In un-irradiated mice a low level of human cells is observed in the peripheral blood by FACScan. The positive cells vary from about 0 - 1.6 percent. Furthermore, injection of IL-3 does not significantly affect the percentage of positive cells. In contrast, the mice irradiated before marrow transplantation showed between 1.5 and 30 percent human cells in the peripheral blood. These cells appear to be of the myelomonocytic lineage. They show no staining with T or B cell markers (CD 3, 4, 8, 19), although they do stain for the myeloid cell marker (CD33) . Furthermore, the human cells have a high side scatter profile indicating that they are of the myelomonocytic lineage. In addition, two of the four mice which were irradiated before implantation show low but significant levels of serum immunoglobulin after six weeks.

The above results demonstrate that one can obtain high levels of human cells in an immunocompromised mouse at least partially ablated of endogenous bone marrow, by implanting relatively large amounts of human bone marrow, particularly with the stromal bone tissue. The human bone marrow implanted after irradiation provides a mouse which results in substantial numbers of cells of human myelomonocytic lineage and can provide for cells of other lineages as well, such as lymphoid. By appropriate combinations of tissue in conjunction with the bone marrow one can provide inside the immunocompromised mouse a hematopoietic system mimicking the human hematopoietic system. Thus, one may study various processes, compounds, drugs, or the like, and their effect on the human hematopoietic system by cytologic or histologic analysis, selection, staining, or the like.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A non-primate genetically immunocompromised mammalian host comprising at least two percent circulating human blood cells of the total number of circulating blood cells in the blood stream of said host, said host being produced by substantially ablating endogenous bone marrow and implanting human bone marrow.

2. A non-primate host according to Claim 1, wherein said ablating is by at least sublethal irradiation.

3. A non-primate host according to Claim 1, wherein said human bone marrow is bone tissue.

4. A non-primate host according to Claim 1, wherein said human bone marrow is dispersed bone marrow.

5. A non-primate host according to Claim 1, wherein said host is murine.

6. A murine genetically immunocompromised host, wherein said host lacks functional B and T lymphocytes as a result of a genetic defect, said host comprising at least five percent circulating human blood cells of the total number of circulating blood cells in the blood stream of said host, said host being produced by substantially ablating endogenous bone marrow and implanting human bone marrow.

7. A murine host according to Claim 6, wherein said host is a CB-17 scid/scid mouse or progeny thereof.

8. A murine host according to Claim 7, comprising at least twenty-five percent of circulating human blood cells.

9. A.murine host according to Claim 6 , comprising human fetal bone marrow.

10. A murine host according to Claim 6 , comprising at least one of human lymph node, liver or thymus tissue.

11. A murine host according to Claim 6, wherein said host is at least six weeks old from the time of implantation of said human bone marrow.

12. A murine host according to Claim 6 comprising circulating human immunoglobulins.

13. A murine host according to Claim 6, wherein said murine host is irradiated with at least 200 rad.