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WO2001030369A1 - Procede d'induction d'une tolerance immunologique pour des greffons heterologues - Google Patents

Procede d'induction d'une tolerance immunologique pour des greffons heterologues Download PDF

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WO2001030369A1
WO2001030369A1 PCT/US2000/028982 US0028982W WO0130369A1 WO 2001030369 A1 WO2001030369 A1 WO 2001030369A1 US 0028982 W US0028982 W US 0028982W WO 0130369 A1 WO0130369 A1 WO 0130369A1
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cells
primate
porcine
tnf
tgf
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PCT/US2000/028982
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Maria A. Giovino
John Douglas Jackson, Jr.
Rodney L. Monroy
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Biotransplant, Inc.
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Priority to AU13378/01A priority Critical patent/AU1337801A/en
Publication of WO2001030369A1 publication Critical patent/WO2001030369A1/fr

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    • C12N5/0602—Vertebrate cells
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    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow

Definitions

  • the present invention relates to methods of enhancing hematopoiesis of donor progenitor cells following xenogeneic transplant, especially into a primate, such as a human.
  • a path for eliminating the shortage of donor organs for allotransplantation is to develop the technologies required to transplant non-human organs into humans, i.e., xenotransplantation.
  • the development of clinical xenotransplantation will also allow for the transplantation of non- human cells and tissues.
  • the species considered to be a primary potential source of such xenogeneic organs is the pig.
  • a certain strain of the domesticated pig denoted miniature swine, is considered ideal partly because of its similar size to humans.
  • the proposed use of pigs as organ donors in xenotransplantation would obviate problems associated with the consideration of non-human primates as donors.
  • xenografts generally, the transplanting of organs, tissues or cells between animals of different species
  • pathogens that cause disease are known to infect both humans and non-human primates, such as the transmission of HIV from the chimpanzee to humans.
  • chimpanzees and orangutans the closest non- human primates phylogenetically, are endangered species and far too rare to be considered for organ transplantation purposes.
  • baboons are too small to be an appropriate donor for most organ transplants. Even the largest baboons weigh less than 40 kg.
  • gestation times and productivity of primates would not allow a commercially significant generation of source animals.
  • MHC self major histocompatibility
  • the present invention solves this problem by providing a means of inducing tolerance (i.e., reducing the severity and/or eliminating the immunological response to the transplant), especially to porcine tissue, using hematopoietic stem cells, especially porcine hematopoietic stem cells (pHSCs).
  • a means of inducing tolerance i.e., reducing the severity and/or eliminating the immunological response to the transplant
  • porcine tissue especially porcine tissue
  • hematopoietic stem cells especially porcine hematopoietic stem cells (pHSCs).
  • the present invention relates to a means of inducing tolerance to cells, tissues, and organs of a xenogeneic source by transplanting pHSCs that subsequently engraft in the recipient bone marrow and proliferate, differentiate, and function immunologically as self cells in the recipient.
  • TGF- ⁇ transforming growth factor beta
  • TNF- ⁇ tumor necrosis factor alpha
  • the present invention relates to the just described process wherein the bone-marrow-derived cells are hematopoietic progenitor cells. In another embodiment these cells may be peripheral blood stem cells.
  • myeloablative regimen comprises removal from said primate of a sample of bone marrow- derived cells followed by in vitro depletion of either CD2 + cells or both CD2 + cells and CD20 + cells from said sample, and subsequent reintroduction of said bone marrow cells back into said primate.
  • Such process will also commonly employ whole body irradiation to destroy all remaining hematopoietic stem cells.
  • Another embodiment of the present invention employs a non- myeloablative conditioning process selected from the group consisting of whole body irradiation, commonly using a non-lethal dose, administration of horse anti-human thymocyte globulin (ATG), thymic irradiation, splenectomy, anti-T cell antibodies as well as co-stimulatory blockade molecules such as anti-CD40 Ligand antibody and CTLA4lg and any combination of the foregoing.
  • a non- myeloablative conditioning process selected from the group consisting of whole body irradiation, commonly using a non-lethal dose, administration of horse anti-human thymocyte globulin (ATG), thymic irradiation, splenectomy, anti-T cell antibodies as well as co-stimulatory blockade molecules such as anti-CD40 Ligand antibody and CTLA4lg and any combination of the foregoing.
  • the present invention relates to a process wherein said xenogeneic bone marrow-derived cells are hematopoietic progenitor cells.
  • the methods of the present invention employ xenogeneic bone marrow-derived cells that are porcine cells.
  • cytokines that are porcine cytokines.
  • these cytokines may be selected from the group consisting of interleulin-3 (IL-3), stem cell factor (SCF), and granulocyte-monocyte- colony stimulating factor (GM-CSF).
  • IL-3 interleulin-3
  • SCF stem cell factor
  • GM-CSF granulocyte-monocyte- colony stimulating factor
  • the primate used in the present invention is a human.
  • the present invention also provides methods for enhancing engraftment that employ a specific TGF- ⁇ antagonist selected from the group consisting of anti-TGF- ⁇ antibodies, soluble TGF- ⁇ receptors, latency-associated peptide (LAP) and lysofilline, including active fragments thereof.
  • a specific TGF- ⁇ antagonist selected from the group consisting of anti-TGF- ⁇ antibodies, soluble TGF- ⁇ receptors, latency-associated peptide (LAP) and lysofilline, including active fragments thereof.
  • the present invention also provides methods for enhancing engraftment that employ a specific TNF- ⁇ antagonist selected from the group consisting of anti-TNF- ⁇ antibodies and soluble TNF- ⁇ receptors, including active fragments thereof.
  • a specific TNF- ⁇ antagonist selected from the group consisting of anti-TNF- ⁇ antibodies and soluble TNF- ⁇ receptors, including active fragments thereof.
  • the present invention utilizes a mixture of antagonists that comprises an anti-TGF- ⁇ antibody, or an active fragment thereof, and an anti-TNF- ⁇ antibody, or an active fragment thereof.
  • both antibodies are monoclonal antibodies, or active fragments thereof.
  • TGF- ⁇ transforming growth factor beta
  • TNF- ⁇ tumor necrosis factor alpha
  • Figure 1 is illustrative of the growth of porcine or primate (cynomolgus monkey) BMC (bone marrow cell) cultures on a primate stroma in the presence of pSCF (porcine stem cell factor) and plL-3 (porcine interleukin-3) over a period of four weeks, (a) Total cell number per well, (b) total colony forming units per well.
  • the space indicates a change in scale with 350 being the end of the first scale and the start of the second scale. Error bars represent ⁇ 1 SEM (standard error of the mean) for six (6 ) separate experiments.
  • Results in Figure 1 show that porcine hematopoiesis is not maintained to the same extent as primate hematopoiesis on a primate stroma.
  • Figure 2 illustrates the growth of porcine and primate BMCs cultured together on a primate stroma in the presence of pSCF and plL- 3 over a period of four weeks. At each week the pig and primate cells were separated from the co-cultures to determine contributions from each BMC population, (a) Total cell number per well, (b) total colony forming units (CFU) per well. Error bars represent ⁇ 1 SEM from six separate experiments. Figure 2 shows porcine and primate cell number and CFU content after separation from mixed co-cultures using immunomagnetic selection with species specific antibodies.
  • Figure 3 depicts the growth of porcine BMCs grown in Transwell ® inserts above no stroma, porcine stroma and primate stroma alone. All cultures were grown in the presence of pSCF and plL-3. (a) Total cell number per well, (b) total colony forming units per well. Error bars represent + 1 SEM for three separate experiments. As demonstrated in Figure 3, porcine progenitors were best maintained and expanded when cultured above porcine stroma, indicating that cells in the lower chamber can influence cell growth in the upper chamber, presumably mediated by soluble factors.
  • Figure 4 depicts the growth of porcine BMCs grown in Transwell ® inserts above: primate stroma alone, porcine BMCs on primate stroma, primate BMCs on primate stroma and porcine BMCs and primate BMCs on primate stroma. All cultures were grown in the presence of pSCF and plL-3. (a) Total cell number per well, (b) total colony forming units per well. Error bars represent ⁇ 1 SEM for three separate experiments. Figure 4 shows that when additional BMCs (porcine, primate or both) were added to primate stroma in the lower chambers, there was a further marked decline in total cell and CFU numbers as compared to primate stroma alone.
  • Figure 5 depicts the growth of porcine BMCs grown on primate stroma in the presence of porcine SCF and porcine IL-3 with no antibody; with blocking antibody to TGF- ⁇ ; with blocking antibody to TNF- ⁇ ; with blocking antibodies to TGF- ⁇ and TNF- ⁇ .
  • Figure 6 depicts the growth of primate BMCs grown on primate stroma in the presence of hSCF (human stem cell factor) and hlL-3 (human interleukin-3) with no antibody; with blocking antibody to TGF- ⁇ ; with blocking antibody to TNF- ⁇ ; with blocking antibodies to TGF- ⁇ and TNF- ⁇ .
  • hSCF human stem cell factor
  • hlL-3 human interleukin-3
  • Figure 7 depicts the growth of porcine BMCs grown on porcine stroma in the presence of pSCF and plL-3 with no antibody; with blocking antibody to TGF- ⁇ ; with blocking antibody to TNF- ⁇ ; with blocking antibodies to TGF- ⁇ and TNF- ⁇ . a) Total cell number per well b) Total colony forming units per well. The addition of the blocking antibodies resulted in a relatively small effect on cell and CFU output.
  • Figure 8 depicts the growth of porcine and primate allogeneic cultures with species specific SCF and IL-3.
  • the figure depicts results for porcine cells with no inhibitory cytokine, or with 1 ng/ml huTGF- ⁇ , or with 1 0ng/ml huTGF ⁇ ; primate cells with no inhibitory cytokine, or with 1 ng/ml huTGF- ⁇ , or with 10ng/ml huTGF- ⁇ .
  • Figure 9 illustrates the growth of porcine and primate allogeneic cultures with species specific SCF and IL-3: porcine cells with no inhibitory cytokine; with 1 ng/ml pTGF- ⁇ ; and with 10ng/ml pTGF- ⁇ ; primate cells with no inhibitory cytokine; with 1 ng/ml pTGF- ⁇ ; and with 1 0ng/ml pTGF- ⁇ .
  • Figure 10 illustrates the growth of porcine and primate allogeneic cultures with species specific SCF and IL-3: porcine cells with no inhibitory cytokine; with 1 ng/ml huTNF- ⁇ ; and with 10ng/ml huTNF- ⁇ ; primate cells with no inhibitory cytokine; with 1 ng/ml huTNF- ⁇ ; and with 1 0ng/ml huTNF- ⁇ .
  • the porcine cells were inhibited to a much greater extent than primate cells, similar to the results with TGF- ⁇ . (m indicates monkey, p indicates porcine).
  • Figure 1 1 illustrates the growth of porcine and primate allogeneic cultures with species specific SCF and IL-3: porcine cells with no inhibitory cytokine; with 1 ng/ml pTNF- ⁇ ; and with 10ng/ml pTNF- ⁇ ; primate with no inhibitory cytokine; with 1 ng/ml pTNF- ⁇ ; and with 1 0ng/ml pTNF- ⁇ .
  • Figure 1 2 demonstrates that addition of either Latency Associated Peptide (LAP) or soluble TGF receptor II (sTGFrll) results in a similar improvement of the growth of porcine BMCs as achieved using the anti-TGF antibody.
  • Figure 1 3 shows that when the addition of soluble TNFrl and II is compared to the use of anti-TNF- ⁇ antibody the results are equivalent.
  • LAP Latency Associated Peptide
  • sTGFrll soluble TGF receptor II
  • Figure 1 4 shows that the addition of LAP to anti-TNF- ⁇ antibody has equivalent results as compared to the anti-TGF- ⁇ /anti-TNF- ⁇ antibodies combination.
  • a state of mixed hematopoietic chimerism be developed in which both donor and recipient hematopoietic stem cells exist in the recipient, thereby allowing the recipient to reestablish immunocompetence, which retaining the ability to recognize the donor antigens as self.
  • the present invention relates to methods of increasing the extent of mixed hematopoietic chimerism as a means of inducing tolerance to xenogeneic organs, tissues and cells, especially following transplant. Induction of such tolerance has been found to be facilitated by the addition of porcine cytokines, which have an effect on pHSC engraftment and enhances the level of engraftment [See: Giovino et al. (1997) Xenotransplantation 4: 1 12-1 19; Hawley et al. US Patent Nos. 5,589,582, 5,858,963, and 5,863,528], the entire contents of which are incorporated herein by reference). It is also necessary to create niches for the progenitor stem cells to engraft.
  • inhibitory factors that influence the growth of pHSCs are present in the primate bone marrow microenvironment.
  • the effects of such inhibitory factors may be overcome by the addition/administration of reagents that inhibit or otherwise eliminate or reduce the activity of the inhibitors.
  • anti-transforming growth factor-beta (anti-TGF- ⁇ ) and anti-tumor necrosis factor alpha (anti-TNF- ⁇ ) antibodies are present in in vitro cultures of pHSCs being cultured on primate stroma the total cell and colony forming unit numbers (CFUs) are increased.
  • Hematopoiesis is known to be regulated by a complex interaction of bone marrow cells, stromal cells and growth factors. Hematopoietic lineage restricted stem cell growth has been shown to be inhibited by the addition of exogenous TGF- ⁇ to long-term bone marrow cultures (LTBMC) Van Ranst et al. 1996. Experimental Hematology 24: 1059- 1 51 5. Treatment of such cultures with anti-TGF- ⁇ antibody has resulted in a five-to-twenty fold increase in non-adherent cells in the cultures by week 4 when compared to untreated controls (Waegell et al. 1994. Experimental Hematology 22: 1051 -1057).
  • TNF- ⁇ a monocyte/macrophage product
  • HPPCFCs high proliferative potential colony forming cells
  • Rubin et al discloses murine monoclonal antibodies to human TNF.
  • Other investigators have described rodent or murine antibodies to recombinant human TNF which had neutralizing activity in vitro (Lian et al. ( 1 986) Biochem, Biophys. Res Comm. 137:847-854; Meager et al. (1 987) Hybridoma 6: 305-31 1 ; Fendly et al. (1 987) Hybridoma 6: 359- 369; Bringman et al. (1 987) Hybridoma 6: 489-507; Hirai et al. (1 987) J. Immunol. Methods 96: 57-62; Jumming et al US Patent 5,91 9,452).
  • Anti-human TNF- ⁇ antibodies are commercially available, e.g. from R & D Systems, Minneapolis, MN).
  • Anti-human TGF- ⁇ antibodies are also well described in the literature and are commercially available, e.g. from R&D Systems, Minneapolis, MN.
  • the term "antibody” is meant to include polyclonal antibodies, monoclonal antibodies (MAbs), chimeric antibodies, humanized antibodies, and anti-idiotypic (anti-Id) antibodies, as well as fragments, regions or derivatives thereof, including Fab and F(ab') 2 , and provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.
  • Such anti-TNF- ⁇ and anti-TGF- ⁇ antibodies of the present invention are capable of binding portions of TNF- ⁇ and TGF- ⁇ , respectively, that inhibit the binding of TNF- ⁇ (or TGF- ⁇ ) to TNF- ⁇ (or TGF- ⁇ ) receptors.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites.
  • MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein. (1 975) Nature 256:495-497; U.S. Pat. No. 4,376, 1 1 0; Ausubel et al, eds., (1 987) 1 992. Current Protocols In Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, N.Y., and Harlow and Lane.
  • Antibodies A Laboratory Manual Cold Spring Harbor Laboratory; Colligan et al. 1 992, 1 993. eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., the contents of which references are incorporated entirely herein by reference.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof.
  • a hybridoma producing a MAb of the present invention may be cultivated in vitro, in situ or in vivo.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable regions derived from a murine MAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine MAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric MAbs are used.
  • Chimeric antibodies and methods for their production are known in the art (Cabilly et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3273- 3277; Morrison et al. (1984) Proc. Natl. Acad. Sci.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen- binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the MAb with the MAb to which an anti- Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein entirely incorporated by reference.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-ld antibody.
  • the anti-anti-ld may be epitopically identical to the original MAb which induced the anti-Id.
  • antibodies to the idiotypic determinants of a MAb it is possible to identify other clones expressing antibodies of identical specificity.
  • the antibodies of the present invention can include at least one of a heavy chain constant region, a heavy chain variable region, a light chain variable region and a light chain constant region, wherein a polyclonal Ab, monoclonal Ab, fragment and/or regions thereof include at least one heavy chain variable region or light chain variable region which binds a portion of TNF- ⁇ (or TGF- ⁇ ) and inhibits and/or neutralizes at least one TNF- ⁇ (or TGF- ⁇ ) biological activity.
  • Preferred antibodies of the present invention are high affinity human-murine chimeric anti-TNF- ⁇ (or TGF- ⁇ ) antibodies, and fragments or regions thereof, that have potent inhibiting and/or neutralizing activity in vivo against human TNF- ⁇ (or TGF- ⁇ ).
  • the immunoglobulin gene can be from any vertebrate source, such as murine, but preferably, it encodes an immunoglobulin having a substantial number of sequences that are of the same origin as the eventual recipient of the immunoreceptor peptide. For example, if a human is treated with a molecule of the invention, preferably the immunoglobulin is of human origin.
  • TNF- ⁇ (or TGF- ⁇ ) receptor constructs for linking to the heavy chain can be synthesized, for example, using DNA encoding amino acids present in the cellular domain of the receptor.
  • TNF- ⁇ (or TGF- ⁇ ) receptor constructs for linking to the heavy chain can be synthesized, for example, using DNA encoding amino acids present in the cellular domain of the receptor.
  • TNF- ⁇ (or TGF- ⁇ ) receptor fusion proteins using portions of the receptor that are known to bind TNF- ⁇ (or TGF- ⁇ ).
  • Anti-TNF- ⁇ (or TGF- ⁇ ) peptides and/or MAbs of the present invention can be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier, diluent or excipient selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1 .0 to 5, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • administration can be provided as a daily dosage of anti-TNF- ⁇ peptides, monoclonal chimeric and/or murine antibodies of the present invention 0.1 to 100 mg/kg, per day, for at least one day, or alternatively, at least one week, or any combination thereof, using single or divided doses of every 24, 1 2, 8, 6, 4, or 2 hours, or any combination thereof.
  • TNF- ⁇ Since circulating concentrations of TNF- ⁇ tend to be extremely low, in the range of about 10 pg/ml in non-septic individuals, and reaching about 50 pg/ml in septic patients and above 100 pg/ml in the sepsis syndrome (Hammerle, A. F. et al., 1 989, ) or can only be detectable at sites of TNF- ⁇ -mediated pathology, it is preferred to use high affinity and/or potent in vivo TNF- ⁇ -inhibiting and/or neutralizing antibodies, fragments or regions thereof, for both TNF- ⁇ immunoassays and therapy of TNF- ⁇ -mediated pathology. Such antibodies, fragments, or regions, will preferably have an affinity for hTNF- ⁇ , expressed as K a , of at least 1 0 8 M ⁇ more preferably, at least 10 9 M " ⁇ most preferably 10 10 MA
  • TGF- ⁇ The circulating concentrations of TGF- ⁇ are in the range of 1 -50 ng/ml (Wakefield et al. (1 995) Clinical Cancer Res. 1 : 1 29-1 36; Kyrtsonis et al. (1 998) Med Oncol. 15: 1 24-1 28).
  • Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5- 95% by weight based on the total weight of the composition.
  • anti-TNF- ⁇ (or anti-TGF- ⁇ ) peptides or antibodies can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
  • the vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) .
  • the formulation is sterilized by commonly used techniques.
  • TGF- ⁇ antagonists include agents which block, diminish, inhibit, or interfere with TGF- ⁇ activity and may include anti-TGF- ⁇ antibodies, soluble TGF- ⁇ receptors, or small molecule inhibitors such as lysofylline.
  • TNF- ⁇ antagonists include agents which block, diminish, inhibit, or interfere with TNF- ⁇ activity and may include anti-TNF- ⁇ antibodies, soluble TNF- ⁇ receptors, or small molecule inhibitors such as lysofylline.
  • anti-TNF- ⁇ and anti-TNF- ⁇ are anti-TNF- ⁇ and anti-TNF- ⁇ and anti-TNF- ⁇ .
  • TGF- ⁇ antibodies are administered together.
  • anti-TNF- ⁇ antibody is administered in conjunction with a anti-TGF- ⁇ agent other than an antibody.
  • anti-TGF- ⁇ antibody is administered in conjunction with an anti-TNF- ⁇ agent other than an antibody.
  • TGF- ⁇ and TNF- ⁇ antagonists other than antibodies are administered.
  • TGF- ⁇ is secreted in the form of an inactive complex non- covalently linked to latency associated peptide (LAP). Once the two components are cleaved TGF- ⁇ becomes active (Ribeiro SM, (1 999) J. Biol. Chem. 274 1 3586-1 3593). LAP has been shown to inhibit the activity of TGF- ⁇ (Bottinger et al. ( 1 996) Proc. Natl. Acad. Sci. USA. 93: 5877-5882). For this reason it is feasible to substitute the addition of anti-TGF- ⁇ antibody with LAP.
  • Soluble TNF receptors have been shown to block the activity of TNF- ⁇ , either occurring naturally in vivo (Aderka et al. ( 1 992) J.Exp.Med. 175: 323-329: Pinckard et al. (1 997) J. Immunology 158: 3869-3873) or by the addition of soluble receptors to block effects of TNF- ⁇ (Eliaz et al. 1 996. Cytokine 8:482-487; Camussi and Lupia. (1 998) Drugs 55: 61 3-620). Since the soluble TNF receptors can inhibit TNF activity it is reasonable to consider their use within our system.
  • Soluble TGF- ⁇ receptor II has been shown to inhibit the activities of TGF- ⁇ (Smith et al. (1 999) Circ. Res. 84: 1 21 2-1 222; Komesli et al. (1 998) Eur. J. Biochem. 254: 505-51 3). These results suggest that sTGFrll could be used to substitute for TGF- ⁇ antibody.
  • the present invention relates to a process for enhancing engraftment of xenogeneic hematopoietic cells in a primate receiving xenogeneic bone marrow- derived cells, comprising administering to said primate, separately or as a mixture, of an effective amount of a transforming growth factor beta (TGF- ⁇ ) antagonist and an effective amount of a tumor necrosis factor alpha (TNF- ⁇ ) antagonist.
  • TGF- ⁇ transforming growth factor beta
  • TNF- ⁇ tumor necrosis factor alpha
  • the present invention includes a process for enhancing engraftment of xenogeneic hematopoietic cells in a primate receiving xenogeneic bone marrow- derived cells, comprising:
  • TGF- ⁇ transforming growth factor beta
  • TNF- ⁇ tumor necrosis factor alpha
  • the xenogeneic cells may be bone marrow cells or bone-marrow-derived cells, including peripheral blood stem cells, and preferably hematopoietic cells, most preferably hematopoietic progenitor cells.
  • step (b) there is optional administration before, during or after step (b), of an effective amount of at least one cytokine derived from the donor species.
  • step (c) there is administration to said primate, separately or as a mixture, of an effective amount of a transforming growth factor beta (TGF- ⁇ ) antagonist and an effective amount of a tumor necrosis factor alpha (TNF- ⁇ ) antagonist, the latter two substances being administered either separately or as a mixture.
  • TGF- ⁇ transforming growth factor beta
  • TNF- ⁇ tumor necrosis factor alpha
  • the conditioning regimen may be either a myeloablative regimen or a non-myeloablative regimen.
  • said regimen is myeloablative
  • the method comprises complete elimination of the recipient's hematopoietic progenitor cells such that without the administration of exogenous cells (in accordance with the present invention this would comprise autologous and xenogeneic cells) the patient would die.
  • the method of the present invention therefore comprises prior removal from said primate, especially a human patient, of a sample of cells, such as bone marrow cells or bone marrow-derived cells, especially autologous hematopoietic cells, followed by, preferably, in vitro depletion of either CD2 + cells or, preferably, both CD2 + cells and CD20 + cells from said sample, and subsequent reintroduction of said cells back into said primate, this latter step being carried out at some point in the overall procedure, along with the sample of xenogeneic cells, with the exact timing left to the sound discretion of the researcher and/or clinician conducting such procedure.
  • a sample of cells such as bone marrow cells or bone marrow-derived cells, especially autologous hematopoietic cells
  • non-myeloablative conditioning regimen may comprise, but is not limited to, whole body irradiation, administration of horse anti-human thymocyte globulin (ATG), thymic irradiation, splenectomy, and/or any combination of the foregoing.
  • non- myeloablative conditioning regimen involves partial elimination of the recipient's (i.e., the primate's) hematopoietic progenitor cells through the use of non-lethal doses of radiation, or anti-T cell antibody, or chemicals such as cyclophosphamide.
  • non-myeloablative steps may be conducted in combination and in any temporal sequence, as dictated by the needs and exigencies of the overall procedure being conducted and as determined by the researcher and/or clinician conducting said procedure.
  • the conditioning regimen may be either a myeloablative or a non- myeloablative procedure, as dictated by the needs of the procedure being carried out.
  • the xenogeneic bone marrow cells introduced into the recipient animal, or primate may be any type of bone marrow-derived cells or hematopoietic progenitor cells as required by the procedure being conducted and as dictated by the needs and preferences of the researcher and/or clinician.
  • the xenogeneic bone marrow cells introduced into the recipient animal as part of the present invention are hematopoietic stem cells, most preferably porcine hematopoietic stem cells.
  • the process of the present invention also optionally provides for the administration to the recipient animal of a sample of a cytokine, preferably a mixture of cytokines, and most preferably a mixture of cytokines derived from the same species as the xenogeneic donor organism, the latter preferably being a pig.
  • a cytokine preferably a mixture of cytokines, and most preferably a mixture of cytokines derived from the same species as the xenogeneic donor organism, the latter preferably being a pig.
  • the donor animal is a pig
  • the xenogeneic bone marrow cells or bone marrow- derived cells, such as hematopoietic progenitor cells, and cytokines are both porcine in origin.
  • the cytokines are selected from the group consisting of interleulin-3 (IL-3), stem cell factor (SCF), and granulocyte-monocyte-colony stimulating factor (GM-CSF).
  • the recipient animal such as a human or other primate, will also receive a dosage of substances comprising TGF- ⁇ and TNF- ⁇ antagonists.
  • such TGF- ⁇ antagonist is selected from the group consisting of anti-TGF- ⁇ antibodies, soluble TGF- ⁇ receptors, and latency-associated peptide (LAP) including active fragments thereof and said TNF- ⁇ antagonist is selected from the group consisting of anti- TNF- ⁇ antibodies and soluble TNF- ⁇ receptors, including active fragments thereof.
  • the TGF- ⁇ antagonist is an anti-TGF- ⁇ antibody, or an active fragment thereof, and the TNF- ⁇ antagonist is an anti-TNF- ⁇ antibody, or an active fragment thereof.
  • active fragments can include, but are not limited to. Fab and F(ab') 2 fragments which commonly retain the antigen specificity and affinity of the complete antibody molecule.
  • Such active fragments can also include heavy-light chain dimers as well as dimers made up solely of the variable regions of the antibodies.
  • Antibodies useful for the present invention also include bispecific antibodies. Such antibodies may be polyclonal or monoclonal and can include chimeric antibodies, humanized antibodies and other types of altered antibody molecules.
  • Antibodies useful in practicing the present invention include recombinant as well as plasma derived antibodies. Where such antibodies are recombinant in nature, these may be produced in cells of any type that can synthesize and secrete antibody molecules, including mammalian cells in culture, especially myeloid and Chinese hamster ovary (CHO) cells, as well as plant cells.
  • the polypeptides making up the antibodies useful for practicing the present invention may also be synthesized by chemical means without the need for intermediary cellular production.
  • the present invention also relates to processes for enhancing the growth rate of bone marrow cells in vitro comprising growing said cells on a stromal layer in a culture medium comprising species specific cytokines (i.e., cytokines derived from the same species as the bone marrow cells) and further comprising a growth-stimulating amount of a transforming growth factor beta (TGF- ⁇ ) antagonist and a growth- stimulating amount of a tumor necrosis factor alpha (TNF- ⁇ ) antagonist.
  • species specific cytokines i.e., cytokines derived from the same species as the bone marrow cells
  • TGF- ⁇ transforming growth factor beta
  • TNF- ⁇ tumor necrosis factor alpha
  • the TGF- ⁇ antagonist is selected from the group consisting of anti-TGF- ⁇ antibodies, soluble TGF- ⁇ receptors, latency-associated peptide (LAP) and lysofilline, and active fragments thereof.
  • the TNF- ⁇ antagonist is selected from the group consisting of anti-TNF- ⁇ antibodies and soluble TNF- ⁇ receptors, and active fragments thereof.
  • the bone marrow cells are hematopoietic stem cells, most preferably porcine hematopoietic stem cells.
  • the stromal cells are either porcine stromal cells or primate stromal cells and, in a most preferred embodiment, the bone marrow cells are porcine hematopoietic stem cells.
  • the primate is selected from the group consisting of baboons, cynomolgus monkeys and humans.
  • the enhanced growth rate is measured as either an increase in the total number of cells or an increase in the number of colony forming units, with the latter being preferred.
  • Said enhanced growth rate may also comprise an increase in both total cell number and colony forming units.
  • the amounts of any and all substances employed either for inducing immunological tolerance or enhancing cell growth rate are understood to be “effective amounts” of said substances and additives.
  • the term “effective amount” means at least the minimum amount of the substance required to produce the desired biological effect and is in no way limited to the particular amounts and dosages recited in the following examples.
  • the "effective amount” of any such substance or additive may need to be determined for the particular experiment or clinical procedure being carried out and may also depend on the identity of the recipient and donor species as well as the species from which the particular substance or additive is derived. Exact dosages are therefore left to the sound discretion, based on the sound experience and knowledge of the prevailing art, of the researcher and/or clinician conducting the procedure.
  • doses of antibodies should not be less than 1 0 ⁇ g/kg/day, which would constitute a minimum effective amount.
  • Pig femurs and humeri were collected from a herd of MHC class I and II inbred miniature swine (Sachs et al. (1 976) Transplantation: 22:559). Primate femurs were obtained from cynomolgus monkeys, Macaca fascularis (Covance, Alice, TX).
  • Porcine bone marrow cells were harvested from the femurs and humeri by aseptically scraping with bone curettes and flushing the cavity with Dulbecco's phosphate buffered saline without calcium and magnesium (D-PBS, Gibco, Grand Island, NY) containing 5% citrate- phosphate-dextrose solution (Sigma, St. Louis, MO) and 20 ⁇ g/ml gentamycin (Gibco). Cells were washed 2 times at 2000 rpm for 5 minutes in D-PBS, resuspended in D-PBS then layered over Histopaque (sp.gr. 1 .077; Sigma) and centrifuged at 400 x g for 25 minutes.
  • D-PBS Dulbecco's phosphate buffered saline without calcium and magnesium
  • Cibco citrate- phosphate-dextrose solution
  • Gibco 20 ⁇ g/ml gentamycin
  • IMDM Iscove's Modified Dulbecco's Media
  • FBS fetal bovine serum
  • DNase U/ml DNase
  • the non-adherent cells were loaded directly into a sterilized elutriator system (Beckman Instruments, Palo Alto, CA) using a Beckman JE-6B rotor system equipped with a 40 ml chamber. Elutriation was performed using a constant rotor speed of 2040 rpm and cells were separated by increasing the flow rate. Cells (1 -9 x 1 0 9 ) were loaded at a flow rate of 45 ml/min. After all the cells had entered into the chamber, the media flow rate was increased to 50 ml/min and a first fraction (700 ml) was collected.
  • a sterilized elutriator system Beckman Instruments, Palo Alto, CA
  • Elutriation was performed using a constant rotor speed of 2040 rpm and cells were separated by increasing the flow rate.
  • Cells (1 -9 x 1 0 9 ) were loaded at a flow rate of 45 ml/min. After all the cells had entered into the chamber, the
  • Fraction 2 (700 ml) was collected by increasing the flow rate to 75 ml/min. After collecting the second fraction, media flow and rotor were turned off and chamber contents (fraction 3) were harvested aseptically under a biological hood. Consistent with previous reports (Monroy et al. 1 984. Exp Hematol: 1 2:384; Wagner et al. 1 988. Blood 72:1 1 68) the 75 ml/min fraction was enriched for CFU-Mix and BFU-E bone marrow progenitors.
  • This population was further enriched by depleting T-cell and myeloid populations utilizing porcine specific anti-CD2 (MSA-4) and anti-myeloid(74-22-1 5A) monoclonal antibodies (provided by David H. Sachs, Massachusetts General Hospital, Charlestown, MA).
  • Antibodies were added at a concentration of 1 5 ⁇ g/1 x10 9 cells in 5 mis of D-PBS- with 0.5% BSA for 30 minutes at 4° C. Cells were then centrifuged for 5 min at 2000 rpm, resuspended in 10 ml of PBS + BSA and washed again. Following the wash cells were then incubated with Dynal M450 magnetic beads according to the manufacturer's instructions. The unbound cells were used in these experiments as a population enriched for porcine progenitor cells.
  • Preformed primary porcine stromal cell cultures were set up using fraction 3 of elutriated BMC. Then 1 x 10 6 cells/well were plated into 24 well plates coated with 1 % gelatin (Sigma) in 1 ml of LTBMC medium (M-199 base medium (Gibco), 10% FBS, 10% horse serum
  • stromal cell cultures from primate bone marrow were established and treated similarly except that 2 x 10 6 cells/well were plated and the cultures were initially grown at 37°C for the first week and at 33°C for subsequent weeks. Media were demi- depleted and supplemented with fresh media at weekly intervals until a confluent adherent cell layer was present ( - 3 weeks).
  • stromal cell cultures were irradiated with a dose of 10 Gy using a cesium irradiator (J.L.Shephard, San Fernando, CA).
  • the enriched pig BMC were seeded onto irradiated stromal cells at 2.5 x 10 5 cells/well in a 1 ml volume.
  • Porcine growth factors stem cell factor (SCF), interleukin-3 (IL-3), GM-CSF) have been cloned (Hawley et al. 1 997. Xenotransplantation 4: 103-1 1 1 ) and expressed in Pichia pastoris.
  • 2ng/ml of rplL-3 (recombinant porcine interleukin-3) and 25ng/ml of rpSCF (recombinant porcine stem cell factor) were added to the LTBMC media at the initiation of the cultures and at each weekly feeding.
  • Porcine cytokine concentrations used in this study were determined in titration experiments in long term bone marrow culture to provide the highest percentage of long term growth of pig cells (data not shown). Cultures were grown at 37°C, 5% CO2. and 95% humidity. At weekly intervals media was demi-depleted and replenished with fresh media containing the appropriate growth factors. Adherent and non-adherent cells from 2 wells for each parameter were combined and enumerated at weekly time points. One aliquot from each harvest was assayed for CFU activity, and a second aliquot was used to assess cell morphology by microscopic examination after Diff- Quik (Baxter, MacGaw Park, ID staining of cytospin preparations.
  • Primate CD34 + cells were seeded onto primate stroma at 5x10 4 cells per well per ml, also in pSCF + plL-3. These cultures were set up as controls for the co-culture experiments where porcine and primate cells were cultured together on a primate stroma to evaluate survival of porcine cells in this mixed environment.
  • Porcine enriched (2.5x10 5 ) and primate CD34 + (5x10 4 ) BMCs were cultured together on a primate stroma in the presence of porcine cytokines to determine if porcine cells could survive in a primate hematopoietic microenvironment. These cell doses contained roughly equivalent number of pig and primate CFUs in the initial inoculum. To determine survival of porcine cells within mixed co-cultures, cells were physically separated from each other. To separate porcine BMCs from primate BMCs after mixed co-culture, an anti-pan pig antibody was used.
  • porcine BMCs were plated on collagen coated Transwell * (Costar, Cambridge MA) inserts at 2.5x10 5 cells per insert above various parameters. Parameters below Transwei s included: no stroma, porcine stroma alone, primate stroma alone, primate cells on primate stroma, porcine cells on primate stroma and primate and porcine cells on primate stroma. Cultures were fed weekly with pSCF and plL-3, cells from the Transwelfs were counted and plated in CFU assays for determination of progenitor content.
  • Recombinant human and porcine TNF- ⁇ and TGF- ⁇ were added to cultures of porcine cells on porcine stroma and to primate cells on primate stroma.
  • FICOLLTM separated BMCs were depleted of mature cells using anti-T-cell and myeloid antibodies.
  • Cells were then plated at 2.5x10 5 cells per well in the presence of pSCF (25ng/ml) and plL-3 (2ng/ml) onto irradiated porcine stromal cells.
  • Recombinant human TNF- ⁇ (R&D Systems), recombinant porcine TNF ⁇ (Endogen, Cambridge, MA), recombinant human TGF- ⁇ (R&D Systems) and recombinant porcine TGF- ⁇ (R&D Systems) were added to cultures at 1 and 1 0 ng/ml.
  • cells were harvested and placed into CFU assays for assessment of progenitors.
  • FICOLLTM separated primate BMCs were depleted of T and B cells by incubation with anti-CD2 and anti-CD20 antibodies (anti-Leu-5b and anti-Leu-1 6, Becton Dickinson, San Jose, CA) followed by depletion with mouse IgG magnetic beads (Dynabeads M450, Dynal). Cells were plated at 2.5x10 5 per well and cultured in 1 00 ng/ml of hSCF and hlL-3 in the presence or absence of human and porcine TNF- ⁇ and TGF- ⁇ .
  • Adherent and non-adherent cells were combined to assess progenitor cell content. Briefly, non-adherent cells were aspirated, wells were then rinsed with PBS and adherent cells were treated with trypsin EDTA (Gibco) for 2 minutes at 37°C and then pooled together.
  • trypsin EDTA Gibco
  • Progenitor cell content of porcine LTBMC was assayed by plating 25,000-1 00,000 cells in 1 .1 % methylcellulose (Stem Cell Technologies, Vancouver, Canada) supplemented with 30% FBS, porcine GM-CSF (5ng/ml), plL-3 (2 ng/ml); pSCF (25 ng/ml), and 2 U/ml human Erythropoeitin (Epo) (R&D Systems). Colonies ( > 50 cells) were counted after 10-1 4 days in culture at 37°C, 5% CO 2 . Each factor was individually titrated for its colony stimulating activity and concentrations that gave maximum stimulation were used in the assay.
  • hlL-3 100 ng/ml
  • hlL-6 lanterleukin-6
  • hSCF 100 ng/ml
  • hEpo 2 U/ml
  • hLIF Leukemia Inhibitory Factor
  • hGM-CSF 10 ng/ml
  • Porcine and primate BMCs were cultured on a primate stroma for
  • porcine CFUs There were fewer porcine CFUs than primate at each time point.
  • porcine BMCs did not flourish as well as primate BMCs on a primate stroma.
  • Previous studies have shown that maintenance of long-term hematopoiesis of porcine cells in these cultures occurs only in the presence of pig cytokines, although not to the same extent as when cultured on porcine stroma (Giovino et al. 1997. Xenotransplantation 4:1 12).
  • FIG. 2 shows porcine and primate cell number and CFU content after separation from mixed co-cultures using immunomagnetic selection with species specific antibodies. Cultures of primate or porcine cells alone on a primate stroma were also analyzed as a control for the separated cultures. A marked inhibition of porcine cell and CFU production in the mixed co-cultures was observed at all time points as compared to porcine cells alone on primate stroma. In contrast, little or no inhibition of primate cells occurred in these cultures. Thus porcine cell growth appears to be actively inhibited in the presence of primate cells.
  • porcine BMCs were cultured in Transwell * inserts above various culture conditions. Throughout the culture period, cells in the upper chamber were evaluated for cell number and the presence of porcine hematopoietic progenitors. Culture systems utilized in the lower chambers were as follows: no stroma, primate stroma and porcine stroma. As demonstrated in Figure 3, porcine progenitors were best maintained and expanded when cultured above porcine stroma, indicating that cells in the lower chamber can influence cell growth in the upper chamber, presumably mediated by soluble factors.
  • porcine cells were cultured in the upper chambers above the following: CD34 + primate BMCs on primate stroma, porcine BMCs on primate stroma and both porcine and primate BMCs on primate stroma.
  • Figure 4 shows that when additional BMCs (porcine, primate or both) were added to primate stroma in the lower chambers, there was a further marked decline in total cell and CFU numbers as compared to primate stroma alone. From these results, it appears that soluble factors released from the primate stroma are inhibitory to porcine BMCs and that this effect is enhanced by the addition of either porcine or primate BMCs to the primate stroma.
  • FIG 10 Shown in Figure 10 are the effects of human TNF- ⁇ on primate and porcine BMCs. Porcine cells were inhibited to a much greater extent than primate cells, similar to the results with TGF- ⁇ .
  • Figure 1 1 shows the effects of porcine TNF- ⁇ on primate and porcine BMCs. Results again show that pig cells were more sensitive to the inhibitory effects of porcine TNF- ⁇ . The results obtained from primate cultures show that porcine TNF- ⁇ appeared to be stimulatory, resulting in 2-fold increases in CFU production at 3 to 6 weeks.
  • Major histocompatibility complex-inbred (MHC-inbred) miniature swine are used in these studies.
  • Baboons Paperio anubis, Biological Resources, TX are recipients of the porcine BM.
  • BM cells are depleted in vitro of CD2 + cells alone or CD2 + and CD20 + cells. Processed BM cells are frozen until infused into the baboon after completion of the conditioning regimen. More details are presented in Kozlowski et al., (1 998) Transplantation. 66: 1 76.
  • the non-myeloablative conditioning regimen consists of (i) whole body irradiation (WBI) in two fractions of 1 .5 Gy on day -6 and day -5; (ii) horse antihuman thymocyte globulin (ATG) 50 mg/kg i.v. daily on days -3, -2, -1 ; (iii) thymic irradiation (TI) of 7 Gy on day -1 ; (iv) splenectomy on day 0; EIA followed by porcine DM infusion on day 0 with subsequent EIAs and porcine BM infusion if necessary.
  • WBI may be replaced by either higher doses of BM.
  • TI may be replaced by antibody treatment.
  • Costimulatory blockade molecules e.g., CTLA4 Ig and anti-CD1 54 (anti-CD40 ligand) antibody may be administered.
  • CTLA4 Ig and anti-CD1 54 (anti-CD40 ligand) antibody may be administered.
  • Such procedures are described in Sykes PCT application WO 97/21836, Sachs US patent 5,876,708; Sykes PCT application WO 99/39726; Sykes and Sayegh, PCT application WO 99/39727 ), the contents of which references are incorporated entirely herein by reference.
  • Pharmacologic immunosuppressive treatment may consist of cyclosporine ( 1 5-20 mg/kg/day for days 0-27 or longer as necessary).
  • Xenotransplantation. 6: 1 7-27 are administered as follows: porcine IL-3 and pSCF are administered at 100 ⁇ g/kg/day from days 0-14 and porcine GM-CSF is administered at 100 ⁇ g/kg/day from days 14-28.
  • Anti-TNF- ⁇ and anti-TGF- ⁇ antibodies are administered from days
  • PCR polymerase chain reaction
  • results show that some combinations do work as well as both antibodies in cultures of porcine cells on a primate stroma.
  • Combinations compared to anti-TGF- ⁇ and TNF- ⁇ were as follows; anti-TNF- ⁇ antibody and LAP; and sTNFrl&ll and sTGFrll.
  • Results in Figure 1 4 show that the addition of LAP to anti-TNF- ⁇ has an equivalent effect as compared to a combination of anti-TGF- ⁇ and anti-TNF- ⁇ antibodies.
  • soluble TNF receptors I & II and soluble TGF receptor II are added to cultures the resulting effects are not improved over either added alone. The cause of this is most likely due to inadequate dosing of soluble receptors.

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Abstract

La présente invention porte sur des procédés visant à induire une tolérance immunologique chez un animal recevant des cellules et des tissus hétérologues, notamment par greffe. Ces procédés comprennent des schémas posologiques de conditionnement à myélosuppression ou sans myélosuppression et consistent à administrer au receveur une quantité efficace d'un point de vue thérapeutique d'un mélange d'antagonistes du facteur de croissance transformant β (TGF-β) et d'antagonistes du facteur de nécrose tumorale α (TNF-α). Ce procédé comprend également l'utilisation d'anticorps anti lymphocytes T ainsi que des molécules de blocage costimulatrices telles que l'anticorps du ligand anti CD40 et CTLA4lg.
PCT/US2000/028982 1999-10-22 2000-10-20 Procede d'induction d'une tolerance immunologique pour des greffons heterologues WO2001030369A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13378/01A AU1337801A (en) 1999-10-22 2000-10-20 Process for inducing immunological tolerance for xenogeneic transplants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16097199P 1999-10-22 1999-10-22
US60/160,971 1999-10-22

Publications (1)

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WO2001030369A1 true WO2001030369A1 (fr) 2001-05-03

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PCT/US2000/028982 WO2001030369A1 (fr) 1999-10-22 2000-10-20 Procede d'induction d'une tolerance immunologique pour des greffons heterologues

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AU (1) AU1337801A (fr)
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036748A2 (fr) * 2000-11-03 2002-05-10 Nexell Therapeutics, Inc. Procedes d'affaiblissement et d'isolement de lymphocytes t, alloreactifs et reactifs vis-a-vis d'antigenes, provenant de cellules donneuses hematopoietiques
WO2004002425A2 (fr) * 2002-06-28 2004-01-08 Bio Transplant, Inc. Methodes permettant d'ameliorer l'acceptation d'une greffe par depletion des cellules souches hematopoietiques
US7285269B2 (en) 2002-12-02 2007-10-23 Amgen Fremont, Inc. Antibodies directed to tumor necrosis factor
US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EMERY D.W. ET AL.: "Enhancement of swine progenitor chimerism in mixed swine/human bone marrow cultures with swine cytokines", EXPERIMENTAL HEMATOLOGY, vol. 27, no. 8, August 1999 (1999-08-01), pages 1330 - 1337, XP002937188 *
JACOBSEN S.E.W. ET AL.: "Ability of flt3 ligand to stimulate the in vitro growth of primitive murine hematopoietic progenitors is potently and directly inhibited by transforming growth factor- beta and tumor necrosis factor-alpha", BLOOD, vol. 87, no. 12, 15 June 1996 (1996-06-15), pages 5016 - 5026, XP002937189 *
SABLINSKI T. ET AL.: "Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors", TRANSPLANTATION, vol. 67, no. 7, April 1999 (1999-04-01), pages 972 - 977, XP002937187 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036748A2 (fr) * 2000-11-03 2002-05-10 Nexell Therapeutics, Inc. Procedes d'affaiblissement et d'isolement de lymphocytes t, alloreactifs et reactifs vis-a-vis d'antigenes, provenant de cellules donneuses hematopoietiques
WO2002036748A3 (fr) * 2000-11-03 2003-10-02 Nexell Therapeutics Inc Procedes d'affaiblissement et d'isolement de lymphocytes t, alloreactifs et reactifs vis-a-vis d'antigenes, provenant de cellules donneuses hematopoietiques
WO2004002425A2 (fr) * 2002-06-28 2004-01-08 Bio Transplant, Inc. Methodes permettant d'ameliorer l'acceptation d'une greffe par depletion des cellules souches hematopoietiques
WO2004002425A3 (fr) * 2002-06-28 2004-08-26 Bio Transplant Inc Methodes permettant d'ameliorer l'acceptation d'une greffe par depletion des cellules souches hematopoietiques
US7285269B2 (en) 2002-12-02 2007-10-23 Amgen Fremont, Inc. Antibodies directed to tumor necrosis factor
US9420770B2 (en) 2009-12-01 2016-08-23 Indiana University Research & Technology Corporation Methods of modulating thrombocytopenia and modified transgenic pigs

Also Published As

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